key: cord-014946-l3vwb2v7 authors: tian, hong; wu, jing-yan; yin, shuang-hui; shang, you-jun; man, zi-ping; zhao, na; jin, ye; liu, xiang-tao title: genetic variation analyses of nsp2 gene of prrsv in ningxia hui autonomous region of china date: 2009-05-28 journal: virol sin doi: 10.1007/s12250-009-3017-4 sha: doc_id: 14946 cord_uid: l3vwb2v7 to gain a better understanding of the genetic diversity and evolution of prrsv in the ningxia hui nationality autonomous region (ningxia) of china, the nsp2 genes from a series of prrsv strains collected from the region in 2007 were partially sequenced. these sequences were then analyzed along with the classical strain (ch-1a) and two other epidemic strains sd (3) and sd2006. comparison of the nucleotide sequence with ch-1a indicated that nsp2 genes of seventeen ningxia isolates (nx strain) have deletions of 87 nucleotides. sequence analysis indicated that homology between the ningxia strain and ch-1a was 60.3%–79.9% in the nucleotide sequence, and homology between the nx strains and sd strains was 80.3%–98.8% in the nucleotide sequence. the nsp2 genes of the seventeen isolates had 74.9%–100% nucleotide sequence identities with each other. this study was undertaken to assess the regional variation of prevalent prrsv and to establish a sequence database for prrsv molecular epidemiological studies. seven orfs located at the 3`-terminus of the genome are postulated to encode viral structural proteins. among the nonstructural proteins, the multidomain protein nsp2 is the largest prrsv replicative protein. genetic analysis of both coronaviridae and arteriviridae (1) originally identified the protein to be spanning amino acids (aa) 384 to 1 363 (nsp2 aa 1 to 981); this was subsequently projected to comprise aa each sequence of the pcr products was determined by both enzyme analysis and sequencing. the viral rna of all clinical samples were extracted using a qiaamp viral rna mini kit (qiagen, germany), following the manufacturer's instructions. in brief, after lysis of the specimens, the mixture was applied to a spin column as described by the manufacturer's protocol. reverse transcription was performed at 42℃ for 1.5 h with 5μl total rna, and then pcr was carried out in a reaction volume of 50 μl containing 1μl of each primer (s1 and r1, 50 pmol/μl), 5 μl of 10×pcr buffer, 4 μl 2.5 mmol/l dntps, 3 μl of 25 mmol/l mgcl 2 , 1μl of rnase inhibitor (40u/μl), 0.5 μl of taq (5u/μl), 10 μl of reverse transcription production and 24.5 μl ddh 2 o. amplification conditions were 94℃ for 4 min, then 30 cycles that each consisted of denaturing at 94℃ 1min, annealing at 57℃ 40 s, and extension at 72℃ for 1 min, the sample was then heated at 72℃ for 10 min, and a final stage of 4℃ for 5 min. the amplified pcr products were analyzed by agarose gel electrophoresis and purified using the qiaquick gel extraction kit (qiagen) per the manufacturer's protocol. all nucleotide sequences were obtained from clinical sample rna by direct sequencing of pcr products. sequence data were analyzed using the method relative to strain ch-1a, the nsp2 genes of seventeen isolates have 87 nucleotides deleted at positions 2 937 to 2 948, 2 952 to 2 978, 2 986 to 3 003 and 3 007 to 3 036, respectively; these deletions are the same as those for the sd (3) and sd2006 strains (fig.2) . ningxia and ch-1a was 60.3%-79.9% in nucleotide sequence, and homology between ningxia and the common sd strain was 80.3%-98.8% in nucleotide sequence. the nsp2 genes of seventeen isolates had 74.9%-100% nucleotide sequence identity with each other (fig.3) . diversity and evolution of a newly emerged north american type 1 porcine arterivirus: analysis of isolates collected between financial evaluation and decision making in the swine breeding herd key: cord-014927-g4r7j8hk authors: shao, yi-ming title: aids research and its role in china’s aids prevention and control policies date: 2008-01-13 journal: virol sin doi: 10.1007/s12250-007-0042-z sha: doc_id: 14927 cord_uid: g4r7j8hk by the end of 2005, the estimated number of hiv infected people in china was 650,000. the seriousness of the epidemic calls for effective control measures to tackle the problems in order to avoid the tragedy in africa from happening in china. “prevention first” is the cornerstone of the country’s health policy. on 2003 world aids day, premier jiabao wen announced a new national aids control policy, “four frees and one care”. this policy clearly shows that the chinese government has once again taken full responsibility to solve public health problems and has profound impact far beyond the aids field. in early 2006, the central government put scientific and technology innovation as a national priority and set the target to build an innovative china by year 2020. since then, the government has been increasing investment in science and technology with major emphasis on both infectious diseases control and new drug research and development. for the first time, development of 100 new drugs and control of major infectious diseases (aids, hbv, tb and other emerging infectious diseases) have been selected as national key scientific projects. china’s best minds in related fields will be pooled to work together in order to remove the technical barriers blocking efficient control of the major infectious disease in china. knowledge on molecular epidemiology, immunology, pathogenesis, haart, as well as hivdr strains will certainly provide urgently needed scientific information for china’s aids control program. only evidence-based strategy from good research will provide long-term effective control of aids. hiv/aids was introduced to china in the mid 1980s by foreign travelers and blood products (12) . the epidemic in china began at the end of the 1980s, when idus in ruili, a small town bordering myanmar in yunnan province, were found to be infected by hiv. the initial epidemics were localized along china's southwest border regions, mostly in idu populations (1, 5) . by the mid 1990s, the hiv/aids epidemic was scaled-up by both further spread of drug abuse in other regions and blood contamination in the illegal plasma collection activities in central china (13, 14) . 422 virologica sinica vol.22, no 6 there-after, a steady increase via sexual transmission has been observed, indicating that the epidemic has entered the general population (6) . by the end of 2005, the estimated number of hiv infected people in china was 650 000 (10) . the seriousness of the epidemic calls for effective control measures to tackle the problems in order to avoid the tragedy in africa from happening in china (8, 11) . national sentinel surveillance of hiv infection in china from 1995 to 1998 towards a new generation of vaccines: the cytokine il-12 as an adjuvant to enhance cellular immune responses to pathogens during prime-booster vaccination regimens progress in the development of an hiv-1 vaccine a call for replicating vector prime-protein boost strategies in hiv vaccine design hiv infected people were first identified in intravenous drug users in china replicating rather than nonreplicating adenovirus-human immunodeficiency virus recombinant vaccines are better at eliciting potent cellular immunity and priming high-titer antibodies state council aids working committee office and un theme group on hiv/aids in china the ministry of science and technology, and the ministry of finance of people's republic of china joint united nations programme on hiv/aids and world health organization hiv-1 drug resistance in china: nation-wide survey and analysis of impacting factors in the national arv treatment program. tupp0302 , 3rd ias conference on hiv pathogenesis and treatment detection of antibody to lav/htlv-iii in sera from hemophiliacs in china control of transmission of hiv among drug users and commercial blood donors the epidemiological study of hiv infection among paid blood donors in one county of china key: cord-338606-6nk2ij20 authors: abdel-moneim, ahmed s.; moore, matthew d.; naguib, mahmoud m.; romalde, jesus l.; söderlund-venermo, maria title: wsv 2019: the first committee meeting of the world society for virology date: 2019-12-19 journal: virol sin doi: 10.1007/s12250-019-00189-y sha: doc_id: 338606 cord_uid: 6nk2ij20 the world society for virology (wsv) was founded and incorporated as a nonprofit organization in the united states in 2017. wsv seeks to strengthen and support both virological research and virologists who conduct research of viruses that affect humans, other animals, plants, and other organisms. one of the objectives of wsv is to connect virologists worldwide and support collaboration. fulfilling this objective, virologists from fourteen countries in north america, europe, africa, asia, and the middle east met on 25–27th august 2019 in stockholm, sweden at the karolinska university hospital for the first committee meeting of wsv. this meeting included compelling keynote and honorary speeches and a series of 18 scientific talks were given encompassing a diverse array of subjects within virology. followed by the scientific session, a business session was held where multiple aspects and next steps of the society were discussed and charted out. the world society for virology (wsv) was founded in 6 may 2017 and incorporated on 14 december 2017 as a non-profit organization, in the state of massachusetts in the united states of america. the overall mission of wsv is to strengthen research on viruses affecting humans, animals, plants and other organisms. the wsv's main objectives are to connect virologists worldwide, to provide educational resources, and to support a network of scientific collaboration among virologists, with a goal of helping members to advance their careers and to obtain funding, resources, and recognition for their achievements. wsv also collaborates with other international organizations and virology societies (abdel-moneim et al. 2017 ). the first committee meeting of the wsv was supported by the swedish research council and organized at the karolinska university hospital (kuh), stockholm, sweden from 25 to 27th august 2019. it included a series of presentations in different fields of virology and also panel discussions of the future plans of the wsv, including the first wsv virology congress. following the welcome dinner on 25 august, the meeting kicked off on 26 august. with an introduction and welcome message from prof. kristina broliden, division of infectious diseases, department of medicine solna karolinska institutet (ki), sweden. this was followed by the presentations of the two keynote speakers: prof. anna martling, dean, ki and prof. anna färnert, head of division for infectious diseases, kuh, on the current and perspective work of ki and kuh. this was followed by a group picture (fig. 1) , a picture of the honorary speakers with meeting chairman (fig. 2) and a series of presentations. the first presentation was delivered by prof. ahmed s. abdel-moneim (taif university, saudi arabia and beni-suef university, egypt) who outlined the origin of the wsv, the stages of foundation, and the recruitment of volunteer teams for different committees. he also outlined the expansion of the wsv from the few early members to the current 920 members from more than 80 countries. he also paid homage to the late managing committee member, prof. michael g. rossmann (30 july 1930-14 may 2019; purdue university), and acknowledged his role in attracting scientists to join the wsv (fig. 3 ). in the scientific session, there was a series of presentations on different topics of virology: emerging and re-emerging viruses in the session break, promotion videos for wsv were recorded in many different languages (wsv 2019). on 27 august, in the morning session, ahmed s. abdel-moneim presented the current and future plans of the wsv, after which matthew d. moore presented the steps that need to be taken to register wsv as a non-profit organization with 501(c) (3) status. following these two presentations, a series of sessions concerning the future plans for wsv were conducted. the board members discussed the wsv bylaws and the election of wsv officers. the bylaws draft and the election criteria and rules have since been approved after extensive modifications. the election is planned to start in the end of-november 2019, when an election call is submitted to all wsv members. other different topics were also thoroughly discussed, including online training, webinars, partnership with national and international societies and organizations, creating wsv educational resources, and determining the suitable time and place for the first wsv congress. sciences, leeds, uk, prof. sayed f. abdelwhab, department of microbiology, minia university, el-minia, egypt. the wsv 2019 came to a close with summary remarks and honoring kristina broliden by a plaque for her great effort to host a successful meeting. this was followed by a tour of ki. launching a global network of virologists: the world society for virology (wsv) wsv (2019) world society for virology the first committee meeting of the world society for virology the following committee members that were unable to join the meeting in person in stockholm, and had participated with advance suggestions and ideas: key: cord-322005-70snojec authors: yao, pingping; zhang, yachun; sun, yisheng; gu, yulin; xu, fang; su, bo; chen, chen; lu, hangjing; wang, dehui; yang, zhangnv; niu, biao; chen, jiancai; xie, lixia; chen, lei; zhang, yajing; wang, hui; zhao, yuying; guo, yue; ruan, juncheng; zhu, zhiyong; fu, zhenfang; tian, dayong; an, qi; jiang, jianmin; zhu, hanping title: isolation and growth characteristics of sars-cov-2 in vero cell date: 2020-06-19 journal: virol sin doi: 10.1007/s12250-020-00241-2 sha: doc_id: 322005 cord_uid: 70snojec nan similar sequences to reported sars-cov-2 strains, except for 8#. all of seven covid-19 cases, from different cities of zhejiang province, were confirmed by real-time rt-pcr around 2020/01/26 (2020/01/24-2020/01/28). basic information of those patients were listed in table 1 . vero cells were seeded overnight in mem containing 10% fbs before samples (processed swabs or sputum) were inoculated. after incubation for an hour, the cells were replenished with fresh maintain medium (mem containing 3% fbs). the cells were observed daily for cytopathic effect (cpe) from the 2nd to the 7th days ( fig. 1a) . at the day 7, the cell culture supernatant was collected for virus titration (table 1) . it is well known that mutation on viral genes may have an impact on virulence, immunogenicity and other characterizations of viruses. in a recent report, 149 mutations were found among 103 sequenced isolates of sars-cov-2 . to investigate whether our viruses show mutations different from other reported sars-cov-2, all the seven isolates were sequenced at the 3rd passage and the sequences were aligned with coding sequence (cds) of sars-cov-2 wuhan-hu-1 (nc_045512). three and four of the seven isolates belong to the l and the s clades, respectively (table 1) . it is interesting to note that these seven patients infected with sars-cov-2 have high viral load in the early stage of clinical sign, which is consistent with previous reports (kim et al. 2020; zou et al. 2020) . as shown in fig. 1b , genomic sequences of the viruses share sequence identity higher than 99.9%. the phylogenetic analysis of the isolated viruses together with other available viruses indicates that the seven isolates are closely related to each other, as well as to the other published sequences, which is consistent with the previous studies ren et al. 2020; zhou et al. 2020) (fig. 1c) . and, each virus has 2-5 mutations except for isolate 12#, which is exactly the same as wuhan-hu-1. it's worth noting that apart from the 5 mutations, sequence of (fig. 1d, 8# p3 ). to confirm this, isolate 8# at passage 2 and 4 was sequenced (fig. 1d, 8# p2, 8# p4) . sequencing result showed that the nesting peak gradually weakened from p2 to p4 (fig. 1d) fig. 1 characteristics of sars-cov-2. a the cytopathic effect was observed in vero cells infected with the isolated viruses at 5 dpi. b nucleotide mutations of isolated viruses at passage 3. the sars-cov-2 infected-cells were homogenized with trizol, and total rna was extracted and was used for rt-pcr as described below. firststrand cdna was synthesized with oligo(dt) primers, then the complete genome was segmented amplified and sequenced. sequencing results were analyzed by snapgene. c phylogenetic tree was generated by clustal omega based on amino acid sequence of seven isolates and part of the available viruses. d sequence results of 8#, from passage 2 to passage 4. e the multiple growth curve of isolated viruses. the virus growth curves were drawn based on the viral titers measured at each indicated time point. the growth curves were obtained by three independent experiments. infected with serial tenfold diluted supernatant. cpe were observed and recorded at 96 h after infection. virus titer was calculated by reed-muench method (kint et al. 2015) . as shown in fig. 1e , all the seven isolated virus showed similar growth character: virus titers are detectable at 1 dpi, peak at 3 dpi, and then begin to decline. among the seven isolates, the peak titer of isolate 12# reached 7.75 tcid 50 /ml, which is much higher than the other ones. in conclusion, seven sars-cov-2 strains were isolated, sequenced and characterized in vero cells, and a deletion mutation was identified after short passage in vero cells. these results shall facilitate the understanding of the characteristics of sars-cov-2 in vitro. viral load kinetics of sars-cov-2 infection in first two patients in korea quantification of infectious bronchitis coronavirus by titration in vitro and in ovo severe acute respiratory syndrome coronavirus2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding identification of a novel coronavirus causing severe pneumonia in human: a descriptive study on the origin and continuing evolution of sars-cov-2 a pneumonia outbreak associated with a new coronavirus of probable bat origin sars-cov-2 viral load in upper respiratory specimens of infected patients acknowledgements this work was partially supported by the national natural science foundation of china (31702248). conflict of interest the authors declare that they have no conflict of interest.animal and human rights statement the study was approved by the ethics committees of prevention and control of infectious disease of zhejiang province. all participants provided written informed consent. written consents were obtained from all parents involved in the study. key: cord-356154-ifb3qiz7 authors: zhang, rong; chen, xiaohua; huang, yuqing; zhang, qi; cheng, yan; zhang, nan; zhang, haibo; yang, bo; liu, fang; liu, yingle; lan, ke title: a study of two cases co-infected with sars-cov-2 and human immunodeficiency virus date: 2020-09-07 journal: virol sin doi: 10.1007/s12250-020-00280-9 sha: doc_id: 356154 cord_uid: ifb3qiz7 nan patient 2, male, 37 years old, unmarried, lived in wuchang district, wuhan city before onset of the disease, developed fever without any cause in early january, when chest pain was intermittent. ct scan results in early february indicated lesions in bilateral lungs (supplementary table s1 ), but the result of the sars-cov-2 nucleic acid test was negative. patient 2 was severely ill and admitted on february 11, with significant wheezing symptoms. low-dose hormonal anti-inflammatory therapy began on february 14 and continued until patient 2 became relieved on february 18. patient 2 was also treated with antiviral, anti-infective, and other symptomatic treatments. however, the patient's condition deteriorated again on february 20, and the nucleic acid test results were single positive for covid-19 sars-cov-2. after that, three more sars-cov-2 nucleic acid test results were negative. the patient was treated with tocilizumab injection on march 5 due to his critical condition. however, two sars-cov-2 antibody tests of patient 2 obtained negative results on march 5 and 6. the patient was tested for hiv antibodies on march 7 and 8, respectively, and the results were both positive (table 3 ). after that, the test results were reported to the wuhan center for disease control and prevention for review, the final reexamination result is still positive. on march 8, the patient was transferred to a specialized hospital for further treatment. we report the early clinical treatment of two covid-19 patients diagnosed with aids. both patients showed typical symptoms of sars-cov-2 infection throughout the disease, such as fever, chest tightness, and shortness of breath. ct scans of both patients can be used as imaging evidence for covid-19 infection. meanwhile, ct scans of both patients during treatment showed disease deterioration. the laboratory test of sars-cov-2 mainly consists of sars-cov-2 virus nucleic acid test and serological test. specific antibodies are generally produced 3-5 days after the onset of covid-19. an increased igm antibody indicates recent acute infection, while an increased igg antibody indicates a previous infection. in this study, we found unnormal changes of viral nucleic acid and antibodies in two notes: nd, no data; ?, positive; -, negative we assumed that hiv infection had damaged their immune systems; this could also explain why the patient tested negative for sars-cov-2 antibodies in the late stages of treatment when the disease became worse. the destruction of the immune system by hiv may have affected the normal humoral immune process. meanwhile, considering the latent period of hiv is usually quite long, 8-9 years, on average, we suspect that co-infection with sars-cov-2 may increase the pathogenicity of both and lead to the destruction of the immune system. the patient's symptoms seemed to ease in the early stages of treatment, but as aids progressed, the immune system was destroyed, making covid-19 condition worse and the treatment less effective. it is also worth noting that the patient was admitted to a designated hospital for covid-19 treatment, where an il-6 inhibitor (tocilizumab injection) was used to relieve severe inflammation. sars-cov-2 infected patients may develop an uncontrolled immune response known as a cytokine storm, which leads to the massive production of proinflammatory cytokines and other inflammatory proteins, such as il-6, tnf-a, and gm-csf, causing severe lung tissue damage, respiratory failure, and even death (vaninov 2020; mehta et al. 2020). interleukin 6 (il-6) is a type of cytokine produced by activated t cells and fibroblasts. it turns the precursor of b cells into antibody-producing cells. synergizing with colony-stimulating factor, it can promote the growth and differentiation of original bone marrow-derived cells and enhance the lysis function of natural killer cells (smits et al. 2011; tanaka et al. 2014) . il-6 appears to be a significant contributor to coronavirus-mediated respiratory failure (herold et al. 2020; liu et al. 2020) . in this study, both severely ill patients presented with abnormally high levels of il-6. the patient 1 began treatment with tocilizumab on march 4. the data indicated that the patient's symptoms were relieved, and there was no fever after 3 days of treatment with tocilizumab. the clinical manifestations of patient 1 after the administration of il-6 inhibitors indicated that il-6 could be a key target for inhibiting covid-19 inflammation. although the patient's condition deteriorated again later, we suspect that this was related to the destruction of the immune system in patient 1 with aids. patient 2 was also treated with tocilizumab on march 5. because 1 day after the start of the medication, the patient was transferred to a specialized hospital for further treatment, the treatment effect still needs to be evaluated by the following study. in general, the blocking of the il-6 receptor with tocilizumab has a particular effect on the treatment of covid-19 patients with severe disease, but it may have little effect on patients with fig. 1 the clinical courses of two cases co-infected with sars-cov-2 and hiv. r. zhang et al.: two cases co-infected with sars-cov-2 and hiv autoimmune diseases, which should be considered comprehensively in clinical practice. covid-19 patients with immunodeficiency disease may cause more severe illness and poor treatment response due to the destruction of the immune system. however, the current screening work for patients with immunological diseases is still not very adequate; the therapeutic effect for an immunocompromised patient without the corresponding targeted treatment may not be evident (castro and gourley 2010) . therefore, we suggest that patients with covid-19 should be tested for immune diseases to achieve targeted treatment. diagnostic testing and interpretation of tests for autoimmunity elevated levels of il-6 and crp predict the need for mechanical ventilation in covid-19 the role of interleukin-6 in monitoring severe case of coronavirus disease 2019 covid-19: consider cytokine storm syndromes and immunosuppression distinct severe acute respiratory syndrome coronavirusinduced acute lung injury pathways in two different nonhuman primate species il-6 in inflammation, immunity, and disease vaninov n (2020) in the eye of the covid-19 cytokine storm acknowledgement this study was supported by the special fund for key: cord-014932-web2tdef authors: li, jian-qiang; liu, ji-xing; lan, xi; cheng, jie; wu, run; lou, zhong-zi; yin, xiang-ping; li, xue-rui; li, bao-yu; yang, bin; li, zhi-yong title: cloning the structure genes and expression the n gene of porcine epidemic diarrhea virus dx date: 2009-05-28 journal: virol sin doi: 10.1007/s12250-009-2982-y sha: doc_id: 14932 cord_uid: web2tdef the structure genes spike (s), nucleocapsid (n), membrane (m), small membrane (sm) of a porcine epidemic diarrhea virus (pedv) strain dx isolated in gansu province, north-west of china, were cloned, sequenced and compared with published sequences of pedv strains. the nucleotide sequences encoding the entire s, sm, m and n genes open reading frame (orf) of dx were 4 152, 231, 681 and 1 326 bases long respectively. there were transcription regulatory sequences (trss) upstream of the initiator atg of the s, n and m genes. the amino acids sequences of s, m and n contained 30, 3 and 7 potential asparagine (n)-linked glycosylation sites. homologous analysis and phylogenetic trees showed that dx had the closest relationship with strains ljb/06, js-2004-2z and ch/hljh/06 that were also isolated from china and indicated the prevalence of some pedv isolates in china were widespread since the js-2004-2z strain originated from the south of the china, and ljb/06 and ch/hljh/06 were isolated from northeast china. the n gene was cloned using two primers which contained nco i and bamh i restriction enzyme sites and subcloned into expression vector pet30a. the recombinant plasmid was then transformed into e.coli rossta. sds-page showed there was a protein of about 55kda as expected and western blot indicated the n protein had biological activity. several subgenomic mrnas for the production of structure and non-structure proteins (6, 18) . (1, 5) . the m and sm proteins are essential for viral envelope formation and release, the m protein also can stimulate the production of interferon (ifn) (1, 7) . the n protein participates in transcription of the viral genome, the formation of the viral core, and packaging of viral rna. in the early stage of pedv infection, the pig produces high levels of antibodies against the protein n. since the n protein is highly conservative in the coronaviruses, it has a good response and immunogenicity, so it is the best candidate protein for early diagnosis reagents and vaccine development. although some nucleotides sequences of pedv isolated from china has been reported, the data of dx structure genes are useful for furthering the study of the molecular biology of pedv strains that are prevalent in china, especially in the north-west. in this study, the structure genes have been cloned and the n protein has been expressed. the pedv dx strain was collected from the feces of piglets suffering from severe diarrhea in gansu north-west china. total rna was isolated from purified feces samples and extracted using a rna extraction kit (qiagene, germany) following the manufacturer's instructions. the rt-pcr amplifications were carried out using five primer sets (table 1) and an rt-pcr amplification kit (toyobo, japan). the products were ligated with the pmd18-t vector (takara) and transformed into the competent e. coli jm109. positive clones were sequenced by the takara biotechology (dalian) co.ltd. phylogenetic analysis was performed for the amino acids sequence of peev dx strain structure genes and compared to the pedv reference strains retrieved from genbank. the sequence data were aligned using two pcr primers of pedv n, which contain specific restriction enzyme digestion sites depending on the multiple cloning sites contained in the expression vector pet30a were used. the sense primer the positive recombinant transformant was grown in lb media containing 100μg/ml amp while shaking at 220 r/min at 37℃ and then induced with iptg. cells were harvested by centrifugation at 12000 r/min for 1 min. total cellular pellets were analyzed by 10% sds-page and western blotting. using rt-pcr, two overlapping products of the s gene of approximately 4.2 kb were amplified ( fig.1 to analyze the phylogenetic relationships between dx and other pedv strains isolated in various parts of the world, we constructed 3 neighbor-joining phylogenetic trees (fig.2, fig.3, fig.4) the recombinant plasmid pet30-pn was digested with ncol i and bamh i producing two fragments of 5 400bp (pet30a) and 1 400bp. pcr identification confirmed the presence of the 1 400bp fragment (fig.6) . to obtain the expressed recombinant protein, the time course expression of recombinant plasmid pet-pn was induced by iptg with ranging from 0.5-1 mmol/l at 2, 3, 4, 5 and 6 h respectively. sds-page revealed that the best condition was the iptg with 1 mmol/l at 6 h. the expressed proteins had a molecular weight of 55kda as expected (fig.7) . western blot showed the the n protein is a rna binding protein, it contains basic amino acids such as arginine and lysine and shows a high degree of alkalinity, it is also the only phosphorylated structure protein in the coronavirus. the serine residue is a potential phosphorylation site (2). thedx n protein contained 37 arginine, 33 lysine and 36 serine. the n protein is the most highly expressed protein in coronavirus infected cells and is about 45kda-60kda (12) . the pedv n protein is about 49kda-58kda in different expression systems (2). in this study, an n protein of about 50kda (the expressed vector was abou 5kda) was obtained using the e. coli system, the result is consistent with other e. coli expression system, although there are differences in the observed weights (16) . western blot results showed the expressed n protein reacted with the antibodies, which indicated that the expressed n protein has the biological activity. our results provide a basis for further development of a diagnosis method. the production of recombinant infectious di-particles of a sequence of the spike protein of the porcine epidemic diarrhea virus characterization of the structural proteins of porcine epidemic diarrhea virus, strain cv777 cloning and sequence analysis of the m gene of porcine epidemic diarrhea virus ljb/03 cloning and sequence analysis of the n gene of porcine epidemic diarrhea virus ljb/03 serologic study of the occurrence of epizootic viral diarrhea in swine in switzerland cloning and sequence analysis of the korean strain of spike gene of porcine epidemic diarrhea virus and expression of its neutralizing epitope in plants cloning and sequence analysis of the nucleocapsid gene of porcine epidemic diarrhea virus chinju99 the molecular biology of coronaviruses erhebungen uber porcine coronaviren in osterreich ii porcine epidemic diarrhea virus (pedv) der schweine complete genome sequence of transmissible gastroenteritis coronavirus pur46-mad clone and evolution of the purdue virus cluster isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages a novel internal open reading frame product expressed from a polycistronic of porcine edpidemic diarrhea virus may not contribute to virus attenuation disease of swine a cell culture vaccine against bovine ephemeral fever cloning and sequence analysis of the spike gene of porcine epidemic diarrhea virus chiju99 the nucleocapsid protein of coronavirus mouse hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein a1 in vitro and in vivo key: cord-332672-fbwz8oxp authors: wang, manli; hu, zhihong title: bats as animal reservoirs for the sars coronavirus: hypothesis proved after 10 years of virus hunting date: 2013-10-30 journal: virol sin doi: 10.1007/s12250-013-3402-x sha: doc_id: 332672 cord_uid: fbwz8oxp recently, the team led by dr. zhengli shi from wuhan institute of virology, chinese academy of sciences, and dr. peter daszak from ecohealth alliance identified sl-covs in chinese horseshoe bats that were 95% identical to human sars-cov and were able to use human angiotensin-converting enzyme 2 (ace2) receptor for docking and entry. remarkably, they isolated the first known live bat sl-cov that replicates in human and related cells. their findings provide clear evidence that some sl-covs circulating in bats are capable of infecting and replicating in human (ge x y, et al., 2013). sars-cov remains elusive. although it is suggested that bats are the natural reservoirs for sars-cov, isolation of a sars like virus (sl-cov) from bats have been unsuccessful. to trace the origin of the sudden emerging sars-cov, molecular epidemiological studies have been conducted by different research groups. in 2003, guan et al. isolated sars-covs from himalayan palm civets and two other species in a live-animal market in guangdong, china (guan y, et al, 2003) . the chinese sars molecular epidemiology consortium suggested that the early-phase human sars-cov strains may have originated from wild animals (the chinese sars molecular epidemiology consortium, 2004) . these and other evidences suggested that palm civets were the direct source since the isolates from civets were highly related to human isolates from 2002 -3 and 2003 -4 sars pandemic (guan y, et al, 2013 song h d, et al., 2005; wang m, et al, 2005) . since 2004, sl-covs have been identified from bats by several research groups including dr. shi's lab lau s k, et al, 2005) . these bat isolates are more genetically diverse and share an overall nucleotide identity of 88% to 92% to the sars-covs from humans or civets, resulting in the hypothesis that bats may be the natural hosts of sars-cov. however, there are still some missing links between previously characterized sl-covs from bats and sars-cov that precipitated the 2002-3 outbreaks. 1) albeit the overall genome sequence similarity, there are significant differences in spike (s) protein between the previously known sl-covs and sars-covs. the sequence identity of s1 fell to 64%, accompanying with insertions and (or) mutations in this region. s1 contains the receptor binding domain (rbd), which plays a key role in receptor recognition and is a major determinant of host range and cross-species infection of sars-cov. it was suggested that the previously known bat sl-cov stains cannot jump from bats to civets or humans owing to the significant differences between their rbds (li f, 2013); 2) although sl-covs have been identified from different bat species, isolation of a live sl-covs from bats never succeed; 3) no native sl-cov from bats could use ace2 as receptors and infect human cells, only when its rbd is replaced with the counterpart from a human sars-cov strain (li w, et al, 2003; becker m m, et al, 2008; ren w, et al, 2008) . therefore, these sl-covs seem unlikely to be the immediate precursors of civet or human sars-covs (li f, 2013). after 10 years virus hunting, ge et al. are now able to fill in important 316 missing links associated with sars origin and epidemic (fig. 1) . firstly, they identified two novel sl-cov stains rsshc014 and rs3367 in chinese horseshoe bats, which show higher sequence similarity (~95% identity) with human sars-cov than observed previously (lau s k, et al, 2005; li f, 2013; ren w, et al, 2006; tong s, et al, 2009 ). particularly, their rbds are more closely related to (85% aa identity for rsshc014 and 96% for rs3367) sars-cov rbd, with perfect sequence alignment and absence of any deletion or insertion. secondly, they reported the first isolation of a live bat sl-cov strain (sl-cov-wiv1) with vero e6 cells. this isolate is almost identical to its parental virus rs3367, with 99% nt identity and 100% aa identity even in the highly variable s1 region. thirdly, by using this valuable isolate wiv1, ge et al. demonstrated for the first time that a bat sl-cov strain can exploit human-, civet-and bat-derived ace2 as its cellular entry receptors and replicated efficiently in hela cells which express these receptors. they further assessed the host ranges of wiv1 with a variety of cell lines and found that cells from different species (human, pig, and bat) are able to support the virus replication at different levels. in addition, wiv1 can be neutralized by different sarspatient sera collected in 2003. these findings suggested a much closer relationship between bat wiv1 strain and sars-cov. interestingly, although not being further discussed in their publication, one of the five key residues in rbd, the 479 aa, is an asparagine in the rs3367. the residue 479 is known to be an asparagine only in human sar-covs, but not in the previously identified bat sl-covs or civet sar-covs. it is proposed that an asparagine at position 479 has a higher binding affinity with human ace2 and is likely to determine whether the virus can infect humans (li f, 2013) . the change of the position 479 may have played a role in the adaptation of rs3367 to an ace2 receptor and the cross-species infections of the virus. in summary, ge et al.'s findings solved fundamental questions surrounding the genesis of the sars-cov 2003 epidemic. they provided a plausible scenario of direct bat-tohuman jump of sars-cov without the transmission by interim hosts. bats are known to be important natural reservoirs of many zoonotic viruses, for example, the recently emerged middle east respiratory syndrome coronavirus (mers-cov) (chan j f, et al, 2012) . the publication by ge et al. emphasized bats as public health threats and highlighted the importance of epidemiological and laboratory studies of bats-harboring pathogens. full-length genome sequences of two sars-like coronaviruses in horseshoe bats and genetic variation analysis difference in receptor usage between severe acute respiratory syndrome (sars) coronavirus and sars-like coronavirus of bat origin cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human molecular evolution of the sars coronavirus during the course of the sars epidemic in china detection of novel sars-like and other coronaviruses in bats from kenya sars-cov infection in a restaurant from palm civet key: cord-352988-9ey3ir5e authors: xiang, yang-fei; ju, huai-qiang; li, shen; zhang, ying-jun; yang, chong-ren; wang, yi-fei title: effects of 1,2,4,6-tetra-o-galloyl-β-d-glucose from p. emblica on hbsag and hbeag secretion in hepg2.2.15 cell culture date: 2010-10-08 journal: virol sin doi: 10.1007/s12250-010-3144-y sha: doc_id: 352988 cord_uid: 9ey3ir5e a polyphenolic compound, 1,2,4,6-tetra-o-galloyl-β-d-glucose (1246tgg), was isolated from the traditional chinese medicine phyllanthus emblica l. (euphorbiaceae) and assayed for its potential as an anti-hepatitis b virus (hbv) agent. the cytotoxicity of 1246tgg on hepg2.2.15 as well as hepg2 cells was determined by observing cytopathic effects, and the effects of 1246tgg on secretion of hbsag and hbeag in hepg2.2.15 cells were assayed by enzyme immunoassay. results indicates that treatment with 1246tgg (6.25 μg/ml, 3.13 μg/ml), reduced both hbsag and hbeag levels in culture supernatant, yet the inhibitory effects tend to decline with the assay time. this study provides a basis for further investigation of the anti-hbv activity and possible mechanism of action of 1246tgg. hepatitis b virus (hbv), an important causative pathogen of cirrhosis-related liver failure and hepatocellular carcinoma (hcc), is a public health problem of worldwide concern, and is responsible for one million deaths each year worldwide [7] . china has the biggest hbsag carrier population with more than one-third of the world's 350-400 million chronic hbv carriers [11] . though and hepatitis b e antigen (hbeag) [12] . currently, two therapies, conventional interferon alfa (ifnα) and used in the southwest of china for treating eczema, wart, diarrhea, and headache after a fever [13, 18] . acyl glucoses have been shown to be potent antiviral agents against herpes simplex virus (hsv) [3, 9] , human immunodeficiency virus (hiv) [2, 10] , severe acute respiratory syndrome coronavirus (sars-cov) [15] as well as other viruses. here, we investigated the anti-hbv activity of 1246tgg by detecting the hbsag and hbeag secretion levels in hepg2.2.15 cell culture, a cell line derived by transfection of cloned hbv dna into human hepatoblastoma cell line hepg2 and used to assay for anti-hbv agents [4] . compound 1246tgg was isolated and its structure was identified by the state key laboratory of phytochemistry and plant resources in west china in the kunming institute of botany, chinese academy of sciences, using procedures as described in a previous paper [18] . briefly, the ethanol extract of the fresh leaves and branches of p. emblica was suspended into water and then extracted with diethyl ether. the diethyl ether layer was partitioned between hexane and methanol, and the methanol layer was further chromatographed successively over sephadex lh-20, silica gel, mci-gel chp 20p and chromatorex ods to obtain the desired compound (purity > 95%) in the form of a pale amorphous powder. its structure was identified by comparison of the physical and spectral data with literature values and the 1h-1h cosy spectrum (fig. 1) . the isolated compound was then dissolved in dimethyl sulfoxide (dmso) before use. the final concentration of dmso was less than 0.2%. hepg2 and hepg2. only 5% fbs was added. cells were cultured at 37°c in a humid atmosphere with 5% co 2 . the cytotoxicity assay was performed by observing cytopathic effect (cpe). hepg2 or hepg2. day. cytopathic effects were classified into five levels as follows: >75%, between 75% and 50%, between 50% and 20%, <25% and no cytopathic effect. the assay was performed in four parallel wells. concentrations without cytotoxicity were used for hbsag and hbeag inhibition assay. for hbsag and hbeag secretion assay, hepg2.2.15 cells were seeded onto 24-well tissue culture plates (corning) 3×10 4 cells/well and incubated at 37℃ in a humid atmosphere with 5% co 2 for 24 h before the test. similarly, 1246tgg at two concentrations (6.25 µg/ml and 3.13µg/ml) were diluted in maintenance media and added every 3 d during the 10 d treatment period, namely, on the 1st, 4th and 7th day. before the second treatment of 1246tgg (on the 4th and 7th day) and at the end of the treatment period (on the 10th day), culture media of each compound concentration was collected and stored at -20℃. hbsag and hbeag levels in culture media were measured using an enzyme immunoassay kit (intec) according to the manufacturer's instructions and absorbance at 450nm was measured using an elisa reader (bio-rad). the assay was performed in four parallel wells. results were expressed as  or ±s.d. of four parallel wells. statistical calculations were carried out with the spss 13.0 for windows software package (statistica). one-way anova was used for statistical analyses; p values < 0.05 were considered to be significant. the results of cytotoxicity assay are listed in table 1 . on the 10th day (after the third treatment), 1246 tgg at concentrations ranging from 200µg/ml to 12.5µg/ml all induced cytophathic effects to different extents. to confirm whether the cytotoxicity caused by 1246tgg was specific to hbv dna transfected to determine the inhibitory effects of 1246tgg on hbv antigen secretion, cells were treated with 1246tgg at concentrations of 6.25µg/ml and 3.13µg/ml every 3 d during the 10 d treatment period. as shown in polyphenols, especially flavonoid, phenolic acids and other derivates might be potential antiviral agents [14] . among these, galloyl glucoses, with various number of galloyl groups in the glucose core structure, penta-o-galloyl-β-d-glucose (pgg), was found to be efficient in inhibiting the ns3 protease of hcv [1] . pgg also decreased extracellular hbv in a dosedependent manner in hepg2.2.15 cell culture [8] . in this study, 1246tgg was isolated from traditional chinese medicine p. emblica and its activity in affecting hbv antigen secretion was reported for the first time. 1246tgg showed cytotoxicity towards hepg antiviral compounds from traditional chinese medicines galla chinese as inhibitors of hcv ns3 protease inhibitory effects of egyptian folk medicines on human immunodeficiency virus (hiv) reverse transcriptase inhibition of herpes simplex virus infection by tannins and related compounds a cell culture assay for compounds which inhibit hepatitis b virus replication human hepatic cell uptake of resveratrol: involvement of both passive diffusion and carrier-mediated process resveratrol in human hepatoma hepg2 cells: metabolism and inducibility of detoxifying enzymes hepatitis b virus infection in vitro antiviral activity of 1,2,3,4,6-penta-o-galloyl-beta-dglucose against hepatitis b virus studies on antiviral activity of several hydrolyzable tannins sulfated pentagalloyl glucose (y-art-3) inhibits hiv replication and cytopathic effects in vitro, and reduces hiv infection in hu-pbl-scid mice management of chronic hepatitis b: experience from china the hepatitis b virus ethnopharmacology of phyllanthus emblica l current status of natural products from plants as anti-herpes simplex virus 1 agents respiratory syncytial virus infection induces matrix metalloproteinase-9 expression in epithelial cells anti-cancer, anti-diabetic and other pharmacologic and biological activities of penta-galloyl-glucose contemporary clinical research of traditional chinese medicines for chronic hepatitis b in china: an analytical review phyllanemblinins a-f, new ellagitannins from phyllanthus emblica key: cord-005340-o7tkxs3s authors: fu, xinliang; wang, lifang; fang, bo; ma, ruirui; zheng, yun; huang, san; zhou, pei; cao, zongxi; tian, jin; li, shoujun; zhang, guihong title: import of rift valley fever to china: a potential new threat? date: 2016-10-27 journal: virol sin doi: 10.1007/s12250-016-3876-4 sha: doc_id: 5340 cord_uid: o7tkxs3s [image: see text] sinica and other journals have highlighted the enormous international challenge of emerging arboviral diseases, such as zika virus disease, dengue, and chikungunya (islam et al., 2015; maurice et al., 2015; dai et al., 2016; deng et al., 2016; song et al., 2016; xu et al., 2016; zhang et al., 2016; . rift valley fever (rvf) is a mosquito-borne disease caused by the rift valley fever virus (rvfv), which causes high levels of mortality and morbidity in domesticated animals and mild to serious disease in humans. since the virus was first described in kenya in 1931 (daubney et al., 1931) , no extensive human outbreaks of this disease were reported until 1951, when approximately 20, 000 individuals were infected in south africa (mundel and gear, 1951) . subsequently, there have been several rvf outbreaks with significant impact on human health in eastern africa and the arabian peninsula (meegan, 1979; cdc, 1998; shoemaker et al., 2002; woods et al., 2002) (http://www.who.int/csr/ don/2007_05_09/en/). at present, rvfv is present or endemic in al-most all african nations, as well as saudi arabia and yemen in the arabian peninsula (figure 1 ), and has the potential to expand to other regions. rvfv can be transmitted by many mosquito species (mainly from the genera aedes and culex), as well as flies and ticks (davies and highton, 1980) , and outbreaks of rvf are closely linked to heavy rainfall or flooding (sindato et al., 2011) . in addition, wildlife and livestock may play an important role in outbreaks in humans. human cases are primarily caused by bites from infected mosquitoes or by direct contact with tissues or fluids from infected animals (anyangu et al., 2010; baba et al., 2016) ; in addition, vertical transmission has also been reported in mosquito vectors, ruminants, and humans (arishi et al., 2006; adam and karsany, 2008) . in humans, the disease can present with fever, headache, arthralgia and muscle pain, acute hepatitis, or hemorrhagic and neurological manifestations, and the fatality rate can reach 50% in patients with hemorrhagic complications (swanepoel et al., 1979; madani et al., 2003) . although there are three approved veterinary vaccines, no vaccines are available for human use (http://www.oie.int/manual-ofdiagnostic-tests-and-vaccines-forterrestrial-animals/). on july 21, 2016, a 45-year-old male who had been working in luanda, angola, returned to china ( figure 1 ). he had been suffering symptoms of headache, fever, arthralgia and muscle pain since july 14, and had received treatment in a local hospital in angola. however, the symptoms persisted, and he arrived in beijing capital international airport in serious condition (http://www.who.int/ csr/don/02-august-2016-rift-valleyfever-china/en/). two days later, the national health and family planning commission of the people's republic of china reported this as the first imported case of rift valley fever in china (http://www.aqsiq. gov.cn/xxgk_13386/tsxx/yqts/20160 8/t20160803_471831.htm). the rvf outbreak that occurred in saudi arabia and yemen in 2000 resulted in 2171 infections and 245 deaths (shoemaker et al., 2002) , and this outbreak is reported to have been caused by the same strain of the rvf virus that caused the outbreak in kenya in 1997-1998 (shoemaker et al., 2002) . the outbreak in saudi arabia and yemen in 2000 was caused by infected animals imported from kenya (baba et al., 2016) . confronted with this imported case, the government in china has implemented surveillance, monitoring, and other measures to prevent the spread of rvf in china (http://www. nhfpc.gov.cn/yjb/s3578/201607/e1fc 91f93d034bd0b952b8745356bd65.s html). however, because conditions for the spread of rvf are currently present in china, there is a risk that rvf may spread from this imported case. firstly, mosquitoes, the rvf vector, are very active in summer, and aedes mosquitoes are widely distributed in china, especially southern china. secondly, there have been several rounds of heavy rainfall since june, and many provinces have experienced flooding. based on the lessons learned during the outbreaks in saudi arabia and yemen, and given these conditions, there is a high risk that the imported case may spread or even cause an outbreak, if proper measures to prevent and control rvf are not implemented. early detection of suspected cases is the key to ensure that timely control measures are implemented to prevent the spread and outbreak of emerging or re-emerging infectious diseases, such as severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and ebola, as well as rvf . however, cases of human infection with rvfv are generally asymptomatic, and most result in no more than a short febrile illness (madani et al., 2003) ; some animals may experience an inapparent infection (mansfield et al., 2015) . this is another challenge to detection of suspected cases. because it is a vector-borne disease, mosquitoes (especially aedes and culex) play an important role in the spread and outbreak of rvf; thus, eliminating the vector and preventing mosquito bites are the most effective means to control the spread of rvf. enhanced surveillance and monitoring of the numbers and infection level of the vector are also important. in addition, infection of livestock is a risk factor, because humans can be infected by direct contact with infected animals (anyangu et al., 2010) and mosquitoes can spread rvfv via infected animals. it is therefore advisable to suspend trading of livestock with countries experiencing an rvf epidemic and to implement a strong quarantine. meanwhile, the effectiveness of strengthening collaboration and coordination across sectors, such as government, the centers for disease control and prevention, researchers, and hospitals, to make prevention and control measures more efficient, was demonstrated when the mers case was imported in china (su et al., 2015; su et al., 2016b) and during the rvf outbreak in saudi arabia (himeidan et al., 2014) . development of an effective vaccine for prevention of rvf is urgent for both humans and animals. china has a large population, and its climate and the wide distribution of the mosquito vectors in south china are high risk factors for rvf outbreak. more importantly, once rvfv is introduced into permissive ecologies, it can become endemic, with the potential to spread into other non-endemic regions (murithi et al., 2011) . if there is an rvf outbreak in china, the disease may spread quickly to southeast asia and to other countries in asia. combined with the import of the zika and yellow fever viruses this year (su et al., 2016a; , importation of arboviral pathogens poses a huge challenge for china. therefore, all necessary measures should be taken to prevent and control rvf import and spread in china in the future. this project was supported in part by the national natural science foundation of china (grant no. 31402224 and 31272564) , the public welfare special funds for agriculture scientific research (grant no. 201203039) , and the modern agro-industry technology research system (cars-36). the authors declare that they have no conflict of interest. this article does not contain any studies with human or animal subjects performed by any of the authors. # these authors contributed equally to this work. emerg microbes infect, 5: e58. centers for disease control and prevention (cdc) zika virus and zika fever key: cord-005397-i7x50taa authors: wang, wen-juan; li, xiong; wang, li-hua; shan, hu; cao, lei; yu, peng-cheng; tang, qing; liang, guo-dong title: preparation and identification of anti-rabies virus monoclonal antibodies date: 2012-06-09 journal: virol sin doi: 10.1007/s12250-012-3242-0 sha: doc_id: 5397 cord_uid: i7x50taa to provide a foundation for the development of rapid and specific methods for the diagnosis of rabies virus infection, anti-rabies virus monoclonal antibodies were prepared and rabies virus nucleoprotein and human rabies virus vaccine strain (pv strain) were used as immunogens to immunize 6–8 week old female balb/c mice. spleen cells and sp2/0 myeloma cells were fused according to conventional methods: the monoclonal cell strains obtained were selected using the indirect immunofluorescence test; this was followed by preparation of monoclonal antibody ascitic fluid; and finally, systematic identification of subclass, specificity and sensitivity was carried out. two high potency and specific monoclonal antibodies against rabies virus were obtained and named 3b12 and 4a12, with ascitic fluid titers of 1:8000 and 1:10000, respectively. both belonged to the igg2a subclass. these strains secrete potent, stable and specific anti-rabies virus monoclonal antibodies, which makes them well suited for the development of rabies diagnosis reagents. these techniques need fluorescent-labeled high potency and specific antibodies to rv antigens. however, this detection reagent is not yet approved for commercial manufacture in china, while the imported products are expensive and therefore are not readily available. this mitigates against the use of these techniques as standards. thus, the preparation of potent and specific monoclonal antibodies against rabies virus will contribute significantly to development of techniques for laboratory diagnosis of rabies. since these will have local independent property rights it will facilitate rabies surveillance in china. in 1978, wiktor [11] reported the preparation of rabies virus monoclonal antibodies. since then, rabies virus monoclonal antibody (mcab) technology has been more and more widely used in basic research, and the testing and diagnosis of rabies. in this study, anti-rv monoclonal antibodies were prepared using a human rv vaccine strain and a baculovirus-expressed rv nucleoprotein as antigens. mouse sp2/0 myeloma cell strains were provided by the xinjiang uygur autonomous region center for disease control and prevention; bsr cells were kept in our laboratory. animals used for immunization were 6-8 weeks old female babl/c mice. concentrated solutions of human rabies virus vaccine pv strain were provided by the chengda bio co., ltd. rabies virus nucleoproteins (cvs-11 strain) were expressed and purified by a baculovirus eukaryotic expression system [3] in our laboratory. antigens used for immunization were rabies virus nucleoproteins (cvs-11 strain) and concentrated solution of human rabies virus vaccine pv strain. the concentrations of two antigen suspensions were in the range of 1-1.5 mg/ml after measurement by uv-spectrophotometry, they were then diluted with pbs to the required concentrations (table 1) . ten 6-8 week-old female babl/c mice were divided into two groups. immunizations were conducted on d 1, 15, 29 and 43 respectively according to methods described elsewhere [9] , four times in total. the first three injections were on both sides of the subcutaneous tissue of the neck, back and into the peritoneal cavity. the final immunization was into the peritoneal cavity, the other details are shown in table 1 . after immunization, blood was collected from the caudal vein and specific anti-rv antibody preparation of myeloma cells: sp2/0 myeloma cells were cultured in rmpi1640 medium containing 10% serum at 37℃ and 5% co 2 , and passaged once every two days. sp2/0 cells from the logarithmic phase were selected for the fusion with spleen cells. preparation of feeder cells: one healthy balb/c mouse was killed by cervical dislocation the day before cell fusion, 5 ml 1640 medium (containing 20% serum) was injected into the peritoneal cavity of the mouse, then the medium was pumped out after gentle massage on the mouse peritoneal cavity and transferred into flasks. the sufficient medium was added and mixed with the feeder cells, 200 μl/well was distributed into 96-well plates and incubated at 37℃, 5% co 2 overnight. a booster immunization was conducted on the immunized mice. three days later, spleen cells of the mice which showed better immune efficacy were selected for the fusion according to the methods described [1, 2, 8] , the spleen cells were mixed with sp2/0 cells at a 5-10:1 ratio. after centrifugation, 1 ml 50% peg4000 was added into the mixed cells drop by drop over 1 min and after 90 sec serum-free rmpi-1640 medium was added to stop the reaction. after centrifugation, the fusion cells were resuspended using rmpi-1640 selective medium containing 20% serum (fbs) and 1% hat, and 100 μl/well of it was added into 96-well plates already loaded with the feeder cells. the plates were then incubated at 37℃, 5% co 2 . using the rffit method [12] , the viruses obtained blood from the tails of immunized mice was used for the immunization assessment and the serum antibody titer was detected using ifa technique. the results showed that the highest antibody titers of 10 immunized mice reached 1:6000, indicating effective immunization. the fusion rate was higher than 80%. clone table 2 . number of positive cells after second screening 6 7 number of positive cells after third screening 1 1 positive proportion of single-well cloning after the first cloning 40.6% 43.8% positive proportion of single-well cloning after the second cloning 100% 100% the passage number after the construction of cell strains was taken as the first generation. the stability of the first eleven generations of cells was detected at every other generation, as shown in table 3 the two monoclonal antibody cell strains, 3b12 and 4a12 were detected for the neutralization titer through rffit, the results are shown in table 4 . the titers of obtained ascitic fluid for the two strains, determined by indirect immunofluorescence, were 1:10000 and 1:8000 respectively. uv spectrophotometric analysis showed the concentration of monoclonal antibodies of purified ascitic fluid were 1.06 mg/ml and 0.80 mg/ml respectively. sds-page analysis after purification by electrophoresis (fig. 2) shows that, compared to the vague band of unpurified samples, the purified monoclonal antibodies from the ascitic fluid present two clear bands near 25 kd and 50 kd, consistent with the expected molecular size of the igg light chain and heavy chain. sub-class identification was conducted using elisa capture with a monoclonal antibodies subclass identification kit (sigma), and both monoclonal antibodies obtained were determined as igg2a monoclonal antibodies. instability of fusion cells [6] . that is why the number of positive hybridoma decreased with time, as shown in the result. timely cloning is a more important consideration for efficient generation of monoclonal antibodies because in the same well, the growth ability of cells which do not secrete antibodies will be greater than those secreting antibodies [4] . the titer of the neutralizing antibodies obtained was detected using the rffit and the results showed that the generated monoclonal antibodies had the ability to neutralize rabies virus. the neutralizing activity needed to be further validated and the details are shown in table 4 . there are many ways to purify monoclonal antibodies [14] : including ammonium sulfate precipitation, octanoic acid precipitation and affinity chromatography. of these, ammonium sulfate precipitation shows a higher purified rate of ascitic fluid but a lower recovery rate, and it has a significant impact on the immune activity of igg [5] , while octanoic acid precipitation achieves the opposite result. therefore, in this experiment, using affinity chromatography, our experimental results verified that this purification development and characterization of neutralizing monoclonal antibody to the sars-coronavirus novel monoclonal antibodies that bind to wild and fixed rabies virus strains identification and baculovirus expression of rabies virus n gene a human monoclonal antibodies cocktail as a novel component of rabies postexposure prophylaxis antibodies: a laboratory manual. cold spring harbor laboratory continuous cultures of fused cells secreting antibody of predefined specificity evaluation of a direct, rapid immunohistochemical test for rabies diagnosis purification and application of the rabies viral monoclonal antibody laboratory techniques in rabies. geneva: world health organization use of neutralizing murine monoclonal antibodies to rabies glycoprotein in passive immunotherapy against rabies monoclonal antibodies against rabies virus produced by somatic cell hybridization: detection of antigenic variants the establishment of a rapid fluorescent focus inhibition test for testing rabies virus neutralizing antibody competitive elisa using a rabies glycoprotein-transformed cell line to semi-quantify rabies neutralizing-related antibodies in dogs the study of purified methods of mice ascitic fluid igg mcab key: cord-315486-pjb5v1tc authors: wu, xiaojun; wang, tong; zhou, yilu; liu, xiaofan; zhou, hong; lu, yang; tan, weijun; yuan, mingli; ding, xuhong; zou, jinjing; li, ruiyun; liu, hailing; ewing, rob m.; hu, yi; nie, hanxiang; wang, yihua title: different laboratory abnormalities in covid-19 patients with hypertension or diabetes date: 2020-09-30 journal: virol sin doi: 10.1007/s12250-020-00296-1 sha: doc_id: 315486 cord_uid: pjb5v1tc nan the pandemic of covid-19, a disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2; previously known as 2019-ncov), has placed an enormous burden on health authorities in 213 countries and territories. at the time of writing, over 26 million cases have been recorded across the world, with more than 870 k associated deaths (www.worldometers.info/coronavirus/). world bank warns covid-19 pandemic risks dramatic rise in poverty, with the majority of economies expecting to suffer from falling levels of gdp in 2020. the main symptoms of covid-19 are onset of a new continuous cough and/or a high temperature with symptoms ranging from mild to life threatening. those who are elderly or have pre-existing health issues are more likely to develop severe disease. the most prevalent comorbidity is hypertension, followed by diabetes (guan et al. 2020a, b) . we reported recently that hypertension is a risk factor for severe cases of covid-19, independent of age and other variables (liu et al. 2020a ). an important question is why patients with hypertension and diabetes yield poorer clinical outcomes than those without. human pathogenic coronavirus sars-cov-2 utilizes angiotensin-converting enzyme 2 (ace2) as a receptor for viral cell entry. since the levels of ace2 are substantially increased in patients with hypertension or diabetes, who are treated with ace inhibitors (aceis) and angiotensin ii type-i receptor blockers (arbs) (ferrario et al. 2005 ), fang and colleagues hypothesized that ace2-stimulating drugs could potentially increase the risk of developing severe covid-19 (fang et al. 2020 ). this was not supported by a recent study led by dr. reynolds (reynolds et al. 2020) , whose analysis showed no positive association for aceis or arbs for either the risk of sars-cov-2 infection or severe illness (reynolds et al. 2020) . what else might explain the poorer clinical outcomes of covid-19 patients with hypertension or diabetes? to explore this question, we re-analysed the same cohort of 99 covid-19 patients discharged from the general wards of renmin hospital of wuhan university between 5 february 2020 and 14 march 2020 (ethics approval no: wdry2020-k124) (liu et al. 2020a, b) . demographic, clinical, laboratory and radiographic findings were extracted from electronic medical records. we asked whether there were any factors associated with covid-19 patients with either hypertension or diabetes (summarised in table 1 ). all data analyses and graphs were done in r (v3.6.1) or graphpad prism (v8.2.1). as expected, covid-19 patients with health conditions like hypertension or diabetes had a longer length of hospital stay than those without, but this was at the limit of statistical significance (fig. 1a , p = 0.07). to check the extent of pulmonary pathology in covid-19 patients, we developed a high-resolution computed tomography (hrct) score system considering both radiographic features and distributions yuan et al. 2020) . in brief, the hrct findings were graded on a 3-point scale: normal attenuation (1 point), ground-glass attenuation (2 points), and consolidation (3 points). each lung zone (6 lung zones in total for each patient) was assigned a following scale according to the distribution of the affected lung parenchyma: normal (0 point), \ 25% abnormality (1 point), 25%-50% abnormality (2 points), 50%-75% abnormality (3 points), and [ 75% abnormality (4 points). the 4-point scale of the lung parenchyma distribution and virologica sinica the radiographic scale described above were then multiplied to produce a weighted score for each zone. points from all zones were then summed for a final total cumulative score (hrct score), with values ranging from 0 to 72. hrct peak scores are the highest hrct score during disease course, which reflex maximal chest hrct abnormalities. we found hrct peak scores for hypertensive covid-19 patients were higher compared to non-hypertensive ones ( fig. 1c ). these findings confirmed an increased disease severity in covid-19 patients with hypertension or diabetes (liu et al. 2020a ). most interestingly, in laboratory findings on admission (table 1) patients compared to non-hypertensive ones; while lymphocyte count was not significantly changed in hypertensive covid-19 patients (median: 0.96 vs. 1.09; p = 0.260). in contrast, there was a significant decrease in lymphocyte count in covid-19 patients with diabetes compared with those without (fig. 1h ; median: 1.08 vs. 0.83; p = 0.019); while changes in white blood cell counts, neutrophil counts, d-dimer and ldh were not significant (p [ 0.05) in covid-19 patients with diabetes. in addition, hypertensive covid-19 patients were older than nonhypertensive ones (p = 0.003), but this was not the case for diabetic covid-19 patients (p = 0.273). there were no significant differences in c-reactive protein, sex and common symptoms (such as fever, cough, dyspnoea, fatigue, anorexia and/or lethargy, myalgia and/or arthralgia and diarrhoea) for both hypertension and diabetes. sars-cov-2 infections lead to a fast activation of innate immune cells, especially in covid-19 patients developing severe disease . it was reported that circulating neutrophil numbers are consistently higher in survivors of covid-19 than in non-survivors, and the infection also induces lymphopenia (low level of lymphocytes in the blood) (zhou et al. 2020) . in our analysis, we found on admission hypertensive covid-19 patients had higher neutrophil numbers compared to non-hypertensive ones, while lymphopenia was more frequently observed in covid-19 patients with diabetes. these results suggest different mechanisms exist for hypertension or diabetes as risk factors for severe cases of covid-19. it is known that both hypertensive and diabetes patients have impaired immune response to infections (lopez gelston and mitchell 2017; zhu et al. 2020) . low-grade inflammation is known to facilitate the development of hypertension, and poor glycaemic control influences cytokine profile and t cell activation, which were reflected by the laboratory findings in this study. however, more lab work is required to confirm these in covid-19. established mouse models of hypertension and diabetes can be applied to the k18-hace2 transgenic mice for this purpose. there are several limitations in this study. (1) interpretation of our findings might be limited by the sample size. (2) this study is limited by its retrospective nature, the non-standardised documentation resulting in the inability to remove the influence of age on laboratory findings. despite these limitations, we were able to identify different laboratory abnormalities on admission in covid-19 patients with hypertension or diabetes, which might shed light on future mechanistic studies. are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? effect of angiotensinconverting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme 2 china medical treatment expert group for c (2020a) comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis clinical characteristics of coronavirus disease 2019 in china risk factors associated with disease severity and length of hospital stay in covid-19 patients temporal radiographic changes in covid-19 patients: relationship to disease severity and viral clearance recent advances in immunity and hypertension renin-angiotensin-aldosterone system inhibitors and risk of covid-19 association of radiologic findings with mortality of patients infected with 2019 novel coronavirus in wuhan clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study sars-cov-2 viral load in upper respiratory specimens of infected patients acknowledgements we acknowledge all the patients involved in this study, and appreciate all the frontline medical and nursing staff involved in the diagnosis and treatment of patients in wuhan. this animal and human rights statement all methods were carried out in accordance with relevant guidelines and regulations. this retrospective study was approved by the ethics committee of renmin hospital of wuhan university, hubei, china (no. wdry2020-k124), and the requirement for informed consent was waived by the ethics committee due to a public health outbreak investigation. key: cord-348467-a2e3f161 authors: alqahtani, amani salem; wiley, kerrie elizabeth; tashani, mohamed; heywood, anita elizabeth; willaby, harold wayne; bindhim, nasser fahad; booy, robert; rashid, harunor title: camel exposure and knowledge about mers-cov among australian hajj pilgrims in 2014 date: 2016-01-18 journal: virol sin doi: 10.1007/s12250-015-3669-1 sha: doc_id: 348467 cord_uid: a2e3f161 [image: see text] more chronic diseases: 8% had diabetes, 7% asthma, 7% hypercholesterolemia and 5% hypertension. most pilgrims (82%) were travelling to hajj for the first time and planned to stay in saudi arabia for a median of 27 (range: 7 to 40) days. about two-thirds of respondents (64%) received 'travel health advice' before hajj, including from general practitioners (gps) (81%), travel clinics (24%) and specialist websites (e.g. saudi ministry of health [moh] website) (12%). many pilgrims consulted other individuals or sources: for example, 67% consulted hajj tour group leaders, 46% consulted their friends and family members who had previously attended hajj, and 17% browsed 'general websites'. about half of the participants (49%) knew about mers-cov before travelling to saudi arabia; of these, 70% reported mass media as the main information source. demographic factors significantly associated with mers-cov knowledge were female gender, age > 40 years, and middle eastern origin ( table 1) . receipt of pre-travel advice from their gps (odds ratio [or] 1.6, 95% confidence interval [ci] 1.0-2.4, p = 0.03) or from hajj tour group leaders (or 1.6, 95% ci 1.0-2.5, p = 0.04) were also significantly associated with mers-cov awareness (table 1) . of those aware of the disease, 77% answered correctly that the virus affects the respiratory system whereas 60% thought the disease was serious only for the elderly. furthermore, 48% of pilgrims who knew about the disease reported that they were either "not concerned" or "slightly concerned" about contracting the disease during hajj. half (51%) of the respondents were aware of the saudi moh advice for 'at risk' individuals to postpone hajj that year; however, none of these 'at risk' individuals adhered to this recommendation (table 2) . participants were acceptable with using preventive measures during hajj for protection from mers-cov infection: 79% were willing to wash hands frequently, 73% planned to avoid contact with sick people showing symptoms of a respiratory infection, 65% were willing to use facemasks, and another 65% planned to avoid touching their eyes, nose, and mouth. most departing pilgrims (62%) were aware of a mod-erate to high infection risk from raw camel milk consumption, yet 21% of participants were willing to drink it. additionally, 27% of participants were willing to visit a camel farm during hajj, or at least stated the possibility of doing so. pilgrims who knew about mers-cov were significantly more aware of the risks of mers-cov from drinking unpasteurized camel milk compared to those who did not know (43% vs. 23%, p < 0.01). nevertheless, among those who were aware of mers-cov, 27% did not fully realize the risk of catching the disease from unpasteurized camel milk, 15% were willing to drink raw camel milk, and 23% were keen to visit camel farm in saudi arabia (table 3) . for the second survey, 150 returning pilgrims aged 18-72 (median 41) years were recruited after hajj; 47% were men, and 16% reported having one or more chronic diseases: 6% had asthma, 3% diabetes mellitus, 2% malignancies, and 5% had other conditions. most participants (71%) had up to high school education level, 65% were employed, and 77% went for hajj for the first time; the median duration of stay at hajj was 25 (range: 7 to 35) days. of 150 pilgrims, 7% reported having actual contact with camels (e.g. taking photographs with camels), or consuming camel products, including meat (5%) and unpasteurized milk (1.3%). although this was the third year of mers-cov circulating in saudi arabia, only 50% australian departing pilgrims knew about the disease. however, this was a clear improvement compared to the findings of another australian survey conducted a few months earlier (only 35% pilgrims were aware of mers-cov in saudi arabia) (tashani et al., 2014) . compared to the ebola outbreak in 2014 about which 90% australian hajj pilgrims were aware (alqahtani et al., 2015b) , fewer pilgrims were aware of mers-cov. the ebola outbreak severity with consequent wider media coverage probably played a role in increasing awareness. for instance, ebola has affected over 28,640 people with a relatively higher fatality rate (40%) compared to fewer mers-cov cases (about 1,621) and a slightly lower fatality rate (36%) (who, 2015) . in this study, 62% of departing pilgrims were aware of the risk of contracting the disease from drinking raw camel milk while 21% were willing to drink camel milk if offered at hajj. this contrasts with the findings of gautret et al. who showed that only 14% french pilgrims knew about the disease risk from drinking raw camel milk but 40% of them were willing to drink camel milk if offered during hajj (gautret et al., 2013a) , indicating that pilgrims' awareness influenced their attitude. a unique finding to emerge from our study was that departing pilgrims with knowledge about mers-cov were significantly more aware of the risk of drinking raw camel milk (43% vs. 23%, p < 0.01), yet a considerable proportions of them were willingness to drink raw milk and visit camel farms (respectively 15% and 23%). this possibly indicates that despite being aware of mers-cov epidemic in saudi arabia, some individuals were not fully informed about the high risk behaviors (al-mohrej et al., 2015) . this could be addressed by public awareness campaigns. providing direct health education to hajj pilgrims at entry points (e.g. airport) in saudi arabia could improve their understanding about the possible modes of transmission of mers-cov and how to avoid exposure risks (turkestani et al., 2013) . in practice, about 7% of returning pilgrims reported actually coming in close contact with camels and 1.3% drank unpasteurized camel milk. this is unsurprising considering that about 8% french hajj pilgrims had a history of consuming raw camel milk in north africa and saudi arabia (gautret et al., 2013a ). an iphone appbased small survey conducted parallel to this survey revealed that 16% of international hajj pilgrims reported contact with camels during hajj (alqahtani et al., 2015a) . consumption of raw camel products (milk, liver, meat, or urine) can lead to outbreaks of other infections such as brucellosis, plague, and rift valley fever (bin saeed et al., 2005; davies, 2006; kalimuddin et al., 2010) . therefore, pilgrims who consume raw milk or other products are at risk of other zoonotic diseases if not mers-cov, and therefore, could benefit from appropriate health education. the current mers-cov guidelines by saudi moh stated that any person with close contact with camels (in the last 14 days), including drinking unpasteurized camel milk and showing respiratory symptoms, is suspected with mers-cov (ccc, 2015) . the recent outbreak in south korea, worsened by the initial failure of heath care providers to recognize the infection, shows the importance of early recognition of symptoms of mers-cov (kim and lee, 2015) . to prevent virus introduction to their lands, some countries (e.g. the uk) have issued a set of guidelines for managing suspected mers-cov cases (kumar et al., 2015) , which is exemplary (phe, 2015) . given that 7% people reported having close contact with camels, we suggest development of a public health strategy to better communicate the risk to pilgrims and information about when to seek medical help should symptoms arise upon return. over half of the departing pilgrims were aware of the saudi moh advice to postpone hajj; nevertheless, none of the high-risk pilgrims decided to cancel their trip. this is consistent with the findings of french researchers who also discovered that none of the at-risk pilgrims had planned to postpone their trip (gautret et al., 2013b) . as mass media was the main source of mers-cov knowledge, we believe mainstream media outlets (e.g. tv) should be used to propagate heath messages immediately before, during, and after the hajj season. tour group leaders can also play an important role in disseminating health advice, as shown here and previously (alqahtani et al., 2015b) . (16) travel agents 11 (6) saudi moh 8 (5) 'smarttraveller' website** 8 (5) other sources 3 (2) very serious 100 (60) moderately serious 43 (25) slightly serious 14 (8) not serious 11 (6) for pilgrims with chronic diseases*** very serious 86 (51) moderately serious 48 (28) slightly serious 15 (9) not serious 20 (12) very serious 47 (27) moderately serious 43 (25) slightly serious 47 (27) not serious 36 (21) very concerned 47 (27) moderately concerned 43 (25) slightly concerned 47 (27) not concerned 36 ( digestive tract 12 (7) brain 11 (6) note: *some pilgrims obtained the information from more than one sources, so the percentage is >100; **smartraveller.gov. au; ***missing data have been excluded. in conclusion, many australian hajj pilgrims are not aware of mers-cov in saudi arabia, and some of them engage in activities that may put them at risk of mers-cov; therefore, there is a need for improved awareness among hajj pilgrims and other travelers to the middle east regarding mers-cov. infection prevention and control guidelines for middle east respiratory syndrome coronavirus (mers-cov) infection vector borne zoonotic dis mers, influenza and respiratory illness in travellers returning from the hajj key: cord-259443-5sv3dwbs authors: banik, gouri rani; alqahtani, amani salem; booy, robert; rashid, harunor title: risk factors for severity and mortality in patients with mers-cov: analysis of publicly available data from saudi arabia date: 2016-01-25 journal: virol sin doi: 10.1007/s12250-015-3679-z sha: doc_id: 259443 cord_uid: 5sv3dwbs [image: see text] however, these studies were relatively small and the risk factors for disease severity were rarely explored (majumdar et al., 2015; korea cdc, 2015; saad et al., 2014) . to this end, we have explored the key risk factors for mortality and severity of mers-cov from a larger dataset of saudi arabian patients. data for this analysis were obtained from the command and control center (ccc) of saudi arabian ministry of health (moh). this center has been publicly reporting the mers-cov cases in saudi arabia since may 2013 on their website (http://www.moh.gov.sa/en/ccc/ pressreleases/pages/default.aspx). the site contains data on > 80% of the saudi arabian mers cases. initially, only limited information such as patients' age, sex, nationality, address, date of diagnosis, presenting symptoms, and presence of any pre-existing condition were made publicly available; however, since 24 th september 2014, additional information on likely exposure to animals and other suspected mers cases were added, and it was recorded whether the exposure likely occurred at health care settings or in community settings. two authors (asa and grb) extracted the data from the saudi moh website and added it into a data extraction spread sheet; the first author (grb) double checked the entries, any disagreement was resolved through verifying and ensuring data integrity. the following data were abstracted in the extraction sheet: date of confirmation of the diagnosis, age and sex of the patient, nationality, city or region of the patient's residence, symptoms, whether the patient required intensive care unit (icu) admission, status of the patient at the time of data entry (recovered, being cared in the hospital or icu, or died) and whether the patient was a health care worker (hcw). it was also noted if the patient had any pre-existing medical condition and, if so, what those medical conditions were. additional information (e.g., history of exposure to animal or contact with suspected cases in health care and/or community settings) were collated in a separate column irrespective of whether or not the information was used in this analysis. the proportion of patients with a particular risk factor, such as pre-existing illness or age ≥ 65 years, was calculated separately for those who died and for those who survived. for this analysis, a case of mers was considered 'severe' if he/she needed admission to icu at any time point during the course of the illness, all other cases were considered 'not severe', irrespective of whether they required hospitalization or not. the proportion of patients with a particular risk factor such as the presence of a named pre-existing medical condition was calculated separately for 'severe' and 'not severe' cases. odds ratios (ors) for mortality and severity in the presence of a potential risk factor were calculated with 95% confidence interval (95%ci) by using an authentic online calculator (http://vassarstats.net/odds2x2.html). a p value of < 0.05 was considered statistically significant. as of 11 october 2015, data on 1060 mers-cov cases aged 9 months to 109 years (median 52 years) were published in the saudi moh website; 65% were male (m/f ratio: 1.5). among them, 81% (846/1049) were saudi residents; the non-saudis (19%, 203/1049) included expatriates and immigrants mainly from the philippines, palestine, pakistan, and egypt. riyadh had the highest number of cases (45.5%, 482/1058), followed by jeddah (18%, 192/1058) ( figure 1 ). one hundred and forty one (14%) of all mers cases were hcws. of 379 patients for whom such data were available, 84 (22%) had a history of exposure to health care settings, and of 595 patients for whom such data were available, 250 (42%) gave a history of exposure to suspected cases in the community. of 350 patients for whom the data were available, 17% (60) gave a history of exposure to anim-als: 3% to camels and 14% to other unspecified animals. the presence or absence of a co-morbidity was recorded in two-thirds of cases (n = 685); of these, 496 (72.4%) were considered as being at risk, either because they were aged ≥ 65 years or had one or more pre-existing medical conditions. of 496, 208 (41.9%) were aged 65 years or older, and apart from seven (3%), all had preexisting medical conditions; in 108 (22%) cases, the conditions were specified: diabetes mellitus (50%, 54/108), hypertension (45%, 49/108), and renal (30%, 32/108), cardiac (19%, 21/108), pulmonary (17%, 18/108), malignant (13%, 14/108), and neurological diseases (5%, 5/108). another 16% (17/108) had other chronic diseases in various combinations. over half (52%) of the patients had multiple comorbidities. of the total 1060 cases, 384 (36%) died by the time the data were analyzed, of which 270 (70%) were male. diabetes, hypertension, renal disease, malignancy, and several other conditions hitherto termed as miscellaneous conditions (e.g., anemia, obesity and congenital abnormalities, diseases of the liver and gall bladder, or steroid use) were significant risk factors for mortality from mers-cov (table 1) . male sex was also a significant risk factor for mortality but the proportions of diabetes, hypertension, renal disease, malignancy, and miscellaneous conditions in men were not significantly different from that in women. of 676 mers patients who survived, 130 (19.2%) required icu admission, therefore were considered to have a severe form of the disease. of those who survived, 428 (63%) had pre-existing conditions, of which diabetes and neurological conditions were significant risk factors for severity (table 2) . these results corroborate the findings of other smaller studies from saudi arabia and south korea that showed that older age and presence of co-morbid conditions were associated with higher mortality (saad et al., 2014; feikin et al., 2015; majumder et al., 2015; korea cdc, 2015) . our study has specifically identified that diabetes, hypertension, renal disease, malignancy, miscellaneous conditions, and male sex were the main risk factors for mortality. another small study also demonstrated that diabetes was significantly associated with mortality (or = 15.7; 95%ci 2.5-100.7) (shalhoub et al., 2015) . in contrast, among south korean patients, underlying respiratory disease and older age were the key risk factors for mortality (korea cdc, 2015) . in our study, presence of a respiratory disease was not a significant risk factor and we did not explore the association of older age with mortality, because essentially all patients aged ≥ 65 years in our cohort had a pre-existing disease, but age itself could be an independent risk factor, as other studies from saudi arabia and south korea demonstrated that age > 60 years (in some studies ≥ 65 years) was significantly associated with mortality (feikin et al., 2015; majumder et al., 2015; saad et al., 2014) . additionally, majumder et al. (2015) demonstrated that for every one year of increase in age, the odds of fatality increased by 12% (or 1.1, 95% ci 1.1-1.2). the risk factors might vary geographically; additionally, the relatively small sample sizes in other studies could have missed other significant risk factors. our study also demonstrates that men had higher odds of dying from mers-cov, which is unsurprising given the fact that the disease predominantly affects men. another saudi study also demonstrated that the case fatality rate was higher for men (52%) than for women (23%) (alghamdi et al., 2014) . in the south korean cohort, even though being male increased the odds of mortality, the association was not significant (majumder et al., 2015) , likely because of small size. in our cohort, 65% of the patients were men, while in some other series, 97% of the cases were men, which is explained by the fact that men more often come in close contact with camels than women in arabian countries (alraddadi et al., 2015) . mers-cov patients with diabetes and neurological conditions were more likely to require icu admission than those without these conditions, which are unique findings. in contrast, a study involving 70 saudi arabian mers patients showed that concomitant infections (or 14.1, 95% ci 1.6-126.1, p = 0.02) and low albumin levels (or 6.3, 95% ci 1.2-31.9; p = 0.03) were independent risk factors for icu admission (saad et al. 2014) . approximately 14% of the mers-cov patients in our cohort were hcws, and exposure to healthcare setting was an important ground for transmission of the virus (over 20%), which is supported by other available data (oboho et al., 2015; petersen et al., 2014; saad et al., 2014) , and mathematical modelling suggests that nosocomial transmission is over four times higher than community transmission (chowell et al., 2014) . considering that pre-existing conditions such as diabetes, hypertension, renal, malignant, neurological, and miscellaneous conditions are risk factors for severe outcomes of mers-cov, due attention should be paid to optimum control of these conditions. co-infection with mers-cov and influenza plus other bacterial and viral infections have been reported (zumla et al., 2015) , and co-infection is seen to increase the severity of the disease (saad et al., 2014) . therefore, vaccinations against influenza and pneumococcal disease should be considered for the high-risk individuals (those with pre-existing disease and/or aged ≥ 65 years). these vaccines could prevent co-infections by influenza and streptococcus pneumoniae as well as the fatal outcomes of mers-cov. they may even reduce the need for hospital visits or even icu admission. the limitations of this study are: data could not be validated from the original source and multivariate analysis was not considered. despite these limitations, the study provides valuable information on risk factors for severity and mortality in mers patients from a relatively large dataset, and has actually ascertained some key risk factors. we hope this study would inspire further research based on even larger datasets. middle east respiratory syndrome coronavirus (mers-cov) -saudi arabia middle east respiratory syndrome key: cord-014923-gfiqjgxg authors: zhang, yi; wang, wei; gao, jin-rong; ye, li; fang, xiao-nan; zeng, ying-chun; wu, zheng-hui; she, ying-long; ye, lin-bai title: the functional motif of sars-cov s protein involved in the interaction with ace2 date: 2009-09-16 journal: virol sin doi: 10.1007/s12250-007-0054-8 sha: doc_id: 14923 cord_uid: gfiqjgxg sars-cov is a newly discovery pathogen causing severe acute respiratory problems. it has been established that the s protein in this pathogen plays an important rule in the adsorption and penetration of sars-cov into the host cell by interaction with the ace2 receptor. to determinant which functional motif of the s protein was involved in the interaction with ace2, seven truncated s proteins deleted from the n or c terminal were obtained by an e.coli expression system and purified by column chromatography to homogeneity. each truncated s protein was fixed on to the well of an elisa plate and an interaction was initiated with the ace2 protein. the adsorption were quantified by elisa, and the results indicated that amino acids from 388 to 496 of the s protein was responsible for the interaction with the ace2 receptor, and the interaction could be completely disrupted by an antibody specific to these amino acids. deletions adjacent to this domain did not appear to have a significant impact on the interaction with ace2, suggesting that the s protein of sars-cov could be developed as a vaccine to prevent the spread of sars-cov. . the virus causes atypical pneumonia with diffuse alveolar damage with an overall mortality of 10% that ranges from 0% in children and 50% in persons over 65, seriously threatening public health worldwide. virologica sinica vol.22 no.1 [ 2 the s protein of sars-cov is a large type i membrane glycoprotein projection from the viral envelope which is responsible for both binding to receptors on host cells and for membrane fusion. angiotensin converting enzyme 2(ace2) was found to be an efficient receptor for the s glycoprotein of sars-cov (9, 4, 12) . the s protein also contains important virus neutralizing epitopes that elicit neutralizing antibody in the host species (3, 2) . to determine the critical sequence of the s protein that interacts with the ace2 receptor, the interactions between ace2 and different truncated s proteins were investigated. our study sheds some light on the interaction mechanism and provides useful insight into the development of a protein vaccine for sars-cov. plasmids s1-1 encoding sars-cov s protein residues 262~606，s1-2 encoding 314~606，s1-3 encoding 388~606，s1-4 encoding 314~387，s1-5 encoding 262~496，s1-6 encoding 262~387，s1-7 encoding 388~496 (fig. 1.) were constructed. the sequences of the primers (fp-forward primer, rpreberse primer) employed in the pcr reaction were： s1-1 fp：5′-gtcagggatccatgctcaagtatgatgaaaatggtac-3′ rp：5′-actcgagaattcctaaacatcagtgcagttaac-3′ s1-2 fp：5′-ctgcagggatccaccatggtgagattccctaatattac-3′ rp：5′actcgagaattcctaaacatcagtgcagttaac-3′ s1-3 fp：5′-ctgcagggatccaccatggtcaagggagatgatg-3′ rp：5′-actcgagaattcctaaacatcagtgcagttaac-3′ s1-4 fp：5′-ctgcagggatccaccatggtgagattccctaatattac-3′ rp：5′-actcgagaattcctatacaaaagaatctgc-3′ s1-5 fp：5′-gtcagggatccatgctcaagtatgatgaaaatggtac-3′ rp：5′-actcgagaattcctaaactctgtaaggttgg-3′ s1-6 fp：5′-gtcagggatccatgctcaagtatgatgaaaatggtac-3′ rp：5′-actcgagaattcctatacaaaagaatctgc-3′ s1-7 fp：5′-ctgcagggatccaccatggtcaagggagatgatg-3′ these forward primers carried a bamh ⅰ 1.5 preparation of rabbit anti-s1-1~ s1-7 sera 0.1 mg each of seven purified s proteins were mixed well with 1% al(oh) 3 respectively, and used to immunized rabbit by injecting hypodermal at three point. three weeks after first immunization. booster shot were performed, 10 days later, the blood samples were collected and titers of antibodies were measured by elisa. 6.1 purified s1-1~s1-7 proteins ， dissolved respec-tively in coating buffer (0.016mol/l na 2 co 3 , in the interaction disruption assay, rabbit anti-s1-7 serum was added into the well before adding ace2, the remainder of the steps were the same as above. the fragments of 1 035bp (s1-1), 879bp (s1-2), 657bp (s1-3), 222bp (s1-4), 705bp (s1-5), 128bp (s1-6) and 327bp (s1-7) were amplified by pcr using plasmid pgem-s as template and were then cloned into prokaryotic expression vector pethis. the corrected clones were confirmed by restriction analysis and sequencing. the results of agarose electrophoresis are shown in fig.2 and fig.3 . proteins s1-1~s1-7 s1-1~s1-7 proteins were successfully expressed in fig.2 . analysis of recombinant plasmid pethis s1-1～s1-6 by bamhⅰ ﹠ ecorⅰ digestion m, 1kb dna ladder; 1,pethis s1-1; 2, pethis s1-2; 3, pethis s1-3; 4, pethis s1-4; 5, pethis s1-5; 6, pethis s1-6; 7, pethis s1-5. fig.3 . analysis of recombinant plasmid pethis s1-7. m, 1kb dna ladder; 1, pcr product; 2, pethis s1-7/bamhⅰ﹠ ecorⅰ; 3,pet-his s1-2/ bamhⅰ. fig.4 . expression and purification of the recombinant protein s1-1～s1-7. m, protein marker; 1, s1-1; 2, s1-2; 3, s1-3; 4, s1-6; 5, s1-5; 6, s1-4; 7, s1-7; e.coli bl21(de3) at a high level and formed insoluble inclusion bodies. as fig.4 shows, the proteins were expressed and purified to a high purity. to investigate whether the induced proteins were the expected proteins, western blot analysis of purified proteins were processed. as fig.5 showed, all purified zhang et al. the functional motif of sars-cov s protein involved in the interaction with ace2 5 s1-1~s1-7 proteins could react with the sars pat ients sera specifically and the molecular weights were consistent with expectations, indicating the appropriate proteins were produced. the recombinant ace2 protein was successfully fig.5 . western blot of purified protein s1-1～s1-7. 1, s1-1; 2, s1-2; 3, s1-3; 4, s1-6; 5, s1-5; 6, s1-4; 7, s1-7. expressed in e.coli dh5. fig.6 showed that ace2 was induced and purified. the purified ace2 protein could react with anti-ace2 serum and the expected 92kda band was observed in western blot (fig.7) . purified recombinant proteins s1-1~s1-7 were fixed in the 96-well plate by coating, and then purified ace2 protein was added to allow their interaction. a well coated with purified ace2 protein was used as positive control. another coated with purified s1-1 protein without adding ace2 protein was used as negative control. then anti-vero-ace2 rabbit serum was added as first antibody and hrp labeled goat-anti-rabbit igg was added as second antibody. elisa showed that the results of s1-1, s1-2, s1-3, s1-5, s1-7 wells were positive, while the results of s1-4, s1-6 were negative (table 1. ). our data showed that the aa 388~396 of s1 protein was essential to the interaction between s protein and ace2. to further confirm this result, an antibody induced by s1-7 which only contains aa 388~396 of s protein was used for disrupting the interaction between s and ace2. the results show that all the interactions we re nearly completely blocked by this antibody (table 2. ). whereas the antibody induced by s1-4 which contained deletions aa 388~396 did not show ability to block this interaction. table 1 . interaction between truncated s protein and ace2 s1-1 s1-2 s1-3 s1-4 s1-5 s1-6 s1-7 ace2 table 2 . interaction blocking by antibody against s1-7 s1-1 s1-2 s1-3 s1-4 s1-5 s1-6 s1-7 ace2 anti-s1-7 engineering a protein vaccine for sars, therefore, is a relatively safer option and has great potential as a future direction in sars vaccine development. the spike protein of sars-cov is composed of two parts: s1 and s2. s1 is involved in viral infection, pathogenesis, host ranges and binding to the receptors (5, 6) . so this study on the interaction between s1 protein and the receptor ace2 may shed light on the development of engineering protein vaccines of sars. in this study, seven truncated s1 sequences (s1~s7) were cloned in the prokaryotic expression vectors and induced to express at a high level. our results showed that s1-1, s1-2, s1-3, s1-5 and s1-7 can interact with purified ace2 protein specifically, but s1-4 and s1-6 cannot interact with this receptor. s1-1, s1-2 and s1-3 are sequential truncated proteins of the n-terminal s protein. they can both bind to ace2, suggesting aa 262~387 is not essential for s1-ace2 interaction. s1-5 can bind to ace2, while s1-6 can't and s1-7 can bind to ace2 while s1 4 can t, both of which suggested that aa 388~496 is essential for the s1 ace2 interaction. in this study, two methods were used for investiga-ting the binding of various truncated s1 proteins to the ace2. the first one was the far-western blot. various truncated s1 proteins were resolved on a 15% sds-page gel and then transferred onto n.c membrane. the blots were overlayed with ace2 and the binding of ace2 protein was detected by x-ray with anti-ace2 rabbit serum as the first antibody and goat-anti-rabbit igg as the second antibody. however, no ace2 protein binding to the membrane was detected. the second method was performed as follows. truncated s1 proteins were fixed onto the plates well by coating without denaturing, and then purified ace2 protein solution was added to allow the interaction to proceed.anti-ace2 rabbit serum was used as the first antibody and goat-anti-rabbit igg was used as the second antibody. hrp activities were assayed and our results showed that s1-1, s1-2, s1-3, s1-5, s1-7 can all bind to purified ace2 protein specifically, while s1-4 and s1-6 can not interact with ace2. the results in these two experiments are different suggesting that denaturing of various truncated s1 proteins in sds-page in the first experiment disrupt the conformation of s1 proteins. but in the second experiments, all these proteins maintained their activated conformation. our data indicated that the zhang et al. the functional motif of sars-cov s protein involved in the interaction with ace2 7 interaction of s1 protein and receptor ace2 perhaps depend on the conformation of s1 protein. our data showed that anti-s1-7 protein rabbit antiserum could block the interactions between ace2 and truncated s1 proteins (s1-1, s1-2, s1-3, s1-5, s1-7). so the recombinant s1-7 protein could induce protective neutralizing antibody against sars-cov. our study sheds light on the development of engineering protein vaccines for sars. inactivated sars-cov vaccine elicits high titers of spike protein specific antibodies that block receptor binding and virus entry antigenicity and receptor-binding ability of recombinant sars coronavirus spike protein identification of two antigenic epitopes on sars-cov spike protein angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus efficient replication of severe acute respiratory syndrome coronavirus in mouse cells is limited by murine angiotensin-converting enzyme 2 receptor and viral determinants of sars coronavirus adaptation to human ace2 the genome sequence of the sars-associated coronavirus the severe acute respiratory syndrome a model pf the ace2 structure and function as a sars-cov-receptor characteri-zation of a novel coronavirus associated with severe acute respiratory syndrome expression cloning of functional receptor used by sars coronavirus key: cord-332861-7b5pzmk6 authors: zhang, zhan; li, xiaochen; zhang, wei; shi, zheng-li; zheng, zhishui; wang, tao title: clinical features and treatment of 2019-ncov pneumonia patients in wuhan: report of a couple cases date: 2020-02-07 journal: virol sin doi: 10.1007/s12250-020-00203-8 sha: doc_id: 332861 cord_uid: 7b5pzmk6 nan dear editor, till january 20, 2020, the 2019-new coronavirus (2019-ncov) has caused more than one hundred cases in wuhan (wmhc 2020). during a retrospective study of recent pneumonia patients in our department, we found two patients who are likely being infected with the 2019-ncov. during the hospitalization, those two patients were appropriately treated, and both were discharged within two weeks. thus, we are reporting the clinical features and treatment regiment, and hope the information and experience can be shared. the two patients were a couple. the male was 38 years old, and was admitted to the hospital due to fever for one week and dyspnea for one day on dec. 27, 2019. on admission, he had slight cough of a little green viscous sputum. he had been treated with normal anti-infective therapy in another hospital for 3 days, but did not respond it. after then, he visited our department. the radiography of the chest at the opd suggested the right lung infection. he was previously healthy, and had a history of allergy to heartleaf houttuynia herb (a traditional chinese medicine). physical examination (pe) on admission: t: 37.4°c; p: 95 bpm; r: 20 bpm; and bp: 129/73 mmhg. the breath sounds of both lungs were coarse, and no dry or moist rales were auscultated. the heart and abdomen were unremarkable. routine urine test: urine glucose: 1?; urine specific gravity: 1.03; protein: 1?; and the others were within the normal ranges. routine stool test: occult blood (chemical method): weakly positive. the creatine kinase was within the normal range; lactate dehydrogenase: 279 u/l:; and procalcitonin was within the normal range. to figure out the potential pathogen of his infection, a panel of extra laboratory tests was performed, and the results are shown in table 1 . through those tests, all clinically frequent pathogens are excluded. during the hospitalization, the ct scans of lungs and the dynamics of immune responses were closely monitored. summary reports of serial ct scans and serial blood tests of the male patient are shown in fig. 1 and table 2 . after admission, according to our clinical experience, the patient was given methylprednisolone 40 mg iv gtt for once, and then the fever subsided. the patient was given human gamma globulin 10 g iv gtt qd for five successive days, and then the dose was changed to 5 g. considering the cause was unknown, we also used drugs to treat atypical pathogens, including moxifloxacin for mycoplasma and chlamydia, and oseltamivir and abidol hydrochloride for influenza a virus; meanwhile, the patient was given chinese patent medicine tanreqing iv gtt for adjunctive therapy. on jan 10, 2020, the male patient was re-examined for all inflammatory indices and all showed normal, and he was discharged from hospital on the same day. the female patient was 38 years old and was admitted due to fever, cough, and vomiting for one day on dec 30, 2019. on admission, she had no dyspnea, no chest distress, no expectoration, no pharyngalgia, no nasal discharge, nor nasal obstruction. she was previously healthy, and had no history of allergy to food or drug. pe on admission: t: 38.5°c; p: 128 bpm; r: 22 bpm; and bp: 107/68 mmhg. the breath sounds of both lungs were coarse, and no dry or moist rales were auscultated. the heart and abdomen were unremarkable. the urine and stool examination results were unremarkable. the creatine kinas, lactate dehydrogenase, and procalcitonin were all within the normal ranges. she was examined for the same panel of known pathogens as her husband did, and all showed negative. during her hospitalization, the ct scan of lungs and the dynamics of immune responses were also closely monitored. summary reports of serial ct scans and serial blood tests of the female patient are shown in fig. 2 and table 3 . after admission, according to clinical experience, the patient was given methylprednisolone 40 mg iv gtt for five days, then the patient was given human gamma globulin 10 g iv gtt qd for seven successive days, and then the dose was changed to 5 g. the other drugs were the same as her husband. we also used drugs to treat atypical pathogens, including moxifloxacin for mycoplasma and chlamydia, and oseltamivir and abidol hydrochloride for influenza a virus; meanwhile, the patient was given chinese patent medicine tanreqing iv gtt for adjunctive therapy. on jan 10, 2020, the female patient was re-examined for all inflammatory indexes and all showed normal, and he was discharged from hospital on the same day. after the news press release of china cdc on january 9, 2020 (china cdc 2020), and the confirmation of the 2019-ncov on january 12 from who (who 2020), we tested the serum samples of those two couple patients with coronavirus antibodies provided by prof. zheng-li shi (wuhan institute of virology, chinese academy of sciences) (wang et al. 2018 ). the tests results are shown in table 4 , which indicate that the couple patients were both positive for coronavirus infections. in this couple, the clinical manifestations were moderate to ardent fever, and decrease in white blood cells and lymphocytes; ct findings of the chest were patchy shadows or ground-glass shadows in multiple segments and lobes; they did not respond to 3-day normative anti. but the clinical manifestations in this couple were not exactly the same. on admission, beside fever, the male was predominately manifested by dyspnea and only had mild abnormality in routine urine and stool test results, while the female had obvious gastrointestinal symptoms such as vomiting and diarrhea. these suggest this virus may be present in urine and stools, but we did not run pathogen isolation and detection, and it is only presumption. as for why the male had only abnormality in urine and stools rather than gastrointestinal symptoms, while the female had no abnormality in urine or stools but had gastrointestinal symptoms on the contrary, we have no definite explanation. before the onset, the couple went to a restaurant one stop away from the huanan seafood wholesale market to eat frozen seafood. maybe they get sick after being exposed to the same pathogen. the male developed symptoms first and had more severe clinical symptoms, which may be related to his immunity was better than the female. after the first exposure to the virus, patients with better immunity have stronger body reactions. innate immune cells play a vital role in effective host responses to various pathogens. neutrophils and monocytes-macrophages are the major innate immune cells that coordinate innate immunity against viral lung infections. respiratory viruses can inhibit the innate immune response, and consequently obtain effective opportunities for virus replication and infection. the affected innate immune response also affects subsequent adaptive immune responses; so, viral innate immune evasion often disrupts the complete protective immunity (kikkert 2020 ). therefore, initially, of the two patients, the female had normal neutrophilic granulocytes initially, and both had normal monocytes. the levels of cd3, cd4, cd8, cd19, and cd16 ? 56 cells were all decreased in both patients. it is speculated that the number of immune cells in the blood decreased due to a large number of them were lost or exuded to the infectious site to participate in the body's defense response. on january 5, 2020, the female' conditions were improved, but the cd3, cd4, and cd8 cells declined further. perhaps the female patient recalled more immune cells to participate in the antiviral process in her body. our observations show that the number of cd3, cd4, and cd8 cells cannot predict disease progression. in this study, the male had a more severe decrease in immune cells and more severe conditions than the female. in addition, after comparing our other similar cases, we found that the lower the number of primary immune cells, the more severe the conditions, which is consistent with previous observations (huang et al. 2004) . it is known that in the resting state nk cells are cd56 ? cd16-. after stimulation, they activate and transform into cytotoxic cd56 ? cd16 ? nk cells and participate in the antiviral immunity (seillet et al. 2016) . after the female's condition improvement, the number of cd19 ? and cd16 ? 56 ? cells increased, and this may suggest that the number of cd19 ? and cd16 ? 56 ? cells can be used as an indicator of the patient's disease status. from a previous study on the dynamic changes of peripheral blood immune cells in some 2003 sars cases reported by dr. jianping zhang et al. from beijing ditan hospital, it was shown that during the onset of sars patients, b cells showed a continuous increase, while nk cells showed a slow and continuous decline, which was statistically significant (zhang et al. 2003) . however, our female patient is not consistent with this observation. may it suggest that the 2019-ncov induce slight different immune response from the sars-cov? to answer this, it requires more samples and further comprehensive studies. the patient had no fever, and the cough, chest distress, and dyspnea were slightly improved the symptoms were improved the patient complained of no discomforts when the symptoms in the male were improved on january 3, 2020, the chest imaging manifestations progressed a little compared with before on the contrary. this condition was previously reported in severe legionella pneumonia. the condition change was manifested mainly by clinical symptoms, while the imaging manifestations may be related to the increased exudation and strengthened responsiveness. but it is interesting that the patient's condition was improved but crp and saa were also increased, which has not attracted attentions in the past. it was the same in the female. on january 5, 2020, the patient's condition was improved, and the image showed the lesions were slightly absorbed when compared with before, while crp and saa were also increased significantly. this aspect is worthy of further observation and research. the treatment of these two patients was successful, and they both were discharged from the hospital within 2 weeks. as for experience of medication, we used large doses of gamma globulin, and hormone at the beginning of the disease. but the duration of hormone was different for different patients. after using 40 mg once, the drug was no longer used for the male, while the female used it for a longer time, mainly according to the clinical symptoms and changes of chest imaging findings. other treatments were antibacterial drugs, drugs for atypical pathogens such as mycoplasma and chlamydia, oseltamivir and abidol hydrochloride for viruses, and chinese herbal medicine tanreqing. in general, even in patients with the same viral infection, the clinical manifestations can be different; the severity of the condition may be related to the number of immune cells; crp and saa may not only be related to the severity of the infection, but may also indicate the outcome of the condition; and human blood gamma globulin and hormone appeared to play roles in the treatment of these two patients. the fever subsided, the diarrhea disappeared, and the cough lasted the fever and diarrhea disappeared, and the cough was improved the patient complained of no discomforts immunological characteristics of 1291 cases of severe acute respiratory syndrome in beijing innate immune evasion by human respiratory rna viruses national health commission of the people's republic of china (2007) national surveillance, investigation and management plan of unexplained pneumonia cases development, homeostasis, and heterogeneity of nk cells and ilc1 serological evidence of bat sars-related coronavirus infection in humans world health organization (who) (2020) novel coronavirus-china the study of b lymphocyte and nk cell in severe acute respiratory syndrome (sars) patients clinical features and treatment of 2019-ncov pneumonia patients in wuhan acknowledgements this work was supported by the strategic priority research program of the chinese academy of sciences (xdb29010101 to zls). conflict of interest the authors declare that they have no conflict of interest.animal and human rights statement all procedures performed in studies involving human participants were in accordance with the ethical standards of the ethical review committee of renmin hospital of wuhan university and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. informed consent was obtained from all participants enrolled in the study. key: cord-333195-m4gvpsf8 authors: lu, renfei; wu, xiuming; wan, zhenzhou; li, yingxue; zuo, lulu; qin, jianru; jin, xia; zhang, chiyu title: development of a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of sars-cov-2 date: 2020-04-01 journal: virol sin doi: 10.1007/s12250-020-00218-1 sha: doc_id: 333195 cord_uid: m4gvpsf8 nan transmission ability, and it is spread by droplets produced by coughing and sneezing, infecting susceptible subjects through direct contacts and other possible transmission routes (e.g. fecal-mouth transmission) (guan et al. 2020; . as of mar. 27, 2020, the outbreak had resulted in 533,464 laboratory-confirmed cases, including 24,097 deaths around the world. currently, there lacks suitable antiviral drugs or vaccines for sars-cov-2. early, rapid, and reliable diagnostic assays for sars-cov-2 is of top priority to facilitate public health interventions that can reduce or avoid further spread of sars-cov-2 (dennis lo and chiu 2020). quantitative real-time pcr (qpcr) is a robust technology for reliable laboratory diagnosis, and routinely used to detect causative pathogens. since the release of genomic sequence of sars-cov-2 in jan. 11, 2020, many taqman probe-based rt-qpcr assays had been developed and used in confirmation of large number of sars-cov-2 infections during the outbreak (chu et al. 2020; dennis lo and chiu 2020) . facing the on-going increase of suspected cases and potential contacts, however, the detection capacity of rt-qpcr assays have shown limitations in speed and volume because these assays require sophisticated equipment and highly trained personnel, and are relatively time-consuming (about 1.5-2 h). therefore, there is an urgent demand for faster and sensitive point-of-care testing (poct) assays to facilitate rapid detection of sars-cov-2 infection. however, there is no poct assay available for sars-cov-2 yet. here, we present a novel visual reverse transcription loop-mediated isothermal amplification (rt-lamp) assay for rapid and sensitive detection of sars-cov-2 using mismatch-tolerant technique. based on sars-cov-2 genomic sequences, we designed six sets of lamp primers (4, 1, and 1 in n, s and rdrp genes, respectively) using the open access primer explorer v.5 software tool (http://primerexplorer.jp/). we firstly excluded primer sets with non-specific amplification, and then selected the primers having high amplification efficiency using the conventional rt-lamp method. the rdrp primers showed higher amplification efficiency, and were selected to establish the sars-cov-2 detection assay using the mismatch-tolerant rt-lamp method zhou et al. 2019 ). the rdrp primer information is shown in fig. 1a ntc template input 1: 3 x 10 5 copies 2: 3 x 10 4 copies 3: 3 x 10 3 copies 4: 3 x 10 2 copies 5: 3 x 10 1 copies 6: 3 copies cycle (mins) 6 d 3 (10 5 10 4 10 3 10 2 10 1 ) ntc clinical samples virologica sinica standard strains hcov-oc43 (vr-1558) and hcov-229e (vr-740) were tested. none or very weak amplification signals were observed for all 17 respiratory viruses after 50 min, indicating good specificity of the assay (fig. 1c) . although we did not test the specificity for sars-cov and mers-cov because of the lack of clinical samples or standard strains, the sequence comparison suggests the rt-lamp can also amplify sars-cov due to high sequence similarity (fig. 1b) . to prepare rna standard, a sars-cov-2 rdrp fragment (15,034-15,274 nt in wuhan-hu-1, genbank: mn908947.3) was amplified from positive clinical sample with t7-promoter-containing primers, and then the products were subjected to in vitro transcription. rna products were quantitated by nanodrop 2000c (thermo, usa). a tenfold serial dilution of rna standard from 10 6 to 1 copies per ll was used to test the sensitivity of the sars-cov-2 rt-lamp assay. the result showed that the rt-lamp can detect as few as 3 copies of sars-cov-2 rna per 25 ll reaction (fig. 1d ). in particular, amplification curves of template inputs [ 300 copies appeared within 20 min, showing a fast amplification (fig. 1d) . for the poct diagnosis of sars-cov-2 infection in the resource-poor settings, we developed the assay into a visual detection using warmstart colorimetric lamp 2 9 master mix (new england biolabs, beverly, ma, united states). a 25 ll reaction system includes 1 9 warmstart colorimetric lamp buffer, 0.15 unit of q5 high-fidelity dna polymerase, 1.6 lmol/l each of primers of fip and bip, 0.2 lmol/l each of primers of f3 and b3, and 0.8 lmol/l of loop primer flp and blp. the reactions were performed at 63°c, and the color change was observed at the 30-, 40-, and 50-min time points. the visual detection system contains a ph-sensitive indicator dye cresol red. because lamp amplification results in a significant ph change of buffer from an initial alkaline condition to a final acidic condition (tanner et al. 2015) , the color change of cresol red from burgundy to orange or yellow indicates a positive reaction. the assay gave a clear color indication for all tested samples during 30-50 min (data not shown). we selected 40 min as the cut-off for the visual assay (fig. 1e) . the sensitivity of the colorimetric rt-lamp assay for sars-cov-2 was 30 copies per reaction, slightly lower than the real-time monitoring (fig. 1e) . to evaluate the performance of the colorimetric rt-lamp assay for sars-cov-2, we compared it with the sars-cov-2 rt-qpcr kit (liferiver bio, shanghai, china) using 24 clinical samples. all 17 positive samples as shown by the rt-qpcr assay were also positive by the novel rt-lamp assay, demonstrating a 100% consistency (supplementary table s1 ). the severity of sars-cov-2 pandemic had exceeded sars-cov. in jan. 30, 2020, the covid-19 pandemic was declared as a public health emergency of international concern by who. early diagnosis and timely implementation of intervention and quarantine measures are very crucial for prevention and control of emerging infectious diseases such as covid-19 (dennis lo and chiu 2020). the rapid identification of sars-cov-2 and subsequent release of its genome sequence are very important in a public health perspective, and these made the development of sars-cov-2 diagnostic assays possible. many rt-qpcr assays have been developed and used in the confirmation of sars-cov-2 infection during the pandemic (chu et al. 2020) . however, high level facility requirement of rt-qpcr assays is a major factor limiting the confirmation speed and causing a large number of suspected cases waiting for tests as these assays are not available in primary care and community hospitals or health care centers where there were no real-time thermal cycler and trained professional. on the other hand, the median incubation period of covid-19 is 4 days (range 0-24.0 days), and the longest incubation period observed is 24 days (guan et al. 2020) . in particular, some infected individuals did not develop obvious clinical symptoms (rothe et al. 2020) . the long incubation period and asymptomatic infections imply a huge risk of virus transmission to other people (wu et al. 2020a, b) . in this respect, poct detection tools that can be performed in fields and even patients' own houses have a huge advantage, and should be developed. lamp is the most commonly used isothermal amplification technique for poct diagnosis due to its high sensitivity, rapid reaction, simple operation, and easy observation of results. recently, we developed a mismatchtolerant lamp method by adding 0.15 u of high-fidelity dna polymerase . the main difference between the mismatch-tolerant lamp and the conventional ones is the inclusion of an additional amount of highfidelity dna polymerase in the former, which largely improves the sensitivity and reaction speed zhou et al. 2019) . using this mismatch-tolerant technique, we developed a one-step single-tube rt-lamp assay for detection of sars-cov-2. the assay can be performed at 63°c for 40 min in a real-time or a regular thermal cycler, or any heating block such as a dry incubator or a water bath. the result is easily judged by the color change from burgundy to orange or yellow. compared to the conventional lamp protocol, the novel sars-cov-2 rt-lamp assay is more rapid and more sensitive. the sensitivity of the assay is 30 copies per reaction. although we did not test the specificity for sars-cov and mers-cov due to a lack of clinical samples, we sound the assay exhibited specificity by testing 17 common human respiratory viruses, including four other human coronaviruses oc43, 229e, hku-1 and nl63. the evaluation with 24 clinical samples showed that all 17 covid-19 patients in nantong city were positive for sars-cov-2 by both the rt-lamp and the rt-qpcr assays, showing a full consistence. in conclusion, the novel visual rt-lamp assay is a simple, rapid, and sensitive approach for detection of sars-cov-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current sars-cov-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of sars-cov-2 from bats to humans. molecular diagnosis of a novel coronavirus (2019-ncov) causing an outbreak of pneumonia racing towards the development of diagnostics for a novel coronavirus (2019-ncov) coronaviridae study group of the international committee on taxonomy of v (2020) the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 clinical characteristics of coronavirus disease 2019 in china the first disease x is caused by a highly transmissible acute respiratory syndrome coronavirus a mismatchtolerant rt-lamp method for molecular diagnosis of highly variable viruses feng z (2020) early transmission dynamics in genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding transmission of 2019-ncov infection from an asymptomatic contact in germany visual detection of isothermal nucleic acid amplification using ph-sensitive dyes compensation of ace2 function for possible clinical management of 2019-ncov-induced acute lung injury a new coronavirus associated with human respiratory disease in china nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study a mismatch-tolerant reverse transcription loop-mediated isothermal amplification method and its application on simultaneous detection of all four serotype of dengue viruses china novel coronavirus i, research t (2020) a novel coronavirus from patients with pneumonia in china acknowledgements we thank the biobank of pathogen discovery and big data center, institut pasteur of shanghai, chinese academy of sciences, for storing the standard strains of hcov-oc43 and hcov-229e that were previously purchased from the american type culture collection (atcc). this work was supported by the grants from the national science and technology major project of china (2019yfc1200603, 2017zx10103009-002). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. conflict of interest the authors declare that they have no conflict of interest.animal and human rights statement the study was approved by the ethics committees of nantong third hospital affiliated to nantong university (e2020003). written informed consents were obtained from all involved patients. key: cord-003246-3ajfb18m authors: liang, zhenli; pan, hongbo; wang, xijing; zhu, yinchuan; dang, yiwu; fan, xiaohui; gao, lingxi; zhang, zengfeng title: histopathological features and viral antigen distribution in the lung of fatal patients with enterovirus 71 infection date: 2018-04-19 journal: virol sin doi: 10.1007/s12250-018-0029-y sha: doc_id: 3246 cord_uid: 3ajfb18m nan enterovirus 71 (ev71) infection causes hand-foot-andmouth disease (hfmd) in infants and children. patients with hfmd usually have good prognosis; however, in some extreme cases the infection can be accompanied by central nervous system diseases, eventually leading to cardiorespiratory failure, and even death. currently, ev71 has become one of the most important pathogens infecting central nervous system, after poliovirus. previous studies have shown that lung injury in patients with hfmd is associated with neurogenic pulmonary edema (npe) after ev71 infection of the brainstem, rather than with direct viral invasion (jiang et al. 2012 ). this might be because ev71 was not isolated and inflammatory cell infiltration rarely occurred in lung and lung injured tissues (kao et al. 2004; jiang et al. 2012) . ev71 can be transmitted through respiratory tract and cause upper respiratory tract infection, such as herpangina; nevertheless, the translocation route from the upper respiratory tract to the lung tissue or through viremia to the lung still remains unclear. viral receptors have an important role in mediating the specific binding of viruses to the target cells. the distribution of viral receptors is usually closely related to the host range and tissue tropism of the viruses, and is considered as one of the key factors for viral pathogenicity. to further explore the causes of lung injury by ev71, we first examined the distribution of ev71 receptor in normal human lung tissues, and then investigated the target cells of ev71 infection, thus providing the molecular basis for the lung infected ev71. consequently, the pathological changes in the lung tissues of fatal ev71 infection were perceived, and the distribution of ev71 antigen and various inflammatory cells in the lung tissues were detected. to investigate whether ev71 could infect the lung tissues of lower respiratory tract, the distribution of ev71 receptor, psgl-1 (human p-selectin glycoprotein ligand-1) and scarb2 (human scavenger receptor class b member 2) (nishimura et al. 2009; yamayoshi et al. 2009 ) were examined in the lung tissue specimens collected form 12 patients with tumor or non-inflammatory diseases. patients including four cases of pneumonic pseudotumor, two cases of pulmonary cyst, 4 cases of pulmonary fibroma and two cases of pulmonary myxoma. we selected normal lung tissue in the specimens of pneumonectomy in these patients. general, the distribution of those receptors is the same as health people. the tissues were fixed by 10% formalin, dehydrated by gradient ethanol, soaked and embedded with wax. paraffin sections were sectioned continuously at a thickness of 4 lm. tissue sections were dewaxed into water, and endogenous peroxidase activity was blocked. primary antibodies of psgl-1 (1:50) and scarb2 (1:100) (products from vector company, california, usa) were added, and incubated overnight in a refrigerator at 4°c. samples were then incubated with horseradish peroxidase labeled (mouse/rabbit) igg polymers (products from vector company, california, usa) at room temperature, developed by dab, re-dyed by hematoxylin and observed under microscope. no histopathological changes and infective lesions were found in the selected tissues. results showed that scarb2 was positively distributed in the intrapulmonary bronchial epithelial cells, bronchiolar epithelial cells, alveolar cells and macrophages ( fig. 1a-1c ). psgl-1 was positively zhenli liang and hongbo pan have contributed equally to this work. the online version of this article (https://doi.org/10.1007/s12250-018-0029-y) contains supplementary material, which is available to authorized users. distributed in a scattered pattern in the intrapulmonary bronchial epithelial cells, bronchiolar epithelial cells and macrophages; while no expression was found in the alveolar cells ( fig. 1d-1f ). these results were consistent with jiao et al. (2014) . furthermore, ev71 receptors scarb2 and psgl-1 were expressed in the lung tissues; scarb2 was more widely distributed than psgl-1. these data suggested that ev71 could infect lung tissues, with respiratory tract as one of the portals of invasion. clinically, ev71 infection could cause fever, and hfmd/herpangina, and it can be accompanied by central nervous system diseases such as brain stem encephalitis or lung injury. previous studies have shown that lung injury lesions are characterized by pulmonary congestion, different degrees of neurogenic pulmonary edema and pulmonary hemorrhage, with no diffuse lung injury (chang et al. 1999; kao et al. 2004; jiang et al. 2012) . neurogenic pulmonary edema is often caused by ev71 infection in the brain stem. it was speculated that ev71 may first destroy the specific regulatory function of the brain stem, causing dysfunction of autonomic nervous system. this subsequently resulted in the excess fluid accumulation in the lung interstitium and/or alveoli, forming interstitial and/or alveolar pulmonary edema syndrome (chang et al. 1999; kao et al. 2004; jiang et al. 2012) . moreover, it is clinically characterized by acute dyspnea and progressive hypoxemia, which is similar to acute respiratory distress syndrome (ards) (jiang et al. 2012) . the pathological changes of ards include dark red coloring of the lung tissue and hyaline membrane formation and organization. in this study, the bronchial and pulmonary tissues from 6 autopsy cases with ev71 infection were obtained from the department of pathology at the first affiliated hospital of guangxi medical university. patients included in this study were between 1 and 3 years old, and there were 4 males and 2 females. ev71 isolation and nucleic acid detection in the cerebrospinal fluid, brain, heart and lung tissues of 6 cases were all positive (table 1 , supplementary figure s1 ). he staining was used to observe the pathological changes. immunohistochemistry was performed to detect the ev71 antigen and kinds of inflammatory cells. antigen retrieval was performed under high temperature and high pressure after dewaxed tissue sections into water and blocking the endogenous peroxidase activity. samples were then incubated with the following primary antibodies: ev71 (1:10,000, to label ev71 antigen, provided by the national infectious disease diagnostic reagent and vaccine engineering technology research center, xiamen university, china), cd3 (to label t cells), cd20 (to label b cells), cd57 (to label nk cells), cd68 (to label macrophages) (all purchased from sigma, new york, usa) at 4°c overnight, following incubation with horseradish peroxidase labeled (mouse/rabbit) igg polymers (sigma products, new york, usa) at room temperature was applied, developed by dab, and observed under microscope. the protein edema in the alveolar space with fibrin exudation was observed. some alveoli were filled with mononuclear cells, alveolar macrophages, exfoliated epithelial cells and cell debris in 5 out of 6 autopsy cases with ev71 infection (fig. 1g) . also, fibrin like exudates and hyaline membrane formation in some alveolar cavities with moderately light pink stained exudation in the alveolar septum and space, fibrinous exudation and pulmonary hyaline membrane were observed. the alveolar septum was widened, and the capillaries in the septum were highly dilated and congested (fig. 1h) . these results confirmed the above speculation of neurogenic pulmonary edema. the mortality of neurogenic pulmonary edema remained high, thus remaining an important b fig. 1 detection of ev71 in fatal patients' lung and the histopathology changes. a-f expression and distribution of ev71 receptor scarb2 and psgl-1 in the normal lung tissues (immunohistochemistry). scarb2 was positively expressed in the bronchial epithelial cells (black arrow) (a), bronchiolar epithelial cells (black arrow) (b), alveolar cells (black arrow) and macrophages (red arrow) (c); psgl-1 produced a scattered pattern and was positively expressed in the bronchial epithelial cells (black arrow) (d), bronchiolar epithelial cells (black arrow) and macrophages (red arrow) (e), while no expression was found in the alveolar cells (f). g-l pathological changes of lung tissues in fatal children with ev71 infection (he staining). protein edema in the alveolar space with fibrin exudation was observed inside, and some alveoli were filled with mononuclear cells, alveolar macrophages, exfoliated epithelial cells and cell debris (g); the alveolar septum was widened, the capillaries in the septum were highly dilated and congested, with infiltration of inflammatory cells (h); intrapulmonary bronchitis and bronchiolitis, diffuse or focal infiltration of inflammatory cells in the wall and surrounding tissues (i, j); compensatory emphysema phenomenon of ruptured alveolar wall and fusion of pulmonary alveoli (k); the lymph nodes near the hilar bronchus indicated a reactive hyperplasia, germinal center enlargement, paracortical zone atrophy, and lymphocyte depletion (l) (black arrow). m-t distribution of ev71 antigen and inflammatory cells in the lung tissues of fatal children with ev71 infection (immunohistochemistry). cd3? t lymphocytes (m) and cd20? b lymphocytes (n) were diffusely distributed in the wall of bronchus and bronchioles and surrounding tissues; cd57? nk cells (o) and cd68? macrophages (p) were scattered in the widened alveolar septum and alveolar spaces; virus antigen was positive in the intrapulmonary bronchial epithelial cells (q), bronchiolar epithelial cells (r), alveolar cells (s) and macrophages (t) (black arrow). table 1 the ev71 nucleic acid detection result of cerebrospinal fluid, brain, heart and lung tissues in 6 cases (positive cases/total cases). cerebrospinal fluid brain heart lung nucleic acid 3/6 6/6 1/6 4/6 complication and main cause of death in children with ev71 viral infection. interestingly, bronchitis and bronchiolitis, diffuse or focal infiltration of inflammatory cells in the wall and surrounding tissues, such as lymphocytes and mononuclear macrophages around the bronchial wall (fig. 1i, 1j) , widened alveolar septum with infiltration of inflammatory cells, and some mononuclear cells in the lumen or/and alveolar space filled, alveolar macrophages and exfoliated epithelial cells (fig. 1g, 1h ) were all found in 4/6 dead infants with ev71 infection. these results were consistent with the pathological changes of viral pneumonia. also, the phenomenon of compensatory emphysema of ruptured alveolar wall, fusion of pulmonary alveoli (fig. 1k) , and the lymph nodes near the hilar bronchus demonstrated reactive hyperplasia, germinal center enlargement, paracortical zone atrophy, and lymphocyte depletion (fig. 1l) . furthermore, the distribution of immune cells in the infected lung tissue was detected. t cells (fig. 1m) and b cells (fig. 1n) were diffusely distributed in the bronchus and bronchioles and surrounding tissues; nk cells ( fig. 1o ) were distributed in a scattered manner in the widened alveolar septum and alveolar spaces; and macrophages ( fig. 1p) were mainly located in the alveolar space. these data suggest that the above immune cells may have an important role in the local immune response of the lung tissue in critically ill patients. literature reported that ev71 infection could cause respiratory diseases, such as pharyngitis, laryngitis, bronchitis and pneumonia (gilbert et al. 1988; merovitz et al. 2000; tsai et al. 2001) . although ev71 was not isolated from the autopsy of lung specimens, ev71 rna remained positive (vallet et al. 2009 ). to further confirm whether ev71 could infect the lung tissues; immunohistochemical method was used to detect ev71 virus antigen. briefly, our data showed that the viral antigen was positive in the bronchial (fig. 1q ) and bronchiolar epithelial cells (fig. 1r ) and positively scattered in the alveolar cells (fig. 1s) and macrophages (fig. 1t ) in 4 out of 6 cases. furthermore, the distribution of viral antigen in the lung tissues was consistent with that of ev71 receptor, indicating that the lower respiratory tract of the lung also remained the target tissue of ev71 infection. therefore, the above results suggested that lung injury was caused by direct injury and/or immunopathologic injury of respiratory tract epithelium after viral infection. lung tissues of ev71 infected patients with severe hfmd showed interstitial pneumonia and positive viral antigens in the pulmonary epithelial cells and macrophages. this in turn suggested that, lung injury, in addition to the neurogenic cause, might also be caused by ev71 infection that in turn invades the upper respiratory tract, spreads to the lower respiratory tract, and then infects the lung tissues. therefore, severe ev71 infection is considered as a systemic virally infective disease. in addition to the involvement of the central nervous system, the respiratory system and lung tissues were also considered as the main target organs of the virus. in critically ill children, neurogenic damage and/or virus that directly infect the lung causes pulmonary edema and interstitial pneumonia, leading to respiratory and circulatory failures and even death. clinical features and risk factors of pulmonary oedema after enterovirus-71-related hand, foot and mouth disease outbreak of enterovirus 71 in victoria, australia, with a high incidence of neurogenic involvement autopsy findings in children with hand, foot, and mouth disease distribution of ev7 1 receptors scarb2 and psgl-1 in human tissues mechanism of fulminant pulmonary edema caused by enterovirus 71 enterovirus 71 infections at a canadian center human p-selectin glycoprotein ligand-1 is a functional receptor for enterovirus 71 respiratory virus infections among pediatric inpatients and outpatients in taiwan from 1997 to 1999 fatal case of enterovirus 71 infection scavenger receptor b2 is a cellular receptor for enterovirus 71 acknowledgements this study was supported by the guangxi natural science foundation grant (2014gxnsfaa118200). conflict of interest the authors declare that they have no conflict of interest.animal and human rights statement the study was approved by the medical ethics committee of guangxi medical university. written informed consents were obtained from all the patients, or their guardians and families prior to the study. key: cord-006752-fmdvwnbw authors: mao, huawei; yen, hui-ling; liu, yinping; lau, yu-lung; malik peiris, j. s.; tu, wenwei title: conservation of t cell epitopes between seasonal influenza viruses and the novel influenza a h7n9 virus date: 2014-06-17 journal: virol sin doi: 10.1007/s12250-014-3473-3 sha: doc_id: 6752 cord_uid: fmdvwnbw a novel avian influenza a (h7n9) virus recently emerged in the yangtze river delta and caused diseases, often severe, in over 130 people. this h7n9 virus appeared to infect humans with greater ease than previous avian influenza virus subtypes such as h5n1 and h9n2. while there are other potential explanations for this large number of human infections with an avian influenza virus, we investigated whether a lack of conserved t-cell epitopes between endemic h1n1 and h3n2 influenza viruses and the novel h7n9 virus contributes to this observation. here we demonstrate that a number of t cell epitopes are conserved between endemic h1n1 and h3n2 viruses and h7n9 virus. most of these conserved epitopes are from viral internal proteins. the extent of conservation between endemic human seasonal influenza and avian influenza h7n9 was comparable to that with the highly pathogenic avian influenza h5n1. thus, the ease of inter-species transmission of h7n9 viruses (compared with avian h5n1 viruses) cannot be attributed to the lack of conservation of such t cell epitopes. on the contrary, our findings predict significant t-cell based cross-reactions in the human population to the novel h7n9 virus. our findings also have implications for h7n9 virus vaccine design. in march 2013, human infections with a novel avian infl uenza h7n9 virus were identifi ed in china . although other h7 subtype infections in humans have been detected before, this is the fi rst report of human infection with an h7n9 infl uenza virus. as of 12 th august 2013, a total of 135 laboratory-confi rmed cases of human h7n9 infection have been reported to who, 44 of these being fatal (who, 2013) . phylogenetic anal-ysis indicated that this novel virus is a reassortant virus, with the hemagglutinin (ha) gene originating from a duck influenza virus, neuraminidase (na) gene from a wild bird virus and six internal genes derived from poultry avian h9n2 viruses (lam t t, et al., 2013; . human infections with h7n9 virus were fi rst clustered around the yangtze river delta followed by occurrence in the neighbouring provinces li q, et al., 2013) . most of hospitalized patients had severe illness, ranging from pneumonia to ards and multi-organ dysfunction sometimes necessitating mechanical ventilation or extra corporeal membrane oxygenation . despite these high fatality rates, a recent study indicated that there may be many milder cases that were not reported and human infections with h7n9 virus may be less severe than previously reported . t cell immunity plays important roles in host defenses against influenza virus infection. while it is generally believed that t-cells do not prevent virus infection in the way that antibody does, cross-reactive t cells, including cd4 and cd8 t cells, are believed to enhance virus clearance and reduce severity of illness, even in the absence of virus-specifi c antibodies (mcmichael a j, et al., 1983) . in the 2009 h1n1 pandemic (pdmh1n1) outbreak, we analyzed the conserved cd8 t cell epitopes in pdmh1n1 virus, and demonstrated that the conserved epitope-specifi c t cells established by seasonal infl uenza virus could cross react against the pandemic virus, which might contribute to the milder pandemic h1n1 illness overall and the lower infection attack rate in young adults even though they did not have detectable cross-neutralizing antibody (tu w, et al., 2010) . in this study, we analyzed the conservation of t cell epitopes of seasonal influenza virus in h7n9 virus, aiming to provide some insight into pre-existing immunity to the h7n9 illness. in the immune epitope database (iedb) (http://www. iedb.org) were retrieved, which is a publicly available database of epitopes for infectious agents (vita r, et al., 2010) . the epitopes that induced a positive t-cell response were included for analysis. the presence of these epitopes in seasonal influenza viruses including a/brisbane/59/2007 (h1n1) and a/brisbane/10/2007 (h3n2) were examined for each virus and then for the two viruses together. the conservation of these pooled t cell epitopes in the novel avian h7n9 (a/anhui/1/2013), 2009 pandemic (a/california/07/2009(h1n1)) (pd-mh1n1), avian h9n2 (a/quail/hong kong/g1/97) and highly pathogenic avian h5n1 (a/common magpie/ hong kong/5052/2007) viruses were further analyzed respectively. the epitopes with 100% conservation were included for analysis. the distribution of epitopes in different viral proteins was checked. chi square test was employed to compare the conservation of t cell epitopes in h7n9 virus with that in other virus. all experimentally determined t cell epitopes were retrieved from iedb database. a total of 223 mhc class i-restricted epitopes with positive t cell response have been reported in influenza a viruses. we examined the presence of these cd8 t cell epitopes in seasonal infl uenza h1n1 and h3n2 virus subtypes (prior to emergence of pdmh1n1). as shown in table 1 , 151 epitopes were detected in seasonal h1n1 and h3n2 viruses. of these, 81 were shared by both of them, 44 were unique to h1n1 and 26 unique to h3n2 virus. most of the epitopes were located in viral internal np, m1 and pb1 proteins, while few were in surface ha and na proteins. among the 151 seasonal infl uenza epitopes, 68.9% (104/151) were conserved in the novel h7n9 virus. a similar conservation level of these epitopes was also detected in pdmh1n1 (69.5%), h9n2 (68.2%) and h5n1 (71.5%) viruses. the conserved epitopes were mainly derived from viral np, m1 and pb1 proteins ( table 2) . as currently, a large number of humans have been exposed to pdmh1n1 prior to h7n9 outbreak, we further examined how many epitopes of seasonal h1n1, h3n2 plus pdmh1n1 viruses are conserved in the avian viruses. as shown in table 3 , 67.7% (113/167), 67.7% (113/167) and 73.1% (122/167) of these cd8 t cell epitopes were invariant in h7n9, h9n2 and h5n1 virus respectively. we next examined the conservation of cd4 t cell epitopes. 687 mhc class ii-restricted epitopes with positive t cell response were obtained from the database, of which 199 and 189 epitopes were present in seasonal h1n1 and h3n2 virus respectively. among them 71 were shared by both seasonal infl uenza viruses. most of the epitopes were derived from ha, np, m2 and na proteins (table 4 ). the conservation of these seasonal infl uenza viral epitopes in avian infl uenza viruses was further analyzed. as shown in table 5 , 23.3% (74/317) of these epitopes were conserved in h7n9 virus. compared to h7n9 virus, a similar conservation level was shown in h9n2 (28.4%, 90/317) and h5n1 (25.6%, 81/317) virus, but a much higher level (31.9%, 101/317, p<0.05) was conserved in pdmh1n1 virus. as with the cd8 t cell epitopes, the conserved cd4 t cell epitopes were mainly located in m1, np and pb1 proteins. few epitopes of surface ha and na proteins were invariant in the avian influenza viruses. we also analyzed the conservation of cd4 t cell epitopes of seasonal h1n1, h3n2 plus pdmh1n1 viruses in avian infl uenza virus. as shown in in this study, we analyzed the t cell epitopes in h7n9 infl uenza virus and found that around 70% and 20% of experimentally determined cd8 and cd4 t cell epitopes of seasonal infl uenza virus were conserved in the novel h7n9 virus. most of the conserved epitopes were derived from viral internal proteins. the degree of conservation of cd8 epitopes between seasonal influenza and novel h7n9 did not differ from that seen with pd-mh1n1, avian h9n2 or avian h5n1 viruses. thus lack of cross-reactive cd8 epitopes could not explain the apparently high susceptibility of humans to novel h7n9 virus. however, the number of cd4 t cell epitopes conserved between seasonal infl uenza h1n1 and h3n2 and h7n9 virus (23.3%) was lower than the conservation seen between seasonal infl uenza and pdmh1n1 (31.9%) but comparable to that seen in h9n2 (28.4%) and h5n1 (25.6%) viruses. thus it is conceivable that there was more cd4 t cell cross-protection against the pdmh1n1 both humoral and t cell immunity are responsible for human defense against influenza virus infection. t cells promote viral clearance and reduce illness severity (mbawuike i n, et al., 2007; mcmichael a j, et al., 1983; webby r j, et al., 2003) . unlike humoral immunity that protects against a specific influenza viral strain alone, t cells could cross-react against across serologically distinct viruses of different ha subtypes. the cross-protection of t cells has been demonstrated in humans in vivo by the cleveland family study (boon a c, et al., 2004; jameson j, et al., 1999) . humans who have not been exposed to avian infl uenza a (h5n1) virus do have cross-reactive memory t cells to a wide range of h5n1 peptides (lee l y, et al., 2008; roti m, et al., 2008) . in 2009 h1n1 pandemic outbreak, we also demonstrated the cross-reactivity of conserved epitope-specifi c t cells against pdmh1n1 virus (tu w, et al., 2010) . for the novel h7n9 influenza virus, although pre-existing humoral immunity is low in the general population (boni m f, et al., 2013) , here we showed that most cd8 and less cd4 t cell epitopes of seasonal infl uenza virus were conserved in h7n9 virus. it could be expected that these conserved epitope-specifi c t cells established by seasonal influenza virus may provide some protection against h7n9 virus, which might explain the presence of mild infection cases. although many of the hospitalized patients with h7n9 virus infection had severe illness, several cases were identifi ed via infl uenza-like illness surveillance network and presented with mild clinical phenotype, even did not require hospitalization. this implied that a substantial number of symptomatic infections with mild to moderate illness remained unconfi rmed. it was estimated that about 1500-27000 symptomatic h7n9 infections might have occurred as of may 28, 2013 . the h7n9 infection may indeed be less serious than previously reported, and the symptomatic case fatality risk was estimated to be 160-2800 per 100 000 symptomatic cases yu h, et al., 2013) . well designed sero-epidemiology studies may help clarify these issues. identifi cation of mild h7n9 infection cases is challenging due to the detection bias. generally, case detection is typically biased to severe patients with mild cases having lower probability of being detected (viboud c, et al., 2013) . therefore, the so far detected several mild cases may be just the tip of an iceberg of h7n9 infections at the community . the case detection bias also leads to an overestimation of fatality risk. in 2009 h1n1 pandemic, the case-fatality estimates rapidly declined with increased data from sero-epidemiological studies defining the true infection attack rate in the population (viboud c, et al., 2013) . similarly, it could be expected that the estimated fatality risk of h7n9 virus may decrease with greater information, which indeed has been demonstrated by a preliminary study . like avian h5n1 infection, most of patients with h7n9 infection who were hospitalized for medical care had severe illness, although both of the two viruses had conserved t cell epitopes that are shared with seasonal infl uenza virus. the inpatient fatality risk of h7n9 infection was estimated to be 36% on admission to hospital . indeed, the clinical phenotype of infl uenza illness depends on host defense mechanisms as well as the virus itself. in comparison to avian h5n1 virus, the h7n9 virus also induced signifi cant hyperinfl ammatory responses in hosts, although the extent of such responses appeared to be less extreme compared with h5n1 virus . substantial increased levels of serum cytokines and chemokines, including ip-10, mig, mip-1β, mcp-1, il-6, il-8 and ifn-α, were detected in patients with h7n9 infection, compared to healthy controls . hypercytokinemia may partly contribute to the clinical severity in h7n9 infection, as seen in h5n1 infection. as the general human population does not have pre-existing humoral immunity against h7n9 and present inactivated seasonal vaccination does not provide cross-protection (boni m f, et al., 2013; development of specific vaccines to prepare for a potential outbreak of h7n9 infection are underway. current influenza vaccine strategy targets an antibody response against surface ha antigen. recent in silico analysis showed that the ha protein of h7n9 virus has low t cell epitope content and poor immunogenicity (de groot a s, et al., 2013) which is compatible with our fi ndings that no cd4 t cell epitopes in ha and na proteins of seasonal influenza virus were conserved in h7n9 virus. the cd4 t cell response is required for the development of antibody response. thus, these data indicate that h7n9 infl uenza vaccine would be of relatively low effi cacy if it were based on current vaccine strategy. however, here we further demonstrate that many t cell epitopes of internal proteins of seasonal infl uenza virus were conserved in the novel h7n9 virus. it has been shown that t cell immunity can provide cross-protection against serologically distinct infl uenza viruses (boon a c, et al., 2004; jameson j, et al., 1999) . therefore, including more conserved t cell epitopes of internal proteins in the vaccine would be an applicable and attractive way to develop an effective h7n9 vaccine. population-level antibody estimates to novel infl uenza a/h7n9 recognition of homo-and heterosubtypic variants of infl uenza a viruses by human cd8+ t lymphocytes tropism and innate host responses of a novel avian infl uenza a h7n9 virus: an analysis of ex-vivo and in-vitro cultures of the human respiratory tract human infections with the emerging avian infl uenza a h7n9 virus from wet market poultry: clinical analysis and characterisation of viral genome preliminary inferences on the age-specific seriousness of human disease caused by avian influenza a(h7n9) infections in china comparative epidemiology of human infections with avian influenza a h7n9 and h5n1 viruses in china: a population-based study of laboratory-confi rmed cases. lancet low immunogenicity predicted for emerging avian-origin h7n9: implication for infl uenza vaccine design clinical fi ndings in 111 cases of infl uenza a (h7n9) virus infection human infection with a novel avian-origin infl uenza a (h7n9) virus detection of mild to moderate infl uenza a/h7n9 infection by china's national sentinel surveillance system for infl uenza-like illness: case series human cd8+ and cd4+ t lymphocyte memory to infl uenza a viruses of swine and avian species the genesis and source of the h7n9 influenza viruses causing human infections in china memory t cells established by seasonal human infl uenza a infection cross-react with avian infl uenza a (h5n1) in healthy individuals preliminary report: epidemiology of the avian infl uenza a (h7n9) outbreak in china origin and diversity of novel avian infl uenza a h7n9 viruses causing human infection: phylogenetic, structural, and coalescent analyses control of mucosal virus infection by influenza nucleoprotein-specific cd8+ cytotoxic t lymphocytes cytotoxic t-cell immunity to influenza healthy human subjects have cd4+ t cells directed against h5n1 infl uenza virus cytotoxic t lymphocytes established by seasonal human influenza cross-react against 2009 pandemic h1n1 influenza virus timely estimates of infl uenza a h7n9 infection severity the immune epitope database 2.0 protection and compensation in the infl uenza virus-specifi c cd8+ t cell response number of confirmed human cases of avian influenza a(h7n9) reported to who human infection with avian infl uenza a h7n9 virus: an assessment of clinical severity all the authors declare that they have no competing interest. this article does not contain any studies with human or animal subjects performed by the any of the authors. hm, yll, jsmp and wt designed the study. hm, hly and yl performed the study and analyzed the data. hm, yll, jsmp and wt wrote the paper. all authors read and approved the fi nal manuscript. key: cord-300466-sk9iilum authors: kong, wen-hua; zhao, rong; zhou, jun-bo; wang, fang; kong, de-guang; sun, jian-bin; ruan, qiong-fang; liu, man-qing title: serologic response to sars-cov-2 in covid-19 patients with different severity date: 2020-07-23 journal: virol sin doi: 10.1007/s12250-020-00270-x sha: doc_id: 300466 cord_uid: sk9iilum the immense patient number caused by coronavirus disease 2019 (covid-19) global pandemic brings the urge for more knowledge about its immunological features, including the profile of basic immune parameters. in this study, eighty-eight reported covid-19 patients in wuhan were recruited from january to february, 2020, including 32 severe/critical cases and 56 mild/moderate cases. their mean age was 56.43 years (range 17–83) and gender ratio (male/female) was 43:45. we tested sars-cov-2 rna with commercial kits, investigated the level of serologic igm and igg antibodies against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) using magnetic particle chemiluminescence immunoassays, and compared the results of serologic tests and nucleic acid test (nat). among 88 patients, 95.45% were confirmed as positive by the combination of nat and antibody test, which was significantly higher (p < 0.001) than by single nucleic acid test (73.86%) or serologic test (65.91%). then the correlation between temporal profile and the level of antibody response was analyzed. it showed that seroconversion started on day 5 after disease onset and igg level was rose earlier than igm. comparison between patients with different disease severity suggested early seroconversion and high antibody titer were linked with less severe clinical symptoms. these results supported the combination of serologic testing and nat in routine covid-19 diagnosis and provided evidence on the temporal profile of antibody response in patients with different disease severity. the outbreak of novel coronavirus disease 2019 has become a global pandemic and caused over 2 million infections by april 15th, 2020 (who 2020). according to the chinese centers for disease prevention and control (cdc) report, among 72,314 covid-19 cases in china's mainland most of cases (81%) presented only mild illness or moderate pneumonia, yet 14% developed severe symptoms such as dyspnea, high respiratory frequency and low blood oxygen saturation, and another 5% were in critical conditions like respiratory failure, septic shock, and multiple organ dysfunction/failure (epidemiology working group for ncip epidemic response and chinese cdc, 2020; wu and mcgoogan 2020) . the global case-fatality rate (cfr) of covid-19 was 6.4% as of april 15, 2020 (who 2020 ), yet the cfr could be even elevated among severe/critical cases, elder population, and patients with preexisting comorbidity chen et al. 2020; wu and mcgoogan 2020) . the reported cfr in icu cases ranged from 22.72% to 38.5% huang et al. 2020; richardson et al. 2020) . it is believed that early adaptive immune response plays an important role in eliminating the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), while the inflammation driven by elevated innate immune response is a major cause of life-threatening respiratory disorders in severe covid-19 cases thevarajan et al. 2020 (lauer et al. 2020; long et al. 2020; thevarajan et al. 2020; xiang et al. 2020; zhang et al. 2020; zhao et al. 2020a, b) , which confirmed the emergence of igm, igg and neutralizing antibodies against sars-cov-2 and suggested the application of serologic test in covid-19 diagnosis (long et al. 2020; zhang et al. 2020) . however, some reports about the temporal profile of sars-cov-2 antibodies were mutually contradictory (thevarajan et al. 2020; xiang et al. 2020; zhao et al. 2020a ) and the immune responses of patients with different demographic and clinical features remain unclear. in this study, we, compared the results of serologic tests and nucleic acid test (nat) from a group of covid-19 patients in wuhan, and analyzed the serologic igm and igg antibody level of patients with different disease severity. in this study, we included confirmed patients with both throat swab and blood sample delivered to the wuhan cdc laboratory before march 1st, 2020. the nucleic acid was extracted from 200 ll of throat swab medium using a generotex automated nucleic acid extraction system (tianlong, xi'an, china). due to medical resource limitations, two commercial quantitative pcr (qpcr) kits were employed in the detection of sars-cov-2 rna. one of the assays (daan gene, guangzhou, china) was used from january to early february, targeting at sars-cov-2 orf1ab and n gene, with a limit of detection (lod) of 500 copies/ml. the other assay (bgi, shenzhen, china) which was used after february 5th targeted at orf1ab fragment only and its lod was 100 copies/ml. the cutoff cycle-threshold (ct) was 40 for both kits. both assays were approved by the national medical products administration (nmpa) of china and had been established in our laboratory. the levels of sars-cov-2-binding igm and igg antibodies were assessed using semi-quantitative magnetic particle chemiluminescence immunoassays (m-clias) on an axceed 260 automated magnetic analyzer (bioscience, chongqing, china) (loeffelholz and tang 2020), as described by long et al.(2020) . both igm and igg assays had received nmpa approvals with registration numbers of 20203400182 and 20203400183, respectively. the sensitivity and specificity of the igm assay in pre-marketing clinical evaluation were 88.30% and 99.50%, while those of the igg assay were 87.25% and 99.25%. fifteen microliter of serum was diluted for 50 times before being used in each test. procedures and cut-off value set-up were performed following the manufacturers' instructions. antibody levels were presented as the measured chemiluminescence values divided by the cutoff (s/co) (long et al. 2020 ). statistical analyses were performed with prism 8.0 (graph-pad, san diego, usa). categorical variables were compared using chi square test. for continuous variables, t test or mann-whitney u test were employed after their normality determined by kolmogorov-smirnov test. a p-value of less than 0.05 was considered statistically significant. a total of 88 covid-19 patients from eleven designated hospitals were included in this study, of whom 43 were male and 45 were female. their mean age was 56.43 years old (range 17-83) and the median interval between initial symptom onset and sample collection was 11 days (range 1-37). thirty-two patients (36.4%) had severe/critical illnesses and required oxygen supplementation or higher life support, while the other 56 patients had mild or moderate symptoms (table 1) . qpcr test confirmed 65 sars-cov-2 infected cases among 88 participants (73.86%). no significant difference was observed between the positive rates of two qpcr kits (37/53 versus 28/35, v 2 = 1.133, p = 0.287). on the other hand, the positive rates of serum igm and igg antibody against sars-cov-2 were 32.95% (29/88) and 65.91% (58/88), respectively (table 2) . altogether, 84 covid-19 cases (95.45%) were identified among all patients by the combination of nat and antibody test, which was significantly more than single nat (v 2 = 15.793, p \ 0.001) or serologic test (v 2 = 24.643, p \ 0.001). the consistency rate between results of antibody test and nat was 48.86% [(39 ? 4)/88]. notably, all the patients that were positive for sars-cov-2 igm were also positive for sars-cov-2 igg. the earliest seroconversion of igg antibody was observed 5 days after the disease onset, and that time interval of igm antibody was 8 days (fig. 1) . for 51 patients with sample collected at 10 days or later after symptom onset, the seroconversion rate was 47.06% for igm (24/51) and 82.35% for igg (42/51). both antibodies were detectable in samples collected over 30 days after onset. when comparing patients with mild/moderate symptoms and patients with severe/critical diseases, no obvious difference was found between their gender ratios (p = 0.136), age composition (p = 0.571) and nat positive rates (p = 0.748), but the mild/moderate group had later sampling time and higher antibody positive rates than the severe/critical group (table 1) . when comparing to the severe/critical cases with the same sampling time, mild/moderate cases presented higher seroconversion rate and higher antibody titer for both igm and igg antibodies (fig. 2) . similar analysis was performed on cases of different genders and in different age groups, but no significant difference was observed between their antibody levels and temporal profiles (fig. 3 ). although nat has been regarded as the gold standard of covid-19 laboratory diagnosis, it is not suitable for low viral load patients and its accuracy is largely depended on the sampling procedure . it makes serologic test a valuable compliment for clinical diagnosis as well as providing information about the disease dynamic. in this study, two nmpa approved m-clia kits were used to detect the level of sars-cov-2 igm and igg antibodies in the serum of covid-19 patients. in addition to the 65 covid-19 patients identified by nat, the antibody tests identified another 19 sars-cov-2 infected patients among the 88-person cohort, which diminished the need of multiple sampling and significantly improved the diagnostic accuracy. meanwhile, the relatively low consistency between nat and antibody test results also indicated those temporal profile of serum antibody is vital for the interpretation of serologic test result and evaluating the immune protection situation of subject. in this study, level of serum antibody against sars-cov-2 started to rise since day 5 after disease onset and remained high in samples collected 1 month after onset. igg level was rose earlier than igm, just like in several other studies (thevarajan et al. 2020; zhang et al. 2020) , but it could be related to the unbalanced sensitivity of igm and igg assays, since earlier igm seroconversion has also been reported zhao et al. 2020a) . the validation and standardization of sars-cov-2 serologic assays in large clinical cohort is crucial for enabling uniform assessment of immunogenicity and efficacy (okba et al. 2020) . we further looked into the possible correlates of serum antibody level with demographic features, including gender, age and disease severity. our results suggested early seroconversion and high antibody titer were likely linked with less severe clinical symptoms. the finding is in consistent with earlier observations of recovered patients thevarajan et al. 2020 ) and the 'twophase immune response' theory in which early adaptive immune response plays protective role in the course of covid-19 , but opposite to a previous study which reported positive correlation between clinical severity and antibody titer (zhao et al. 2020a ). such conflicted data warrant further study of the immunological characteristics of covid-19 patients with different disease severity. there are several limitations in this study. first, only one blood sample was collected from each patient, causing a delay between the reported seroconversion time and the actual seroconversion time. second, two different nat assays were exploited in the detection of sars-cov-2 rna due to the urgent procurement in the outbreak, which leaded to a potential difference in test sensitivity. finally, participants' comorbid conditions were not included in the analysis, because we did not have access to the clinical information of participants. in summary, this study supported the combination of serologic testing and nat in routine covid-19 diagnosis and provided evidence on the temporal profile of antibody response against sars-cov-2 in patients with different disease severity. more insights about the immunological features of covid-19 patients are important for disease prognosis prediction and vaccine development. animal and human rights statement all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and with 1964 helsinki declaration and later amendments or comparable ethical standards. this study was reviewed and approved by the ethics committee of wuhan centers for disease prevention and control. since samples were collected with medical necessity and in accordance to the government guidance, written informed consent was waived. fig. 3 comparison of serologic test results between covid-19 patients of different genders and age groups. a the serologic test results of males (black dots) and females (grey dots) were compared. every dot represented one patient and the vertical axis reported the s/co values of sars-cov-2 igm and igg antibody tests. b the serologic test results of \ 60 years group (black dots) and c 60 years group (grey dots) were compared. every dot represented one patient and the vertical axis reported the s/co values of sars-cov-2 igm and igg antibody tests. the dash line represented the threshold of each test. the early phase of the covid-19 outbreak in epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study characterization of anti-viral immunity in recovered individuals infected by sars-cov-2 chinese center for disease control and prevention (2020) the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application laboratory diagnosis of emerging human coronavirus infections-the state of the art the diagnosis and treatment plan of the novel coronavirus-infected pneumonia, 3rd edn national health commission of the people's republic of china (2020b) the prevention and control plan of new coronavirus pneumonia association of public health interventions with the epidemiology of the covid-19 outbreak in wuhan zanos tp (2020) presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area covid-19 infection: the perspectives on immune responses. version 2 breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study world health organization (2020) coronavirus disease 2019 (covid-19) situation reports characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications antibody detection and dynamic characteristics in patients with covid-19 molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 serological diagnostic kit of sars-cov-2 antibodies using cho-expressed full-length sars-cov-2 s1 proteins acknowledgements the work was supported by the emergency scientific research project for covid-19 from wuhan city (grant no. 2020020101010008). author contributions whk and mql designed the study. whk, rz, jbz, dgk, js, qr, and mql collected samples and clinical information and analyzed data. whk, rz and mql analyzed and interpreted the data. whk and mql wrote and revised the manuscript. mql finalized the manuscript. all authors had full access to the final version of the report and agreed to the submission. conflict of interest the authors declare that they have no conflict of interest. key: cord-261415-qxl14j2m authors: fu, yajing; cheng, yuanxiong; wu, yuntao title: understanding sars-cov-2-mediated inflammatory responses: from mechanisms to potential therapeutic tools date: 2020-03-03 journal: virol sin doi: 10.1007/s12250-020-00207-4 sha: doc_id: 261415 cord_uid: qxl14j2m currently there is no effective antiviral therapy for sars-cov-2 infection, which frequently leads to fatal inflammatory responses and acute lung injury. here, we discuss the various mechanisms of sars-cov-mediated inflammation. we also assume that sars-cov-2 likely shares similar inflammatory responses. potential therapeutic tools to reduce sars-cov-2-induced inflammatory responses include various methods to block fcr activation. in the absence of a proven clinical fcr blocker, the use of intravenous immunoglobulin to block fcr activation may be a viable option for the urgent treatment of pulmonary inflammation to prevent severe lung injury. such treatment may also be combined with systemic anti-inflammatory drugs or corticosteroids. however, these strategies, as proposed here, remain to be clinically tested for effectiveness. syndrome coronavirus-2 (sars-cov-2), causes fatal acute respiratory disease (ard) resembling that of sars-cov guan et al. 2020; huang et al. 2020) . the pathophysiology for sars-cov-2 has not been well studied, but likely resembles that of sars-cov; the acute lung injury caused by sars-cov infection mainly results from aggressive inflammation initiated by viral replication (wong et al. 2004 ). similar to sars-cov infection, sars-cov-2 infection also causes increased secretion of il-1b, ifn-c, ip-10, mcp-1, il-4, and il-10 (huang et al. 2020 ). in addition, compared with non-icu (intensive care unit) patients, icu patients with severe disease had higher plasma levels of il-2, il-7, il-10, gcsf, ip-10, mcp-1, mip-1a, and tnf-a, suggesting a possible cytokine storm associated with disease severity . nevertheless, the causes of these exuberant inflammatory responses in sars-cov-2 infection remain largely unknown. in this review, we attempt to discuss and summarize possible mechanisms of sars-cov-2-mediated inflammatory responses (fig. 1 ). in addition, given that uncontrolled pulmonary inflammation is likely a leading cause of fatality in sars-cov-2 infection, we also attempt to speculate possible therapeutic interventions that may be applied to attenuate inflammatory responses in order to reduce mortality (fig. 2) . enzyme 2 (ace2), as sars-cov for infection, suggesting the likelihood of the same set of cells being targeted and infected zhou et al. 2020) . the early onset of rapid viral replication may cause massive epithelial and endothelial cell apoptosis and vascular leakage, triggering the release of exuberant pro-inflammatory cytokines and chemokines (yang 2020) . in addition, sars-cov-2 infection may also cause pyroptosis in macrophages and lymphocytes (yang 2020) . a vast majority of patients (82.1%) have been found to experience sars-cov-2-induced peripheral blood lymphopenia (guan et al. 2020) , suggesting possible pulmonary infiltration of lymphocytes and/or cell damage through apoptosis or pyroptosis . in sars-cov infection, viroporin 3a has also been shown to trigger the activation of nlrp3 (nod-like receptor protein 3) inflammasome and the secretion of il-1b in bone marrowderived macrophages, suggesting the induction of cell pyroptosis , which can cause the release of large amounts of proinflammatory factors (fink and cookson 2005) . ace2-associated lung injury has been suggested in sars-cov infection (imai et al. 2008; kuba et al. 2005) ; sars-cov s protein can downregulate ace2 (glowacka et al. 2010; wang et al. 2008) , and induce the shedding of catalytically active ace2 ectodomain (haga et al. 2008; jia et al. 2009; lambert et al. 2005) . loss of pulmonary ace2 function has been suggested to be associated with acute lung injury (imai et al. , 2008 kuba et al. 2005 kuba et al. , 2006 ; the reduction in ace2 function can cause dysfunction of the renin-angiotensin system (ras) and enhance inflammation and vascular permeability. in a murine ard model, loss of ace2 expression resulted in enhanced vascular permeability, increased lung edema, neutrophil accumulation, and diminished lung function ). in addition, in human airway epithelia, ace2 is constitutively shed by the action of disintegrin and metalloprotease 17 (adam17, also known as tnf-a cleavage enzyme, tace) to release enzymatically active soluble ace2 (sace2) (lambert et al. 2005) . both sars-cov infection and inflammatory cytokines such as il-1b and tnf-a can enhance ace2 shedding (haga et al. 2008; jia et al. 2009; lambert et al. 2005) . the biological function of sace2 remains largely unknown. however, sars-cov s protein-induced ace2 shedding has been found to be tightly coupled with tnf-a production in cell culture conditions (haga et al. 2008) . intriguingly, the s protein from another coronavirus, hnl63-cov, does not induce ace2 shedding, although the virus also binds to ace2 to mediate nhl63-cov entry (haga et al. 2008 ). hnl63-cov infection only causes the common cold, suggesting a potential pathogenic role of sace2 in sars-cov infection. these previous studies suggest that sace2 antiviral neutralizing antibodies play an important role in viral clearance. however, previous studies in animal models have shown that in sars-cov infection, such anti-s protein-neutralizing antibodies (anti-s-igg) can also cause severe lung injury by altering inflammatory responses (liu et al. 2019) . in sars-cov/macaque models, it has been found that s-igg present in infected lungs can facilitate severe lung injury; in these sars-cov s proteinvaccinated chinese macaques, acute lung injury was more pronounced than in unvaccinated control animals that showed only minor to moderate lung inflammation (liu et al. 2019) . consistent with this observation, adoptive transfer of purified anti-s-igg-neutralizing antibody (i.v. injection) to macaques, despite the fact that it reduced viral loads following subsequent challenge with sars-cov pumc , led to acute diffuse alveolar damage in all infected animals, whereas in the control group (injected with non-specific igg), only minor to moderate inflammation in the lungs was observed (liu et al. 2019) . this animal study suggests that despite viral suppression, the presence of anti-spike protein antibody at the acute stage of sars-cov infection can actually cause severe acute lung injury that persists until the late stages. similar observations of sars-cov vaccine-induced pulmonary injury have also been reported in multiple animal models using mice and african green monkeys (bolles et al. 2011; clay et al. 2012; tseng et al. 2012) . results from these animal studies also appear to mirror some of the clinical observations in sars-cov infected patients: the development of acute respiratory disease coincides with antiviral igg seroconversion in 80% of patients (peiris et al. 2003 ). in addition, it was found that patients who developed the anti-s-neutralizing antibody faster had a higher chance of dying from the disease; it took an average of only 14.7 days for the deceased patients to reach their peak levels of neutralizing antibody activities, as opposed to 20 days for the recovered patients (zhang et al. 2006) . the mechanisms of the anti-s neutralizing antibodyinduced inflammation and lung injury remain only partially understood. it has been proposed that the presence of s-igg prior to viral clearance alters the functional polarization of alveolar macrophages in acutely infected macaques. anti-s-igg can promote proinflammatory monocyte/ macrophage accumulation and the production of mcp-1 and il-8 in the lungs. such anti-s-igg-initiated proinflammatory responses appear to be mediated through the binding of the virus-anti-s-igg complex to the fc receptors (fcr) present on monocytes/macrophages, as fcr blockade reduces the production of inflammatory cytokines (liu et al. 2019) . it is also possible that such a virus-anti-s-igg complex may additionally activate the classical pathway of the complement system, leading to cellular damages, although this has not been investigated. alternatively, antibody-dependent cell-mediated cytotoxicity (adcc) may also be involved. a major question in sars-cov-induced pulmonary disease is why a small percentage of patients, particularly those who produce neutralizing antibody early, experience persistent inflammation, ard, and eventually succumb, while other patients survive the inflammatory responses and clear the virus. we speculate that a possible underlying mechanism may be related to antibody-dependent enhancement (ade) of viral infection that occurs in some patients with early, sub-optimal antibody activity that cannot completely clear the virus, but instead leads to persistent viral replication and inflammation (fig. 2) . ade is a well-known virology phenomenon that has been demonstrated in the infections of multiple viruses such as dengue, flavivirus, and influenza virus (halstead and o'rourke 1977; haslwanter et al. 2017; ochiai et al. 1992; . ade promotes viral cellular uptake of infectious virus-antibody complexes following their interaction with fcr or other receptors, leading to enhanced infection of target cells (halstead and o'rourke 1977; haslwanter et al. 2017; ochiai et al. 1992; . thus, interaction of fcr with the virusanti-s-igg complex may facilitate both inflammatory responses and persistent viral replication in the lungs of some patients (fig. 2) . given that there are very few mechanistic studies on inflammatory responses in sars-cov infection, we focus only on discussing limited mechanisms that might be involved. for the convenience of further discussion of potential therapeutics, here we separate sars-covmediated inflammatory responses into two different stages (fig. 1) : the primary response and the secondary response. primary inflammatory responses occur early after viral infection, prior to the appearance of neutralizing antibodies (nab). these responses are mainly driven by active viral replication, viral-mediated ace2 downregulation and shedding, and host antiviral responses, which can lead to increased cytokine/chemokine production and cellular damage through apoptosis and/or pyroptosis. most patients can tolerate primary inflammatory responses with a positive outcome of viral load reduction or even viral clearance, followed by receding of inflammation. secondary inflammatory responses begin with the generation of adaptive immunity and the appearance of nab that further diminish viral replication. however, as described above, the appearance of nab can also trigger fcr-mediated inflammatory responses and cause severe lung injury. in sars-cov infected patients, the appearance of antiviral igg coincides with the onset of acute respiratory disease in 80% of patients (peiris et al. 2003) . a possible underlying mechanism is likely antibody-dependent enhancement (ade) of viral infection that leads to persistent viral replication and inflammatory responses from macrophages. given that most patients can survive primary inflammatory responses, we mainly focus on discussing secondary inflammatory responses that frequently lead to fatality. there are several potential therapeutic approaches that may be applied or developed (fig. 2) . these approaches focus primarily on blocking fcr receptor to prevent virus-nab complex binding to fcr to trigger inflammatory responses (nimmerjahn and ravetch 2008a, c) . first, fcr can be blocked by specific antibodies to inhibit its activation. small-molecule inhibitors can also be developed to interact with the ig-binding domains of fcr to block fcr activation. second, the inhibitory fcr, fccriib, may also be targeted to inhibit fcr activation. several fccriib specific antibodies are now being developed for potential use as novel immune suppressors (van mirre et al. 2004; veri et al. 2007 ). third, fcr activation can also be inhibited by targeting the neonatal fc receptor (fcrn), which is essential for extending the half-life of igg. antibody or small molecule-mediated blockage of fcrn can prevent igg interaction with fcrn, which can decrease circulating igg levels (nimmerjahn and ravetch 2008b) . in addition, intravenous immunoglobulin (ivig) can be used to saturate the igg recycling capacity of fcrn and proportionately reduce the levels of antiviral nab. ivig can also competitively block the binding of antiviral nab to other fcrs (kurlander and hall 1986) . although we mainly focus on strategies targeting virus-nab complex binding to fcr, it is possible that the virus-nab complex may also initiate inflammatory responses through the classical pathway of the complement system, which may be blocked through c5-and c5a-targeted inhibition (horiuchi and tsukamoto 2016) . in sum, at present, there is no effective antiviral therapy for sars-cov-2 infection, which frequently leads to fatal inflammatory responses and acute lung injury. here, we discuss the various mechanisms of sars-cov-mediated inflammation. we also assume that sars-cov-2 likely shares similar inflammatory responses. potential therapeutic tools to reduce sars-cov-2-induced inflammatory responses include various methods to block fcr activation. in the absence of a proven clinical fcr blocker, the use of intravenous immunoglobulin to block fcr activation may be a viable option for the urgent treatment of pulmonary inflammation to prevent severe lung injury. such treatment may also be combined with systemic anti-inflammatory drugs or corticosteroids. however, these strategies, as proposed here, remain to be clinically tested for effectiveness. conflict of interest the authors declare that they have no conflict of interest. animal and human rights statement this article does not contain any studies with human or animal subjects performed by any of the authors. a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in 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endocytosis of the receptor-binding domain of sars-cov spike protein together with virus receptor ace2 plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome cell pyroptosis, a potential pathogenic mechanism of 2019-ncov infection antibody responses against sars coronavirus are correlated with disease outcome of infected individuals single-cell rna expression profiling of ace2, the putative receptor of wuhan 2019-ncov acknowledgments the authors wish to thank feng li for helpful discussions and jennifer guernsey for editorial assistance. key: cord-273608-dxx3p1x5 authors: deng, jikui; ma, zhuoya; huang, wenbo; li, chengrong; wang, heping; zheng, yuejie; zhou, rong; tang, yi-wei title: respiratory virus multiplex rt-pcr assay sensitivities and influence factors in hospitalized children with lower respiratory tract infections date: 2013-04-11 journal: virol sin doi: 10.1007/s12250-013-3312-y sha: doc_id: 273608 cord_uid: dxx3p1x5 multiplex rt-pcr assays have been widely used tools for detection and differentiation of a panel of respiratory viral pathogens. in this study, we evaluated the qiagen resplex ii v2.0 kit and explored factors influencing its sensitivity. nasopharyngeal swab (nps) specimens were prospectively collected from pediatric inpatients with lower respiratory tract infections at the time of admission in the shenzhen children’s hospital from may 2009 to april 2010. total nucleic acids were extracted using the ez1 system (qiagen, germany) and 17 respiratory viruses and genotypes including influenza a virus (flua), flub, parainfluenza virus 1 (piv1), piv2, piv3, piv4, respiratory syncytial virus (rsv), human metapneumovirus (hmpv), rhinoviruses (rhv), enteroviruses (env), human bocaviruses (hbov), adenoviruses (adv), four coronaviruses (229e, oc43, nl63 and hku1), and flua 2009 pandemic h1n1(h1n1-p) were detected and identified by the resplex ii kit. in parallel, 16 real-time taqman quantitative rt-pcr assays were used to quantitatively detect each virus except for rhv. influenza and parainfluenza viral cultures were also performed. among the total 438 nps specimens collected during the study period, one or more viral pathogens were detected in 274 (62.6%) and 201(45.9%) specimens by monoplex taqman rt-pcr and multiplex resplex, respectively. when results from monoplex pcr or cell culture were used as the reference standard, the multiplex pcr possessed specificities of 92.9–100.0%. the sensitivity of multiplex pcr for piv3, hmpv, piv1 and bov were 73.1%, 70%, 66.7% and 55.6%, respectively, while low sensitivities (11.1%–40.0%) were observed for flua, env, oc43, rsv and h1n1. among the seven viruses/genotypes detected with higher frequencies, multiplex pcr sensitivities were correlated significantly with viral loads determined by the taqman rt-pcr in flua, h1n1-p and rsv (p=0.011−0.000). the qiagen resplex ii multiplex rt-pcr kit possesses excellent specificity for simultaneous detection of 17 viral pathogens in nps specimens in pediatric inpatients at the time of admission. the sensitivity of multiplex rt-pcr was influenced by viral loads, specimen process methods, primer and probe design and amplification condition. multiplex rt-pcr assays have been widely used tools for detection and differentiation of a panel of respiratory viral pathogens. in this study, we evaluated the qiagen resplex ii v2.0 kit and explored factors influencing its sensitivity. nasopharyngeal swab (nps) specimens were prospectively collected from pediatric inpatients with lower respiratory tract infections at the time of admission in the shenzhen children's hospital from may 2009 to april 2010. total nucleic acids were extracted using the ez1 system (qiagen, germany) and 17 respiratory viruses and genotypes including influenza a virus (flua), flub, parainfluenza virus 1 (piv1), piv2, piv3, piv4, respiratory syncytial virus (rsv), human metapneumovirus (hmpv), rhinoviruses (rhv), enteroviruses (env), human bocaviruses (hbov), adenoviruses (adv), four coronaviruses (229e, oc43, nl63 and hku1), and flua 2009 pandemic h1n1(h1n1-p) were detected and identified by the resplex ii kit. in parallel, 16 real-time taqman quantitative rt-pcr assays were used to quantitatively detect each virus except for rhv. influenza and parainfluenza viral cultures were also performed. among the total 438 nps specimens collected during the study period, one or more viral pathogens were detected in 274 (62.6%) and 201(45.9%) specimens by monoplex taqman rt-pcr and multiplex resplex, respectively. when results from monoplex pcr or cell culture were used as the reference standard, the multiplex pcr possessed specificities of 92.9-100.0%. the sensitivity of multiplex pcr for piv3, hmpv, piv1 and bov were 73.1%, 70%, 66.7% and 55.6%, respectively, while low sensitivities (11.1%-40.0%) were observed for flua, env, oc43, rsv and h1n1. among the seven viruses/genotypes detected with higher frequencies, multiplex pcr sensitivities were correlated significantly with viral loads determined by the taqman rt-pcr in flua, h1n1-p and rsv (p=0.011-0.000). the qiagen resplex ii multiplex rt-pcr kit possesses excellent specificity for simultaneous detection of 17 viral pathogens in nps specimens in pediatric inpatients at the time of admission. the sensitivity of multiplex rt-pcr was influenced by viral loads, specimen process methods, primer and probe design and amplification condition. multiplex rt-pcr; respiratory viral loads; cell culture; lower respiratory tract infection lower respiratory tract infections (lrtis) are the most frequent cause of hospitalization among children worldwide (ahn k m, et al., 1999; garbino j, et al., 2004; ruuskanen o, et al., 2011; sung c c, et al., 2011; thompson w w, et al., 2003; van woensel j b, et al., 2003) . a large proportion of lrtis are caused by respiratory viruses including influenza virus a (flua) and b (flub), parainfluenza viruses 1-4 (piv1-4), respiratory syncytial virus (rsv), rhinoviruses (rhv), enteroviruses (env), and adenoviruses (adv) (juven t, et al., 2000; legg j p, et al., 2005; weigl j a, et al., 2005) . over the past decade, the viral pathogen list has been expanded to several newly discovered viruses including human metapneumovirus (hmpv) (van den hoogen b g, et al., 2001) , some coronaviruses (nl63, hku1, sars) (pyrc k, et al., 2007) , human bocaviruses (hbov) (allander t, et al., 2005) , and pandemic influenza a/h1n1 2009 virus (h1n1-p) (anonymous, 2009) . various viruses with different shedding levels may result in a large disease severity range from common bronchitis to fatal pneumonia martin e t, et al., 2012; takeyama a, et al., 2012; torres j p, et al., 2010) . rapid and accurate etiologic diagnosis of lrtis is essential to patient management. several multiplex rt-pcr-based devices are commercially available for detection and differentiation of a panel of respiratory viral pathogens (zhang s, et al., 2011) . they are being increasingly used in the clinical setting as a cornerstone technique in the clinical virology laboratory as they possess sensitivities greater than rapid viral antigen testing and test turnaround time shorter than standard respiratory virus culture (balada-llasat j m, et al., 2011; kim s r, et al., 2009; rand k h, et al., 2011; schindera c, et al., 2010) . varying sensitivities have been reported between different devices for specific viral pathogens in different studies (balada-llasat j m, et al., 2011; kim s r, et al., 2009; rand k h, et al., 2011; schindera c, et al., 2010) . in this study, we evaluated the resplex ii v2.0 kit (qiagen, germany), which uses a target enriched multiplexing rt-pcr amplification coupled with a suspension array detection, for detection and identification of a panel of respiratory specimens in pediatric inpatients with lrtis. clinical accuracy of the resplex ii assay was validated on a panel of prospectively collected consecutive nasopharyngeal swab (nps) specimens in comparison to viral culture and a monoplex real-time taqman rt-pcr. we also correlated the resplex ii assay sensitivity with viral loads determined by the quantitative, monoplex real-time taqman rt-pcr. patients recruited in this study were pediatric inpatients aged less than 14 years old with clinical diagnosis of lrtis in shenzhen children's hospital as part of routine clinical care at division of respiratory disease between may 2009 and april 2010. samples used in this study were nps specimens collected by an eswab (482c, copan diagnostics, inc., murrietta, ca) prospectively from these patients at the time of admission. consecutive nps specimens were collected and included except for low volumes after routine diagnostic tests were performed. each sample was divided into three 1 ml aliquots and stored immediately in -80 °c. the shenzhen children's hospital institutional review board (irb) classified this study as non-human research without clinical history review; the study was exempted from irb approval and informed consent requirements were waived. one nps aliquot was used to perform influenza and parainfluenza viral cultures. specimens were inoculated into mdck (madin-darby canine kidney) cells in 96-well microplates (corning, usa) using enhanced cell culture with fluorescent antibody detection for primary viral isolation of influenza viruses a and b. plates were incubated at 37 °c in 5% co 2 and inspected daily after inoculation for the presence of cytopathic effect (yang z f, et al., 2010) . hemagglutination assays (ha) using mdck cell culture supernatants 2-3 days post-infection were performed for confirmation of flua infections (yang z f, et al., 2010) . all cultures were screened at 5 to 7 days post-inoculation by direct immunofluorescence using imagen reagents (chemicon, usa). in cultures with positive cytopathic effect or ha results, direct immunofluorescence was performed to identify the types of influenza viruses (landry m l, et al., 2000) . one nps aliquot was sent to the state key laboratory of respiratory diseases of the guangzhou medical university where several quantitative monoplex real-time taqman rt-pcr assays were performed as previously described (liu w k, et al., 2011) . pathogens detected individually by this platform included flua, h1n1-p, flub, piv1, piv2, piv3, piv4, rsv, hmpv, env, hbov, adv, four coronaviruses (229e, oc43, nl63 and hku1) as well as two atypical bacterial pathogens (mycoplasma pneumonia and chlamydia pneumonia). total nucleic acids from nps were extracted using a qiaamp minelute virus kit (qiagen, germany), in accordance with the manufacturer's protocol and previously described (sefers s e, et al., 2006) . primers and the probe were synthesized by takara (dalian, china). one step rt-pcr reaction buffer (primescript one step rt-pcr kit ver.2) was also purchased from takara. amplification was conducted using 10 pmol of primers, 3 pmol of probe and 5 μl specimen extract in a final volume of 25 μl on the abi-7500 real-time pcr instrument (applied biosystems, foster city, ca). cycling conditions included an initial incubation at 50 °c for 30 min, 94 °c for 2 min, followed by 40 cycles of 94 °c for 10 sec and 55 °c for 35 sec (liu w k, et al., 2011) . the amplified gene target sequence was inserted into the pmd18-t vector (takara) and used as a positive control for quantification analysis. sensitivity of the pcr assay was calculated to be 10 copies of plasmid dna using positive control plasmid diluted titrations. five standards covering viral loads from 10-100,000 copies/ml were included to generate a quantification standard curve for absolute viral load determination of each tested nps specimen. one nps aliquot was used for a panel of respiratory viral pathogen detection by the resplex ii panel (qiagen, germany) (li h, et al., 2007) . version 2.0 covers viruses and types of flua, flub, rsv, piv1, piv2, piv3, piv4, hmpv, coxsackieviruses/echoviruses, rhv, human bocaviruses, adenoviruses b and e, and four coronaviruses (nl63, hku1, 229e, and oc43). a newer version (resplex ii plus panel pre) was used later in the study which also includes flua 2009 pandemic h1n1 (h1n1-p). total nucleic acids were extracted from 200 μl of nps specimen using the qiagen q-card ez1 virus mini kit (cat. no.955314) in an ez1 extraction system and eluted in 50 μl of water. the rt-pcr amplification was performed on the abi-7500 real-time pcr system with 10 μl of extract included in each reaction. the amplification product was then detected and identified by using a suspension bead array for multiplex hybridization in the liquichip 200 workstation with the qiaplex mdd software (qiagen, germany). controls were included on each test run including a positive control (in vitro transcribed rna corresponding to a human genomic dna sequence), an internal control to check for viral rna isolation and pcr inhibition, and a sample control to detect traces of human genomic dna present in each specimen. a positive result was determined using a cut-off median fluorescence intensity (mfi) of 250 or the mean plus 2 standard deviations of the negative controls (gharabaghi f, et al., 2011) . the combined results of the viral culture and monoplex real time rt-pcr was used as a reference standard. a sample was determined to be positive for the tested virus when viral culture or real time rt-pcr was positive. for comparison of qualitative categorical data,  2 test or fisher's exact test and mantel-haenszel  2 test were used wherever appropriate. a p<0.05 was considered statistically significant. trend analysis and rank test was used to determine the correlations between resplex assay sensitivities and viral loads. 3) 6/9 (66.7) 4/5 (80.0) 1/6 (16.7) 6/7 (85.7) 0/2 (0) 1-9.9×10 3 1/33 (3.0) 0/12 (0) 2/3 (66.7) 2/4 (50.0) 0/6 (0) 1/3 (33.3) 0/1 (0) 1-9.9×10 2 0/13 (0) 0/11 (0) 0/0 (0) 3/6 (50.0) 0/2 (0) 0/3 (0) 0/1 (0) negative 6/300 (2.0) 2/402 (0.5) 3/408 (0.7) 5/412 (1.2) 0/289 (0) 3/420 (0.7) 0/420 (0) a total of 438 qualified nps specimens collected during a one full year study period were included in the final testing and analysis. the male ratio was 77.6%. the mean age of patients was 1.4 years, ranging from one month to 11 years. among the total specimens tested, 263 (60.0%), 134 (30.6%) and 41 (9.4%) were from children of <1, 1-3 and >3 years old, respectively. among the total 438 nps specimens, one or more viral pathogens were detected in 274 (62.6%) and 201(45.9%) specimens by monoplex taqman rt-pcr and multiplex resplex rt-pcr, respectively. viral culture was positive for 56 (12.8%) specimens including 17 flua, 3 flub, 14 piv1, and 22 piv3. among these culture positive specimens, monoplex real-time taqman rt-pcr results were fully concordant while resplex results were negative for 13 specimens including six flua, four piv1 and three piv3. when results from monoplex rt-pcr or cell culture were used as the reference standard, the multiplex pcr possessed specificities ranging from 92.9% to 100.0% for the viruses tested. the sensitivities of multiplex pcr varied with high sensitivity observed for piv3, hmpv, piv1 and hbov (55.6-73.1%) and low sensitivity for flua, env, oc43, rsv and h1n1-p (11.1-40.0%) ( table 1) . rhinoviruses were detected from 36 specimens (8.3%) by the resplex assay including 22 rhinovirus alone and 14 co-detected with other viruses including rhv/env (n=10), rhv/rsv (n=2), rhv/hmpv (n=1), and rhv/229e (n=1). since rhinoviruses were not detected by the monoplex taqman rt-pcr, these data were not further analyzed. we further correlated viral load information determined by the taqman rt-pcr with the resplex diagnostic sensitivities for seven viruses/genotypes detected with higher frequencies (positive rate >4% detected by monoplex rt-pcr). the resplex multiplex pcr sensitivities correlated significantly with viral loads for rsv, flua and h1n1-p (p=0.011-0.000). significant correlation was not observed for piv1, piv3, hbov, and hmpv (p>0.05) ( table 2) . during the study, a new version of resplex, resplex ii plus panel pre, was manufactured to increase coverage of h1n1-p. there were 124 and 314 specimens which were tested by resplex ii panel and resplex ii plus panel pre, respectively (table 1) . for the seven non-h1n1-p viruses/genotypes detected with higher frequencies, the sensitivities dropped (range from 3.6% to 43.7%) for five viruses including flua, rsv, hbov, piv1 and piv3. the several multiplex rt-pcr-based devices have been commercially available for detection and identification of a panel of viral pathogens causing lrtis. we evaluated one of the platforms (resplex ii), which combines a target enriched multiplexing rt-pcr amplification and a suspension array detection (balada-llasat j m, et al., 2011; li h, et al., 2007) , using prospectively collected nps specimens from pediatric inpatient patients with clinical diagnosis of lrtis. the resplex ii multiplex rt-pcr kit possessed excellent specificity and varied sensitivities ranging from 11.1 to 73.1%, but which was relatively lower than two recently reported studies on children populations (forman m s, et al., 2012; hayden r t, et al., 2012) . significantly lower sensitivities by the resplex ii assay were observed especially for flua, env, oc43, rsv and h1n1-p. we observed the following factors which might explain the discrepancies between our and previously reported studies. first, based on the patient populations and reference standards, the test sensitivities could vary significantly. mak g et al (mak g c, et al., 2011) reported the sensitivity of resplex ii for flua and h1n1-p were only 31.8% and 42.9% when monoplex rt-pcr was used for comparison. however in previous studies in which virus culture or dfa was used as the main reference standard, the sensitivities of resplex ii were observed to be from 84%-100% (baladallasat j m, et al., 2011; li h, et al., 2007) . in our pediatric inpatients with lower respiratory tract infections at the time of admission, the monoplex rt-pcr detected significantly more flu and piv than in cell culture. secondly, different extractions vary significantly in quality and quantity of nucleic acid recovery. in our study, nucleic acids were extracted using the qiagen ez-1 system for the resplex ii, a system with reported low quantity and quality of nucleic acid recovery (tang y w, et al., 2005; yang g, et al., 2011) . finally, the multiplex pcr condition requires careful optimization; any addition or modification of primers/probes may significantly alter the amplification efficiency. in our study, when the target of h1n1-p was added, the sensitivities of resplex ii plus panel pre assay dropped for most of the viral targets, especially for flua detection. previous studies revealed that the detection of flua (h1n1) viruses correlated with the time after symptom onset and viral load (cheng p k, et al., 2010) . in our study, among the seven viruses/genotypes detected with higher frequencies, resplex ii sensitivities correlated significantly with viral loads determined by the taqman rt-pcr in rsv, flua and h1n1-p. in contrast, no viral load correlation was observed in piv3, hmpv, piv1 and hbov. since viral load did not correlate with lower sensitivities for some of the targets, the pcr amplification and hybridization efficiencies may also play a role. it is reasonable that multiplex pcr sensitivities decreased with decreasing viral load of target virus. in the resplex ii, primers and probes were designed from the f genome of hmpv in which significant sequence variations were revealed by the recent finding of four geno/ subtypes (mackay i m, et al., 2006) . whether the poor correlation between viral load and pcr sensitivity in hmpv is related to the primer/probe design merits further investigation. varied analytical sensitivities were noticed in other viral targets in a recent study in immunocompromised children (hayden r t, et al., 2012) . clinical characteristics of acute viral lower respiratory tract infections in hospitalized children in seoul, 1996-1998 cloning of a human parvovirus by molecular screening of respiratory tract samples swine influenza a (h1n1) infection in two children--southern california evaluation of commercial resplex ii v2.0, multicode-plx, and xtag respiratory viral panels for the diagnosis of respiratory viral infections in adults performance of laboratory diagnostics for the detection of influenza a(h1n1)v virus as correlated with the time after symptom onset and viral load diagnostic performance of two highly multiplexed respiratory virus assays in a pediatric cohort respiratory viruses and severe lower frequency of detection of picornaviruses and seven other respiratory pathogens in infants correlation of pandemic (h1n1) 2009 viral load with disease severity and prolonged viral shedding in children simultaneous detection and high-throughput identification of a panel of rna viruses causing respiratory tract infections detection of human bocavirus from children and adults with acute respiratory tract illness in guangzhou, southern china genetic diversity of human metapneumovirus over 4 consecutive years in australia evaluation of qiagen resplex ii for the detection of pandemic influenza a (h1n1) 2009 and influenza a (h3n2) virus multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children identification of new human coronaviruses comparison of two multiplex methods for detection of respiratory viruses: filmarray rp and xtag rvp viral pneumonia. lancet immunofluorescence versus xtag multiplex pcr for the detection of respiratory picornavirus infections in children qiaamp minelute virus kit effectively extracts viral nucleic acids from cerebrospinal fluids and nasopharyngeal swabs viral etiology of acute lower respiratory tract infections in hospitalized young children in northern taiwan rhinovirus load and disease severity in children with lower respiratory tract infections comparative evaluation of three commercial systems for nucleic acid extraction from urine specimens mortality associated with influenza and respiratory syncytial virus in the united states respiratory syncytial virus (rsv) rna loads in peripheral blood correlates with disease severity in mice a newly discovered human pneumovirus isolated from young children with respiratory tract disease viral lower respiratory tract infection in infants and young children the descriptive epidemiology of severe lower respiratory tract infections in children in kiel comparison of commercial systems for extraction of nucleic acids from dna/rna respiratory pathogens a pathogenic and clinical study of 882 cases of adult influenza-like illness in guangzhou molecular diagnosis of viral respiratory infections key: cord-354529-k8p2u7iq authors: wu, yongran; hong, ke; ruan, lianguo; yang, xiaobo; zhang, jiancheng; xu, jiqian; pan, shangwen; ren, lehao; chen, lu; huang, chaolin; shang, you title: patients with prolonged positivity of sars-cov-2 rna benefit from convalescent plasma therapy: a retrospective study date: 2020-08-31 journal: virol sin doi: 10.1007/s12250-020-00281-8 sha: doc_id: 354529 cord_uid: k8p2u7iq convalescent plasma therapy has been implemented in a few cases of severe coronavirus disease 2019. no report about convalescent plasma therapy in treating patients with prolonged positivity of sars-cov-2 rna has been published. in this study, we conducted a retrospective observational study in 27 patients with prolonged positivity of sars-cov-2 rna, the clinical benefit of convalescent plasma therapy were analyzed. qrt-pcr test of sars-cov-2 rna turned negative (≤ 7 days) in a part of patients (early negative group, n = 15) after therapy, others (late negative group, n = 12) turned negative in more than 7 days. pulmonary imaging improvement was confirmed in 7 patients in early negative group and 8 in late negative group after cp therapy. viral load decreased in early negative group compared with late negative group at day 3, 5, 7 after implementing convalescent plasma therapy. patients in early negative group had a shorter median length of hospital stay. in conclusion, convalescent plasma therapy might help eliminate virus and shorten length of hospital stay in patients with prolonged positivity of sars-cov-2 rna. the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) emerged in late 2019 (zhu et al. 2020) , has been rapidly spreading and causing a worldwide pandemic (kirby 2020; saglietto et al. 2020) . the pneumonia induced by sars-cov-2 is known as coronavirus disease 2019 (ivers and walton 2020) . to date, the virus has infected millions of people all over the world. recently, many studies about long-term viral duration in covid-19 patients have been published wan et al. 2020; zhou et al. 2020; shi et al. 2020; yang jr et al. 2020; li et al. 2020) . the longest duration observed was 83 days in one patient's upper respiratory tract samples . although the association between viral duration and disease severity or older age was inconsistent in different studies, some studies reported longer viral duration correlated with severe disease or older age in covid-19 patients zhang yc et al. 2020; hu et al. 2020) . besides, there are limited evidences about the infectivity of sars-cov-2 for patients with prolonged positivity of sars-cov-2 rna (walsh et al. 2020) . thus, treating prolonged positive patients might be necessary during this covid-19 pandemic. by now, no anti-viral therapy has been proven effective in treating covid-19. convalescent plasma (cp) therapy is a classical passive antibody therapy used to treat viral pandemic historically, such as influenza a (hin1) (hung et al. 2011) , ebola virus disease (sahr et al. 2017 ) and sars (cheng et al. 2005) . recently case reports showed that cp collected from recovered patients might be effective to treat critically ill patients with covid-19 (zhang b ahn et al. 2020; duan et al. 2020; shen et al. 2020) . however, in these studies, critically ill patients at the early stages of illness were mainly aimed. there is not any report about implementing cp therapy in patients with prolonged positivity of sars-cov-2 rna. we noticed that convalescent plasma was sometimes given to these patients with prolonged positivity of sars-cov-2 rna. herein, we perform a retrospective study to analyze the clinical benefit of cp therapy in patients with prolonged positivity of sars-cov-2 rna. this single-center retrospective observational study was performed in jinyintan hospital, which is one of the earliest designated hospitals for covid-19 in wuhan, china. patients who confirmed covid-19 admitted into jinyintan hospital from january 1 to april 20, 2020, were included for initial screen. we investigated all patients with covid-19 who received cp therapy during hospitalization without enrolled in any other random control trial. patients were excluded if their sars-cov-2 tests were negative before infusion of cp. the clinical outcomes (discharges, mortality, length of hospital stay) were monitored up to april 25, 2020. the discharged patients in jinyintan hospital must need to meet the following criteria: patients with two consecutive negative tests of respiratory specimens; patients' symptom resolved; no hospitalization was required as assessed by clinicians. in our study, all discharged patients still need to transport to other isolation sites for medical observation for 2 weeks. in our study, we defined patients with tests of sars-cov-2 turned negative b 7 days after the first infusion of cp to be in early negative group (en group), others were defined to be in late negative group (ln group). clinical information of patients was collected from the electronic medical information system of jinyintan hospital, including the following factors: demographic data; date of symptom onset, admission, first cp infusion and discharge; laboratory data before and after infusion of cp, including white blood cell count, neutrophil count, lymphocyte count, liver and kidney function test, and inflammatory factors such as high sensitive c-reaction protein (hscrp); results of sars-cov-2 test and cycle threshold value (ct value) of quantitative reverse transcription-polymerase chain reaction; patients' status and treatments before or after the cp therapy, including the vital signs, anti-virus therapy, oxygen therapy, and other treatments; total volume dose of cp; pulmonary imaging examination data; information on complications such as transfusion-related adverse reactions. test of sars-cov-2 were performed in a laboratory in jinyintan hospital. respiratory tract specimens (including nasopharyngeal, sputum, bronchial alveolar lavage fluid) collected from patient were transferred to the laboratory within 4 h. the quantitative reverse transcription-polymerase chain reaction (rt-pcr) of sars-cov-2 has already been described previously (corman et al. 2020) . total nucleic acid extraction was performed on the specimens using the rna viral kit (life river). the e gene, n gene, and rdrp gene of sars-cov-2 (located in orflab reading frame) was detected using a specific kit (life river), which was approved by the china food and drug administration. ct value is the number of cycles required for the fluorescent signal to cross the threshold for a positive test, and a higher ct value is correlated with lower viral load. according to the instruction of the kit, ct values of specimens with e gene, n gene, and rdrp gene \ 43 were considered to be positive, and results were highly reliable. the imaging artificial intelligence (ai)-assisted diagnostic system can quickly identify covid-19, delineate and quantify lesions designed by the chinese academy of sciences, national biological information center, tsinghua university, and hospital of zhongshan university. it is developed by applying advanced ai technologies, such as deep learning, transfer learning, and using the multiple neural network architecture training models. besides ai, x-ray and computed tomography (ct) images of all patients have been manually reviewed by a group, consisting of three experienced imaging specialists. all cp were obtained from donors recovered from covid-19, the interval between symptom onset and donation of donors were [ 3 weeks; all donors must meet the discharge standard of the seventh trial version of the new coronavirus pneumonia prevention and control program (chinese national health commission 2020). all the donors were confirmed without transfusion-related infectious diseases before donation. interval between discharge and donation must be [ 10 days. the neutralizing antibody titer was evaluated before transfusion, convalescent plasma with titer of [ 1:160 were used for patients in our study. after the clinician applying for a blood transfusion, convalescent plasma would be transferred from the blood center to the isolation ward on the same day. data were expressed as categorical variable and continuous variable. to compare the en group and ln group, chisquare test was used to analyze the categorical variable. for the continuous variable, results of data were shown as median and inter-quartile range (iqr). mann-whitney test was used to perform nonparametric test. a two-sided p value \ 0.05 were considered statistically significantly different between the two groups. spss 22.0 is used for statistical analysis. demographics and baseline characteristics of patients with prolonged positivity of sars-cov-2 rna before cp therapy as shown in table 1 , a total of 27 adult patients, with a median 44.0 (30.0-47.0) days between symptom onset and last positive test of sars-cov-2 rna before cp therapy, were included. their median age was 64.0 (57.0-72.0) years and 15 (55.5%) patients were male. we conducted a subgroup analysis between patients of en group and ln group. demographic data was shown as table 1 , median age, percentage of male patients, coexisting chronic diseases of patients in both groups were not significantly different (table 1) . each patient of both groups underwent laboratory tests before cp therapy including white cell count, neutrophil count, lymphocyte count, platelet count, hematocrit, serum creatinine test, serum total bilirubin, serum alanine aminotransferase, serum aspartate aminotransferase, and hscrp test, and the results were shown as table 1 . as shown in table 2 , patients in both groups have a longer median interval between symptom onset and date of cp transfusion as compared to former reports [40.0 (26.0-47.0) days in en group and 45.5 (41.2-57.0) days in ln group]. the median body temperature and oxygen therapy before cp transfusion were not significant different. the median fraction of inspiration o 2 (fio 2 ), peripheral oxygen saturation and anti-viral therapies of both groups before cp therapy are shown in table 2 . before transfusion, eight patients in en group and seven in ln group received broad-spectrum antibiotic therapy, three patients in en group and two in ln group received corticoid therapy after admission. four patients in en group and two in ln group received infusion of immunoglobulin after admission. as shown in tables 1, 2, demographics and baseline characteristics of patients in en group and ln group were not significant different before cp therapy. clinical benefit and outcome of patients with prolonged positivity of sars-cov-2 rna after cp therapy as shown in table 3 , the median and interquartile ranged total volume of cp transfusion was 400 (200-400) ml in en group and 400 (400-800) ml in ln group. no adverse reactions related to blood transfusion were found during infusion in both groups. the median interval between transfusion and discharge was 7.0 (4.0-11.0) days in en group and 24.0 (14.7-28.7) days in ln group. most patients underwent x-ray or ct scan before and after transfusion (en group = 8; ln group = 12), and pulmonary imaging improvement was confirmed in 7 patients in en group and 8 in ln group after cp therapy. the median length of hospitalization in en group was 37.0 days and 52.0 days in ln group, as shown in table 3 . due to the definition of en group and ln group, the median length of hospitalization in ln group was much longer than en group, thus we didn't make a comparative analysis. three patients died in ln group within 60 days, two died from refractory hypoxemia and one in ln group died from severe septic shock. no patients died in en group within 60 days. comparison of lung imaging before and after cp therapy in patients with prolonged positivity of sars-cov-2 rna five patients in our study underwent ct scan before (within 3 days) and reexamination after transfusion (within 5-8 days). for these patients, comparison and analysis of ct images were performed before and after transfusion by using the ai-assisted diagnostic system described above (fig. 1a, 1b) . three (60%) patients showed as consolidation of ct images before cp therapy (fig. 1c) , five all showed as ground-glass opacity (ggo) (fig. 1d ) before cp therapy, which were similar to the former report about ct findings in covid-19 patients (adair and ledermann 2020). after transfusion, the total consolidation percentage decreased after transfusion in three patients (fig. 1c) , and the total ggo percentage decreased in five all patients' ct images (fig. 1d ). variation trend of viral load before and after cp therapy in patients with prolonged positivity of sars-cov-2 rna after admission, patients in both groups underwent sars-cov-2 tests by using rt-pcr as described above. the variation trend of ct value in both groups are show as fig. 2 . the median ct value on admission is 33.0 (28.7-38.2) in en group and 32.5 (22.1-38.1) in ln group, without significant differences (p = 0.591, fig. 2) . besides, the median ct values was not significant between en group and ln group before transfusion [34.0 (26.4-38.3) vs. 30.9 (26.7-35.7), p = 0.591, fig. 2 ]. after transfusion, ct values of patients in en group increased and [ 43 within 7 days gradually and most of them (n = 13) discharged within 10 days and were unable to detect at 9, 12, and 15 days. conversely, the median ct values of patients in ln group remained \ 43 at 3, 5, 7, 9, and 12 days after transfusion, 6 patients in ln group still remained \ 43 at 15 days after transfusion (fig. 2 ). our study explored the efficiency of cp therapy in covid-19 patients at a later stage of the illness. all patients with prolonged positivity of sars-cov-2 rna in our study were implemented cp therapy including mild cases. we confirmed that the viral load rapidly decreased after cp therapy in some patients (en group), whereas others remain positive 7 days after cp therapy (ln group). the difference in baseline information, viral load, and other interventions was not significant before transfusion. after cp therapy, more than half patients obtained a rapid decrease of viral load. cp therapy is a classic therapy against pandemic that can be traced back to the early twentieth century and clinicians treated the 1918 spanish influenza with convalescent sera (luke et al. 2006) , which was found to be effective in decreasing the mortality of 1918 spanish influenza pandemic. in the 21st century researches have shown that cp therapy effectively and safely treats h1n1 and sars at the early stage of illness. besides sars and h1n1, there is also some anecdotal information on the use of convalescent serum in seriously ill individuals. two patients diagnosed with ebola viral disease received cp therapy on the early stage of illness (day 8 and 3 after symptom onset) and recovered without serious long-term sequelae to date (kraft et al. 2015) . one patient diagnosed with h5n1 received cp therapy on day 8 after symptom onset recovered and successfully discharged (zhou et al. 2007 ). thus, cp therapy might be an effective therapy for certain viral diseases, especially in early stage of illness. however, to our knowledge, no reports about convalescent plasma therapy in viral disease at later stage have been published. recently one case report suggests efficiency of cp therapy in treating 5 patients with severe covid-19 (corman et al. 2020) , the interval between the symptom onset and transfusion was \ 20 days. however, the interval in our study is much longer than the previously mentioned report. in our study, empirical anti-viral therapies were already implemented in these patients, but ct value of respiratory tract specimens collected from these patients were still \ 43 before cp therapy, represented a high viral load. these patients with prolonged positivity of sars-cov-2 rna [44.0 (30.0-47.0) days] still needed to hospitalize and separate, which may cause a huge cost during covid-19 pandemic. in fact, individual human case studies reported long periods of viral shedding that . recently, a report have shown long-term coexisting of sars-cov-2 in some patients, and one of them did not produce any sars-cov-2-specific igg with a positive test of sars-cov-2 in sputum after 46 days of illness . no specific therapies have proven to treat patients with prolonged positivity of sars-cov-2 rna. in our study, more than half of the prolonged positive patients (en group) met the discharge standard within 7 days and discharged rapidly after cp therapy. thus, patients with prolonged positivity of sars-cov-2 rna might benefit from cp therapy with shorter length of hospitalization and less cost. besides, rt-pcr of sars-cov-2 turned negative within 15 days after first infusion in five patients in ln group, one turned fig. 1 ct images before and after cp therapy. a results of ai-assisted diagnostic system in patient 2 before cpt, blue areas represent ggo in ct images, red areas represent consolidation in ct images. b results of ai-assisted diagnostic system in patient 2 after cpt. c consolidation of ct in patients 1, 2, and 3 decreased after the transfusion. d ggo of ct in patients 1, 2, 3, 4, and 5 decreased after cp therapy. median ct value in early negative group was significantly greater than late negative group on day 5, p = 0.008. ***: the median ct value in early negative group was significantly greater than late negative group on day 7, p = 0.003. negative at 21 days, and six were still positive until the deadline. our research showed that the viral load of the respiratory tract specimen in two groups differed after cp therapy. viral load may correlate with transmission potential in covid-19 (little et al. 2020) . recently, a report have shown viral load that was detected in the asymptomatic patient was similar to that in the symptomatic patients, which suggested the transmission potential of asymptomatic or minimally symptomatic patients . besides, cp treatment may discontinue sars-cov-2 shedding although didn't reduce mortality in critically end-stage covid-19 patients (zeng et al. 2020 ). in our study most patients might still remain transmission potential even though symptom were mild before cp therapy. thus, implementing cp therapy in patients with prolonged positivity of sars-cov-2 rna might help to decrease viral road and potential of transmission in our study. in addition, reports suggests the mortality of severe covid-19 patients was more than 60% at 28 days (yang x et al. 2020a (yang x et al. , 2020b wang z et al. 2020) . whether cp therapy can reduce mortality of patient with covid-19 is unclear in our study. symptoms of most patients in our study were mild, so we didn't do a mortality analysis. after cp therapy, three patients in ln group died in our study. we assumed that virus duration might be a vital reason of death in these three patients for their viral durations were quite long (59, 53, 53 days). this study has several limitations. first, it is a retrospective observational study, a randomized double-blind trial would be more accurate to assess the efficacy of cp therapy in covid-19. second, symptoms of most patients in our study were mild, including more critically ill patients would be helpful in determining whether cp therapy could reduce mortality in covid-19. third, no studies have reported the appropriate time and dosage of cp implemented in patients with covid-19; therefore, all decisions of cp therapy intervention were made by clinicians empirically. forth, we did not monitor the neutralizing antibody in patients after cp therapy. in conclusion, this retrospective observational study on cp therapy shows that patients with prolonged positivity of sars-cov-2 rna can benefit from a rapid decrease of viral load and improvement in pulmonary images. the appropriate time to implement cp therapy and the optimal cp dosage are still to be explored in the future. chest ct findings of early and progressive phase covid-19 infection from a us patient use of convalescent 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from commercial pig farms in colombia date: 2014-08-08 journal: virol sin doi: 10.1007/s12250-014-3471-5 sha: doc_id: 5372 cord_uid: 7x8ro8p2 porcine respiratory disease complex (prdc) is a serious health problem that mainly affects growing and finishing pigs. prdc is caused by a combination of viral and bacterial agents, such as porcine reproductive and respiratory syndrome virus (prrsv), swine influenza virus (siv), mycoplasma hyopneumoniae (myh), actinobacillus pleuropneumoniae (app), pasteurella multocida and porcine circovirus 2 (pcv2). to characterize the specific role of swine influenza virus in prdc presentation in colombia, 11 farms from three major production regions in colombia were examined in this study. nasal swabs, bronchial lavage and lung tissue samples were obtained from animals displaying symptoms compatible with siv. isolation of siv was performed in 9-day embryonated chicken eggs or madin-darby canine kidney (mdck) cells. positive isolates, identified via the hemagglutination inhibition test, were further analyzed using pcr. overall, 7 of the 11 farms were positive for siv. notably, sequencing of the gene encoding the hemagglutinin (ha) protein led to grouping of strains into circulating viruses identified during the human outbreak of 2009, classified as pandemic h1n1-2009. serum samples from 198 gilts and multiparous sows between 2008 and 2009 were obtained to determine antibody presence of app, myh, pcv2 and prrsv in both siv-h1n1p-negative and -positive farms, but higher levels were recorded for siv-h1n1p-positive farms. odds ratio (or) and p values revealed statistically significant differences (p<0.05) in prdc presentation in gilts and multiparous sows of farms positive for siv-h1n1p. our findings indicate that positive farms have increased risk of prdc presentation, in particular, pcv2, app and myh. swine infl uenza is an acute, highly contagious respiratory disease resulting from infection with type a infl uenza virus, a member of the orthomyxoviridae family. influenza a viruses have been isolated from different species, including humans, pigs, dogs, horses, sea mammals and birds (kuntz-simon g, et al., 2009; webster r g, et al., 1987) . the viruses are classified into subtypes based on antigenic properties of the external glycoproteins, hemagglutinin (ha) and neuraminidase (na). influenza a viruses have been further classified into 16 ha and 9 na subtypes (webster r g, et al., 1987; fouchier r, et al., 2005) . the proteins are highly variable and critical for the induction of antibody response in the host (gramer m, 2008) . although other subtypes have been identifi ed, infl uenza a virus h1n1, h1n2 and h3n2 subtypes are the most prevalent in pig populations worldwide (kuntz-simon g, et al., 2009) . in pigs, the disease is characterized by sudden onset, coughing, dyspnea, fever and prostration, followed by rapid recovery. lesions generally develop rapidly in the respiratory tract and regress quickly. the course and severity of disease are likely to vary with the strain of the virus, age and immune status of the host (easterday b c, et al., 1999) . morbidity is high (near 100%), but mortality is low (usually less than 1%). recovery begins 7 to 10 days post-infection. subclinical infections are common, and most pigs can be reinfected with other strains without showing clinical signs (reeth v k, et al., 2006) . porcine respiratory disease complex (prdc), a multifactorial condition, is a serious health problem in growing and finishing pigs, and poses a threat to the swine industry worldwide. prdc results from a combination of viral and bacterial agents, including porcine reproductive and respiratory syndrome virus (prrsv), swine influenza virus (siv), mycoplasma hyopneumoniae (myh), actinobacillus pleuropneumoniae (app), pasteurella multocida and porcine circovirus type 2 (pcv2) (kim j, et al., 2003) . although the etiology of prdc involves multiple pathogens and varies among farms, several researchers maintain that etiology is mainly associated with myh and prrsv (dee s, 1996; thacker e l, et al., 1999) . pneumonia caused by prdc is characterized by slow growth, decreased feed effi ciency, lethargy, anorexia, fever and cough. while farms and slaughter houses in colombia have tested positive for siv over the years, the role of siv in prdc is yet to be established. there is evidence of classic h1n1 and infl uenza a h1n1 pandemic 2009 circulation in swine populations, but surveillance reports on prdc are scarce to date. the main goal of the current research was to generate surveillance, epidemiological, antigenic as well as phylogenetic data to ascertain the presence of swine influenza (h1n1) pandemic virus and determine its association with prdc (prrsv, myh, app and pcv2) in sows from production farms in colombia. a total of 198 gilts and multiparous sows distributed in 11 farms from three major swine-producing areas in colombia (antioquia, valle del cauca and cundinamarca) were included in the present study. blood samples were obtained via venipuncture of the jugular vein using red top vacutainer ™ tubes. serum samples were identifi ed and stored at -20°c until analysis. two hundred and forty-two nasal swabs, 8 bronchial lavage (bl) and 25 lung tissue samples of animals displaying symptoms compatible with siv (easterday b c, et al., 1999) were collected from the 11 farms. samples were collected in brain-heart infusion medium (bhi) medium (bd®) supplemented with 2% antibiotic and anti-mycotic solution (sigma®), filtered through a 0.22 μm filter, and stored at -70 0 c until processing for siv isolation, either in chicken embryo eggs or the madin-darby canine kidney (mdck) cell line. briefl y, 9 day-old spf embryonated chicken eggs were inoculated via the allantoic cavity with 200 μl filtered sample, incubated at 37°c for 72 h, and monitored daily. allantoic fluid was collected and evaluated for hemagglutination activity following standard procedures (who, 2005) . twenty four-well plates were seeded with 8×10 5 cells/well of mdck and grown in dulbecco's modifi ed eagle medium (dmem) (gibco®) supplemented with 5% fetal bovine serum (fbs, gibco®), 1% antibiotic (sigma®) and 1% l-glutamine (sigma®). complete growth medium was removed from confl uent monolayers and washed three times with pbs supplemented with 2 μg/ml tpck trypsin (sigma®). each well was infected with 200 μl of the original fi ltered sample and incubated for 1 h at 37°c, 5% co 2 , followed by the addition of 2 ml/well complete dmem. cells were incubated at 37°c, 5% co 2 , for 3-6 days and monitored daily for cytopathic effect (cpe). following the incubation period, cell culture supernatant (cs) and allantoic fl uid (af) were collected and tested with the hemagglutination assay (ha) using chicken erythrocytes according to the standard office international des epizooties (oie) protocol (swine infl uenza, 2005) . all ha-positive samples from egg inoculation or cell culture isolation were further analyzed for effi cient subtyping of virus. viral rna was extracted using a commercial rna extraction kit (qiamp viral rna®, qiagen, ca, usa) following the manufacturer's protocol. amplification of ha and na gene segments was performed with a duplex reverse transcriptionpolymerase chain reaction (rt-pcr) assay targeting the respective genes of h1n1 swine infl uenza virus (choi y, et al., 2002) . initial reverse transcription was performed with m-mvl reverse transcriptase (invitrogen) using 4 μl viral rna as template and the uni-12 primer (invitrogen, maryland, usa) to generate cdna. the conditions for reverse transcription (rt) were as follows: 70°c for 5 min, 94°c for 5 min, 42°c for 30 min, and a fi nal step of 4°c. amplifi cation of 1006 bp ha (primers h1f and h1r) and 754 bp na (primers n1f and n1r) genes of siv was performed (table 1 ) under the following reaction conditions: 94°c for 5 min, 35 cycles of 94°c for 1 min, 53.5°c for 1 min, 72°c for 1 min, followed by 72°c for 15 min, and a fi nal step of 4°c to terminate amplifi cation. pcr products were run on an agarose gel, and isolated and gel-purifi ed using the qiaquick gel extraction kit (qiagen ® ). gel-purified products were sequenced by macrogen ® , usa, using big-dye ® terminator cycle sequencing. dna sequences were combined and edited using the lasergene sequencing analysis software package (dnastar ® , madison, wisconsin). multiple sequence alignments were made using clustal w to identify related reference infl uenza genes. sequence comparisons and phylogenetic relationships through a bootstrap trial of 1000 were determined with the mega 5.1 program using the clustal w alignment algorithm, and evolutionary history inferred using the neighbor-joining method (saitou n, et al., 1987) for tree construction. gene sequences of colombian strains were compared with those of swine, avian and human infl uenza viruses, which were retrieved from the ncbi infl uenza virus resource. serum samples were subjected to the hemagglutination inhibition test to identify antibodies against siv using strain a/puerto rico 8/34 (h1n1) as antigen, according to the who protocol (2005). prdc was diagnosed based on detection of antibodies against prrsv, myh, app and pcv2 in serum. specifi c antibodies were measured with elisa kits (table 2) . data were analyzed using the statistix 8.0 program. the chi-square test was initially performed to determine the probability of presentation of respiratory disease complex with and without the presence of siv, and logistic regression analysis subsequently utilized to determine the risk factors that increase the presentation of disease when prdc farms and animals are positive or negative for siv. data were considered signifi cant at p<0.05, and graphs plotted using graphpad software. among the 11 farms surveyed, 7 were positive for swine flu pandemic virus, either from embryo chicken egg or mdck cell culture isolates (table 3) . positive the hemagglutination inhibition test revealed positivity to siv-h1n1p in 36.3% gilts, 33.3% sows with 1-2 births and 30.3% sows with multiparous (>3) births. the data suggest a trend in reactivity to siv-h1n1p, as the antibody response appears to decrease through time. prdc presentation was lower in siv-h1n1p-negative than siv-h1n1p-positive farms ( table 4 ). the odds ratio value and p value revealed significant differences (p<0.05) in prdc risk presentation in gilts and multiparous sows of siv-h1h1p-positive farms (figure 2) . analysis was completed using logistic regression to de-termine the likelihood of increase in prdc disease presentation in siv-h1n1p-positive farms. according to our results, siv-h1n1p-positive farms had 3.19, 1.75 and 5.14 times greater risk of introduction of app, myh and pcv2, respectively, compared with siv-h1n1p-negative farms. in contrast, for prrsv, reduced risk of disease presentation (or, 0.05) was observed in farms positive for siv-h1n1p. the presence of siv-h1n1p in positive farms increases the risk of introduction of prcd especially pcv2, app and myh. when analyzed by age group, the gilts are more susceptible to pcv2, myh and app, respectively, while multiparous sows are more susceptible to pcv2, app and myh. logistic regression analysis was performed to determine the risk of prdc presentation in farms positive and negative for siv-h1n1p. the odds ratio and p value revealed significant differences (p<0.05) in risk prdc presentation per individual. in animals positive for siv-h1n1p, major risk of mycoplasma hyopneumoniae, pcv2 and app presentation was 2.42, 2.08 and 1.98 times that of siv-negative animals, respectively. in contrast, for prrsv, the risk of presentation of disease (or, 0.74) was decreased in animals positive for siv-h1n1p. many groups have defined prdc as a multifactorial respiratory disease involving several pathogens (harms p a, et al., 2002; kim j, et al., 2003; opriessing t, et al., 2007; fachinger v, et al., 2008) , while other investigators (thacker e ,l, 2006) argue that prdc is enzootic pneumonia produced by mycoplasma spp. and other opportunistic bacteria, aggravated by the respiratory virus. the main goal of the present study was to conduct a systematic analysis of swine influenza virus infection and determine its role in prdc presentation in the major swine-producing areas of colombia. serological reactivity determined using hi disclosed that swine influenza virus h1n1 is prevalent in gilts and multiparous sows from farms in colombia, although the antibody response decreased over time. we expected to fi nd isolates from infl uenza of classical h1n1 virus origin in the fi eld. surprisingly, however, sequencing results led to grouping of viruses isolated from the farms within the original h1n1 pandemic of 2009, four of which shared 99% homology. therefore, it is important to consider swine infl uenza virus of pandemic origin as a pathogen playing an infl uential role in the presentation of prdc in commercial pig farms in the country. the pandemic h1n1/09 virus (a/ca/4/09) is a swine infl uenza a virus subtype h1n1 strain responsible for the 2009 fl u pandemic. the virus originates from swine, but was never established as circulating in pig populations before its detection in humans (reeth v k, et al., 1996) . siv-h1n1p presentation in colombia may have been attributed to human transmission from pigs, as reported in various countries (weingartl h m, et al., 2010; sreta d, et al., 2010; pereda a, et al. 2010) . this hypothesis is based on the presentation of disease outbreaks in pigs and humans, as well as reports by the world organization for animal health (oie), which documented the presence of the virus in various countries, including argentina, canada, australia, ireland and united states. our results indicate that siv h1n1p positivity in animals increases the risk of presenting prdc, particularly pcv2, app and myh. primiparous and multiparous sows are more susceptible to pvc2, followed by myh and app in gilts, and app and myh in multiparous sows. a study by hansen m s, et al. (2010) in denmark revealed the presence of pcv2 in most lung samples from animals presenting prdc and indicated associations of this virus with the majority of viral and bacterial pathogens. the most frequently detected pathogens were in the order pcv2, myc hyopneumoniae, pasteurella multocida and myc hyohinis, identified in different proportions in pigs with pneumonia in germany (palzer a, et al., 2008) , taiwan (chiou m t, et al., 2004) and the usa (choi y, et al., 2003) . this diversity in the presentation of pathogens involved in prdc may reflect the different diagnostic methods, health status of animals, farm management factors or simply the complex nature of pneumonia in pigs. siv-h1n1p-positive farms were more susceptible to prdc presentation for both gilts and multiparous sows, and interactions were mainly observed among pcv2 and siv. research in animals between 18 and 20 weeks of age showed that siv acts together with pcv2 in pigs under fi eld conditions. prdc induces severe clinical disease lesions presenting both infl uenza and wasting syndrome post-weaning (dorr p, et al., 2007; pallares f j, et al., 2002) . wei h, and co-workers (2010) concluded that subclinical pcv2 infection results in increased severity of subsequent siv-h1n1 infection and siv can trigger the severe form of pcv2, although the percentage of animals affected is comparable to that reported for other co-infections known to trigger severe pcv2. myh is associated with presentation of swine enzootic pneumonia, characterized by high morbidity and low mortality in affected farms (opriessing t, et al., 2012) . myh acts as an immunosuppressant by increasing its pathogenicity as well as that of siv at the time of replication of the two agents (opriessing t, et al., 2012 , yazawa s, et al., 2004 . thacker e and colleagues (2001) reported that the percentage of lung lesions and clinical presentation of disease in pigs inoculated with siv 21 days after inoculation with myh is higher than that in pigs inoculated with myh only. in our study, higher risk (1.7%) of introduction of myh in siv-h1n1p-positive farms was observed, compared to siv-h1n1p-negative farms. app, a highly contagious infectious agent, causes pleuropneumonia in pigs, characterized by increased susceptibility to secondary infections (gutierrez c, et al., 2000) . app is considered an obligate parasite of the respiratory tract of pigs (taylor d j, 1999) . this report is consistent with the present study, where siv-h1n1ppositive farms displayed 81.1% positivity for app. app has two different biotypes, specifically, biotype 1 containing 12 serotypes and biotype 2 with six serotypes. all biotypes are capable of causing disease, with some serotypes being more aggressive than others (bossé j t, et al., 2002) . app is reported to increase the incidence of stress associated mainly with pleuropneumonia and interactions with viral and bacterial agents involved in the introduction of prdc (bossé j t, et al., 2002; kim j, et al., 2003) . regarding prrsv presentation in siv-h1n1ppositive and -negative farms, the incidence of prrsv was lower in positive (1.6%) than negative farms (15.3%). co-infection studies of prrsv and siv have yielded conflicting results in terms of clinical disease. in animals experiencing swine influenza infection and prrsv infection as a secondary pathogen, the disease is exacerbated, leading to prolonged fever, increased cough and weight loss ( van reeth k, et al. 1996; kitikoon p, et al., 2009 ). on the other hand, prior infection with prrsv does not aggravate acute siv presentation but can generate subsequent chronic infection (pol j m, et al., 1997) . prrsv is transmitted transplacentally through direct contact after birth, and piglets from infected mothers are infectious during subsequent stages of production, particularly the fattening and finishing stages (prieto c, et al., 1997) . similarly, siv is transmitted by direct contact between infected and uninfected animals via aerosol and can affect animals at any stage, particularly during fattening and fi nishing, as the pigs are raised in very close proximity to each other ( van reeth k, et al., 1996) . for this reason, we expected to find significant interactions between the two viruses in multiparous sows. however, this was not observed in our study. regarding tropism, prrsv has high affinity for differentiated macrophages. destruction of these cells in the lung by prrsv is suggested as the key event causing lung susceptibility to secondary pathogens (gucht s v, et al., 2014; van reeth k, et al., 1994) . prrsv and siv are the primary etiologic agents associated with respiratory disease of pigs in the united states (choi y, et al., association of siv-h1n1p with prdc in sows from commercial pig farms in colombia 2003) and co-infection could explain this role in porcine respiratory disease complex presentation. primary siv infection of epithelial cells of the airways results in cellular necrosis, production of infl ammatory mediators and rapid infiltration of phagocytic cells, including lung alveolar macrophages (pam) susceptible to prrsv infection. siv infection-infl ammation may increase the target cells for initial infection of prrsv, resulting in increased and prolonged pneumonia (kitikoon p, et al., 2009) . based on results from the current study, we propose that siv-h1n1p blocks prrsv replication or vice versa, and presentation of the disease is dependent on virulence and the time interval between infections (kitikoon p, et al., 2009) . prdc results from a combination of infectious agents as well as environmental stressors and challenges that affect the health of pigs, resulting in reduced performance, increased medication costs and high rates of mortality. clearly, the different forms of presentation of respiratory disease in pigs are attributed to the interactions of viral and bacterial agents, and although the etiology of prdc involves multiple pathogens with variations among farms, limited information is available on the spread of the virus in our pig population. the current study showed that pcv2 and app are the main viral-bacterial agents interacting with siv-h1n1p in positive animals. although it is difficult to establish whether swine flu virus is a pandemic primary, secondary or opportunistic agent, our data clearly indicate that siv-h1n1p plays an important role in the presentation of prdc, signifying the need to increase agricultural surveillance to prevent future outbreaks. this study was supported by colombia's agriculture ministry, colombian association of swine producers, cercafe and national university of colombia. we are grateful to mirela norho for statistical analysis and farms of antioquia y valle del cauca that participated in the study. the authors declare no conflicts of interest in terms of financial relationships with the industry or directly with the academy. all experiments were performed in accordance with the 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potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia immunology of the porcine respiratory disease complex mycoplasmal diseases dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine infl uenza virus: a clinical and virological study influenza virus a pathogenicity: the pivotal role of hemagglutinin infection of cesarean-derived colostrum-deprived pigs with porcine circovirus type 2 and swine infl uenza virus genetic and pathobiologic characterization of pandemic h1n1 2009 infl uenza viruses from a naturally infected swine herd who, 2005. manual on animal influenza diagnosis and surveillance experimental dual infection of pigs with an h1n1 swine infl uenza virus (a/sw/hok/2/81) and mycoplasma hyopneumoniae key: cord-014920-mmykzj9w authors: wang, shi-qun; wu, jian-guo title: biological effects of hbv x protein on hepatocellular carcinogenesis in association with cellular factors date: 2008-05-10 journal: virol sin doi: 10.1007/s12250-008-2952-9 sha: doc_id: 14920 cord_uid: mmykzj9w the x protein (hbx) of human hepatitis b virus (hbv) acts as an indirect transcriptional transactivator to regulate the expression of many viral and cellular genes, as well as playing a critical role in pathogenesis and the development of hepatocellular carcinoma (hcc). here we described the biological effects of hbx in association with four cellular factors, including inflammatory factors (cox-2 and inos), oncoprotein (ras), and a newly identified tumor suppressor (yuef). the characteristics of these effectors, which might be associated with hepatocellular carcinoma, are also discussed. the genome of hepatitis b virus (hbv) is approximately 3 000 to 3 300 nucleotides in length, and molecular studies have suggested the presence of at least four genes in the viral genome: the s gene coding for the glycosylated envelope protein, c gene for the core protein, p gene for the hbv polymerase, and x gene for the x protein that is associated with transactivating properties (19) . hbv infection causes liver diseases that vary greatly in severity from person there is accumulating evidence suggesting a close association between chronic hbv infection and an increased risk for the development of primary hepatocellular carcinoma (hcc). persistent infection by hbv results in sustained low level liver damage of infected hepatocytes. almost all such damage can be attributed to attack by the host's immune system. the rate of hepatocyte proliferation must increase in order to compensate for cell loss. it is generally accepted that such an increased rate of proliferation over long periods is a major contributor to the development of liver cancer (7, 23, 25) . in addition, the inflammation and phagocytosis that are integral to the immune response can lead to increased local concentrations of received: 2008-02-12, accepted: 2008-03-02 superoxides and free radicals that are highly carcinogenic (1). in addition, the viral x region appears to have pleiotropic effects that could also be involved in the oncogenic processes, including transcriptional activation of cellular growth regulatory genes, modulation of apoptosis and inhibition of nucleotide excision repair of damaged cellular dna (8, 14, 16) . the effects of hbx are mediated by interaction with cell proteins and activation of cell signaling pathways. here we focus on the roles of four representative cellular factors (cox-2, inos, ras, and yuef) that are associated with hbx and discuss their contributions to hepatitis b virus-associated hepatocarcinogenesis according mainly to our recent work. cyclooxygenase-2 (cox-2), a rate-limiting enzyme in the pathway of prostaglandin (pg) synthesis, is one of the important cellular factors that have been suggested previously to be associated with inflammation caused by microbes infections (5, 15, 18) . our tumor suppressors are a specific group of proteins that appear to prevent formation of cancer and loss-offunction mutations in related genes enhance susceptibility to cancer. in a previous study, we used hbv x protein as a bait protein for screening a human liver cdna library using a yeast two-hybrid system (27) . many human hepatocellular carcinomas contain integrated hbv, and the viral x protein appears to have pleiotropic effects that could be involved in the oncogenic process. through interaction with cell proteins, hbx also alter mechanisms of apoptosis and interfere with nucleotide excision repair of damaged dna. together with an influence on cellular signaling, these mechanisms may favor the cell's malignant clonal expansion. but we cannot forget that tumor formation in mammals is a much more complex process and we must make the best endeavors to make more apparent the role of viruses in the development of human cancer. hepatocellular carcinoma caused by iron overload: a possible mechanism of direct hepatocarcinogenicity the immune response during hepatitis b virus infection expression of inducible nitric oxide synthase and cyclooxygenase-2 in angiogenesis and clinical outcome of human gastric cancer tumour overexpression of inducible nitric oxide synthase (inos) increases angiogenesis and may modulate the anti-tumour effects of the vascular disrupting agent zd6126. microvasc res cox-2 inhibitors--ibc conference nitric oxide synthase expression and activity in the tight-skin mouse model of fibrosis survival and apoptosis: a dysregulated balance in liver cancer the upregulation of expressed proteins in hepg2 cells transfected by the recombinant plasmid-containing hbx gene putative tumor suppressor yuef affects the functions of hepatitis b virus x protein in hepatoma cell apoptosis and p53 expression activated ras oncogene collaborates with hbx gene of hepatitis b virus to transform cells by suppressing hbx-mediated apoptosis the hepatitis b virus x protein promotes tumor cell invasion by inducing membrane-type matrix metalloproteinase-1 and cyclooxygenase-2 expression spike protein of sars-cov stimulates cyclooxygenase-2 expression via both calcium-dependent and calcium-independent protein kinase c pathways hepatitis b virus replication is associated with an hbxdependent mitochondrion-regulated increase in cytosolic calcium levels cyclooxygenase-2 expression inhibits trophic withdrawal apoptosis in nerve growth factor-differentiated pc12 cells cyclooxygenase 2 expression in rat corneas after ethanol exposure hepatitis b virus x protein overcomes oncogenic rasinduced senescence in human immortalized cells borna disease virus p protein inhibits nitric oxide synthase gene expression in astrocytes cyclooxygenase 2 expression in vessels and nerves in reversal reaction leprosy hepatitis b virus biology cyclooxygenase regulates angiogenesis induced by colon cancer cells functional switch of viral protein hbx on cell apoptosis, transformation, and tumorigenesis in association with oncoprotein ras host cyclooxygenase-2 modulates carcinoma growth hepatic tumor necrosis factor signaling and nuclear factor-kappab: effects on liver homeostasis and beyond nucleocapsid protein of sars-cov activates the expression of cyclooxygenase-2 by binding directly to regulatory elements for nuclear factor-kappa b and ccaat/enhancer binding protein hepatocyte-restricted constitutive activation of ppar alpha induces hepatoproliferation but not hepatocarcinogenesis mitotic aberration coupled with centrosome amplification is induced by hepatitis b virus x oncoprotein via the ras-mitogenactivated protein/extracellular signal-regulated kinasemitogen-activated protein pathway human hepatitis b virus x protein promotes cell proliferation and inhibits cell apoptosis through interacting with a serine protease hepsin key: cord-284549-edliu3it authors: zhou, hui; qian, qi; shu, ting; xu, jiuyue; kong, jing; mu, jingfang; qiu, yang; zhou, xi title: hepatitis c virus ns2 protein suppresses rna interference in cells date: 2019-11-27 journal: virol sin doi: 10.1007/s12250-019-00182-5 sha: doc_id: 284549 cord_uid: edliu3it rnai interference (rnai) is an evolutionarily conserved post-transcriptional gene silencing mechanism and has been well recognized as an important antiviral immunity in eukaryotes. numerous viruses have been shown to encode viral suppressors of rnai (vsrs) to antagonize antiviral rnai. hepatitis c virus (hcv) is a medically important human pathogen that causes acute and chronic hepatitis. in this study, we screened all the nonstructural proteins of hcv and found that hcv ns2 could suppress rnai induced either by small hairpin rnas (shrnas) or small interfering rnas (sirnas) in mammalian cells. moreover, we demonstrated that ns2 could suppress rnai via its direct interaction with double-stranded rnas (dsrnas) and sirnas, and further identified that the cysteine 184 of ns2 is required for the rnai suppression activity through a serial of point mutation analyses. together, our findings uncovered that hcv ns2 can act as a vsr in vitro, thereby providing novel insights into the life cycle and virus-host interactions of hcv. electronic supplementary material: the online version of this article (10.1007/s12250-019-00182-5) contains supplementary material, which is available to authorized users. hcv is a positive-sense, single-stranded rna virus that belongs to the genus hepacivirus of the family flaviviridae. its genome is approximately 9.6 kb in length and encodes a polyprotein that is cleaved by host and viral proteases to produce ten mature viral proteins, including structural core protein, envelope proteins e1 and e2, the p7 ion channel protein, and nonstructural (ns) proteins ns2, ns3, ns4a, ns4b, ns5a and ns5b (yin et al. 2018) . hcv infection causes acute and chronic hepatitis in humans with a high propensity for chronicity. a large number of hcv-infected patients fail to clear this virus and remain asymptomatic for years to develop severe liver diseases such as cirrhosis and hepatocellular carcinoma (paboriboune et al. 2018) . the capability of establishing chronic infection by hcv is partly due to its ability to evade host innate immune responses (horner and gale 2013) . rnai is an evolutionarily conserved mechanism in all eukaryotes for post-transcriptional gene silencing. rnai serves as an important antiviral immune response in a wide range of organisms, including fungi, plants, and invertebrates (guo et al. 2019; maillard et al. 2019) . moreover, recent studies have demonstrated that rnai also exerts antiviral effects in mammals maillard et al. 2013; li et al. 2016; qiu et al. 2017; xu et al. 2019) . the antiviral rnai is triggered by the viral replicative intermediate double-stranded rnas (vri-dsrnas) produced in the course of viral replication. these vri-dsrnas can be recognized and cleaved by host endoribonuclease dicer into virus-derived small interfering rnas (vsirnas) of approximately 21-23 nucleotide (nt). argonaute (ago) protein within rna-induced silencing complexes (riscs) electronic supplementary material the online version of this article (https://doi.org/10.1007/s12250-019-00182-5) contains supplementary material, which is available to authorized users. incorporates one of the strands of vsirna duplex, which directs the risc-mediated degradation of cognate viral rnas (maillard et al. 2019) . to counteract the antiviral rnai, viruses have evolved to encode viral suppressors of rnai (vsrs) targeting various steps of the rnai pathway (wu et al. 2010) . one common strategy utilized by vsrs, including nodamura virus (nov) b2, ebola virus (ebov) vp35, influenza a virus (iav) ns1, and human enterovirus 71 (ev-a71) 3a, is to prevent viral dsrna from cleavage by dicer (li et al. 2004; sullivan and ganem 2005; haasnoot et al. 2007; qiu et al. 2017) . alternatively, multiple vsr proteins have been shown to directly target the key components of the rnai pathway, such as dicer or ago proteins. for example, wuhan nodavirus (whnv) b2 can directly bind to drosophila dicer-2 to inhibit vsirna production (qi et al. 2011 (qi et al. , 2012 , while cricket paralysis virus 1a has been found to directly inhibit drosophila ago2 (nayak et al. 2018) . in the case of hcv, the core protein was found to suppress rnai by interacting with dicer (chen et al. 2008) . moreover, hcv structural e2 protein was also identified to antagonize rnai by targeting ago2 (ji et al. 2008) . consistent with the findings in hcv, a number of rna viruses were shown to encode more than one viral protein to counteract rnai. for instance, vp35, vp30 and vp40 of ebov were found to suppress rnai through different mechanisms (haasnoot et al. 2007; fabozzi et al. 2011) . severe acute respiratory syndrome coronavirus (sars-cov) encoded 7a and nucleocapsid contain rnai suppression activities (karjee et al. 2010; cui et al. 2015) . besides, tat and nef proteins of human immunodeficiency virus-1 (hiv-1) were identified to suppress rnai through the dsrna-binding and ago interaction, respectively (bennasser et al. 2005; aqil et al. 2013) . the phenomena of viruses encoding multiple vsrs as well as vsrs targeting different steps of the rnai pathway highlight the importance of suppressing rnai during the viral life cycle. although the structural core and e2 proteins of hcv were shown to suppress rnai, it is unclear if any hcv nonstructural protein contains rnai suppression activity. in this study, we uncovered that hcv nonstructural ns2 protein possessed a potent in vitro vsr activity that suppressed the rnai induced by short hairpin rna (shrna) and sirna in mammalian cells. we also confirmed the conserved residue in hcv ns2 that could disrupt its activity to suppress rnai. overall, our findings demonstrated that hcv ns2 contains vsr activity, thereby providing insights into the life cycle of hcv. for expression of hcv nonstructural proteins including ns2, ns3, ns3/4a, ns4a, ns4b, ns5a and ns5b in hek293t cells, their orfs were cloned into the prk-flag, respectively. the templates expressing hcv proteins were kindly provided by prof. ying zhu (wuhan university, china). the point mutations were introduced into the ns2 coding region by pcr-mediated mutagenesis with the appropriate primers. for expression of recombinant ns2 in sf9 cells, the orfs of ns2 and its mutant were constructed into the vector pfastbac htb-mbp as previously described (yang et al. 2017) . the resulting plasmids were subjected to the bac-to-bac baculovirus expression system to express the fusion proteins with an mbp tag at the n-terminus. the egfp-sirna was chemically synthesized by guangzhou ribobio co., ltd., china. all the primers and oligonucleotides used in this study are shown in supplementary table s1 . hek293t, 293t-nodice (kindly provided by prof. bryan cullen, durham, nc, usa) and huh7.5 cells were maintained in dulbecco's modified eagle's medium (dmem, gibco) containing 7% fetal bovine serum (fbs) (gibco), 100 u/ml penicillin and 100 lg/ml streptomycin at 37°c in an incubator with 5% co 2 . cells were seeded in 6-well plates and grown overnight to reach 50% confluence. before transfection, the medium was changed to dmem containing without serum and antibiotic. cells then were transfected with the indicated plasmids by using fugene hd reagent (roche, basel, switzerland) according to the manufacturer's instructions. cells were harvested in lysis buffer [50 mmol/l tris-hcl (ph 7.4), 150 mmol/l nacl, 1% np40, 0.25% deoxycholate and a protease inhibitor cocktail (rhoche)]. then the lysates were subjected to 15% sds-page and western blotting analysis. the antibodies used in this study are as follow: anti-tubulin (proteintech group, 1:5000), anti-flag (proteintech group, 1:5000) and anti-myc (proteintech group, 1:3000). total cellular rnas were extracted using trizol reagent (thermo) according to the manufacturer's instructions. for virologica sinica the detection of egfp mrna, 5 lg of total rnas were subjected to denatured 1.5% agarose gels with 2.2 mol/l formaldehyde. the separated rnas were transferred onto the hybond-a nylon membrane (ge healthcare), and fixed at 120°c for 15 min. then the membranes were hybridized with dig-labeled probes in hybridization ovens at 65°c overnight. finally, the membranes were incubated with anti-dig antibody conjugated with alkaline phosphatase and exposed to the luminescent image analyzer las4000 (fuji film). the probes for detection of egfp and gapdh mrna were complementary to their orf region of 500-720 nt and 760-1060 nt, respectively. for detection of small rnas, 20 lg of total rnas were subjected to 7 mol/l urea-15% page and transferred to hybond-a nylon membrane (ge healthcare). the membrane was chemically cross-linked in 1-ethly-3-(3dimethylaminopropyl) carbodiimide (edc) at 60°c for 30 min. the dig-labeled rna probes targeting egfp sirna and u6 were synthesized by takara. the expression and purification of mbp alone and mbpfusion proteins were performed in sf9 cells using baculovirus expression system as previously described (yang et al. 2017) . briefly, cells were infected with mbp-tagged hcv ns2 expressing baculoviruses for 72 h. infected cells were resuspended, lysed via sonication and then centrifuged at 11,000 9g for 30 min to remove debris. the protein in the supernatant was purified using amylose affinity chromatography (new england biolabs, ipswich, ma) according to the manufacturer 0 s protocol and then concentrated using amicon ultra-15 filters (millipore, schwalbach, germany). all purified proteins were quantified with a bicinchoninic acid (bca) protein assay kit (cwbio, china) and stored at -80°c in aliquots. proteins were separated on 10% sds-page and visualized by coomassie blue. we generated 200-nt dig-labeled dsrna and 22-nt sirna via in vitro transcription using dig rna labeling mix (roche). mbp-fusion ns2 or mutant proteins were reacted with dig-labeled rnas (0.2 lmol/l 200-nt dsrna or 22-nt sirna) in a binding buffer [50 mmol/l hepes (ph 8.0), 15 mmol/l nacl, 0.5 mmol/l mgcl 2 , 10% glycerol and 1 u of rnase inhibitor (promega)] at 22°c for 45 min; the total volume was 10 ll. then the reaction mixtures were separated on 6% (for dsrna) or 12% (for sirna) tbe-page and transferred to hybond-a nylon membrane (ge healthcare). the membranes were incubated for 30 min with anti-dig antibody conjugated with alkaline phosphatase (roche). hek293t cells were lysed in a lysis buffer containing 20 mmol/l tris-hcl (ph 7.4), 200 mmol/l nacl, 2.5 mmol/l mgcl 2 , 0.5% triton x100, 0.5 u/ll rnase inhibitor (promega) and a protease inhibitor cocktail (roche). after centrifugation for 15 min at 12,000 9g, the supernatant was pre-cleared via incubation with protein-a/ g agarose beads (roche) at 4°c for 4 h. then the precleared lysates were incubated with antibodies (anti-flag, anti-myc or anti-igg as a negative control) together with protein-a/g agarose beads (roche) at 4°c for 12 h. the antibody-bound complexes were washed for five times with the same lysis buffer except that nacl concentration was raised to 600 mmol/l. finally, rnas were extracted using trizol reagent (thermo) according to the manufacturer 0 s protocol. to identify whether hcv has any nonstructural protein which works as a potential vsr, we screened them via the reversal-of-silencing assay in hek293t cells (fig. 1a) . in brief, cells were co-transfected with the plasmids encoding egfp and egfp-specific shrna (shegfp), together with the vectors for divers hcv nonstructural proteins. nov b2 (nb2), a well-characterized vsr was used as a positive control. the expression of the hcv-encoded proteins and nb2 were detected by western blotting with anti-flag and anti-myc antibodies (fig. 1b) . at 48 h post transfection (hpt), the mrna levels of egfp were detected by northern blotting with a digoxigenin (dig)-labeled rna probe targeting 500-720 nt of egfp orf. egfp-specific shrna can effectively eliminate the egfp transcripts (fig. 1a, lane 2) . as shown in fig. 1a , hcv ns2 protein can effectively restore the expression of rnai-silenced egfp (lane 4). expectedly, expression of nb2 or genetic ablation of dicer (nodice) suppressed shrna-induced rnai in hek293t cells (fig. 1a, lanes 3 and 11) . importantly, hcv ns2 did not affect the transcription efficiency of egfp in the absence of shrna (fig. 1c) , excluding out the possibility that hcv ns2 can directly promote egfp transcription. we sought to examine whether the rnai suppression activity of hcv ns2 is dependent on the protein expression levels. thus, hek293t cells were co-transfected with the plasmids for egfp and egfp-specific shrna, together with the increasing amount of ns2 plasmid as indicated. our findings showed that the reversal effect of egfp silencing increased progressively at the mrna levels, along with the growing amount of ns2 plasmid being transfected ( fig. 2a, 2b) . we further examined the vsr activity of hcv ns2 at different time points. our results showed that the reversal effect of egfp silencing could be observed at 48 hpt (fig. 2c) , indicating that the vsr activity was dependent on the expression level of hcv ns2 protein. taken together, our findings showed that hcv ns2 displays vsr activity in mammalian cells. having established that hcv ns2 contains rnai suppression activity, we sought to examine the detailed mechanism through which hcv ns2 inhibits rnai. in the process of rnai, dsrna/shrna requires the dicer-mediated cleavage into sirna (maillard et al. 2019) . to investigate whether hcv ns2 can inhibit this step, small rnas harvested from hek293t cells co-expressing egfp-specific shrna together with ns2 were subjected to northern blotting with a dig-labeled rna probe targeting egfp sirna produced from shrna by dicer. as shown in fig. 3a , the accumulation of dicer-cleaved sirna was reduced in the presence of ns2 compared to that in cells expressing empty vector, indicating that hcv ns2 can suppress dicer-mediated sirna production. these results are consistent with the previous findings that hcv ns2 effectively restored shrna-induced silencing of egfp transcript in hek293t cells (fig. 1a) . in the process of shrna-induced rnai, shrna is cleaved by dicer into sirna. after identifying that hcv ns2 suppressed the sirna production in cells expressing shrna, we performed rna immunoprecipitation (rna-ip) assay to test whether hcv ns2 sequestrated dsrna in hek293t cells. briefly, cells expressing flag-tagged ns2, myc-tagged nb2 or empty vector, together with egfpspecific dsrna (1-196 nt of egfp orf) were lysed and immunoprecipitated with the indicated antibodies, respectively. rnas extracted from the rna-ip precipitates were examined via northern blotting with the rna probe targeting the 196-nt dsrna of egfp. as shown in fig. 3b , our findings show that ns2 can associate with dsrna in cells (lane 9). we sought to examine whether hcv ns2 could directly bind to dsrna. to this end, we performed gel shift assays by incubating the recombinant mbp-fusion hcv ns2 (mbp-ns2; supplementary fig. s1a ) and the in vitrotranscribed 200-nt dig-labeled dsrna. as shown in fig. 3c , the mobility of dsrna was inhibited in a dosedependent manner by the hcv ns2 protein (lanes 4-7) , compared to the control reaction with mbp protein alone. of note, mbp-fusion whnv b2 (mbp-wb2), another well-characterized vsr that contains the dsrna-binding activity, was used as the positive control (fig. 3c, lane 3) . taken together, our findings indicate that hcv ns2 can suppress rnai by sequestrating dsrna. northern blotting with dig-labeled rna probe targeting the 500-720 nt of egfp orf. gapdh mrna was used as the loading control. b the expression of hcv proteins was detected by western blotting. arrow indicates the unrelated bands in the bot. c hek293t cells were co-transfected with the plasmid encoding egfp, together with hcv ns2 or nov b2 plasmid, respectively. at 48 hpt, egfp mrna levels were examined via northern blotting. we sought to examine whether hcv ns2 protein can inhibit rnai induced by sirna. to this end, hek293t cells were co-transfected the plasmid for egfp and the chemically synthesized egfp-specific sirna (siegfp, 50 nmol/l), together with the ns2 plasmid. egfp-specific sirna induces a significant reduction of egfp expression (fig. 4a, lane 2) . our results showed that hcv ns2 efficiently restored the egfp mrna levels, indicating that hcv ns2 can inhibit sirna-induced rnai in mammalian cells (fig. 4a, lane 4) . because hcv ns2 can directly bind to dsrna, we sought to determine whether ns2 suppressed sirna-induced rnai by directly binding to sirna. to this end, we conducted gel shift assay by incubating mbp-ns2 together with dig-labeled synthetic 22-nt sirna. as shown in fig. 4b , ns2 can directly bind to sirna in a dose-dependent manner. taken together, these results indicate that hcv ns2 can suppress rnai by sequestering sirna. after determining the vsr activity of hcv ns2, we sought to identify the critical residues required for its rnai suppression activity. thus, we performed multiple sequence alignments of ns2s encoded by multiple hcv isolates (supplementary fig. s1b ). because the dsrna/ sirna-binding and protein dimerization are critical for vsr's activity (maillard et al. 2019) , the positivelycharged and highly conserved residues, including arginine (r), lysine (k), histidine (h), glutamic acid (e) and cysteine (c) were subjected to single-point mutation to alanine (a), and the resulting mutant ns2 proteins were then examined via the reversal-of-silencing assay in hek293t cells (fig. 5a-5c ). our findings show that the c184a mutation (ns2 c184a ) significantly suppressed the activity of hcv ns2 to suppress rnai (fig. 5c, lane 7 and fig. 5d, lane 5) . moreover, we further confirmed that the c184 is critical for the vsr activity of hcv ns2 via the reversal-of-silencing assay in huh7.5 cell line (fig. 5e) , which is usually used for hepatitis c research. moreover, we found that c184a abolished the dsrna-and sirnabinding activities of hcv ns2 in vitro (fig. 6a, 6b ). hek293t cells were co-transfected with the plasmids encoding egfp (0.1 lg) and egfp-specific shrna (0.3 lg), together with the increasing amounts of the plasmid encoding hcv ns2 (0.5 lg, 1.0 lg and 1.5 lg, respectively). at 48 hpt, egfp mrna levels were determined by northern blotting (a). gapdh mrna was used as the loading control. the mrna levels of egfp normalized to that of gapdh were quantified with bio-rad quantity one software and were graphed in (b) with that in the presence of the hcv ns2 (0.5 lg) defined as 100%. error bars represent sd values from the results of three independent experiments. *p \ 0.05, as measured by two-way anova (graphpad prism). cell lysates were harvested and analyzed by western blotting with anti-flag, anti-myc and anti-tubulin antibodies. c hek293t cells were co-transfected with plasmids encoding egfp (0.1 lg) and egfp-specific shrna (0.3 lg), together with either empty vector or the plasmid encoding hcv ns2 (1 lg) or nb2 (1 lg) as indicated. at 24, 36, and 48 hpt, total rnas were extracted and the level of egfp mrna was examined by northern blotting. cell lysates were harvested and analyzed by western blotting with anti-flag, anti-myc and anti-tubulin antibodies. besides, we also found that ns2 c184a failed to suppress the processing of shrna into sirna in hek293t cells (fig. 6c ). rnai is an antiviral immune mechanism conserved in both invertebrates and mammals. as a counter-defense to rnai, viruses evolved to encode vsrs that were identified to target different steps of the rnai pathway. in the present study, we screened the viral nonstructural proteins of hcv for rnai suppression activity and found that hcv ns2 protein is a vsr. the gel shift and rna-ip analyses show that hcv ns2 possesses both dsrna-and sirna-binding activities that are required for rnai suppression. thus, we speculate that hcv ns2 may function as vsr by shielding virus-derived dsrna from dicer cleavage and binding to viral sirna to interfere with risc assembly. in addition to ns2, hcv structural proteins core and e2 were also shown to contain rnai suppression activity by directly targeting the protein components of the rnai pathway (chen et al. 2008; ji et al. 2008) . hcv core interacts with dicer and inhibits the sirna production, while hcv e2 can block the risc assembly by interacting with ago2. interestingly, we showed that hcv ns2 as a vsr can target the rna components of the rnai pathway by directly binding to dsrna and sirna. these results indicate that hcv can suppress antiviral rnai at different stages by encoding multiple vsrs. this phenomenon can also be found in other rna viruses, such as ebov, sars-cov, hiv-1 and denv (bennasser et al. 2005; karjee et al. 2010; fabozzi et al. 2011; aqil et al. 2013; kakumani et al. 2013; cui et al. 2015; kakumani et al. 2015) . thus, encoding multiple vsrs that target different steps of the rnai pathway may offer advantages in the mode of action required for efficient suppression of rnai, thereby highlighting the importance of the antiviral rnai in the hostvirus interactions. hcv ns2 protein is a * 23 kda transmembrane protein that has multiple functions during the hcv replication. it contains a protease domain that functions as a cysteine protease to catalyze a cleavage between ns2 and ns3 (pieroni et al. 1997) . besides, ns2 colocalizes with e1, e2 ns3 and ns5a near the core protein and lipid droplet during the virion assembly . moreover, ns2 can work as a potent interferon antagonist and apoptosis inhibitor (erdtmann et al. 2003; kaukinen et al. 2013 ). here we found that hcv ns2 contains an rnai fig. 5 c184 is critical for the vsr activity of hcv ns2. a-d hek293t cells were co-transfected with the plasmids encoding egfp (0.1 lg) and egfp-specific shrna (0.3 lg), together with either empty vector or the plasmids encoding different hcv ns2 mutants (1 lg each) as indicated, at 48 hpt, total rnas were extracted and the levels of egfp mrna were determined via northern blotting. 293t-nodice cells were used as a control. gapdh mrna was used as the loading control. cell lysates were also subjected to western blotting with anti-flag, anti-myc and anti-tubulin antibodies. e huh7.5 cells were co-transfected with the plasmids encoding egfp (0.25 lg) and egfp-specific shrna (0.75 lg), together with either empty vector or a plasmid encoding ns2 or ns2 c184a as indicated (1.5 lg each). at 48 hpt, total rnas were extracted and the level of egfp mrna was examined via northern blotting. suppression activity, adding a novel function to its multiple roles in the viral life cycle. hcv ns2 is identified to possess dsrna-and sirna-binding activities, similar to many other vsrs such as nov b2 and sars-cov nucleocapsid. however, although these vsrs use the same strategy of suppressing rnai by sequestrating dsrna and/ or sirna, they do not share sequence identity or structural conservation, suggesting that vsrs of distinct virus families are evolved independently during the viral evolution. hcv ns2 forms a dimer with an n-terminal alpha-helical subdomain and a c-terminal antiparallel beta-sheet (lorenz et al. 2006 ). in addition, the conserved residues h143, e163 and c184 have been found to important for hcv ns2's dimerization (lorenz et al. 2006) . our mutational analyses showed that mutation of c184 abolished the dsrna-and sirna-binding activities of hcv ns2 and eliminated its vsr activity, implying the dimerization of hcv ns2 is required for its rnai suppression activity. our findings are consistent with the previous observations that dimerization is important for vsr activities of many viruses. for example, ev-a71 3a forms a dimer and disrupting its dimerization blocks the vsr activity (qiu et al. 2017) . in conclusion, our findings demonstrate that hcv ns2 can act as a vsr by sequestrating dsrna from dicer cleavage and interfering with risc assembly by sirnabinding. moreover, these results add hcv ns2 as a new member of hcv-encoded vsrs, which suppressing rnai by a different mechanism. and it provides insight into the life cycles and virus-host interactions of hcv. fig. 6 the dsrna-and sirna-binding activities of hcv ns2 are required for its rnai suppression. mbp-ns2 wt and mbp-ns2 c184a were incubated with 0.2 lmol/l 200-nt dig-labeled dsrna (a) or 22-nt sirna (b) at 22°c for 45 min. complexes were separated on 6% (a) or 12% (b) tbe-page, respectively. c hek293t cells were co-transfected with egfp specific shrna (0.3 lg) and either the plasmid encoding ns2 wt or ns2 c184a (1 lg of each). total rnas were subjected to small rna northern blotting. u6 was used as the loading control. the hiv-1 nef protein binds argonaute-2 and functions as a viral suppressor of rna interference evidence that hiv-1 encodes an sirna and a suppressor of rna silencing hcv core protein interacts with dicer to antagonize rna silencing the nucleocapsid protein of coronaviruses acts as a viral suppressor of rna silencing in mammalian cells the hepatitis c virus ns2 protein is an inhibitor of cide-b-induced apoptosis ebolavirus proteins suppress the effects of small interfering rna by direct interaction with the mammalian rna interference pathway small rna-based antimicrobial immunity the ebola virus vp35 protein is a suppressor of rna silencing regulation of hepatic innate immunity by hepatitis c virus suppression of short interfering rna-mediated gene silencing by the structural proteins of hepatitis c virus role of rna interference (rnai) in dengue virus replication and identification of ns4b as an rnai suppressor dengue ns3, an rnai suppressor, modulates the human mirna pathways through its interacting partner the 7a accessory protein of severe acute respiratory syndrome coronavirus acts as an rna silencing suppressor hepatitis c virus ns2 protease inhibits host cell antiviral response by inhibiting ikkepsilon and tbk1 functions interferon antagonist proteins of influenza and vaccinia viruses are suppressors of rna silencing rna interference functions as an antiviral immunity mechanism in mammals induction and suppression of antiviral rna interference by influenza a virus in mammalian cells structure of the catalytic domain of the hepatitis c virus ns2-3 protease hepatitis c virus ns2 protein serves as a scaffold for virus assembly by interacting with both structural and nonstructural proteins antiviral rna interference in mammalian cells slicing and dicing viruses: antiviral rna interference in mammals a viral protein restricts drosophila rnai immunity by regulating argonaute activity and stability hepatitis c in laos: a 7-year retrospective study on 1765 patients in vitro study of the ns2-3 protease of hepatitis c virus rna binding by a novel helical fold of b2 protein from wuhan nodavirus mediates the suppression of rna interference and promotes b2 dimerization targeting of dicer-2 and rna by a viral rna silencing suppressor in drosophila cells human virus-derived small rnas can confer antiviral immunity in mammals a virus-encoded inhibitor that blocks rna interference in mammalian cells viral suppressors of rna-based viral immunity: host targets zika virus infection induces rnai-mediated antiviral immunity in human neural progenitors and brain organoids cypovirus capsid protein vp5 has nucleoside triphosphatase activity nap1l1 regulates hepatitis c virus entry and interacts with ns3 acknowledgements we wish to thank prof. ying zhu (wuhan, china) and prof. bryan cullen (durham, usa) for kindly providing materials. this work was supported by the strategic priority research program of chinese academy of sciences (xdb29010300 to x.z.), the national natural science foundation of china (81873964 to y.q., 31670161 to x.z. and 31800140 to j.m.), the science and technology bureau of wuhan (2018060401011309 to x.z.), and the advanced customer cultivation project of wuhan national biosafety laboratory (2018accp-ms11 to y.q.). x.z. is supported by the newton advanced fellowship from the academy of medical sciences, uk (naf005\1002). author contributions hz performed the experiments, analyzed the data and drafted the manuscript; qq, ts, jx, jk and jm helped to perform the experiments; qq and ts helped to analyze the data; yq and xz designed the experiments, analyzed the data, and finalized the manuscript. all authors approved the final manuscript. conflict of interest the authors declare that there are no conflict of interest.animal and human rights statement this article does not contain any studies with human or animal subjects performed by any of the authors. key: cord-269021-juh2qkm0 authors: bai, zhihua; gong, yue; tian, xiaodong; cao, ying; liu, wenjun; li, jing title: the rapid assessment and early warning models for covid-19 date: 2020-04-01 journal: virol sin doi: 10.1007/s12250-020-00219-0 sha: doc_id: 269021 cord_uid: juh2qkm0 human beings have experienced a serious public health event as the new pneumonia (covid-19), caused by the severe acute respiratory syndrome coronavirus has killed more than 3000 people in china, most of them elderly or people with underlying chronic diseases or immunosuppressed states. rapid assessment and early warning are essential for outbreak analysis in response to serious public health events. this paper reviews the current model analysis methods and conclusions from both micro and macro perspectives. the establishment of a comprehensive assessment model, and the use of model analysis prediction, is very efficient for the early warning of infectious diseases. this would significantly improve global surveillance capacity, particularly in developing regions, and improve basic training in infectious diseases and molecular epidemiology. since the early twenty-first century, human beings have experienced several serious public health events caused by pathogens shared with wild or domestic animals, such as severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), and influenza a (h1n1) virus et al. emerging zoonoses pose a growing threat to global health, having caused hundreds of billions of dollars in economic losses in the past two decades (karesh et al. 2012) . the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) which can be deadly was first identified in wuhan, china in december 2019 (cheng and shan 2020; wu p et al. 2020b) . it evolved from wildlife and can cause fever and severe respiratory syndrome in human being which were named covid-19 by the world health organization (who) xu et al. 2020) . as of march 27, 2020, the still ongoing outbreak of around 82,098 confirmed cases and 3298 deaths in mainland china (http://2019ncov. chinacdc.cn/2019-ncov/), and most new coronavirus cases are now outside of china, with 200 countries reporting cases up to now. on january 30, 2020, the who announced that the epidemic constituted a public health emergency of international concern. these vulnerabilities emphasize the need for a systematic, preemptive approach that aims to prevent the spread, or even the initial emergence of pandemics. the systematic preemptive approach refers to the rapid assessment and an early warning of outbreaks of infectious diseases. early warning is an important measure to control and prevent outbreaks and epidemics of infectious diseases. the core is to find the abnormal distribution of infectious diseases and then estimate the risk of an outbreak. its main work is to collect and analyze relevant information and data on the incidence of infectious diseases, and to explore the spatiotemporal transmission and epidemic laws of infectious diseases by using genomics, statistics and mathematical methods. it is indispensable to use mathematical methods to establish appropriate warning models in zhihua bai and whenever a new pathogen appears, its origin always causes widespread concern. most of the emerging infectious diseases that affect humans are zoonotic, which can be traced back to some specific wildlife, but there have been few analytical tools to determine which host species may carry the next virus that will infect humans, or which viruses may cross species boundaries directly. however, with the advent of the era of big data, it no longer seems to be a difficult problem (fig. 1 ). in order to understanding patterns of viral diversity in wildlife and determinants of successful cross-species transmission, or spillover, kevin et al. created a database of 2805 mammal-virus associations, including 754 mammal species (14% of global mammal diversity) from 15 orders and 586 unique viral species (recognized viruses found in mammals) from 28 viral families. they used generalized additive models to identify and rank hostspecific predictors of the number of total and zoonotic viruses in mammals. the model showed that bats are host to a significantly higher proportion of zoonoses than other mammalian orders (after controlling for reporting effort and the other predictor variables). moreover, the phylogenetic host breadth and other viral traits are significant predictors of zoonotic potential, providing a novel framework to assess if a newly discovered mammalian virus could infect people (olival et al. 2017 ). identifying the origin of the virus can help health authorities carry out accurate health surveillance. undoubtedly, the development of genome sequencing technology has contributed a great deal to sars-cov-2 traceability. wassenaar and zou (2020) demonstrated that the noncoding flanks of the viral genome can be used to correctly separate the recognized four betacoronavirus subspecies by whole-genome sequence comparisons, which has implications for rapid classification of new viruses. comparing the sequence of the genomic sequence obtained with various mammalian coronavirus sequences suggests that bats may be a natural reservoir for sars-cov-2 (paraskevis et al. 2020; zhou et al. 2020) , and pangolins are potential intermediate hosts zhang et al. 2020) . chen et al. (2020) used a low-input metagenomic nextgeneration sequencing (mngs) approach on rna extracted from bronchoalveolar lavage fluid (balf) from two patients. the mngs methodology used to investigate infectious microorganisms from original clinical samples is currently achievable (gu et al. 2019) . firstly, they found that a majority of the viral reads (99.9% and 99.7% respectively for sample 1 and 2) were associated with coronaviruses and identified sars-cov-2 as the sole pathogen. the genome comparisons indicated that sars-cov-2 shared a 98.7% nucleotide identity with bat coronavirus strain btcov/4991 while it was quite divergent from sars-cov (79.7%). furthermore, phylogenetic trees were reconstructed based on the nucleotide alignment of key viral genes by using the maximum likelihood method, employing a best fit substitution model and a spr branch swapping algorithm. an understandable phylogeny showed sars-cov-2 has the most recent common ancestor with the neighboring bat coronaviruses, supporting the bat origin. another way to help identify the origin and evolutionary trends of viral hosts is to study codon usage bias given that the use of viral codons is subject to different selection pressures in different host environments. ji et al. (2020) found that the codon usage bias of sars-cov-2 is most similar to that of snakes by using relative synonymous codon usage (rscu) analyses. in fact, there are two levels of codon usage biases, one is at the amino acid level and the other is at the synonymous codon level. amino acid composition can also introduce confounding effects if we only focus on the variation of synonymous codon usage. in order to overcome the above problem, another researchteam performed global correspondence analysis (ca), within-group correspondence analysis (wca) and between-group correspondence analysis (bca) among different genes in the coronavirus viral sequences . the results showed that the sars-cov-2 had amino acid usage similar to bats, but the synonymous codon usages were relatively different, which indicated similar protein characteristics but maybe different evolutionary histories. however, these analytic approaches have not been used in studying viral sequences widely, so the information only provided some insights into wildlife reservoirs and that the further validation by animal model experiments is indispensable. moreover, as a potential intermediated mammal, virus in civets share * 99.8% genome identity to sars-cov in humans. as the origin for sars-cov-2 is most possibly the bats, and we should also be concerned about other mammals, while are likely to be predicted as intermediate hosts. in the case of a gradually improved infectious disease surveillance system, the research on forecasting and early warning of epidemics based on models has become the focus of the public health system. the principle aim is to establish a suitable mathematical model based on the dynamic characteristics of infectious disease transmission, and to conduct qualitative as well as quantitative analysis and computer simulations of the transmission process. currently, the models used for forecasting and warning at home and abroad mainly include the time series model, the linear regression model, the grey dynamics model, the artificial neural network model, the markov model, the bayesian model and the complex network model (jennings et al. 2008; zhu et al. 2011; wesolowski and suchacz 2012; aghdam et al. 2013; shen et al. 2013) . practice has proved that in public health emergencies, it is important to establish suitable prediction models to change passive prevention into active prevention. for example, during the outbreak of foot and mouth disease in the uk, the politicians relied heavily on mathematical modeling in their selection of epidemic control measures with great success (ferguson et al. 2001; keeling et al. 2001) . in response to the current epidemic of sars-cov-2, many researchers have developed mathematical models with varying degrees of complexity, aiming to assess the capacity of pathogen transmission and which interventions are most likely to be effective (fig. 2) . in the early stages of the outbreak, it is important to gain an understanding of the transmission pattern and potential of sars-cov-2. if more than one secondary case is produced for each primary case on average, the chain of transmission events within an outbreak is extended. the basic reproduction number (r 0 ) is the most important index to measure and directly explain the level of virus transmission (anderson et al. 2004) . it refers to the average number of people who will be infected with an infectious disease under natural conditions. due to the facts that (1) the infected patients might die, (2) some patients may develop immunity after recovering, and (3) that the infected population is of a limited size, the r 0 value will decrease with the decrease in the infected population, and the transmission speed is significantly reduced (anderson et al. 1992) . essentially, r 0 determines how intensive a policy will need to be to control the epidemic, whereas both the generation time (tg) and r 0 determine the time available to implement suitably intensive controls. one way to estimate r 0 is to model an infectious disease curve that obeys exponential growth (de silva et al. 2009 ). the nonlinear least square (nls) framework has been adopted for data fitting and parameter estimation (wallinga and lipsitch 2007; zhao et al. 2019) . since the transmission chain of sars-cov-2 remains unclear, many researches adopted the tg information from sars and mers, which is similar to sars-cov-2. zhao et al. (2020) reviewed the sars-cov-2 cases and suggests they might have been under-reported roughly from the 1st to the 15th of january 2020 because the number of cases appeared inconsistent with the following rapid growth of the epidemic curve since the 16th of january 2020. they modeled the epidemic curve of sars-cov-2 cases, in mainland china from december 1, 2019 to january 24, 2020 through the exponential growing poisson process. the number of unreported cases was determined by the maximum likelihood estimation. as a result, they estimated the r 0 of sars-cov-2 was 2.56 (95% ci: 2.49-2.63) and the number of unreported cases was 469 (95% ci: 403-540). this study helped us understand what might be happening in the early stages of outbreak. another algorithm estimated r 0 using the markov chain monte carlo (mcmc) method with gibbs sampling and a non-informative flat prior. wu et al. estimated the r 0 for sars-cov-2 was 2.68 (95% ci: 2.47-2.86) (wu jt et al. 2020a) . although the r 0 values estimated by different methods were different to some extent, it was not difficult to find that the r 0 value remained larger than 1 at the early stage of the outbreak indicating the possibility of sustained human-to-human transmission . adam et al. estimated that the median daily r 0 , declined from 2.35 (95% ci: 1.15-4.77) 1 week before travel restrictions were introduced on january 23 to 1.05 (95% ci: 0.413-2.39), which may have reflected the outbreak control efforts or a growing awareness of sars-cov-2 during this period (kucharski et al. 2020 ). population estimates of r 0 can obscure considerable individual variation in infectiousness, as highlighted during the global emergence of sars by numerous super spreading events in which certain individuals infected unusually large numbers of secondary cases (dye and gay 2003; leo et al. 2003; lipsitch et al. 2003; riley et al. 2003; shen et al. 2004; bauch et al. 2005) . while super spreading always remains a rare event, it can result in a large and explosive transmission event and have a major impact on the course of an epidemic. for each primary case, riou et al. generated secondary cases according to a negative-binomial offspring distribution with mean r 0 and dispersion k (the lower the value of k, the higher the impact of super spreading). after 1000 stochastic simulations for each individual combination, their simulations suggest that very low values of k are less likely, and the establishment of sustained transmission chains from single cases cannot be ruled out (riou and althaus 2020) . therefore, the author emphasized the importance of screening, surveillance and control efforts, such as early detection and isolation, contact tracing and the use of personal protective equipment. the most classic infectious disease prediction and early warning models are the susceptible-infectious-recovered (sir), susceptible-infectious-susceptible (sis) and susceptible-infectious-recoverd-susceptible (sirs) models. the sir model is used to predict diseases in which individuals can obtain permanent immunity after infection. the sis model is used for diseases in which the individual is not immune after the infection is cured. the sirs model is used for diseases in which individuals can acquire immunity for a certain period of time after infection. however, these common predictive models are only applicable when there is a non-drug prevention intervention. the estimated numbers of infected people far exceed reported cases in the available literature which used these models virologica sinica (anastassopoulou et al. 2020; peng et al. 2020) . so it is necessary to improve these traditional models according to the actual situations. joseph et al. used the susceptible-exposed-infectiousrecovered (seir) model to simulate the wuhan city epidemic since it was detected in december, 2019 (wu jt et al. 2020a) . they estimated that 75,815 individuals (95% ci: 37 304-130,330) individuals had been infected in wuhan city as of january 25, 2020. and then the seir model was extended into a seir-metapopulation model to simulate the spread of sars-cov-2 across mainland china. given that public health professionals and the general public have realized the threats caused by virus, the transmissibility of the epidemic might be reduced compared with its nascent stage at wuhan city. so they assumed that the transmissibility of sars-cov-2 was reduced by 0%, 25%, and 50% after wuhan city was quarantined on january 23, 2020 and the rate of transmissibility was similar across all cities. the estimated result was that if there was no reduction in transmissibility, the wuhan city epidemic would peak around april of 2020, and local epidemics across cities in mainland china would lag by 1-2 weeks. a 50% reduction in transmissibility would push the r 0 to 1.3, in which case the epidemic would grow slowly without peaking during the first half of 2020. these analytical data gave us an early warning that sars-cov-2 could be about to become an epidemic in the absence of mitigation on its present trajectory. the sir structure was modified based on the unique characteristics of the outbreak of sars-cov-2. in the modified sir model, recovered population r was extended to include those cured, died, and isolated in hospitals because under those conditions the virus does not spread . this model was used to predict the trend under three possible scenarios, such as the current trend maintained, control efforts expanded, and person-to person contact increased due to work resuming. by march 12, 2020, the total infectious estimates in each of the three scenarios are 72,172, 54,348, and 149,774. the model also predicted that the probability of no new cases in other cities at the end of february is 95.4%, 100%, and 75.7%, under these three scenarios respectively. the simulation suggested that strict quarantine of inner or inter-city population movement would have a significant effect on the suppression of virus spreading. the severe measures such as extending holidays and limiting group activities played an important role in stopping the spread of the epidemic. significant results have been accompanied by high social costs. therefore, in order to plan for the gradual recovery of social production activities after the epidemic is under control, it is essential to scientifically predict the future development of the epidemic. in epidemiological studies, the moving average method is mostly used to provide early warning of infectious diseases (petukhova et al. 2018) . the main idea is to calculate based on the data of infectious disease incidence over a long period of time and then establish an early warning line. by comparing the incidence data of a certain period with the early warning line, the epidemic trend of the disease can be judged and the risk of incidence can be estimated. however, this method can only be applied to the outbreak warning of existing infectious diseases. to substitute for it, a moving average prediction limit (mapl) method was proposed, its standard deviation can be used to establish a prediction limit to predict the epidemic trend, so as to timely predict the epidemic risk grading in the affected area. the data has shown that the number of new cases in the whole country has been moving towards a faster decline zone in the 11 days after february 12, 2020 outside hubei province, and stabilized near the prediction limit of the faster decline zone in the 5 days up to february 23, which is a low risk level. it indicates that after the nationwide anti-epidemic campaign, the epidemic situation in the country has been reduced as of the end of february. the overall trend of new suspected cases is consistent with the number of new confirmed cases. it is speculated that as long as the previous effective measures for outbreak prevention and control are adhered to, there is a great hope to keep the outbreak at a low risk level, and the risk of the epidemic in hubei province in the future will be less serious. there is no doubt that understanding the real-time risk levels of the epidemic has a great guiding role in planning measures to gradually lift restrictions and restore normal economic production and social life. the term case fatality rate (cfr) is widely used to describe the proportion of infected people who eventually die from a disease caused by pathogens. assessing cfr can help understand the severity of infection and anticipate the likely number of deaths by the end of the epidemic. sars-cov-2 is the virus that can cause death in infected people. jung et al. (2020) estimated the risk of death from confirmed cases (ccfr), while using data from confirmed cases outside mainland china and a right-censored likelihood to model the number of deceased cases and process the determination bias. the estimated ccfr value was 5.3% when the index case onset date was fixed on december 8, 2019 and 8.4% when epidemic exponential growth occurred to fit the data with other model parameters. although the ccfr of covid-19 is not high compared to the 17% cfr of sars in hong kong ghani et al. 2005) and 20% cfr of mers in south korea (mizumoto et al. 2015) , the 5%-8% risk of death is by no means negligible given the overall scale of the ongoing epidemic. however, the ccfr estimation models also have limitations, such as the ccfr only addressed fatality among confirmed cases. more precise infection fatality risk (ifr) estimates including infected individuals other than confirmed cases. in addition to quantifying the overall risk of death, future studies should identify groups at risk of death such as the elderly and potential comorbidities. at present, in the context of global economic integration, countries around the world are getting close. on the one hand, human beings enjoy the convenience brought by economic development, and on the other hand, they bear the survival challenges brought by ecological destruction. with the continuous destruction of the environment and growth of population, human beings have been frequently attacked by infectious diseases. the covid-19 epidemic caused much suffering, significant mortality, great disruption to social and work activities and considerable economic losses. to mitigate the spread of the virus, the chinese government has progressively implemented a metropolitan-wide quarantine of wuhan city and several nearby cities since january 23, 2020. numerous domestic airports and train stations, as well as international airports, have adopted temperature screening measures to detect individuals with fevers. strict control measures are being implemented in densely populated and remote areas, including extended holidays, cancellation of crowd gatherings, calls for home isolation, and so on (xiong and yan 2020) . fortunately, with the efforts of the chinese government and people, the outbreak has been brought under control relatively quickly. the outbreak has not only caused huge losses, but also reminded us of the need to strengthen public health surveillance and management. at the micro level, it is important to find the agent that is the cause of observed morbidity and mortality (gardy and loman 2018) . when used with phylogenetic analysis, it is possible to find the natural reservoir and intermediate host of the virus, which can help us isolate the source of the infection accurately. furthermore, codon usage bias analysis provides us with another method and perspective to trace the source of the virus, although the results are still open to question. at the macro level, to solve the surveillance and detection problem, the next set of issues concern data capture, the development of diagnostic tests and treatment algorithms and the identification of public health measures to control the epidemic spread. real-time data capture and associated analysis to reveal how the epidemic is expanding and how interventions are acting to slow its spread is essential. based on the mathematical frameworks, we can better assess the infectious capacity of pathogens and the effects of public health control measures. but the existing prediction models still have great limitations. first of all, the establishment of the model cannot be separated from the support of a large amount of data. in other words, the key to establish a reliable model is to track the epidemic dynamics and release the clinical information and epidemiological data in a timely manner. however, official data is often uncertain because medical resources are limited. the available data only reports confirmed cases in hospitals and ignores infected people who do not have access to medical services. this makes it difficult to accurately predict the development of the epidemic. secondly, we need to make it clear that fitting all factors into a model is not easy and models are often built on strong assumptions so the data estimated by the model may be biased in some cases. in this case, simple public health measures are unlikely to be effective, and other measures are needed, such as tighter restrictions on movement, greater availability of antivirals drugs and expanded vaccine development and production facilities. these questions suggest that there is still much room for improvement in rapid risk assessment and accurate early warning of emerging infectious diseases. it is clearly in everyone's interest to greatly enhance global surveillance capabilities, especially in developing regions, and concomitantly to improve basic training in infectious disease and molecular epidemiology. a clustering 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modeling of its spike protein for risk of human transmission simple framework for real-time forecast in a data-limited situation: the zika virus (zikv) outbreaks in brazil from 2015 to 2016 as an example estimating the unreported number of novel coronavirus (2019-ncov) cases in china in the first half of january 2020: a data-driven modelling analysis of the early outbreak discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin the application of time series analysis in predicting the influenza incidence and early warning. zhonghua yu fang yi xue za zhi spatially explicit modeling of 2019-ncov epidemic trend based on mobile phone data in mainland china acknowledgements jl is supported by grants from the national science and technology major project (2018zx10101004) and the youth innovation promotion association of cas (2019091). key: cord-014938-7evmiuv5 authors: wei-ming, yan; jia-quan, huang; xiao-ping, luo; qin, ning title: expression of prothrombinase/fibroleukin gene fg12 in lung impairment in a murine severe acute respiratory syndrome model date: 2008-01-13 journal: virol sin doi: 10.1007/s12250-007-0020-5 sha: doc_id: 14938 cord_uid: 7evmiuv5 to evaluate the role of murine fibrinogen like protein 2 (mfgl2) /fibroleukin in lung impairment in severe acute respiratory syndrome (sars), a murine sars model induced by murine hepatitis virus strain 3 (mhv-3) through trachea was established. impressively, all the animals developed interstitial pneumonia with extensive hyaline membranes formation within alveoli, and presence of micro-vascular thrombosis in the pulmonary vessels. mhv-3 nucleocapsid gene transcripts were identified in multiple organs including lungs, spleen etc. as a representative proinflammatory gene, mfgl2 prothrombinase expression was evident in terminal and respiratory bronchioles, alveolar epithelia and infiltrated cells in the lungs associated with fibrin deposition and micro-vascular thrombosis. in summary, the established murine sars model could mimic the pathologic characteristics of lungs in patients with sars. besides the physical damages due to virus replication in organs, the up-regulation of novel gene mfgl2 in lungs may play a vital role in the development of sars associated lung damage. . all animal studies were carried out according to the animals were acclimatized to laboratory conditions for 1 week before experiments and all procedures that involved animals were conducted according to the hospital guidelines. culture collection (atcc) and plaque purified on monolayers of dbt cells and titred on l2 cells using a standard plaque assay (12, 16) . to setup the murine sars model twenty balb/cj mice were inoculated with 100pfu of mhv-3 through trachea. another twenty balb/cj mice injected with sterilized saline through trachea in the same volume were set up as a control. animal survival rate was then observed. additionally, 10 balb/cj mice were injected with sterilized saline as a control. in a separate experiment, tissues including lungs, spleen, liver, kidneys, intestine, heart, brain were collected at a series of time points from balb/cj mice infected with mhv-3 through trachea. the samples were snap freezed and kept at a -80 freezer or fixed with 10% formalin for further experiments. the tissues that had been fixed with 10% formalin were dehydrated in graded alcohol solutions and xylene and embedded in paraffin. 4-m sections were cut and tissues were then stained with hematoxylin and eosin (he). approximate 40mg of each frozen organ was homogenized in 0.1 mol/l pbs (ph7.4), and the supernatants were subjected to titer analysis on l2 cells by a standard plaque assay as described elsewhere (12, 16) . the titration examination in each organ was repeated for three times in duplicate. quantitative data were reported as the means standard deviation for at least three separate experiments. differences of virus titers between organs at 48h following mhv-3 infection were analyzed by using one-way analysis of variance, and a p value of less than 0.05 was considered statistically significant. balb/cj mice inoculated with mhv-3 through trac in a separate set of experiment, tissues including lungs, spleen, liver, kidneys, intestine, heart and brain from balb/cj mice infected with mhv-3 through trachea were examined as described above. various histopathological changes were found in these organs (shown in fig.2 intestine, heart and brain: no significant pathological changes were found in these organs ( fig.3 . d4, e4, f4). to determine viral titers, tissues were homogenized and the supernatants were subjected to titer determination on l2 cells as described previously (12, 16) . virus plaques were found in all collected organs and increased with the progress of the infection widespread fibrin deposition in association with the mfgl2 expression, especially in microvasculars (fig.7 . a) and hyaline-membranes ( fig.7 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syndrome in patients suffering from sars. chin critica1 care med severe acute respiratory syndrome and its lesions in digestive system effect of sars-associated coronavirus on peripheral blood picture and liver function. chin critica1 care med immune responses against sars-coronavirus nucleocapsid protein induced by dna vaccine key: cord-298734-h286m32c authors: xia, siyu; wu, ming; chen, si; zhang, tao; ye, lina; liu, jun; li, hui title: long term culture of human kidney proximal tubule epithelial cells maintains lineage functions and serves as an ex vivo model for coronavirus associated kidney injury date: 2020-06-29 journal: virol sin doi: 10.1007/s12250-020-00253-y sha: doc_id: 298734 cord_uid: h286m32c the mechanism of how sars-cov-2 causes severe multi-organ failure is largely unknown. acute kidney injury (aki) is one of the frequent organ damage in severe covid-19 patients. previous studies have shown that human renal tubule cells could be the potential host cells targeted by sars-cov-2. traditional cancer cell lines or immortalized cell lines are genetically and phenotypically different from host cells. animal models are widely used, but often fail to reflect a physiological and pathogenic status because of species tropisms. there is an unmet need for normal human epithelial cells for disease modeling. in this study, we successfully established long term cultures of normal human kidney proximal tubule epithelial cells (kptecs) in 2d and 3d culture systems using conditional reprogramming (cr) and organoids techniques. these cells had the ability to differentiate and repair dna damage, and showed no transforming property. importantly, the cr kptecs maintained lineage function with expression of specific transporters (slc34a3 and cubilin). they also expressed angiotensin-converting enzyme 2 (ace2), a receptor for sars-cov and sars-cov-2. in contrast, cancer cell line did not express endogenous slc34a3, cubilin and ace2. very interestingly, ace2 expression was around twofold higher in 3d organoids culture compared to that in 2d cr culture condition. pseudovirion assays demonstrated that sars-cov spike (s) protein was able to enter cr cells with luciferase reporter. this integrated 2d cr and 3d organoid cultures provide a physiological ex vivo model to study kidney functions, innate immune response of kidney cells to viruses, and a novel platform for drug discovery and safety evaluation. electronic supplementary material: the online version of this article (10.1007/s12250-020-00253-y) contains supplementary material, which is available to authorized users. in december 2019, sars-cov-2, a novel coronavirus zhou p et al. 2020) , caused an outbreak of acute pneumonia in wuhan, china singhal 2020; ye et al. 2020; zhou f et al. 2020) . the world health organization (who) declared sars-cov-2 infection outbreak as a ''public health emergency of international concern'' on january 30, 2020. later on march 11, who defined covid-19 as a pandemic (who 2020a). until june 11, it has been reported globally 7,273,958 covid-19 cases and 413,372 deaths in 215 countries (who 2020b) . the complete clinical picture of covid-19 is only partially understood. covid-19 cases have ranged from very mild (including asymptomatic), mild, moderate to severe and critical severe including illness resulting in death. covid-19 common siyu xia and ming wu contributed equally to this work electronic supplementary material the online version of this article (https://doi.org/10.1007/s12250-020-00253-y) contains supplementary material, which is available to authorized users. symptoms include fever, cough and shortness of breath. muscle pain, sputum production and sore throat are less common. while the majority of the cases show mild symptoms, some progress to severe pneumonia and multiorgan failure including lung, heart, and kidney. the rate of deaths per number of diagnosed cases is estimated to be 3.4% but varies by age and other health conditions singhal 2020; wu and mcgoogan 2020; , yang x et al.2020 ye et al. 2020; , zhang h et al.2020 zhou c et al. 2020 , zhou f et al. 2020 zou l et al. 2020) . the mechanisms of how sars-cov-2 cause severe multiorgan failure are largely unknown. acute kidney injury (aki) is the second leading frequent organ damage in coronavirus infected cases, which goes just after respiratory system injuries (chafekar and fielding 2018) . the epidemiological and clinical studies showed that 3%-19% covid-19 cases were reported with aki li et al. 2020) . the latest study also reported the detection of viral rna in urine samples of covid-19 patients . however, whether aki of covid-19 patients are caused by coronavirus-induced cytopathic effect or immunopathogenic damage remains unclear. previous studies have shown that sars-cov-2 enters cell through its receptor, angiotensin converting enzyme 2 (ace2) (zhou p et al. 2020 ). single-cell rna sequencing data analysis showed that ace2 is expressed in kidney proximal convoluted tubules pan et al. 2020) . these data indicated that renal tubule cells could be the potential host cells targeted by sars-cov-2. due to the lack of specific detection of ace2 mrna and protein expression in human kidney tubule cells, it is hard to confirm the direct infection of sars-cov-2. since expression of ace2 may play roles in the regulation of renal function, it's of interest to uncover the mechanisms by which sars-cov-2 infection damages kidney function. animal models for study of sars-covs include african green monkeys, macaques and mice (kong et al. 2009; smits et al. 2011) . however, there are some limitations because of species difference. the viral tropism correlates to expression of the viral receptor of host cells and intracellular restriction as well. for example, human influenza viruses preferentially bind to a2-6-gal linked sialic acids, while avian influenza viruses use a2-3-gal linked sialic acids as receptors (guo et al. 2009; chan et al. 2013) . human kidney proximal tubule epithelial cells (kptecs) are important for many functions of human kidney, such as clearing blood and resorption of essential end-products from metabolism, including protein, sodium, etc. (smith et al. 2006) . these cells are highly useful for studying basic cell biology and modeling kidney diseases (phillips 2003) . however, primary kptecs in vitro undergo a very limited number of population doublings (pds), thus it would be difficult to obtain reproducible results due to differences of primary cells. previous studies have been focused on the immortalization of kptecs using viral oncogenes hpv16 e6/e7, or a hybrid adeno-12-sv40 virus, or sv40 and htert (ryan et al. 1994; racusen et al. 1997; kowolik et al. 2004; orosz et al. 2004; wieser et al. 2008 ). however, due to genetic manipulation, these immortalized cells and traditional cancer cell lines largely loss differentiation capacity and normal physiological function. there is an unmet need for normal epithelial cells for disease modeling. recently, conditional reprogramming (cr) allows for indefinite proliferation of human epithelial cells with no transduced viral or cellular genes . cr cells (crcs) maintain differentiation potential and lineage function in vitro suprynowicz et al. 2012; zhu et al. 2017) . in this study, we firstly established long term cell cultures of kptecs using 2d cr and 3d organoids technologies, which maintained the lineage function, and the ability to differentiate and repair dna damage. they also did not have transforming property as cancer cells did. importantly, these cells expressed endogenous specific transporters slc34a3 and cubilin, and ace2. the latter is a receptor for entry of sars-cov and sars-cov-2. in contrast, cancer cell line did not express these proteins endogenously. very interestingly, ace2 expression was around twofold higher in 3d organoids compared to that in 2d cr condition. this integrated 2d cr and 3d organoid cultures provide a novel ex vivo model to study kidney functions, innate immune response of kidney cells to viruses, and a novel platform for drug discovery and safety evaluation. cryopreserved primary kptecs were purchased from lonza (catalog #: cc-2553). cells were cultured in cr condition on irradiated 3t3-j2 fibroblasts as described previously and passaged in dulbecco's modified eagle medium (dmem)/f12 containing 5 lmol/l y-27632 (yongtech, shenzhen). cr kptecs were trypsinized in two steps by using 0.05% trypsin-edta. the initial 30 s trypsinization to remove feeders was followed by a wash of pbs and another 3 min trypsinization to detached cells. cells were subsequently reseeded at a 1:4-6 ratio in the cr condition and cultured for 3-4 days before passaging. kptec-crc cultures were established in matrigel organoids 3d condition as previously described saenz et al. 2014) . briefly, 40 ll of cold matrigel (bd bioscience cat #354230) was added to the surface of sterile pre-chilled, slide chambers (lab-tek ii chamber slide, thermofisher scientific inc.). the slides were then incubated at 37°c for 15 min to allow polymerization of the substratum. subsequently, 3 9 10 4 cells/ml were added to the slides and resuspended in assay media (yongtech, shenzhen) containing 2% fbs, 2% matrigel, and 20 ng/ml egf and incubated at 37°c until 3d organoid formation. 0.5 lg/ml dapi (d3571) was used to stain the nuclei. bright field microscopy, confocal microscopy, immunostaining were carried out as described previously saenz et al. 2014 ). cells were grown on sterile glass cover slips and treated with 5 gy of gamma irradiation and fixed in 4% (w/v) paraformaldehyde at indicated time points, permeabilized with 0.1% saponin in pbs containing 0.2% gelatin, and labeled with the primary (mouse anti-p63, santa cruz, sc-863; goat polyclonal ace2 antibody, r&d systems, abingdon, uk) and secondary (alexa fluor 488 donkey anti-mouse igg, anti-goat alexa fluor 488, molecular probe) antibodies, according to the manufacturer's protocol. 0.5 lg/ml dapi (d3571) was used to stain the nuclei dna. a zeiss axioskop microscope (carl zeiss, inc., thornwood, ny) equipped with a 9 63 objective lens and a hammamutsu charge-coupled device camera was used to image the cells. images were processed using openlab 3.0.7 software. anchorage-independent growth assays with 4 9 10 4 cells in 0.3% low-melting point agarose were performed according to the previous study (feng et al. 2015) . colony images were captured and analyzed with the evos flat screen microscope (life technologies). total cellular rnas were extracted using trizol reagent (thermofisher) and real time rt-pcr was performed as described previously . the primer sequences for detection of ace2 mrna were 5 0 -cattggagcaagtgttggatctt-0 3 and 5 0 -gagc-taatg-catgccattctca-3 0 ; for slc34a3 were 5 0 -cgcaggcgcccgacatcc-3 0 and 5 0 -acctgtttgcg ggcacggagctca-3 0 ; for human cubilin were 5 0 -gc tcatccaggctcccgactctac-3 0 and 5 0 -ttgaag cctgccctggttacactg-3 0 (wieser et al. 2008) . b-actin was used as an internal control (5'-gagcaca gagcctcgccttt-3', 5'-tcatcatccatggtgag ctgg). a total of 5 9 10 3 hela cells in complete dmem or cr pktecs were seeded into 96-well plate. hek293t cells were co-transfected with pspike (sars-cov s), pspax, and lenti-luc for 4 h and pseudovirions were collected. hela cells or cr pktecs were rinsed with pbs and infected with pseudovirions (simmons et al. 2004; wang et al. 2008) . cells were lysated with cell culture lysis reagent and the luciferase activity was read using luciferase assay system from promega (e1500). relative luciferase units (rlu) were plotted by using cell lysates without pseudovirion infections as negative controls. publicly usable online rna sequencing datasets of total rna from 20 human tissues reported in srp056969 were used to analyze the level of ace2 expression. normalized expression level rpkm (reads per kilobase per million reads) and raw counts were available directly online. single cell rna sequencing (scrna-seq) dataset for kidney was retrieved from www.kidneycellatlas.org or a special website portal (www.covid19cellatlas.org) (stewart et al. 2019) . we collected online rna sequencing datasets and analyzed the level of ace2 expression in different organ tissues. as shown in supplementary figure s1 , kidney tissue expresses the highest level of ace2 mrna among the 20 organs reported in srp056969 dataset, and ace2 mrna was predominantly expressed in proximal tubule cells (supplementary figure s2 ). next, we tested whether cr technique could propagate normal kidney epithelial cells. cr technique has been used to efficiently establish long term cell cultures from variety of tissue types without gene manipulation . as far as we know, there is no report on long term culture of human normal kidney cells. as shown in fig. 1 , typical kptecs colonies were observed under cr condition (co-cultured with irradiated j2 fibroblast feeder cells in the presence of the rock inhibitor y-27632). kptecs were observed in 1 day after initial culture, and proliferated rapidly to reach 80%-90% confluence in 4-5 days. kptecs maintained a normal morphology in the cr system for [ 50 passages after one year in culture. the typical cobblestone-like morphology was shown (fig. 1) . when grown in commercial serumfree medium, cr kptecs senesced after 3-4 passages (data not shown). the differentiation potential of normal cells is important for their physiological function. normal epithelial cells could be able to maintain their differentiation potential under in vitro and in vivo differentiation conditions, while transformed or malignant cells usually loss their ability to differentiate to functional cells. previous studies already demonstrated that cr cells from airway, prostate, breast, cervical and skin tissues were able to form well differentiated structures under in vitro organoids or in vivo renal capsule experiments (suprynowicz et al. 2012; liu et al. 2017) . matrigel basement membrane matrix is an important regulatory factor to maintain homeostasis of cells and tissue morphogenesis (lee et al. 2007 ). thus, we next investigated whether the later passage (p30) cr cells from normal kptecs were able to maintain the differentiation properties of normal cells when grown in 3d matrigel organoid cultures. as shown in fig. 2a , cr kptecs formed acinar structures with a well-defined cell/matrigel interface and showed polarized periphery of the colony in matrigel organoids. anchorage-independent growth is a hallmark of transformed or malignant cells. the transformed or malignant cells can form colonies when cultured in soft agar, while normal cells are not able to grow under this stringent culture condition. thus, we tested whether our cr kptecs were able to form colonies in soft agar. as shown in fig. 2b , cr kptecs did not formed colonies in soft agar, indicating that they had no anchorage-independent growth properties as cancer cell line did. it is of special interest to further test if cr kptecs retain characteristic of original normal tissue at the molecular level. for that purpose, we used immunofluorescence to visualize phosphorylated h2ax foci in cr kptecs at various times after irradiation (2 h and 24 h). foci are believed to represent the sites of dna double-strand break (dsb) and h2ax phosphorylation are the early events in dna damage response and repair (ddr). after dsbs repair, foci resolution occurs by dephosphorylation of the p-h2ax protein, while persistence of the foci at late time points is thought to represent residual unrepaired dsb. by comparing the formation and resolution of the foci in the cells at the different time points after irradiation, we can determine the dsb repair kinetics and the status of the ddr response. we analyzed the time-dependent change of gamma irradiation induced-h2ax foci in cr kptecs. the results showed that the ddr response occurred in cr kptecs at 2 h after irradiation (fig. 2c) . however, almost undetectable fraction of the initial foci persist after 24 h were observed, suggesting that the cells had the ability to repair dna damage. since transport functions are unique property of kidney tubule cells, we tested the expression of kptecs specific sodium-dependent phosphate transporters in cr kptecs. we performed rt-pcr for detection of slc34a3 mrna. our results demonstrated that cr kptecs expressed slc34a3, while hela cells did not express slc34a3 as a negative control (fig. 3a) . another specific transport system expressed in kptecs is the megalin/cubilin multiligand receptor, which plays an essential role in endocytotic uptake of filtered proteins by the proximal tubule in kidney. we observed similar pattern for expression of cubilin in cr kptecs using rt-pcr (fig. 3a) . these results suggested that cr kptecs maintain lineage function with expression of specific functional genes in the proximal tubules. as we described above, some cases with covid-19 progress to severe pneumonia and multi-organ failure including kidney. to study whether our long term culture system can be used for studies of sars-cov-2 and covid-19, we performed immunofluoresence and rt-pcr to test the expression of ace2 in 2d and 3d cultures. the ace2 mrna and protein were detectable in both 2d and 3d cultures ( figs. 2a, 3b, 3c) . interestingly, our rt-pcr demonstrated expression of endogenous ace2 in cr kptecs, not in hela cells (fig. 3c) . we also observed a relative higher (around twofold) expression of ace2 in 3d organoids compared to that in 2d cr conditions (fig. 3c) . finally, we generated pseudovirions in hek293t cells transfected with sars-cov s plasmid, pspax, and lenti-luc. cr kptecs and hela cells were infected by pseudovirions to investigate whether these cells could be infected by sars-cov pseudovirions. as shown in fig. 4 , cr kptecs infected with pseudovirions showed rlus of 6890 ± 490, while cr kptecs without pseudovirion infection had rlus of only 28 ± 8. rlus were below 100 in hela cells with or without sars-cov s pseudovirion infection. thus, cr kptecs were permissive for sars-cov infection. these data suggested that our integrated 2d cr and 3d organoids would be an appropriate physiological human cell system for study of srar-cov and sars-cov-2 and their associated kidney injuries, especially for virus entry to kptecs, innate immune response of host cells, drug screening for anti-sars-cov-2 and safety evaluation of kidney functions. in this study, we firstly established long term cultures of human kptecs using cr technology. these cr cells maintained their differentiation potential and the ability to repair dna damage induced by irradiation. these cells also expressed lineage functional markers of kidney proximal tubule epithelial cells, including slc34a3 and megalin/cubilin. these data suggested that cr kptecs would be used as an ex vivo model for studies of kidney diseases or kidney injury associated with other systemic diseases (e.g., diabetes), and discovery of novel biomarkers and targets. as we discussed above, mortality of severe patients with covid-19 are relative high due to preexisting conditions and multi-organ failure wu and mcgoogan 2020; yao et al. 2020; ye et al. 2020; zhang h et al. 2020) . as sars-cov-2 enter host cells via ace2 protein receptor, it is highly possible that sars-cov-2 infect all ace2 expressing cell types. it was reported that ace2 is highly expressed in intestine, testis, kidney, heart, etc., and this might help to explain multiorgan injury (zou x et al. 2020) . in particular, a number of studies have reported kidney injury in severe covid-19 cases li et al. 2020) . furthermore, (ling et al. 2020; rothe et al. 2020; xie et al. 2020; young et al. 2020) , indicating the possible replication of sars-cov-2 in urological system, especially in kidney and bladder. thus, it would be important to study the possibility of sars-cov-2 infection with urological system. in terms of the questions above, we need model systems to study infection of sars-covs in ace2 expressing cell types, especially in kidney epithelial cells (hamming et al. 2004; zou x et al. 2020) . transgenic mice with human ace2 (yang et al. 2007; netland et al. 2008 ) would be used for modeling disease in vivo, however, it's hard to mimic human diseases due to species-related restriction conditions. animal models are usually very expensive and need much longer time to complete, thus they cannot be used for any high throughput screening. another option is human cell lines. cancer cell lines usually gain the ability to proliferate in very stringent conditions, such as soft agar, and they loss their specific differentiation potential. thus, those cancer cell lines are not suitable to study ''normal'' or ''physiological'' response of host cells to viral infections, although viruses may lead lytic infections in some cancer cell lines. therefore, much efforts have been made for generating immortalized cell lines (ryan et al. 1994; racusen et al. 1997; kowolik et al. 2004; orosz et al. 2004; wieser et al. 2008 ). as we described above, hpv16 e6/e7, sv40, and htert have been widely used for immortalization of human primary cells (ryan et al. 1994; racusen et al. 1997; kowolik et al. 2004; orosz et al. 2004; wieser et al. 2008) . these cell lines have been widely used in many study fields, including cell biology, production of research reagents and modeling human diseases. however, due to pathways altered by viral or cellular genes or integration of viral/cellular gene to host genome, these immortalized cell lines cannot reflect the physiological status of normal human cells. a related study reported the susceptibilities of a panel of cell lines to sars-cov (kaye 2006) . among the 21 cell lines, including several a expression of endogenous slc34a3 and cubilin. expression of two transporters was measured using quantitative rt-pcr. b-actin was used as an internal control. both slc34a3 and cubilin were expressed in cr kptecs, but not in hela cells. expression of endogenous ace2 in cr kptecs was measured using if (b) and quantitative rt-pcr (c). scale bar, 10 lm. virologica sinica cell lines from monkey and other species, only three human cell lines (hek293, hep g2 and huh-7) are susceptible (kaye 2006) . besides, sars-cov can achieve high viral load in some cell lines without specific cpe (cytopathic effect) (kaye 2006) . however, none of these cell lines retain physiological functions. a recent study demonstrated that active sars-cov-2 replication was detected in tissues of the upper respiratory tract, especially virus load and shedding were very high during the first week of symptoms ). this suggests an urgent need to establish normal airway cells model from upper (nasal cavity) to lower (lung) respiratory tract to study interactions between sars-cov-2 and host epithelial cells. cr has been widely used for generation of various patient-derived cell models that can be used for modeling human disease, drug discovery and tissue regeneration (borodovsky et al. 2017; wolf et al. 2017; yuan et al. 2017; alkhilaiwi et al. 2018; brewington et al. 2018; jin et al. 2018; moorefield et al. 2018; peters-hall et al. 2018; sayej et al. 2018; wang et al. 2018; zhang et al. 2018; alkhilaiwi et al. 2019; nicolas et al. 2019; palechor-ceron et al. 2019; wan 2019; wang et al. 2019; chai et al. 2020; krawczyk et al. 2020; peters-hall et al. 2020; ). in the current study, we established long term cell cultures of kptecs using cr technology. our cr kptecs maintain lineage differentiation potential and express cell type-specific function markers and ace2 protein. these cells can serve as a physiological cell model for the study of sars-covs and kidney functions, innate immune response of kidney cells to viruses and a novel platform for drug discovery and safety evaluation. long-term expansion of primary equine keratinocytes that maintain the 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adult inpatients with covid-19 in wuhan, china: a retrospective cohort study ex vivo 2d and 3d hsv-2 infection model using human normal vaginal epithelial cells sars-cov-2 viral load in upper respiratory specimens of infected patients single-cell rnaseq data analysis on the receptor ace2 expression reveals the potential risk of different human organs vulnerable to 2019-ncov infection acknowledgements this work was supported by the national natural science foundation of china (81571396 and 81771528); science, technology and innovation commission of shenzhen municipality (jcyj20170411090932146, jcyj20170818110544730). key: cord-295640-mhfu0e9r authors: wang, wenling; huang, baoying; wang, xiuping; tan, wenjie; ruan, li title: improving cross-protection against influenza virus using recombinant vaccinia vaccine expressing np and m2 ectodomain tandem repeats date: 2019-06-25 journal: virol sin doi: 10.1007/s12250-019-00138-9 sha: doc_id: 295640 cord_uid: mhfu0e9r conventional influenza vaccines need to be designed and manufactured yearly. however, they occasionally provide poor protection owing to antigenic mismatch. hence, there is an urgent need to develop universal vaccines against influenza virus. using nucleoprotein (np) and extracellular domain of matrix protein 2 (m2e) genes from the influenza a virus a/beijing/30/95 (h3n2), we constructed four recombinant vaccinia virus-based influenza vaccines carrying np fused with one or four copies of m2e genes in different orders. the recombinant vaccinia viruses were used to immunize balb/c mice. humoral and cellular responses were measured, and then the immunized mice were challenged with the influenza a virus a/puerto rico/8/34 (pr8). np-specific humoral response was elicited in mice immunized with recombinant vaccinia viruses carrying full-length np, while robust m2e-specific humoral response was elicited only in the mice immunized with recombinant vaccinia viruses carrying multiple copies of m2e. all recombinant viruses elicited npand m2e-specific cellular immune responses in mice. only immunization with rvj-4m2enp induced remarkably higher levels of il-2 and il-10 cytokines specific to m2e. furthermore, rvj-4m2enp immunization provided the highest cross-protection in mice challenged with 20 mld(50) of pr8. therefore, the cross-protection potentially correlates with both np and m2e-specific humoral and cellular immune responses induced by rvj-4m2enp, which expresses a fusion antigen of full-length np preceded by four m2e repeats. these results suggest that the rational fusion of np and multiple m2e antigens is critical toward inducing protective immune responses, and the 4m2enp fusion antigen may be employed to develop a universal influenza vaccine. electronic supplementary material: the online version of this article (10.1007/s12250-019-00138-9) contains supplementary material, which is available to authorized users. seasonal influenza is an acute respiratory infectious disease that can cause serious health problems. seasonal influenza epidemics caused by influenza a and b viruses result in 3-5 million severe cases and 290,000-650,000 deaths worldwide; 5%-10% of adults and 20%-30% of children suffer from flu annually (world health organization, who 2019). seasonal influenza viruses undergo antigenic drift, which makes the conventional seasonal vaccines ineffective when the vaccine strains mismatch with the epidemic strains. therefore, seasonal influenza vaccines must be updated regularly for effective prevention of influenza. the production time for conventional influenza vaccines can be lengthy. it takes 1-2 months to obtain vaccine strains using conventional reassortment technology or reverse genetics technology, followed by several more months for large-scale expansion of influenza vaccine using an ample supply of specific pathogen-free (spf) chicken embryos. generally, influenza vaccine production takes 3-6 months from the prediction of the epidemic strain to the ultimate production of the vaccine (emanuel and wertheimer 2006) . for more effective prevention of influenza, new vaccines are warranted to induce crossprotection and long-lasting immune responses (erbelding electronic (np) and extracellular domain of matrix protein 2 (m2e) can induce cross-protection against influenza a virus, showing promise as candidate antigens for the development of a broad-spectrum influenza vaccine (erbelding et al. 2018; zhang et al. 2015; kui et al. 2015) . previously, we expressed a fusion protein of np and m2e (nm2e) in escherichia coli and showed that immunization with nm2e formulated with aluminum hydroxide gel protected mice from a lethal challenge with heterologous influenza virus . therefore, vaccination with recombinant nm2e fusion protein is a promising strategy for the development of a universal influenza vaccine. however, a new expression system is required to further optimize the np-and m2e-based vaccine. the vaccinia virus (tiantan strain) was developed in china as a vaccination agent against smallpox, as documented by who (fenner et al. 1988 ). this strain was used to inoculate several individuals for *50 years, playing a key role in the eradication of smallpox in china (ruan 2013) . the tiantan strain has favorable gene vector and vaccine characteristics, including a powerful multiplication capacity, wide host range, high capacity for insertion of a foreign gene, noncarcinogenicity, and induction of longlasting immunity. using the tk gene system of the tiantan strain, this vaccinia virus has been developed successfully as a viral vector for various vaccine applications (ruan 2013) , and severe acute respiratory syndrome coronavirus vaccines (yan et al. 2009 ). we have developed recombinant vaccinia viruses based on np, m1, m2, and pb1 of influenza a virus wang et al. 2007 wang et al. , 2015 . several of our np-based recombinant vaccinia viruses have induced a protective immune response in balb/c mice. to develop a vaccine with broader protection, we constructed recombinant vaccinia viruses expressing various combinations of np and m2e to exhibit their maximal antigenicity. balb/c mice were immunized with the recombinant viruses to measure np-and m2e-specific humoral and cellular immune responses as well as protective effect against lethal challenge with a heterologous influenza virus. materials b-gal (40-1a) monoclonal antibody (sc-65670) and np monoclonal antibody (5d8) (sc-80481) were purchased from santa cruz biotechnology (santa cruz, ca, usa). m2 monoclonal antibody (14c2) (ab5416) and alexa fluor ò 488-conjugated goat anti-mouse polyclonal antibodies were from abcam (cambridge, ma, usa). bd cytometric bead array (cba) mouse th1/th2/th17 cytokine kit was purchased from bd (franklin lakes, nj, usa). peptides np55-69 (rliqnsltiermvls; h-2drestricted th epitope), np147-155 (tyqrtralv; h-2drestricted ctl epitope), and m2e pooled peptides (peptides of residues 1-15, 5-19, and 9-23) were synthesized by beijing scilight biotechnology ltd. co. (beijing, china). vaccinia virus (tiantan strain) and influenza a virus a/puerto rico/8/34 (pr8) (h1n1) were used in this study. vaccinia virus (tiantan strain) was amplified and titrated in primary chicken embryo fibroblasts (cefs). the pr8 influenza virus was propagated in 9-day-old chicken embryos at 34°c for 48 h, and the allantoic fluid was then collected and stored at -70°c until use. mouse 50% lethal dose (mld 50 ) titer of the influenza virus pr8 was assessed in balb/c mice before conducting the challenge experiments. plasmid pjsc1175 contains two dna fragments (tkl and tkr) derived from vaccinia virus tiantan strain to facilitate homologous recombination with vaccinia virus in host cells. between the two homology arms, there are p11 late promoter of vaccinia virus to regulate the lacz gene and p7.5 early/late promoter of vaccinia virus to regulate the target genes. the pjsc1175 was linearized at a bamh i restriction enzyme site immediately downstream the p7.5 promoter and further treated with calf intestinal alkaline phosphatase. plasmid pet30a-nm2e contains the fusion gene of fulllength np (498 amino acids) and the succeeding m2e (23 amino acids) from influenza a virus, a/beijing/30/95 (h3n2) (bj95) . the np and m2e fusion gene (referred to as npm2e) was amplified from plasmid pet30a-nm2e by polymerase chain reaction (pcr) to introduce bamh i site at each end. the npm2e was digested by bamh i, and inserted into the linearized plasmid pjsc1175. the resulting plasmid carrying npm2e at the same orientation as the p7.5 promoter was designated as pjsc1175-npm2e. from plasmid pjsc1175-npm2e, m2e and np genes were amplified respectively, and fused (m2e preceding np) by pcr to generate a fusion gene, m2enp. dna fragment containing four copies of m2e (from bj95) was amplified from plasmid, pet9a-h4m2e, and fused to the 5 0 terminus of full-length np gene by pcr, resulting in fusion gene, 4m2enp. the dna fragment containing four copies of m2e was also fused to the 5 0 terminus of a np gene (from bj95) truncated at both ends (referred to as nps, encodes 314 amino acids) that was amplified from plasmid pet9a-npsm2e by pcr. the resulting fusion gene was named 4m2enps. as the fusion genes m2enp, 4m2enp, and 4m2enps were engineered with bamh i site at each end through pcr, they were cloned into linearized pjsc1175 as described above. the resulting plasmids were pjsc1175-m2enp, pjsc1175-4m2enp, and pjsc1175-4m2enps, respectively. recombinant vaccinia viruses were generated via homologous recombination, as described by wang et al. (2015) . briefly, cefs were infected with vaccinia virus tiantan strain at a multiplicity of infection of 0.01-0.1, followed by transfection with recombinant pjsc1175 plasmids containing fusion genes of np and m2e. subsequently, recombinant vaccinia viruses were screened by blue-white selection. viral dna was isolated from each recombinant vaccinia virus to confirm the presence of fusion genes (npm2e, m2enp, 4m2enp, and 4m2enps) by pcr and sequencing of pcr products. the confirmed recombinant vaccinia viruses were produced on a large scale in cefs. the control recombinant vaccinia virus rvj1175 was generated using the same method with the empty pjsc1175 vector. to confirm the expression of np and m2e by the recombinant vaccinia viruses using indirect immunofluorescence, a549 cells grown in 48-well culture plates were infected with each strain of virus at 30-40 plaque-forming unit (pfu)/well. at 48 h post infection, the cells were fixed with 4% paraformaldehyde, and the expression of b-gal, np and m2e was confirmed using mouse monoclonal antibody (mab) against b-gal (40-1a), mouse mab against influenza a virus m2 (14c2), and mouse mab against influenza a virus np (5d8). the signals were then visualized using alexa fluor ò 488-conjugated goat anti-mouse. spf female balb/c (5-6 weeks old) mice were purchased from beijing vital river laboratory animal technology co., ltd. (beijing, china). all mice were bred under spf conditions at the institute of laboratory animal science, chinese academy of medical science and peking union medical college, beijing, china. mice were randomly grouped (16 mice/group), inoculated intramuscularly with 1 9 10 7 pfu recombinant vaccinia viruses (rvj1175, rvj-npm2e, rvj-m2enp, rvj-4m2enp, or rvj-4m2enps) at week 0 and week 4 in the bilateral gastrocnemius without the use of reagents or equipment. ten days after the second immunization, three mice from each group were retro-orbital bled and sacrificed. the spleens were removed aseptically and ground through a 200-mesh sieve. spleen mononuclear cells (smncs) were obtained as described by wang et al. (2015) . the cellular immune responses induced in mice were evaluated by enzyme-linked immunospot (elispot) assay ) and the bd cba mouse th1/th2/th17 cytokine kit according to the manufacturer's instructions. peptides np55-69, np147-155, or m2e pooled peptides were used in the elispot and cba assays. the sera were separated by centrifugation, and serum igg titers were determined. two weeks after the second immunization, mice were anesthetized using sodium pentobarbital (10 mg/ml) at a dose of 60 mg/kg of body weight. subsequently, 10-12 mice in each group were challenged with 20 times of mouse 50% lethal dose (mld 50 ) of pr8 intranasally. weight loss and mortality were monitored daily for 3 weeks after the challenge. mice that lost 30% of their initial weight were euthanized and recorded as having died. statistical analysis was performed using spss (ver. 17.0; spss inc., chicago, il, usa) and prism (ver. 5.0a; graphpad inc., la jolla, ca, usa). log conversion was performed for antibody titers. differences in antibody titer, elispot, and cba results among groups were analyzed using one-way anova. the paired t test and log-rank test were used to analyze differences in weight loss curves and survival rate curves, respectively. differences with p values \ 0.05 were considered statistically significant. to generate fusion antigens with the highest potency for inducing cross-protection, we designed a variety of fusion antigen configurations by adjusting the fusion order of np and m2e, increasing the copy number of m2e (4m2e), and truncating the length of nps. the np and m2e gene fragments of influenza a virus a/beijing/30/95 (h3n2) were joined to create npm2e, m2enp, 4m2enp, and 4m2enps using pcr (fig. 1) . each amplified fusion gene fragment was cloned into plasmid pjsc1175 under the regulation of p7.5 early/late promoter. the resulting plasmids pjsc1175-npm2e, pjsc1175-m2enp, pjsc1175-4m2enp, and pjsc1175-4m2enps were confirmed by restriction endonuclease digestion and dna sequencing (data not shown). each confirmed plasmid was transfected into cefs infected by vaccinia virus tiantan strain to facilitate homologous dna recombination between plasmid and viral genomic dna. recombinant vaccinia viruses rvj-npm2e, rvj-m2enp, rvj-4m2enp, and rvj-4m2enps were screened by blue-white selection. the vaccinia virus dna was extracted to amplify the target gene fragments using pcr. the inserted target genes were confirmed by sequencing of the pcr products. a549 cells were infected with recombinant vaccinia viruses, and expression of the target proteins (np and m2e) was identified by immunofluorescence. as a selective marker, b-galactosidase (b-gal) was expressed at similar levels in a549 cells infected by all recombinant vaccinia viruses. except the control virus rvj1175, all other recombinant vaccinia virus expressed m2e and np in the infected cells. it seemed that rvj-4m2enps showed lower expression of truncated np than other recombinant vaccinia viruses carrying full-length np gene (fig. 2) . flow cytometry analysis of the target protein expression showed similar results, and western blot analysis confirmed target protein expression at the expected molecular weights (supplementary materials). to characterize the immunogenicity of fusion antigens expressed by the recombinant vaccinia viruses, five groups of balb/c mice were immunized with 10 7 pfu of recombinant vaccinia virus at week 0 and 4. ten days after the second immunization, sera were taken to measure the antibody titer, and the spleens were removed aseptically to measure the cellular immune response. mice immunized with the recombinant vaccinia virus rvj-npm2e and rvj-m2enp showed strong antibody responses against np, with lower titers of antibodies against m2e (fig. 3a) . rvj-4m2enp induced strong antibody responses against both np and m2e. while rvj-4m2enps only induced a strong humoral immune response against m2e but not np. elispot analysis (fig. 3b ) revealed that rvj-npm2e, rvj-m2enp, rvj-4m2enp, and rvj-4m2enps induced more np147-155-specific spot-forming cells (sfcs) than np55-69-specific sfcs. rvj-m2enp induced significantly more np147-155-specific sfcs than rvj-npm2e (p \ 0.05) and rvj-4m2enps (p \ 0.01). rvj-4m2enp also induced significantly more np147-155-specific sfcs than control virus, nevertheless there is no significant difference compared with rvj-m2enp. except the control virus, all other recombinant vaccinia virus seemed to induce m2e-specific cellular immune responses (fig. 3b) . however, only m2e-specific sfcs induced by rvj-m2enp are significantly more than those induced by control virus (p \ 0.01) and by rvj-npm2e (p \ 0.05). cytokine measurements (fig. 3c) showed that m2e pooled peptides stimulated smncs to secrete several cytokines including interleukin (il)-2, il-6, il-10, and tumor necrosis factor. however, only immunization with rvj-4m2enp induced significantly higher levels of il-2 and il-10 cytokines than that with control (rvj1175) when stimulation with the m2e pooled peptides. we assessed the protective efficacy of the recombinant vaccinia viruses in the balb/c mouse model. two weeks after the second immunization, balb/c mice were challenged with 20 mld 50 of influenza a virus pr8, and then weight loss and survival rates were monitored for 3 weeks. the weight loss curve (fig. 4a) showed that mice immunized with rvj1175, rvj-npm2e, and rvj-m2enp experienced rapid weight loss, and all mice died by 9 days post-challenge. mice immunized with rvj-4m2enps also experienced rapid weight loss; several of these mice died soon after the pr8 challenge, while others experienced maximum weight loss by day 9 post-challenge followed by gradual recovery. mice immunized with rvj-4m2enp experienced the lowest average weight loss (5%) among the groups observed on day 6 post-challenge, followed by rapid recovery to the initial body weight at day 9 postchallenge. the survival rate results (fig. 4b) showed that mice immunized with rvj1175, rvj-npm2e, and rvj-m2enp died on days 7-8, 7-9, and 7-10, respectively, post-challenge. in contrast, 1/12 mice in the rvj-4m2enp died on day 10 post-challenge, showing a final survival rate of 93%. mice immunized with rvj-4m2enps died on days 7-11 post-challenge with a final survival rate of 30%. statistical analysis showed that mice immunized with rvj-4m2enp had significantly highest survival rate, and mice immunized with rvj-4m2enps also had significantly higher survival rate than the remaining three groups (fig. 4b ). the extracellular domain of m2e is highly conserved among influenza a virus subtypes (muñoz-medina et al. 2015; zebedee and lamb 1988) ; thus, it was adopted as a target antigen in the development of a universal influenza vaccine. in the present study, a single m2e induced a poor m2e-specific humoral response (fig. 3a , rvj-npm2e, rvj-m2enp). four tandem repeats of m2e induced robust antibody response against m2e (fig. 3a , rvj-4m2enp, rvj-4m2enps), leading to a much stronger cross-protection than that of other groups against heterosubtypic h1n1 influenza virus challenge (fig. 4b , rvj-4m2enp, rvj-4m2enps). it had been proved that more robust m2especific immune response can be induced with multiple copies of m2e (zhang et al. 2011; zhao et al. 2010b; zhou et al. 2012) , and further enhanced by fusion with an appropriate protein carrier (ma et al. 2013; zhao et al. 2010a; ebrahimi et al. 2012; alvarez et al. 2013; schotsaert et al. 2009 ). our data suggest that m2e-specific antibody and cellular response played important roles in protection, and 4 9 m2e is more favorable than 1 9 m2e to induce protective immune response. m2e specific antibody may play a protective role by antibody-dependent cell-mediated cytotoxicity (adcc), although it could not neutralize influenza virus (kim et al. 2017; lee et al. 2015) . besides, m2e specific cellular immune response may stimulate cd4? t cells to protect animals (adlermoore et al. 2011). np has also been used as a target antigen to develop universal influenza vaccines li et al. 2013; wang et al. 2014; nahampun et al. 2015; lei et al. 2015; zheng et al. 2015) . in the present study, a truncated np protein lacking 50 amino acids at the n terminal and * 70 amino acids at the c terminal was designated as nps. although nps retaining the middle part of np with multiple conserved epitopes, it showed weaker immunogenicity compared with full-length np (fig. 3a , rvj-4m2enp, rvj-4m2enps), and less protection in this experiment (fig. 4b , rvj-4m2enp vs rvj-4m2enps, p \ 0.01), probably because of less np expression (fig. 2, middle panel) , suggesting that full-length np is critical toward inducing protective immunity at least in balb/c mouse. previous research proved that human mab against influenza np played an important role in fighting low-dose infection of influenza virus (fujimoto et al. 2016) , and np might exert a protective effect through adcc (kui et al. (hayward et al. 2015) . the present study showed that npspecific antibody and cellular response played important roles in protection. np-specific antibody could not neutralize influenza virus just as m2e. however, it may protect animals against challenge by adcc, antibody-dependent cellular phagocytosis, or antibody-dependent complement deposition, and np cellular immune response may stimulate cd4? and cd8? t cells for protection (guo et al. 2010; lamere et al. 2011) ; the related mechanism is worthy of further investigation. humoral and cellular immune responses in balb/c mice immunized with recombinant vaccinia viruses. mice were immunized intramuscularly with 10 7 plaque-forming units of recombinant vaccinia viruses at weeks 0 and 4. group 1 (g1) mice immunized with rvj1175 were served as negative controls. a serum was obtained from three mice at 10 days post the second immunization and analyzed by enzyme-linked immunoassay (elisa) for the presence of igg antibodies specific for np (left) or m2e (right), as described in ''materials and methods''. the columns show geometric mean antibody titers, and the bars indicate the 95% confidence interval in each group (n = 3 mice per group). comparative results between two groups are shown in the upper table (ns not significant, *p \ 0.05; **p \ 0.01; ***p \ 0.001 by one-way anova). b, c cellular immune response in mice immunized with recombinant vaccinia viruses. cytokines secreted from stimulated spleen mononuclear cells (smncs) were measured by enzyme-linked immunospot (elispot) assay (b) and mouse th1/th2/th17 cba kit (c). all mice in each treatment group were sacrificed at 10 days post the second immunization. smncs were separated from mouse spleen samples, and 5 lg/ml np55-69 (left b), np147-155 (middle b), and m2e pooled peptides (right b, and c) were used as stimulants in the elispot and cba assays. after stimulation for 24 h, the numbers of smncs producing interferon (ifn)-c (b) are presented as spotforming cells (sfcs)/10 6 smncs. the columns show the average sfcs/10 6 smncs, and the bars indicate the standard deviation of each group. lines above two or more groups indicate that there was no significant difference. *p \ 0.05; **p \ 0.01; ***p \ 0.001 by one-way anova. furthermore, we determined whether the immunogenicity of fusion antigens is influenced by the order of np and m2e. our data showed that rvj-npm2e and rvj-m2enp induced similar antigen-specific igg response (fig. 3a , rvj-npm2e vs rvj-m2enp). however, rvj-m2enp induced significantly more np147-155-as well as m2e-specific sfcs than the former one (fig. 3b , rvj-npm2e vs rvj-m2enp), suggesting that m2e at n terminal of the fusion antigen is a favorable configuration to induce np-and m2e-specific cellular immunity. moreover, our data indicated that four copies of m2e preceding np (4m2e-np) is more robust toward inducing np-and m2especific humoral immune response (fig. 3a , rvj-m2enp vs rvj-4m2enp) and protection (fig. 4b , rvj-m2enp vs rvj-4m2enp). in addition, we observed that only rvj-4m2enp immunization induced significantly higher levels of il-2 and il-10 cytokines in mice when stimulation with the m2e pooled peptides (fig. 3c, rvj-m2enp ). significant progress is made to develop influenza vaccine using viral vectors (kim et al. 2013; li et al. 2013; tutykhina et al. 2018; dhanwani et al. 2016) . several recombinant viral vaccines have been constructed based on modified vaccinia virus ankara expressing ha, np, m1, and pb1 antigens (coughlan et al. 2018; mullin et al. 2016; di mario et al. 2017; altenburg et al. 2017) . vaccinia virus tiantan strain, developed in china as a vaccine against smallpox, has also been used as viral vector in numerous studies to develop recombinant viral vaccines. in this study, the vaccinia virus tiantan strain was used to construct recombinant vaccinia viruses expressing fusion antigens with different configurations. the recombinant vaccinia virus expressing 4m2e and full-length np fusion antigen induced strong cross-protection (92%) against a lethal heterosubtypic pr8 challenge at 20 mld 50 and thus regarded as the optimal one among the four constructs. in the previous study, we expressed the fusion protein nm2e in an e. coli system, and determined its immunogenicity in balb/c mice. however, it is inconvenient and time consuming to express and purify much more alternatives. moreover, it is impossible to express target antigens in an e. coli system successfully sometimes. compared with an e. coli system, the recombinant vaccinia virus system has characteristics such as high multiplication capacity, no requirement to purify proteins, and high-efficiency to determine the immunogenicity of target antigens in animal models (coughlan et al. 2018; mullin et al. 2016) . in fact, we succeeded in constructing and determining the antigenicity of several fusion antigens of np and m2e efficiently. in summary, immunization with a 4 9 m2e and np fusion antigen not only induced np-specific humoral and cellular immune response, but also increased m2e-specific immunity, which together led to superior cross-protection against heterosubtypic pr8 at 20 mld 50 . we concluded that the cross-protection potential correlates with both np and m2e-specific humoral and cellular immune responses induced by rvj-4m2enp. however, the recombinant rvj-4m2enp containing 4 9 m2e and full-length np could not induce significant neutralizing antibodies, which help to reduce the severity and duration of the illness. therefore, it is necessary to introduce conserved b-cell epitopes of ha to induce an enhanced cross-neutralizing response and protective potency (koday et al. 2017) . future studies should focus on the development of universal influenza mice were monitored daily for 21 days after pr8 challenge. a mice were weighed daily to monitor morbidity. average weights in each treatment group were followed for the duration of the study, and the percentage of the original body weight 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induces robust immunity in macaques world health organization (who) (2019) influenza (seasonal) fact sheet sars-cov spike proteins expressed by the vaccinia virus tiantan strain: secreted sq protein induces robust neutralization antibody in mice influenza a virus m2 protein: monoclonal antibody restriction of virus growth and detection of m2 in virions vaccination with different m2e epitope densities confers partial protection against h5n1 influenza a virus challenge in chickens advancements in the development of subunit influenza vaccines induction of protection against divergent h5n1 influenza viruses using a recombinant fusion protein linking influenza m2e to onchocerca volvulus activation associated protein-1 (asp-1) adjuvant an h5n1 m2e-based multiple antigenic peptide vaccine confers heterosubtypic protection from lethal infection with pandemic 2009 h1n1 virus cross-protection against influenza virus infection by intranasal administration of nucleoprotein-based vaccine with compound 48/80 adjuvant immunization with high epitope density of m2e derived from 2009 pandemic h1n1 elicits protective immunity in mice acknowledgements this work was supported by grant from the national key plan for scientific research and development of china (2016yfc1200200). the authors gratefully acknowledge professor xiangmin zhang (wayne state university, detroit, mi usa) for the revision of the manuscript in english. key: cord-025232-5itrsfmk authors: yan, yuqian; jing, shuping; feng, liqiang; zhang, jing; zeng, zhiwei; li, min; zhao, shan; ou, junxian; lan, wendong; guan, wenyi; wu, xiaowei; wu, jianguo; seto, donald; zhang, qiwei title: construction and characterization of a novel recombinant attenuated and replication-deficient candidate human adenovirus type 3 vaccine: “adenovirus vaccine within an adenovirus vector” date: 2020-05-26 journal: virol sin doi: 10.1007/s12250-020-00234-1 sha: doc_id: 25232 cord_uid: 5itrsfmk human adenoviruses (hadvs) are highly contagious and result in large number of acute respiratory disease (ard) cases with severe morbidity and mortality. human adenovirus type 3 (hadv-3) is the most common type that causes ard outbreaks in asia, europe, and the americas. however, there is currently no vaccine approved for its general use. the hexon protein contains the main neutralizing epitopes, provoking strong and lasting immunogenicity. in this study, a novel recombinant and attenuated adenovirus vaccine candidate against hadv-3 was constructed based on a commercially-available replication-defective hadv-5 gene therapy and vaccine vector. the entire hadv-3 hexon gene was integrated into the e1 region of the vector by homologous recombination using a bacterial system. the resultant recombinants expressing the hadv-3 hexon protein were rescued in ad293 cells, identified and characterized by rt-pcr, western blots, indirect immunofluorescence, and electron microscopy. this potential vaccine candidate had a similar replicative efficacy as the wild-type hadv-3 strain. however, and importantly, the vaccine strain had been rendered replication-defective and was incapable of replication in a549 cells after more than twenty-generation passages in ad293 cells. this represents a significant safety feature. the mice immunized both intranasally and intramuscularly by this vaccine candidate raised significant neutralizing antibodies against hadv-3. therefore, this recombinant, attenuated, and safe adenovirus vaccine is a promising hadv-3 vaccine candidate. the strategy of using a clinically approved and replication-defective hadv-5 vector provides a novel approach to develop universal adenovirus vaccine candidates against all the other types of adenoviruses causing ards and perhaps other adenovirus-associated diseases. human adenovirus (hadv) is a wide spectrum virus that causes multiple systemic infections, such as acute respiratory disease (ard), epidemic keratoconjunctivitis, hemorrhagic cystitis, gastroenteritis, as well as other diseases (lion 2014; zhao et al. 2014) . using the whole genome sequence as a means of typing, 103 genotypes are currently recognized and classified into seven species a-g (seto et al. 2011; zhang et al. 2017) . among the genotypes of hadv causing ard, type 3 (hadv-3) is the most common one reported to who (echavarría 2009 hadv-3 infections are reported globally and recently, including the united states (lebeck et al. 2009 ), brazil (pereira et al. 2016) , the united kingdom (alkhalaf et al. 2015) , korea (lee et al. 2010) , australia (harley et al. 2001) , canada (yeung et al. 2009 ), singapore (coleman et al. 2019) , malaysia , japan (fujimoto et al. 2008) , and china (zhang et al. 2006; tsou et al. 2012; wo et al. 2014; chen et al. 2016; yu et al. 2016; lu et al. 2017; lin et al. 2019; zhao et al. 2019) . despite these widespread and recurring outbreaks, there is no specific drug or vaccine approved for use against hadv-3 infections. the protein capsid of adenovirus comprises 240 hexon trimers and 12 penton bases and fibers, of which the hexon protein contains the major neutralizing epitope (''epsilon'') (crawford-miksza and schnurr 1996; singh et al. 2015) . each hexon monomer is encoded by approximately 900 amino acid residues that form a base (rux et al. 2003) , which is relatively sequence-conserved, and three tower domains that occur as loops lying on the exterior surface of the virion (rux and burnett 2000) . these loops contain several hypervariable regions, noted as hvr1-hvr6 in loop 1 and hvr7 in loop 2 (cheng et al. 2018) , that are noted collectively as the epsilon epitope. this epsilon epitope provokes the host immune system into producing adenovirus serotype-specific antibodies that are highly effective and long-lasting (rux et al. 2003; russell et al. 2006; pichla-gollon et al. 2007; radin et al. 2014; tian et al. 2015) . paradoxically, hadv vectors have been widely and safely used in gene therapy protocols (stecher et al. 2003; holterman et al. 2004; stone et al. 2005; toth and wold 2010; wold and toth 2013; duffy et al. 2017) . several drugs incorporating adenoviruses as delivery vectors have been clinically approved and used in patients successfully (liu and kirn 2008; räty et al. 2008) . additionally, hadvs are the vector foundations of vaccines developed against many pathogens, including ebola virus (milligan et al. 2016; shukarev et al. 2017) , hiv (schooley et al. 2010; baden et al. 2014; gu et al. 2014 ), avian influenza virus (peters et al. 2013; scallan et al. 2013) , and foot-andmouth disease virus (pena et al. 2008) , as well as highly contagious bacteria associated with diseases, e.g., tuberculosis (smaill et al. 2013) . in this study, the commercially-available and gene therapy use approved replication-defective hadv-5 vector was used to construct a recombinant attenuated human adenovirus type 3 vaccine (ginn et al. 2018) . the complete hexon gene of hadv-3 gz01 was cloned into the adeasy tm adenoviral vector, and this type-specific antigen was expressed when the recombinant adenovirus vaccine was inoculated into mice. this complete hexon protein with native conformation in theory was expressed successfully and neutralizing antibodies were produced. the recombinant vaccine is expected to be used in the prevention of ard outbreaks caused by hadv-3 infections, and to serve as a model using adenovirus vectors for the construction of other vaccines against additional important serotypes of adenoviral respiratory pathogens. replication-deficient adenoviruses were cultured in ad293 cells (purchased from stratagene; ca, usa), which were grown in dulbecco's modified eagle's medium (dmem) supplemented with 100 iu penicillin/ml, 100 lg streptomycin/ml, and 10% (v/v) fetal calf serum. the hadv-3 gz01 strain (accession number dq099432) was originally isolated from a child with ard (zhang et al. 2006 ) and cultured in a549 cells. viral genomic dna was extracted by our modified protease k digestion method. e. coli strains dh5a and bj5183 are laboratory stocks. plasmids pshuttle and padeasy were purchased from adeasy tm adenoviral vector system (stratagene; ca, usa). platinum ò taq dna polymerase with high-fidelity and restriction enzymes were purchased from life technologies corporation (carlsbad, ca, usa) and new england biolabs (ma, usa), respectively. containing the hexon gene of hadv-3 in brief, the complete hexon gene of hadv-3 gz01 was cloned into pshuttle and recovered as recombinant psad3h. this pshuttle vector was then subjected to homologous recombination with padeasy, using the highly efficient homologous recombination system in e. coli bj5183 according to the manufacturer's protocol. finally, the rad3h recombinant virus was rescued by transfection with prad3h in ad293 cells and cultured for 20 generations. this construction strategy is presented as fig. 1 , with the detailed steps as follows. first, platinum ò taq dna polymerase with high-fidelity was used to pcr amplify the complete hexon gene of hadv-3 gz01, using the primer pairs ad3-gz01-ecor v-hexf (5 0 -tgagatatcgccaccatggccaccccatc-3 0 ) and ad3-gz01-hexr-xho i (5 0 -agactcgagttatgtgg tggcgttgcc-3 0 ). following, two restriction sites, ecor v and xho i, were added onto opposite ends of this amplified hexon gene and the pcr products were purified by pcr clean up kit (axygen; new york, usa). both the pcr products and the pshuttle plasmid were digested by ecor v and xho i in 37°c, respectively, permitted to reanneal, and then ligated using t4 dna ligase at 16°c for 8 h. after ligation, the recombinant was transformed into 200 ll e. coli dh5a competent cells. in the next 12-16 h, the cells were grown in lb agar plates, selected by ampicillin. after selection and amplification of each single clone, the plasmid was extracted and verified by pcr and sequencing, and archived as psad3h. second, approximately 100 ng of padeasy and 700 ng of psad3h linearized with pme i both were transformed into e. coli bj5183 competent cells which contained recombinase, and then selected on lb-kanamycin agar plates. as the small colonies were potential candidate recombination clones (zhang et al. 2009 ), they were selected and further identified by pcr screening, using hadv-3 type-specific primers (ad3f 5 0 -aaga-cattaccactactgaaggagaagaa-3 0 and ad3r 5 0 -cgctaaagctcctgcaacagcat-3 0 ) (han et al. 2013) and producing a pcr product of approximately 300 bp. this pcr-positive plasmid dna was then amplified in dh5a cells, and additionally identified by sequencing and restriction enzyme digestion (ecor i, ecor v, not i, sal i, and xho i). plasmid miniprep kit (axygen; new york, usa) was used to purify this recombinant plasmid prad3h. third, the prad3h plasmid was digested with pac i to release the linear adenovirus genomic dna, which was then transfected into ad293 cells using lipofectamine ltx (life technologies corporation; carlsbad, ca, usa). the cells were examined daily for 10 days for evidence of increased fluorescence and cytopathic effect, which signaled successful transfection and packaging. after 10 days, the cultures were frozen and thawed thrice and then centrifuged at 12,000 9g for 10 min. the supernatant was used to infect the ad293 cells. cells were cultured for about 7 days. fluorescence and cytopathic effect were monitored each day. the recombinant adenovirus rad3h was rescued successfully when both gfp and cpe were observed. the transcription of hadv-3 hexon gene of rad3h was confirmed by rt-pcr analysis as follows: total rna was extracted by rnaiso (takara; tokyo, japan) and reverse transcribed into cdna using the primer hvrr (5 0 -tttctgaagttccactcgtaggtgta-3 0 ). this resultant cdna was used as the template in a pcr amplification with the primer pairs, hvrf1 (5 0 -caggatgcttcg-gagtacctgag-3 0 ) and hvrr (zhang et al. 2020 ). the pcr product was identified using agarose gel electrophoresis and dl10000 markers. subsequent western blot analysis confirmed expression of the hexon protein. a culture of rad3h was frozen and thawed three times, centrifuged at 1000 9g for 10 min, and the supernatant was collected. the samples were added to loading buffer, boiled for 10 min, electrophoresed in 5% concentrated gel, and 10% tris-tricine sds-page. after page, the proteins were transferred onto nitrocellulose membrane by electrophoresis at 25 v for 40 min. the membrane was probed first by binding with mouse anti-hadv-3-hexon antibody (guangzhou huyansuo medical technology; guangzhou, china) as the epitope-specific antibody hexon hadv-3 gz01 pshuttle (1:1000 dilution), and followed with goat anti-mouse igg-hrp (bioworld; georgia, usa) as the secondary amplifying antibody. luminol and hydrogen peroxide were used as chromogenic substrates to score the fluorescence signal. indirect immunofluorescence assay was performed to detect the virus titer of hadv-3. 2 9 10 4 ad293 cells were seeded into each well of a 96-well plate. after an 18-24-h culture, the cells were infected with 100 ll hadv-3. at 48 h post infection, the cells were fixed in methanol, precooled in -20°c, and incubated at 37°c for 30 min with 1% bsa. the mouse anti-hadv-3-hexon monoclonal antibody, at a dilution of 1:1000 in pbst, was added to wells and incubated at 37°c for an hour. fitcconjugated goat anti-mouse igg (1:10000) was then added and the mixture incubated at 37°c for an hour. the number of green fluorescence cells were counted under reversed fluorescence microscopy and virus titer were calculated by fluorescence forming unit (ffu) with the formula of ffu/ml = 10 n 9 average number of gfp positive cells/well 9 10 (n: dilution times). ad293 cells infected with rad3h viruses were grown and harvested when 90% cpe was observed. these cells were frozen and thawed three times, centrifuged at 12,000 9 g for 10 min. supernatant containing viral particles was collected, upon which a grid covered with carbon support formvar film was floated, for 2 min. this was stained with 10 ll sodium phosphor-tungstate, dried at 40°c for 10 min, and the vaccine strained rad3h was observed using a tem technique (fei tecnai tm electron microscope). ad293 cells were seeded into 6-well plates with 5 9 10 5 cells per well. after 16-24 h, the cells were infected with 5 9 10 4 dna copies of rad3h, rad5 or hadv-3 strain gz01, respectively. cultures were harvested after 12, 24, 36, 48, 72, 84, 96, 108, or 120 h post-infection. these cultures were frozen and thawed three times, then the supernatant was collected after centrifugation. the viral dna copies at all the time-points were quantified by realtime pcr using the primers pentonf (5 0 -accaccgt-cagtgaaaacg-3 0 ) and pentonr (5 0 -tatgcc-cagkgccttgt-3 0 ) to quantify the genomic dna copy number. using hadv-5 dna as the template and pentonf and pentonr as primers, the pcr product was ligated into the pmd18-t vector (takara; tokyo, japan). the resultant plasmid was used as the standard for real-time pcr quantification. the viral stability of the recombinant vaccine strain rad3h after continuous passages was further tested. briefly, the virus was cultured in ad293 cells and passaged for more than 20 generations. at every five generations, adenovirus genomic dna was extracted; these served as templates for the pcr amplification of hadv-3 hexon gene, which was then further characterized by dna sequencing. the 20th generation of rad3h harvested from the ad293 cells culture was further inoculated into a549 cells and re-cultured for 7 days to determine if there were replication-competent mutants resulting from the continuous passages of rad3h in ad293 cells. if no green fluorescence was observed during the culturing in a549 cells, it meant that no replication-competent mutants occurred. therefore, the stability of the recombinant vaccine strain was verified. discontinuous density-gradient centrifugation was used to purify and concentrate the recombinant adenovirus rad3h as follows: 6 ml 1.2 g/ml cscl was first added into the centrifuge tube with 8 ml 1.4 g/ml cscl slowly added to the bottom subsequently, resulting in a stratification. on the top of the cscl, 20 ml of the virus culture was then carefully layered, and the tube was centrifuged at 141,000 9g at 4°c for 3 h. the virus band was then extracted and transferred to a dialysis bag (mwco: 14,000 da) and dialyzed in a buffer at 4°c, with two buffer changes, to remove the cscl (10 mmol/l tris-cl ph 8.0, 2 mmol/l mgcl 2 , and 5% sucrose). purified virus was titrated by a modified fluorescence-forming unit assay (philipson 1961) . briefly, ad293 cells were infected with ten-fold diluted rad3h virus in 96 well-plates. after 48 h of incubation, rad3h infected cells are directly observed under fluorescent microscopy. wells with suitable virus dilution were selected for counting the number of fluorescent cells. the virus titer of ffu was calculated as described above. four to six-week-old female specific-pathogen-free balb/c mice were purchased from the laboratory animal center of southern medical university (guangzhou, china) and kept in individual ventilated cage system. six mice in each group were either inoculated with 1.8 9 10 8 ffu/kg of hadv-3 gz01 or immunized with the rad3h recombinant vaccine by the intranasal route or intramuscular route, respectively. the negative control group was inoculated with pbs of the same volume. the mice were boosted with the same virus or vaccine strain at day 14 post inoculation. at days 21, 28, 35 and 42, sera from three mice in each group were collected and the 50% neutralizing antibody titer was determined by microculture neutralization test. a microculture neutralization test was performed to determine the hadv-3 neutralizing antibody titers using a previously described protocol (zhang et al. 2009 ). sera from mice immunized with rad3h by either intramuscular injection or intranasal inoculation were tested for the presence of hadv-3 neutralizing antibody. the 50% virus neutralization titers were calculated by reed-muench method. a pshuttle plasmid psad3h containing the complete hexon gene, which contains the epsilon epitope of hadv-3, was constructed. this construct also contained the left and right homologous regions that enable homologous recombination with padeasy to produce infectious clone prad3h (fig. 1) . the resultant plasmid was confirmed by dna sequencing, which not only provided confirmation of the complete hexon gene sequence but also verified that no mutations were introduced. prior to sequencing, as a rapid check, restriction endonuclease analysis (rea) was performed to characterize the recombinant plasmid rapidly. restriction maps generated with five restriction endonucleases were consistent with the in silico restriction maps predicted by the vector nti 11.5.1 software (invitrogen corp; san diego, ca, usa) (zhao et al. 2014; yu et al. 2016; zhang et al. 2017; pan et al. 2018) (fig. 2) . recombinant plasmid prad3h was linearized by pac i and transfected into ad293 cells to rescue the recombinant adenovirus rad3h. green fluorescence was observed under fluorescence microscopy and the fluorescence density was noted to increase each subsequent day, an indication that the recombinant adenovirus was replicating. at day 10 post-transfection, the virus culture was frozen and thawed three times; after centrifugation, the supernatant was inoculated into ad293 cells. green fluorescence and cpe were observed and recorded at day 8 post-infection (fig. 3) . the increased fluorescence and cpe suggested the transcription of the rad3h hexon gene was confirmed by rt-pcr. a rad5 virus that containing adenovirus type 5 genome except for e1 and e3 regions was used as a positive control. viral rna was extracted from a rad3h culture and digested with dnase. after digestion, the presumed viral rna was used as a pcr template for amplifying the hexon gene as a check for any residual dna. no pcr products were found from these samples, which indicated that the extracted rna did not contain viral genomic dna (fig. 4a) . after reverse transcription, the cdna was used as a pcr template to validate the insert. the resultant amplification of the hexon gene confirmed the rad3h recombinant adenoviruses. (fig. 4b) . expression of the hadv-3 hexon protein in the culture supernatant was also detected by western blots (fig. 4c) , in which hadv-3 specific mouse monoclonal antibody was used to identify hadv-3 hexon. the rad5 adenovirus was used as the negative control, as it contained the hadv-5 hexon gene but not the hadv-3 hexon gene. the hadv-3 hexon protein of 108 kda was confirmed in the rad3h recombinant vaccine strain while no band was found in the rad5 and mock samples, indicating that during the culture, the hadv-3 hexon protein of rad3h can be expressed successfully. the harvested viral culture from rad3h was negativestained by sodium phosphor-tungstate and observed under electron microscopy. typical adenovirus particles, 70-90 nm in diameter, were clearly visible (fig. 5) . this further confirmed the successful infection and replication of the recombinant hadv-3 vaccine in ad293 cells. the replication efficiencies of rad3h, rad5 and hadv-3 strain gz01 were compared by quantification of genomic dna copies using a real-time pcr method. primers targeting the conserved region of the penton base gene to represent the genomic dna number were used in the realtime pcr amplification. the two one-step growth curves of rad3h and hadv-3 strain gz01 showed that the replication efficacy of rad3h viruses were similar to that of hadv-3 wild-type strain in ad293 cells, both of which increased similarly during the 60 h post infection. the peaks of dna copy number of both rad5 and rad3h strains were higher than hadv-3 gz01 strain, which might be because hadv-5 genomic dna is the backbone of both strains and the viruses are cultured in ad293 cells which highly expressed hadv-5 e1a and e1b genes that promote the hadv-5 dna replication (fig. 6) . to assess the stability of the insert, recombinant vaccine strain rad3h was passaged in ad293 cells for at least 20 generations. the virus stability of rad3h harvested from the 20 generations of culture was verified by pcr amplification and dna sequencing of the hadv-3 and hadv-5 hexon genes. there were no amino acid mutations detected in the hadv-3 and hadv-5 hexon gene of rad3h upon sequence verification. this indicated that both hexon genes in this recombinant vaccine strain are well compatible with each other during the viral replication. as expected, both fluorescence and cpe were observed in the ad293 cells infected by rad3h from the first to the twentieth generations (fig. 7a-7d) . however, no green fluorescence was detected in the a549 cells infected by rad3h which was the culture from the first to the twentieth generations of rad3h in a549 cells (fig. 7e-7h) , which indicated no infectious viral particles produced in a549 cells. pcr amplification of the hexon gene from the virus culture in a549 cells showed no product, which indicated that rad3h could only replicate in ad293 cells, but not in the a549 cells due to the e1 deletion. no reverse mutations occurred during the continuous passages of rad3h in ad293 cells. all the results presented confirmed the stability of the vaccine stain rad3h in ad293 cells (fig. 7) . mice were either inoculated with hadv-3 wild-type strain gz01 or immunized with the rad3h recombinant vaccine by either the intranasal route or intramuscular route, respectively, to assess the antibody titer. at day 14 post inoculation/immunization, the mice were boosted with the same inoculant. mouse sera were collected at day 21, 28, 35, and 42 after the prime inoculation/immunization. the 50% neutralizing antibody titers of mouse sera in the intranasal or intramuscular groups collected at different hadv-3 gz01 120 fig. 6 replication dynamics curves of rad3h bearing hadv-3 hexon gene, rad5 and hadv-3 wild-type strain gz01. ad293 cells were infected by the two viruses and harvested at 12, 24, 36, 48, 60, 72, 84, 96, 108 , and 120 h post infection. viral genomic dna copy numbers in cells and supernatants were determined by real-time pcr for the penton base region. time points were determined by the microculture neutralization test ( table 1 ). all of the mice immunized with rad3h or hadv-3 gz01 virus intranasally or intramuscularly produced neutralizing antibodies. however, the mice immunized intramuscularly produced higher and more lasting antibody titers than the intranasally immunized mice. therefore, apparently the intramuscular immunization was more effective than the intranasal immunization in provoking the immune response. in the wild-type gz01-innoculated mice, higher neutralizing antibody titers were produced in both intranasal and intramuscular groups, which might be associated with the replication-competence of wild-type adenoviruses as compared with the replication-deficient rad3h vaccine strain. human adenoviruses are highly contagious pathogens that are associated with several severe and fatal diseases including ard (jing et al. 2019; zhang et al. 2019) . ard associated with hadv-3 often results in severe morbidity and some fatalities. additionally, among all of the adenovirus types causing ard, hadv-3 appears to be the most common found in pediatrics cases (chang et al. 2008) . there is no vaccine currently available for preventing hadv-3 outbreaks. in fact, the only two vaccines available against hadvs (types 4 and 7) are not available to the public. in this study, we provide a novel strategy for the construction of a candidate adenovirus type 3 vaccine that may serve as a template for developing similar vaccines for use against other pathogen hadvs. the time-tested and safe hadv-5 replication-deficient vaccine vector was used for the construction of this candidate vaccine strain that harbors the immunogenic epitope from human adenovirus type 3 hexon. we demonstrate that the administration of this recombinant and replication-deficient rad3h strain could elicit the significant increase of neutralizing antibodies against hadv-3; therefore, it could be considered a candidate vaccine strain to be used for the prevention of hadv-3 infection or epidemics. there are three methods used to construct an adenovirus vector: in vitro ligation, cre/loxp system, homologous recombination in eukaryotic cells or in prokaryote cells method. the genome of human adenoviruses is about 35 kb. compared with the other methods, the method of homologous recombination in prokaryote cells has the advantages of a simpler protocol, higher recombination efficiency, and easier screening of positive clones. in this study, we used the homologous recombination in e. coli bj5183 to construct a recombinant adenovirus vector to serve as the basis for a vaccine against a commonly circulating adenoviral respiratory pathogen. this recombinant dna vector construction protocol consists of two vectors: an adenovirus backbone plasmid, padeasy, and a pshuttle plasmid. the pshuttle plasmid contains a multiple cloning site and a kanamycin resistance gene, which support the insertion and maintenance of a foreign gene, as well as provides for the rapid screening for recombination. a pcrderived product comprising the complete hexon gene (epsilon epitope) of adenovirus type 3 was directly cloned into the multiple cloning site of pshuttle using the ecor v and xho i restriction sites. the linearized psad3h and padeasy were transformed into an engineered bacterial strain bj5183 that was reca proficient and supplied the machinery necessary to execute the recombination event. homologous recombination was carried out to obtain the vaccine vector bearing the complete hadv-3 hexon gene. to overcome the undue homologous recombination during the bacterium culture, the recombinant time in bj5183 was restricted to no more than 8 h, then recombinant bacterial clones were screened by kanamycin resistance. following the construction of the recombinant plasmid, the putative size of the insert allows for an initial screen, with additional identification, confirmation, and characterization by pcr, rea, dna sequencing, and other assays. the rad3h vaccine contains most genes of hadv-5 except the e1 and e3 regions, as well as the complete hadv-3 hexon gene. the e1 region is essential for the the mice were either immunized with the rad3h recombinant vaccine or inoculated with hadv-3 wildtype strain gz01 intranasally or intramuscularly. at day 21, 28, 35, and 42 after the prime inoculation/ immunization, the neutralizing antibodies against hadv-3 were titrated. virologica sinica assembly of infectious virus particles, so the replication of rad3h can only take place in adenovirus packaging cell line like ad293 cells which express the e1 proteins of hadv-5, while rad3h cannot replicate in any kind of human cells. therefore, the safety of rad3h recombinant adenovirus vaccine is guaranteed. the recombinant vaccine rad3h expresses the complete hadv-3 hexon gene in infected cells, which can maintain the native conformation of the whole hadv-3 hexon protein. in theory, this will guarantee a better immune effect. however, mice immunized with recombinant vaccine rad3h produced lower neutralizing antibody titers than wild-type hadv-3 strain. one of the causes may be that the capsid proteins of fiber and penton base in the wildtype viruses also provoke certain immunogenicity (gahery-segard et al. 1998; feng et al. 2018) . in rad3h vaccine strain, no hadv-3 fiber and penton protein were expressed, while there was in hadv-3 wild-type strain. another cause is the rad3h vaccine strain is replication deficient, which cannot replicate and express proteins well in normal cells. as the result, the induced humoral immunity by the wildtype strain in mice was stronger than replication deficient vaccine strain. in summary, a recombinant and attenuated adenovirus vaccine candidate against hadv-3 was constructed based on a commercially-available, replication-defective hadv-5 vector that has been widely used in previous gene therapy protocols and vaccine development. this potential vaccine candidate had a similar 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adenovirus epidemical features of hadv-3 and hadv-7 in pediatric pneumonia in adenovirus vectors for gene therapy, vaccination and cancer gene therapy characterization of culture-positive adenovirus serotypes from respiratory specimens in fatal community-acquired pneumonia in children caused by re-emergent human adenovirus 7d associated with higher severity of illness and fatality rate comparative genomic analysis of two strains of human adenovirus type 3 isolated from children with acute respiratory infection in southern china construction and characterization of a replication-competent human adenovirus type 3-based vector as a livevaccine candidate and a viral delivery vector comparative genomic analysis of two emergent human adenovirus type 14 respiratory pathogen isolates in china reveals similar yet divergent genomes a survey of recent adenoviral respiratory pathogens in hong kong reveals emergent and recombinant human adenovirus type 4 (hadv-e4) circulating in civilian populations molecular typing of human respiratory adenoviruses with universal pcr and sequencing primers for three major capsid genes: penton base, hexon, and fiber re-emergent human adenovirus genome type 7d caused an acute respiratory disease outbreak in southern china after a twenty-one year absence comparison of viral and epidemiological profiles of hospitalized children with severe acute respiratory infection in beijing and construction and characterization of a novel hadv-3 vaccine author contributions qz designed the experiments. yy, sj and lf carried out the experiments. yy, sj, lf, jz, zz, ml, sz, jo, wl, wg, xw, jw, ds and qz analyzed the data. yy, ds, and qz wrote the paper and finalized the manuscript. all authors read and approved the final manuscript. conflict of interest the authors declare that they have no conflict of interest.animal and human rights statement the whole study was approved by the southern medical university animal ethics committee. all institutional and national guidelines for the care and use of laboratory animals were followed. key: cord-025181-eg108wcd authors: zheng, zhihang; li, min; liu, zhihua; jin, xia; sun, jin title: establishment of murine infection models with biological clones of dengue viruses derived from a single clinical viral isolate date: 2020-05-25 journal: virol sin doi: 10.1007/s12250-020-00229-y sha: doc_id: 25181 cord_uid: eg108wcd dengue virus (denv) is a single-stranded rna virus transmitted by mosquitoes in tropical and subtropical regions. it causes dengue fever, dengue hemorrhagic fever and dengue shock syndrome in patients. each year, 390 million people are estimated to be infected by four serotypes of dengue virus, creating a great burden on global public health and local economy. so far, no antiviral drug is available for dengue disease, and the newly licensed vaccine is far from satisfactory. one large obstacle for dengue vaccine and drug development is the lack of suitable small animal models. although some denv infection models have been developed, only a small number of viral strains can infect immunodeficient mice. in this study, with biologically cloned viruses from a single clinical isolate, we have established two mouse models of denv infection, one is severe lethal infection in immunocompromised mice, and the other resembles self-limited disease manifestations in balb/c mice with transient blockage of type i ifn responses. this study not only offers new small animal models of dengue viral infection, but also provides new viral variants for further investigations on dengue viral pathogenesis. dengue virus (denv) is an arbovirus transmitted by aedes aegypti and aedes albopictus mosquitoes. it belongs to flavivirus genus of flaviviridae family and contains a positive single-stranded rna genome. denv can be antigenically classified into four serotypes (denv-1-4), which are often co-circulating in the endemic regions with similar clinical manifestations. though the majority of denv infections are asymptomatic, a minority of which can result in self-limited disease including dengue fever (df), and less than 1% may develop into dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss). because of the global warming, increased air travel, and the lack of an efficacious vaccine, denv has become the most prevalent mosquito-borne viral pathogen in recent decades (guzman et al. 2010) . each year, denv is estimated to infect 390 million people globally, affecting nearly half of the world population. asia accounts for 75% of the dengue disease burden, followed by latin america and africa (bhatt et al. 2013) . as an imported pathogen in china, dengue virus had caused sporadic outbreaks in southern china before 2013. however, an unexpected large dengue outbreak attacked guangzhou in 2014, resulting in 46,864 national reported cases that year (jin et al. 2015) , in which the majority was caused by dengue virus serotype 1 (denv-1), and the others by serotype 2 (denv-2) (zhao et al. 2016; li et al. 2017) . since then, 2000-5000 confirmed cases were reported to china cdc (chinese centers for disease control and prevention) each year. in nature, non-human primates and human are the only mammalian hosts for denv infection. naturally acquired dengue virus does not infect any outbred or inbred mice, unless viruses are inoculated with extremely high dose (e.g. 10 -9 pfu of denv-2 16681 in 4-week old c57/bl6) (chen et al. 2007) or through intracerebral injection. in some other models, mouse-brain adapted strains are used. but such infection of adapted strain or intracerebral infection usually results in neurological manifestations, which are not routine clinical signs of human diseases. and, after passaging in mice, characteristics of these mouse brain-adapted viral strains may differ from strains naturally acquired, attenuation in their ability to cause human disease was documented (sabin and schlesinger 1945; hotta 1952; sarathy et al. 2015) . thus, the inability of dengue virus to replicate in murine model had hampered the development of dengue vaccine and antiviral therapy. monkey models, while more useful, are not suitable for routine preclinical testing of vaccine candidates or drug prototypes because of their high cost, limited animal availability and ethical concerns. the species specificity of denv infection may be partially attributed to the differences of innate immunity between primates and mice. interferon (ifn) signaling plays an essential role in the protection against dengue disease in humans, nevertheless denv has also evolved many mechanisms to antagonize ifn signaling and ifn production in vivo (green et al. 2014) . one of them is the binding and degradation of human stat2 by dengue viral protein ns5 (ashour et al. 2010) . another is the cleavage of human sting molecules by dengue viral protease ns2b3 (aguirre et al. 2012; yu et al. 2012) . interestingly, both of these two antagonistic mechanisms only exist in human. denv ns5 does not bind to murine stat2, neither does ns2b3 cleave murine sting proteins, because the target sequences on these two molecules are highly species specific (ashour et al. 2010; aguirre et al. 2012; stabell et al. 2018) . but deletion of mstat2 or msting could enhance the replication of denv in mouse or mouse derived primary cells (ashour et al. 2010; aguirre et al. 2012) . these observations also imply the necessity of ifn signaling and ifn production in restricting denv infection in mice. therefore, immunocompromised mice without complete ifn responses or humanized mice are presumed more susceptible to denv infection. as humanized mice are laborious to prepare, and often introduce mouse-to-mouse variation, they are less suitable for application in vaccine development (akkina et al. 2011) . in contrast, knockout mice are easier to manipulate and a series of ifn deficient mice have been tested in the development of dengue infection model, including those of ifna/br -/-, ifncr -/-, ifna/b/cr -/-, mavs -/-, stat1 -/-, stat2 -/-, irf3 -/and irf7 -/(shresta et al. 2006; perry et al. 2011; prestwood et al. 2012 ). among them, type i/ii ifn receptors double knock out mice usually develop lethal diseases when challenged with suitable denv strains, thus are used more frequently. however, only a limited number of dengue virus strains are able to replicate in mice and reproduce the severe symptoms (e.g., vascular leakage) of human disease. these viruses are usually based on clinical isolates (denv-3 c0360/94, denv-3 703-4, denv-4 tvp-376), obtained through alternate passage between mosquito c6/36 cells and ag129 mice (denv-2 d2s10), or isolated after plaquepurification (denv-2 d2y98p-pp1, denv-2 s221) (sarathy et al. 2015) . such mouse models are valuable in testing vaccine efficacy and studying viral pathology or transmission. notably, there are an increasing number of investigations that suggest t cell immunity is an essential component for flavivirus vaccine (st john and rathore 2019). but, type i interferon are vital for cd4 ? and cd8 ? t cell generation and maturation (crouse et al. 2015) . and ifn-c is also one of the major cytokines mediating th1/ctl function and t cell development. therefore, ifn deficient transgenic mice are not ideal for studies of t cell immunity or t cell vaccines in an active immunization model, for the lacking of cross-talk between ifn pathway and adaptive immunity. in this study, we have purified two dengue virus strains of serotype 2 exhibiting different plaque sizes from one clinical isolate denv-2 gz. through subcutaneous injection of low doses of these viruses to type i/ii ifn receptor knock-out mice, we successfully established mouse models of lethal infection. the pathogenicity of the two variants in ag6 was demonstrated to be different. further, we compared the infectivity of these two viral variants in a self-limited infection model, in which type i ifn receptor of wild-type balb/c mice had been transiently blocked before infection, and found only the virus strain exhibiting larger plaque size caused infectious viral particles in sera. our research here provides two kinds of dengue virus infection mouse models, which reproduce both severe and self-limited manifestations of human diseases, expanding the current choice of dengue viral infection small animal models. dengue virus serotype 2 clinical strain, denv-2 gz, was originally isolated from a patient in guangzhou in 2012, and was kindly provided by dr. xiaoyan che of zhujiang hospital in guangzhou, china. the virus was propagated in mosquito c6/36 cells for no more than three passages in our lab. virus titer was determined in vero cells by foci forming assay. vero cells were passaged in dulbecco's modified eagle's medium (dmem, gibco, ny, usa) supplemented with 10% fetal bovine serum (fbs, gibco, auckland, new zealand) and 1% penicillin/streptomycin (p/s, gibco, ny, usa), and cultured at 37°c in a 5% co 2 incubator. mosquito c6/36 cells were cultured in minimum essential medium (mem, gibco) supplemented with 10% fbs, 1% penicillin/streptomycin and 1% non-essential amino acids (gibco, ny, usa) in a 28°c incubator under 5% co 2 atmosphere. to purify viruses of different plaque morphology from the original clinical isolate, virus stock of denv-2 gz was serially diluted, and used to infect c6/36 cell at 8, 4, 2, 1, and 1/2 pfu per well in a 96-well plate. seven days post infection, supernatant of each well was harvested. the foci size of virus that produced in each well was determined subsequently in vero cells by foci forming assay in 48-well plate. only supernatant containing virus with uniform foci size was kept for further propagation. after three passages, two viral clones maintained different plaque sizes, herein named as denv-2 1d4-5-sp (variant with small plaque size) and denv-2 8h2-7-lp (variant with large plaque size), were stored in a -80°c freezer. titer of virus stock and infectious viral particle in serum of mice were determined using foci forming assay in vero cells. specifically, vero cells were seeded at 7 9 10 4 cells/ well in a 48-well tissue culture plate and incubated overnight at 37°c, 5% co 2 . on the second day, sera of infected mice or viral stocks were serially diluted in dmem and added into wells containing vero cells for a 2 h incubation period. subsequently, the viral inoculum was removed and dmem supplemented with 1.5% fbs, 1% p/s, 1.5% carboxymethylcellulose (cmc, sigma, mo, usa) was added. after incubation for 96 h at 37°c, cells were fixed with 4% paraformaldehyde (pfa). following the removal of pfa, infected cells were stained with antidengue monoclonal antibody d1-11 (1:500, santa cruz biotechnology, ca, usa) for 2 h, followed by addition of biotin labeled anti-mouse igg (santa cruz biotechnology, ca, usa), and then streptavidin-alkaline phosphatase (sigma, mo, usa). finally, the foci of dengue virus were visualized by adding bcip/nbt substrate (beyotime biotechnology, jiangsu, china). neutralizing activity of mouse sera was evaluated using foci reduction neutralization test (frnt) as previous described (sun et al. 2017 ). genome information of two viruses was acquired through sanger sequencing. briefly, viral genomic rna was extracted from the virus stocks. after synthesis of viral cdna with specific primers, 14 fragments covering the whole coding region of genome were amplified using phusion high-fidelity pcr polymerase (thermofisher, lithuania). when the pcr product concentration was high enough, they were sent directly for sequencing. otherwise, fragments were inserted into the peasy-blunt plasmid (transgene, beijing, china), which was then used to transform e. coli bacteria. three colonies containing each of the fragments were sequenced. full-length sequence of the single open reading frame was achieved based on the consensus sequence of each fragment, and then stringed together by overlapping them with each other. after that, sequence information of these two viruses was submitted to genbank, and the access numbers are mn952966 (denv-2 8h2-7-lp) and mn952967 (denv-2 1d4-5-sp) respectively. ifn-c elispot assay was performed according to manufacturer's protocol (mabtech, nacka strand, sweden). splenocytes of infected or control mice were isolated by homogenizing and filtering through 40 lm cell strainers. after depleting erythrocytes, cells were re-suspended in 10% fbs/prmi-1640 and added to elispot plates (millipore, co. cork, ireland) pre-coated with ifn-c antibodies. then pbs, 10 lg/ml peptide, ns1 or e80 protein or 5 lg/ ml cona was added to each well to stimulate splenocytes, and the plates were incubated at 37°c for 40-48 h. after being washed with pbs for 5 times, plates were stained with biotin conjugated antibody, and subsequently streptavidin-horse radish peroxidase (hrp). finally, immune spots were visualized with the addition of tmb substrate. the image of spots was captured and analyzed by immu-nospot analyzer (cellular technology ltd. usa). balb/c mice were purchased from beijing vital river laboratory animal technology. type i and type ii ifn receptor double knock-out c57bl/6 mice (ag6) were originally provided by dr. guangxun meng of institut pasteur of shanghai, and raised in our laboratory. all mice were bred and maintained in specific pathogen-free barrier facilities and used at 6-8 weeks of age. for virus infection experiments, all procedures were completed under bsl-2 and absl-2 laboratory conditions as approved by the biosafety committee of institut pasteur of shanghai. female balb/c mice of 6-8 weeks old were injected with 2 mg anti-type i ifn receptor monoclonal antibody (mar1-5a3, bioxcell, usa) intraperitoneally (i.p.) at one day prior to challenge with 1.8 9 10 6 pfu parental strain, 8h2-7-lp or 1d4-5-sp through subcutaneous (s.c.) injection. blood samples were collected though retro-orbital bleeding daily from the 1st day post infection (1 dpi) to 7th day post infection (7 dpi). whole blood was lysed and homogenized within trizol-ls reagent (invitrogen, ca, usa) for measuring viral rna copy. meanwhile, for further detection of infectious viral particles in serum, sera were isolated from the blood through centrifugation. female ag6 mice of 6-8 weeks old were infected with 1 9 10 3 pfu, 1 9 10 4 pfu or 1 9 10 5 pfu denv viruses through subcutaneous (s.c.) injection on day 0. body weight and survival rate were monitored from 1 dpi to 15 dpi, during which moribund mice with weight loss over 20% were euthanatized and recorded as death. vascular leakage was determined following intravascular administration of evans blue dye (sigma, louisiana, usa). briefly, 200 ll evans blue dye (1% in pbs) was intravenously injected to mice on the 7th days post-infection of the two viral variants. after 1 h dissemination, the mice were anaesthetized and perfused with 50 ml pbs. digestive tracts and livers were collected and photographed. to quantify viral genomic copy in blood, rna was extracted following the user's guide of trizol ls reagent. a two-step quantitative rt-pcr (qrt-pcr) was then performed. first, cdna was synthesized using the fas-tquant rt kit (with gdnase) (tiangen biotech, beijing, china). second, amplifications were carried out using the fastfire qpcr premix (probe) kit (tiangen biotech, beijing, china) with the primer pairs designed according to the conserved sequence in denv 3 0 utr: (forward, 5 0 -tgayaagcartcagacac-3 0 ; reverse, 5 0 -tcac-carrctccctttgc-3 0 ; fluorescence probe 5 0 -cca-gagatcctgctgtctc-3 0 ). amplification cycles consisted of an initial incubation step of 95°c for 1 min, 40 cycles at 95°c for 5 s, 55°c for 10 s, and 72°c for 20 s. standard rna were in vitro transcribed from a dna template that contains a t7 promoter and the target polynucleotide fragment for the primers indicated above. to obtain the standard curve, serially diluted standard rna (10 2 -10 9 copy) were used as templates in parallel with sample rnas. copy of viral rna was calculated according to standard curves. all data were analyzed using graphpad prism software. t test or two-way anova were used to analyze the significance as indicated in legends. all results were expressed as means plus and minor standard errors of mean (sem). p values of 0.05 or less were considered statistically significant (*p \ 0.05; **p \ 0.01, ***p \ 0.001; ****p \ 0.0001). during in vitro propagation of a clinical strain of denv-2 obtained from guangzhou, china, heterogenous foci of different plaque sizes were observed in vero cell cultures (fig. 1a) . through limiting dilution and culture in mosquito c6/36 cells, two viral variants were separated. after subsequent passage for three rounds in c6/36 cells, two virus strains with stable foci morphology were obtained. one of them forms larger foci of about 1.25 mm diameter after 80 h culture, and it was termed as denv-2 8h2-7-lp (fig. 1a) ; the other forms smaller foci of about 0.25 mm diameter and was termed as denv-2 1d4-5-sp (fig. 1a) . we next compared the replication abilities of these two viruses in both vero cells and c6/36 cells using the onestep growth curve. denv-2 8h2-7-lp had a higher replicating efficiency in vero cells than denv-2 1d4-5-sp during the first 72 h after infection, followed by a decline afterwards, due to the probability of running out target cells for infection; in comparison, denv-2 1d4-5-sp rose slowly to a peak at 96 h post infection (hpi), and kept its plateau until 120 hpi (fig. 1b) . distinct from what was observed in vero cells, there was no obvious growth difference between the two viruses in mosquito cells (fig. 1c) . taken together, these data demonstrated that two viral variants with different foci sizes are isolated from one clinical virus isolate. and these two variants have different replication abilities in vero but not mosquito c6/36 cells. to determine whether the different replication phenotypes reflect underlying genetic divergence between the lp and sp viruses, we sequenced the whole coding sequence of denv-2 8h2-7-lp and denv-2 1d4-5-sp, and aligned their envelope sequence with those of representative viruses belonging to different denv-2 genotypes, including asian genotype, american asian genotype, cosmopolitan genotype and sylvatic genotype. additionally, denv-2 strains isolated in guangdong, china, from 2005 to 2014 were also included for comparison. the phylogenetic analysis showed that denv-2 8h2-7-lp and denv-2 1d4-5-sp belonged to the cosmopolitan genotype and were clustered together with many strains isolated in west pacific regions, with percentages of homology ranging from 99.5% to 100% (fig. 2) . further comparison of genomic information between these two viruses also indicated that they had sequence variations in coding regions as illustrated in table 1 . collectively, we found 14 sequence variations in e, ns3, ns4b and ns5 regions, including 7 synonymous mutations and 7 amino acid changes. among the 7 nonsynonymous mutations, two are in domain i/ii regions of e protein, two are in peptidase domain and helicase domain of ns3 respectively, another is located in a transmembrane helix of ns4b, and the rest two are in ns5 rdrp domain. all of these domains are components of viral binding, replication or processing machinery, and highly related to viral antagonisms to host immunity. the conservation of mutations was also analyzed in comparison with representative strains from different genotypes, interestingly, though most synonymous mutations were also observed in other dengue-2 viruses, the seven nonsynonymous mutations were unique, and these sites seem more conserved in dengue-2 viruses (table 1) . these genomic characteristics may contribute to the different infectivity both in vitro and in vivo. as type i/ii interferon deficient mice are more susceptible to dengue virus infection, we determined the in vivo infectivity of these two viruses in type i and type ii ifn receptors double knock-out ag6 mice. ag6 mice between 6 and 8 weeks old were infected through s.c. injection with 10 3 pfu, 10 4 pfu and 10 5 pfu denv-2 8h2-7-lp or denv-2 1d4-5-sp (fig. 3) . it is observed that infection of either virus led to 100% mortality within 7-11 days, even at a dose as low as 10 3 pfu, though denv-2 8h2-7-lp caused more weight loss and faster death than denv-2 1d4-5-sp at the same dosage. specifically, weight loss began on 3 dpi, 4 dpi and 5 dpi in mice infected with 10 5 , 10 4 , and 10 3 pfu denv-2 viruses were used to infect cells at moi 2.5, supernatant of infected cells were collected at the indicated timepoints. titers of infectious virus in supernatant were measured using foci forming assay in vero cells. detection limits of infectious virus titer were indicated with dashed lines. significance of the differences was calculated with two-way anova test, ***p \ 0.001, n.s., not significant. 8h2-7-lp, respectively (fig. 3a , 3b, 3c), and death began on 7 dpi, 8 dpi and 9 dpi correspondingly (fig. 3d, 3e, 3f ). in contrast, weight loss was first observed 1-3 days later in mice infected with denv-2 1d4-5-sp, on 5 dpi or 6 dpi with inoculation of 10 4 -10 5 pfu or 10 3 pfu, respectively. the death was also delayed, began on 8 dpi or 9 dpi correspondingly. as a mixture of two types of viral variants, the parental strain was also able to cause weight loss and lethal diseases in ag6 mice. comparing to the two purified variants, the parental quasispecies presented intermediate level of pathology, which was most obvious in the dose groups of 10 5 pfu. through evans blue staining, we also detected plasma leakage in sick mice on day 7 post infection by either denv-2 8h2-7-lp or denv-2 1d4-5-sp viruses (fig. 4) . consistent with the progress of weight loss and survival curves, leakage of evans blue in liver or digestive tract was more severe in mice infected with 8h2-7-lp on 7 dpi. the evidence above demonstrated that the two viral isolates show differences on the pathology and disease kinetics, though they both have capabilities for causing lethal infection in ifn deficient mice. although both viruses can cause lethal infection in type i and type ii double knock-out mice, whether the lp and sp viruses behave differently in type ii ifn competent host is unknown. we have recently developed a zikv infection model in balb/c mice with transient blockage of type i ifn fig. 2 phylogenetic analysis of denv-2 1d4-5-sp and denv-2 8h2-7-lp with representative serotype-2 dengue viruses of different genotypes isolated from different geographical regions. the phylogenetic tree was obtained after analyzing envelope sequences of denv-2 with mega x software, using the maximum likelihood method and kimura 2-parameter model. members of six reported genotypes of denv-2 were included. denv-2 1d4-5-sp (mn952967) and denv-2 8h2-7-lp (mn952966) were denoted by red dots in the tree. the scale bar denotes an evolutionary distance of 0.02 nucleotides per position in the sequence. virologica sinica receptor (ifnar) (liang et al. 2018) , we thus used the same strategy to examine these two dengue viruses for further comparison. wt balb/c mice were injected with antibody specific for ifnar at one day prior to s.c. infection with 10 6 pfu of either viral strain, and monitored for seven days. the viral rna of denv-2 8h2-7-lp could be detected throughout the week after infection, and the peak rna level of about 10 6 genomic copy per microgram total rna was reached on day 3 post infection, followed by gradually dropping to 10 4 copy on the 7th day (fig. 5a) . in contrast, viral rna of denv-2 1d4-5-sp was not detected until day 4 post infection, when merely 10 4 copy per microgram of total rna was transiently detected for 2 days. consistently, infectious virus was detected in sera of denv-2 8h2-7-lp infected mice from 2 to 4 dpi, during which peak viremia of 10 3 pfu/ml appeared on day 3 post infection. meanwhile, infectious virus was not detectable in mice infected by denv-2 1d4-5-sp (lower than detection limit of 20 pfu/ml) (fig. 5b) . moreover, 8h2-7-lp viral rna was also detected in liver, spleen and eyes on 3 dpi (fig. 5c ), further confirming replication of 8h2-7-lp in susceptible organs. the parental strain also replicated in balb/c mice ( fig. 5a and 5b) , and the kinetics of viremia were closer to that of 8h2-7-lp, except for the delay of peak viremia to the 4th day post infection in both assays. to examine whether the above observed different virological properties of the two denv variants affect adaptive immunity in host, we evaluated adaptive immune responses induced by their infection in balb/c mice. denv-2 8h2-7-lp infection elicited antibodies crossneutralizing the reference strain denv-2 16681 (fig. 6a ) and it also stimulated t cell responses specific to peptides of denv-2 ns1 and zikv ns3 (fig. 6b) . similarly, neutralizing antibody and t cell responses were also detected in mice which were previously infected by denv-2 1d4-5-sp, though both responses were weaker than those elicited by denv-2 8h2-7-lp. considering genome similarity between the two variants, such different magnitudes of adaptive immune responses are mainly related to the different viral replication of two variants and different expression level of immunogens in vivo. in summary, we isolated from the same viral quasispecies two denv variants that showed distinct in vitro replication phenotype in vero cells, and different in vivo infectivity and pathogenicity in mice. using either of these two viral variants, we have established a lethal infection model in immune deficient ag6 mice. with the variant denv-2 8h2-7-lp, we have also developed a self-limited infection model in wt mice pretreated with type-i ifn receptor antibodies. these two closely-related viruses not only offer new tools for in vivo studies, but also provide table 1 sequence variation between 8h2-7-lp and 1d4-5-sp. in this study, we have successfully separated two viral variants that show different foci sizes in vero cells from a common serotype-2 clinical isolate of dengue virus. using these two viral variants, we established mouse infection models that reproduce severe manifestations of dengue diseases in ag6 mice. such lethal model with 8h2-7-lp or 1d4-5-sp viruses in ag6 mice can be used in pathology study, for example, to explore the viral and host factors that mediate hemorrhagic manifestation of dengue viruses. on the other hand, denv-2 8h2-7-lp was used in another infection model which resembles self-limited infection of dengue virus in balb/c mice. and this transient infection model is more suitable for study of t cell responses, since the immune cell lineages are intact and the ifn-c responses are normal during the development of adaptive immunity. additionally, this self-limited infection model is also suitable for investigating how murine host resolves dengue infection. in previous investigations of dengue infection, only a limited number of viral strains were found to infect immune deficient mice, in which human illness without neurological disease can be studied, and lethal doses of these viruses ranged from 10 4 to 10 7 pfu (sarathy et al. 2015) . one often used virus is dengue serotype-2 strain d220, which was obtained from a neuropathic strain pl046 through alternative passage between c6/36 mosquito cells and ag129 mice. the ld50 of this virus in ag129 is 10 5 -10 6 pfu (orozco et al. 2012) . another is d2y98p-pp1, a double-plaque purified clone from a laboratory strain d2y98p, which had been passaged in c6/36 cell for 20 times. and the lethal dose of d2y98p-pp1 in ag129 mice fig. 3 weight loss and survival curve of ag6 mice after infection with denv-2 8h2-7-lp and denv-2 1d4-5-sp. ag6 mice between 6-8 weeks old were infected with two viral variants and the parental isolate through s.c. injection. three different dose of viruses, 10 3 pfu (a, d; n = 3), 10 4 pfu (b, e; n = 3), and 10 5 pfu (c, f; lp and sp n = 4, parental n = 3), were inoculated into mice. mice injected with pbs were included within each panel for control (ct, n = 6). weight change was recorded and presented as a ratio to the initial weight on day 0. moribund mice with weight loss over 20% were euthanatized and recorded as death. two-way anova was used in statistical analysis of differences among groups, *p \ 0.05, **p \ 0.01, ***p \ 0.001,**** p \ 0.0001, n.s., not significant. was documented higher than 10 5 pfu (tan et al. 2011) . in contrast, the virus strains in this study had 100% mortality within 11 days after inoculation in ag6 mice of 6-8 weeks old even when the dose was reduced to 10 3 pfu, i.e., approximately 100 times more lethal than the d220 and d2y98p-pp1 viral strains, and thus making denv-2 8h2-7-lp virus a unique tool for studying the pathogenesis of dengue virus. through evans blue staining, we also confirmed the manifestation of plasma leakage in infected mice, which is a characteristic of severe dengue diseases in human. collectively, we show the highly virulent dengue strain 8h2-7-lp is ideal for establishing lethal infection models at low inoculum. to increase the utility of the new dengue viral variants in the context of relatively normal immune system, we have also adapted a recently developed model that transiently blocks ifnar with antibodies in balb/c mice. high level of viral rna was maintained within the first week after infection by denv-2 8h2-7-lp, but only low level of transient viral rna was detected at 3-4 dpi in mice administered with denv-2 1d4-5-sp. though no clinical symptom was observed, in mice infected with denv-2 8h2-7-lp, we successfully detected infectious virus in sera, and viral rna in multiple organs including liver, spleen and eyes. the viruses might have been eliminated from mice after 7 days of infection, accompanied by the maturation of denv adaptive immunity. thus, such characteristic and progress of infection is consistent with what was observed in patients with mild disease (who 2009). in previous animal models, denv infection of adult wt mice required extremely high inoculum, intracranially injection, or mouse adapted viruses. a portion of these models led to neurological symptoms or additional clinically irrelevant manifestations, limiting their use for pathology and therapeutic study (plummer and shresta 2014; sarathy et al. 2015) . to our knowledge, this is the first study which developed a denv infection model in wt mice, using a moderate dose of a non-adapted virus strain through physiologically more relevant s.c. injection route. it is often observed that many viruses form heterogenous plaques of different sizes in vitro, indicating that viruses exist as quasispecies during transmission or passaging (kato et al. 2017; moser et al. 2018 ), but the virological differences among various phenotypic variants have not been carefully examined. clinically, such viral diversity may assist arbovirus to survive under different selection pressure from two fig. 4 vascular leakage in ag6 mice induced by infection of denv-2 8h2-7-lp and denv-2 1d4-5-sp. ag6 mice of 8 weeks old were infected with 10 5 pfu denv-2 8h2-7-lp or 1d4-5-sp through s.c. injection. seven days post infection, mice were injected intravenously with evans blue and denv-induced vascular leakage was visualized in different tissues. pbs infected mice were used as negative controls. female balb/c mice of 6-8 weeks old were injected through i.p. with 2 mg antibody to type i ifn receptor (mar1-5a3) at one day prior to infection. on the next day, 10 6 pfu of viruses were inoculated into mice through s.c. route. then, blood samples were collected daily after infection. a viral rna copies in blood were measured using qrt-pcr, two-way anova was used in statistical analysis of the differences among groups, **p \ 0.01, ****p \ 0.0001, and b titers of infectious virus in serum samples were determined through foci forming assay in vero cells; n = 5. t test was used in statistical analysis of the differences among groups, *p \ 0.05, **p \ 0.01, *** p \ 0.001, ****p \ 0.0001. c eye, liver and spleen were collected from 8h2-7-lp virus-infected mice, and viral rna loads were determined, n = 4. detection limits of rna copy and infectious virus titer were indicated with dotted lines. n.d. not detected. distinct hosts, invertebrate and vertebrate. denv-2 8h2-7-lp exhibits better replication capability than denv-2 1d4-5-sp in vero cells, whereas their growth in mosquito cells is similar, reflecting possibility that the large-plaque variants in the original isolate contribute more to viral fitness in mammalian host. consistently, in mouse model, denv-2 8h2-7-lp presents higher pathogenicity in ag6 mice and better replication in balb/c mice. comparing the coding sequence of denv-2 8h2-7-lp and denv-2 1d4-5-sp, we found 7 amino acid variations on e, ns3, ns4b and ns5 proteins. e protein is responsible for virus attachment and entry; ns3, ns4b and ns5 are all the components of replication complex and essential antagonists for type i ifn responses (green et al. 2014 ). thus, further investigation of the underlying mechanism for the different phenotypes of these two viruses is valuable for understanding the molecular basis of dengue virulence and denv-host interaction. denv inhibits type i ifn production in infected cells by cleaving human sting humanized rag1-/-gammac-/-mice support multilineage hematopoiesis and are susceptible to hiv-1 infection via systemic and vaginal routes mouse stat2 restricts early dengue virus replication the global distribution and burden of dengue both virus and tumor necrosis factor alpha are critical for endothelium damage in a mouse model of dengue virus-induced hemorrhage regulation of antiviral t cell responses by type i interferons innate immunity to dengue virus infection and subversion of antiviral responses dengue: a continuing global threat experimental studies on dengue. i. isolation, identification and modification of the virus at 40 days post infection, frnt (foci reduction neutralization test) a and ifn-c elispot assay (b) were performed with mouse sera and splenocytes, respectively. reference strain denv-2 16681 was used in frnt assay, peptides and proteins derived from denv-2 16681 (d2-ns1 265, d2-ns1, d2-e80) and zika virus (z-ns3 43, z-ns3 291) were included in elispot assay to measure specific and crossreactive responses dengue fever in china: an emerging problem demands attention characterization of large and small-plaque variants in the zika virus clinical isolate zikv/hu/s36/chiba/2016 molecular epidemiology demonstrates that imported and local strains circulated during the 2014 dengue outbreak in guangzhou recombinant zika virus envelope protein elicited protective immunity against zika virus in immunocompetent mice growth and adaptation of zika virus in mammalian and mosquito cells characterization of a model of lethal dengue virus 2 infection in c57bl/6 mice deficient in the alpha/beta interferon receptor stat2 mediates innate immunity to dengue virus in the absence of stat1 via the type i interferon receptor mouse models for dengue vaccines and antivirals gamma interferon (ifngamma) receptor restricts systemic dengue virus replication and prevents paralysis in ifn-alpha/beta receptor-deficient mice production of immunity to dengue with virus modified by propagation in mice mouse models of dengue virus infection for vaccine testing murine model for dengue virus-induced lethal disease with increased vascular permeability adaptive immune responses to primary and secondary dengue virus infections dengue viruses cleave sting in humans but not in nonhuman primates, their presumed natural reservoir elaboration of tetravalent antibody responses against dengue viruses using a subunit vaccine comprised of a single consensus dengue envelope sequence subcutaneous infection with non-mouse adapted dengue virus d2y98p strain induces systemic vascular leakage in ag129 mice in: dengue: guidelines for diagnosis, treatment, prevention and control, new edn. who guidelines approved by the guidelines review committee dengue virus targets the adaptor protein mita to subvert host innate immunity epidemiological and virological characterizations of the author contributions js and xj designed and supervised the study. zhz and js carried out the experiments. zhz, js and xj analyzed the data and wrote the paper. zhl and ml assisted with animal experiments on ag6 mice. all authors read and approved the final manuscript. conflict of interest all authors declare that they have no conflict of interest. key: cord-273712-r2akpce8 authors: wang, jingjing; deng, feng; ye, gang; dong, wanyu; zheng, anjun; he, qigai; peng, guiqing title: comparison of lentiviruses pseudotyped with s proteins from coronaviruses and cell tropisms of porcine coronaviruses date: 2016-02-19 journal: virol sin doi: 10.1007/s12250-015-3690-4 sha: doc_id: 273712 cord_uid: r2akpce8 the surface glycoproteins of coronaviruses play an important role in receptor binding and cell entry. different coronaviruses interact with their specific receptors to enter host cells. lentiviruses pseudotyped with their spike proteins (s) were compared to analyze the entry efficiency of various coronaviruses. our results indicated that s proteins from different coronaviruses displayed varied abilities to mediate pseudotyped virus infection. furthermore, the cell tropisms of porcine epidemic diarrhea virus (pedv) and transmissible gastroenteritis virus (tgev) have been characterized by live and pseudotyped viruses. both live and pseudoviruses could infected veroccl-81 (monkey kidney), huh-7 (human liver), and pk-15 (pig kidney) cells efficiently. ccl94 (cat kidney) cells could be infected efficiently by tgev but not pedv. overall, our study provides new insights into the mechanisms of viral entry and forms a basis for antiviral drug screening. [image: see text] coronaviruses (covs) are important infectious pathogens that are associated closely with respiratory and enteric diseases in humans and animals (perlman and netland, 2009; belouzard et al., 2012; li, 2013) . covs have a single-strand positive sense rna genome and consist of four groups: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus (table 1) . viral entry into cells is highly dependent on the interaction between viral particles and the host cells. covs use a variety of cellular receptors and co-receptors, including proteins and sugars, to facilitate their entry into cells. infection begins with an interaction between the virus and its specific receptors (li, 2015) . severe acute respiratory syndrome coronavirus (sars-cov) of group beta uses a type i integral membrane protein, angiotensin-converting enzyme 2 (ace2), as a receptor . there is no structural homology between sars-cov and the human coronavirus nl63 (hcov-nl63) receptor-binding domain (rbd), but they recognize the same receptor, ace2 (wu et al., 2011) . although the rbd crystal structures of transmissible gastroenteritis virus (tgev) and hcov-nl63 are similar, they use different receptors (reguera et al., 2012) . aminopeptidase n (apn), also known as cd13, is a type ii transmembrane protein (tusell et al., 2007) . tgev and porcine epidemic diarrhea virus (pedv) infect cells through interaction with porcine (p) apn (li et al., 2007; nam and lee, 2010) . human (h) apn is a receptor for hcov-229e (belouzard et al., 2012) . in the same beta group, the receptors for mouse hepatitis virus (mhv) and bovine coronavirus (bcov) are carcinoembryonic antigen-related cell adhesion molecule 1 (ceacam1) and a sugar, respectively, despite their high sequence homology peng et al., 2012) . ceacam1, the first identified coronavirus receptor (dveksler et al., 1991) , is a type i transmembrane multifunctional protein of the immunoglobulin superfamily (de haan et al., 2005) . cell entry and interspecies transmission of covs are mediated by the spike protein (s). cov s is a class i fusion protein (bosch et al., 2003) . in covs, s is a significant surface protein and plays an important role in mediating infection of virions (schwegmann-wessels et al., 2009) . s is also responsible for receptor binding and the fusion of viral and cellular membranes. all cov s share the same functional component in two domains: an nterminal domain called s1 that is responsible for receptor binding, and a c-terminal s2 domain that is responsible for fusion. s1 contains two subdomains, an n-terminal domain and a c-terminal domain ( figure 1a ). both function as rbds and bind a variety of proteins and sugars (belouzard et al., 2012) . because some covs have strong pathogenicity, the lentiviral pseudotype system is a reliable tool to study the proteins of highly pathogenic viruses under conventional biosafety conditions. hiv-luc is an hiv-1 based lentiviral vector bearing the luciferase reporter gene and has been used in the production of pseudotyped viruses lu et al., 2012; tang et al., 2012) . pseudovirus entry efficiency is characterized based on the luciferase activity. viral particles pseudotyped by various s proteins have been described for several viruses. in our study, we compared the efficiency of pseudotyped viruses with s proteins from different groups of covs. furthermore, the cell tropisms of tgev and pedv were characterized by live and pseudotyped viruses. 293t cells were cultured and maintained in rpmi 1640 medium (invitrogen, carlsbad, ca, usa) supplemented with 10% fetal bovine serum (fbs) at 37 °c in an atmosphere of 5% co 2 . pk-15, huh-7, vero-ccl-81, mdbk, ccl94, bsr, and mdck cells were cultured and maintained in dulbecco's modified eagle's modium (dmem) (invitrogen) supplemented with 10% fetal bovine serum (fbs) at 37 °c in an atmosphere of 5% co 2 . the pedv and tgev strains (genbank accession numbers: kt021232.1 for pedv, hq462571.1 for tgev) were isolated from a suckling piglet. rabbit anti-pedv n protein and rabbit anti-tgev n protein polyclonal antibodies are stored by the lab. the s protein sequences of different groups of covs (genbank accession numbers: abd72982.1 for sars-cov-s, aar92025.1 for mhv-s, np_073551.1 for hcov-229e-s, abg89335.1 for tgev-s, np_598310.1 for pedv-s, aat84362.1 for hcov-oc43-s, abm66810.1 for bcov-s, and np_040831.1 for avian infectious bronchitis virus (ibv) s) were codon-optimized and synthesized (genscript, nanjing, china). the s fragments were cloned into the pcdna3.1 (+) vector with a c-terminal his-tag. on the day before transfection, 293t cells were seeded in six-well plates. the next day, the cells were transfected with s plasmids plus the lentiviral vector hiv-luc lentiviruses pseudotyped with s proteins were solubilized by boiling in sodium dodecyl sulfate sample buffer. the s proteins were fractionated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. the nitrocellulose membrane was incubated with a 1:4000 dilution of a mouse anti-his antibody (proteintech group, chicago, il, usa). goat anti-mouse igg (boster, wuhan, china) was used as the secondary antibody. a plasmid (vector pcdna 3.1(+)) with each viral receptor gene (ace2, ceacam1, hapn, and papn) was transfected into 293t cells with lipofectamine 2000. after incubation for 5-6 h at 37 °c, the supernatant was replaced by dmem containing 2% fbs. after 48 h, the cells were infected by lentiviruses pseudotyped with the s proteins from covs of different groups. for analysis of the lentiviruses pseudotyped with the s proteins from covs of different groups, different cell lines were seeded in 96-well plates. 293t cells expressing receptors were infected by the same lentiviruses and incubated at 37 °c for 2 h. dmem (150 µl) containing 2% fbs was added subsequently to all cells and incub-ated for an additional 48-72 h. the pseudovirus entry efficiency was characterized based on luciferase activity. pedv and tgev were used to infect various cell lines from different species, including pk-15 (pig kidney), huh-7 (human liver), vero-ccl-81 (monkey kidney), mdbk (bovine kidney), ccl94 (cat kidney), bsr (hamster kidney), and mdck (canine kidney) cells at a multiplicity of infection of 0.01. trypsin (10 µg/ml) was included in the cell culture medium to facilitate live virus infections. cells infected with viruses were fixed with 4.0% (v/v) paraformaldehyde at 24 or 48 h post-inoculation. pedv or tgev was detected with fluorescein isothiocyanate-labeled rabbit anti-pedv or anti-tgev n protein antibodies, respectively, and observed under a fluorescence microscope. all experiments were done at least twice (in most cases three or more times). data were analyzed statistically using two-tailed student's t-tests for comparison of pcdna3.1 and cov s pseudovirus. the pseudovirus entry efficiency is reported as the luciferase activity. statistical analyses were performed to compare the treatment groups: ***p < 0.001; **0.001 < p < 0.01. cov diversity is reflected in the variable s proteins (heald-sargent and gallagher, 2012). therefore, we compared the s protein amino acid sequences of covs from different groups, including sars-cov, mhv, hcov-229e, tgev, pedv, hcov-oc43, bcov, and ibv (gen-bank accession numbers abd72982.1, aar92025.1, np_073551.1, abg89335.1, np_598310.1, aat84362.1, abm66810.1, and np_040831.1, respectively) using clustalw ( figure 1b ). because s1 is responsible for receptor binding, the amino acid sequences of the s1 domain were aligned ( figure 1c ). the s proteins from covs of the same group shared sequence similarities of greater than 20%, with some similarities of up to 40%. the s1 sequence similarities of covs from different groups were less than 12%. to determine the success of pseudovirus construction, the s proteins of pseudoviruses were confirmed by western blotting (figure 2a ). the pseudoviruses were quantified with an hiv-1 p24 eia kit and used to infect cells expressing the corresponding receptor. lentiviruses pseudotyped with sars-cov s protein could efficiently infect 293t cells expressing ace2, and the pseudovirus level after entry reached 10 6 relative light units (rlu) ( figure 2b ). the s proteins of hcov-229e, tgev, and pedv exhibit high homology. however, their pseudovirus infection efficiency varies, and they are approximately 50-fold less efficient than the sars-cov-s pseudovirus. the tgev-s and pedv-s pseudoviruses showed similar infection efficiencies. although hcov-oc43, bcov, and ibv utilize a sugar as their receptors, their pseudovirus infection efficiency was not clear. tgev-s and pedv-s pseudoviruses were used to infect pk-15, huh-7, vero-ccl-81, mdbk, ccl94, bsr, and mdck cells ( figure 2c ). the tgev-s and pedv-s pseudoviruses could enter pk-15, huh-7, and vero-ccl-81 cells with a similar efficiency. however, there was an intriguing finding that ccl94 cells could be infected efficiently by tgev-s but not pedv-s pseudoviruses. to further study the cellular entry of covs, we used live pedv and tgev to infect different cell lines. pedv efficiently infected vero-ccl-81 (monkey kidney), huh-7 (human liver), and pk-15 (pig kidney) cells (figure 3) . infection was not evident in mdbk (bovine kidney), ccl94 (cat kidney), bsr (hamster kidney), or mdck (canine kidney) cells. this is consistent with the data in figure 2c . tgev efficiently infected cells from pigs (pk-15), humans (huh-7), monkeys (vero-ccl-81), and felines (ccl94) (figure 4) . overall, our study demonstrates that pedv and tgev can infect cells from different species. covs recognize a variety of cell-surface molecules as their host receptors, including proteins, sugars, and heparan sulfate (li, 2013) . the cell entry mechanisms of these viruses are mediated by the viral s proteins that bind cellular receptors and mediate the fusion of viral and cellular membranes (heald-sargent and gallagher, 2012) . we demonstrated that s proteins from covs of different groups played an important role in mediating viral infection at different efficiencies. sars-cov-s pseudoviruses efficiently entered cells expressing ace2. this result is consistent with previous reports demonstrating that ace2 is a receptor for sars-cov (schwegmann-wessels et al., 2009; wu et al., 2011) . hcov-229e-s and mhv-s pseudoviruses showed strong infectivity, in part because they use proteins as a receptor belouzard et al., 2012) . moreover, pedv-s and tgev-s pseudoviruses had similar infection efficiencies in pk-15, huh-7, or vero-ccl-81 cells, and tgev-s pseudovirus-infected ccl94 cells. the sugar moiety 5-n-acetyl-9-o-acetylneuraminic acid (neu5, 9ac2), found on cell-surface glycoproteins or glycolipids, is recognized by bcov and hcov-oc43. in addition, two other types of sugars, 5-n-glycolylneuraminic acid (neu5gc) and 5-n-acetylneuraminic acid (neu5ac), can serve as receptors or co-receptors for some alpha-and gamma-covs (cavanagh and davis, 1986; krempl et al., 1997; peng et al., 2012; shahwan et al., 2013) . the s-to-sugar binding affinity is lower than the s-protein interaction, which partially explains why the bcov and ibv pseudoviruses have low entry efficiency. although pedv and tgev use the same receptor (papn) and the s proteins present high homology, live pedv and tgev prefer different cell lines; pedv prefers vero-ccl-81, huh-7, and pk-15 cells. this is consistent with a previous report (liu et al., 2015) . however, tgev prefers vero-ccl-81, huh-7, pk-15, and ccl94 cells. this indicates that pedv and tgev may have different species preferences. these results are consistent with the observation that their receptor-binding domains are located in non-homologous regions (belouzard et al., 2012) . however, the mechanism underlying different cell tropisms requires further investigation. the s protein plays a crucial role in entry into host cells by mediating receptor binding and membrane fusion. covs use a variety of receptors and triggers to activate fusion (belouzard et al., 2012) . the first and most important step of virus infection is the interaction between the virus and its cellular receptor. infection of lentiviruses pseudotyped shows that s proteins from different covs have varied abilities to mediate pseudotyped virus infection in different cell types from different tissues. these findings further our understanding of the mechanisms underlying viral invasion and contribute to the development of drugs against covs. this work was supported by the national natural science foundation of china (grant no. 31372440). this article does not contain any studies with human or animal subjects performed by any of the authors. there is no competing interest in this research. g.p. and j.w. designed the experiments and wrote the paper. j.w. performed the experiments. f.d. and q.h. contributed to critical discussions of the data. all authors approved the final manuscript. mechanisms of coronavirus cell entry mediated by the viral spike protein the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex coronavirus ibv: removal of spike glycopolypeptide s1 by urea abolishes infectivity and haemagglutination but not attachment to cells murine coronavirus with an extended host range uses heparan sulfate as an entry receptor cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence ccr5 antagonist td-0680 uses a novel mechanism for enhanced potency against hiv-1 entry, cell-mediated infection, and a resistant variant point mutations in the s protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus porcine aminopeptidase n is a functional receptor for the pedv coronavirus receptor recognition and cross-species infections of sars coronavirus receptor recognition mechanisms of coronaviruses: a decade of structural studies structure of sars coronavirus spike receptor-binding domain complexed with receptor receptor usage and cell entry of porcine epidemic diarrhea coronavirus f18, a novel small-molecule nonnucleoside reverse transcriptase inhibitor, inhibits hiv-1 replication using distinct binding motifs as demonstrated by resistance selection and docking analysis contribution of the porcine aminopeptidase n (cd13) receptor density to porcine epidemic diarrhea virus infection crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor crystal structure of bovine coronavirus spike protein lectin domain coronaviruses post-sars: update on replication and pathogenesis structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies comparison of vesicular stomatitis virus pseudotyped with the s proteins from a porcine and a human coronavirus sialic acid binding properties of soluble coronavirus spike (s1) proteins: differences between infectious bronchitis virus and transmissible gastroenteritis virus a single residue substitution in the receptor-binding domain of h5n1 hemagglutinin is critical for packaging into pseudotyped lentiviral particles mutational analysis of aminopeptidase n, a receptor for several group 1 coronaviruses, identifies key determinants of viral host range a virus-binding hot spot on human angiotensin-converting enzyme 2 is critical for binding of two different coronaviruses key: cord-273373-5elel6qo authors: wang, haofeng; xue, song; yang, haitao; chen, cheng title: recent progress in the discovery of inhibitors targeting coronavirus proteases date: 2016-02-19 journal: virol sin doi: 10.1007/s12250-015-3711-3 sha: doc_id: 273373 cord_uid: 5elel6qo coronaviruses (covs) can cause highly prevalent diseases in humans and animals. the fatal outbreak of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) highlights the threat posed by this unique virus subfamily. however, no specific drugs have been approved to treat cov-associated diseases to date. the cov proteases, which play pivotal roles in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, are attractive targets for drug design. this review summarizes the recent advances in biological and structural studies, together with the development of inhibitors targeting cov proteases, particularly main proteases (m(pro)s), which could help develop effective treatments to prevent cov infection. [image: see text] before the outbreak of the severe acute respiratory syndrome (sars) epidemic worldwide in the winter of 2002 to the summer of 2003, coronaviruses (covs) had long been considered to cause merely mild symptoms in humans despite being attributed to severe diseases in animals (dominguez et al., 2003; drosten et al., 2003; franco-paredes et al., 2003; kuiken et al., 2003; rota et al., 2003) . the sars epidemic, caused by the sars coronavirus (sars-cov), resulted in a total of 775 deaths out of 8,273 cases of infection (dominguez et al., 2003; franco-paredes et al., 2003) . in 2012, another new cov called the middle east respiratory syndrome coronavirus (mers-cov) emerged, which originated from the middle east (zaki et al., 2012) . according to the world health organization, this new cov has thus far resulted in 1,633 cases of infection causing 587 deaths (http:// www.who.int/emergencies/mers-cov/en/). both sarsand mers-covs have zoonotic origins (woo et al., 2009; woo et al., 2012; chan et al., 2013; ge et al., 2013; lu et al., 2015; st john et al., 2015) , strongly suggesting that animal covs remain a potential threat to humans. however, no specific drugs have been approved to treat cov-associated diseases to date. in the wake of the sars outbreak, in-depth research has revealed multiple aspects of the life cycle of cov infection. covs mainly take advantage of spike proteins to bind their receptors for attachment to the host cell membrane (ng et al., 2003) . upon entering host cells, covs start to release genomic rna. subsequent expression of two polyproteins responsible for viral replication and transcription, respectively, requires hijacking of host ribosomes to facilitate translation . the two resulting polyproteins are further processed by two viral-encoded proteases into 16 mature nonstructural proteins (nsps): nsp1, a host gene expression suppressor; nsp2, which functions in interacting with the two host proteins; nsp3,a multi-domain complex; nsp4/nsp6, transmembrane proteins; nsp5, the m pro ; nsp7/nsp8, primases; nsp9, a single-stranded rna-binding protein; nsp12, an rna-dependent rna polymerase; nsp13, 5′to-3′ rna helicase/ntpase/rna 5′-triphosphatase; nsp14, 3′-to-5′ exoribonuclease; nsp15, uridylate-specific endoribonuclease; and nsp16, 2′-o-methyltransferase (cornillez-ty et al., 2009; te velthuis et al., 2012; ivanov et al., 2004; narayanan et al., 2008; snijder et al., 2003; sutton et al., 2004; ziebuhr, 2005) . these nsps can then assemble into the replication-transcription complex and facilitate viral rna replication and transcription (zhao et al., 2013a) . currently, several viral proteins are considered candidate targets for drug development, including proteases, helicases, rdrp, and methyltransferase, which are all involved in the aforementioned viral life cycle (adedeji and sarafianos, 2014) . among them, m pro is an important target, owing to its essential role in the processing of polyproteins (ziebuhr et al., 2000) . in this review, we will focus on recent progress in the development of inhibitors of these proteases, with a particular focus on m pro (table 1) . papain-like protease (pl pro ) ( figure 1a ) exists as a functional domain within the large nsp3, which also encodes a ubiquitin-like fold, an adp-ribose-1d-phosphatase domain, a sars-cov-unique domain, and a transmembrane domain (lee et al., 2015) . pl pro recognizes a specific site comprising the consensus cleavage sequence lxgg between nsp1/2, nsp2/3 and nsp3/4 (lee et al., 2015) . proteolytic cleavage of the peptide bond after the glycine residue can result in the release of nsp1, nsp2, and nsp3 from the viral polyproteins, which is a critical step for viral replication (ziebuhr et al., 2000) . pl pro has been found to have deubiquitination and deinterferon stimulated gene-ylation (isgylation) functions . this can help the virus to escape the host immune response via the interferon signaling pathway (baez-santos et al., 2015) . in mers-cov, pl pro has been reported to protect polyproteins from cleavage through the interferon regulatory factor 3 pathway (sun et al., 2012) . pl pro s from different cov species may also vary in substrate specificity (lee et al., 2015) , which may contribute to the difficulty in developing a wide-spectrum drug targeting covs. nonetheless, some molecules have been found to effectively inhibit cov infection, including zinc ion (zn 2+ ) and zinc conjugate inhibitors, which were reported to target pl pro (han et al., 2005) . however, these inhibitors appear to be unique, because no other metal ions have thus far been found to possess such inhibitory capacity (baez-santos et al., 2015) . in addition, thiopurine compounds could suppress the activity of pl pro s by covalently binding to the cysteine residue at the active site (chen et al., 2009) . some natural products have also been found to inhibit pl pro activity, including tanshinones (park et al., 2012b) , diarylheptanoids (park et al., 2012a) , and geranylated flavonoids (cho et al., 2013) . recently, several crystal structures of sars-cov pl pro in complex with inhibitors have been reported (ratia et al., 2008; ghosh et al., 2009; ghosh et al., 2010; baez-santos et al., 2014) ( figure 1a-1d ). sars-covpl pro contains two blocking loops, bl1 and bl2, which have proven to be vital in blocking access to the catalytic site (lee et al., 2015) . upon binding to the bl2 loop, these inhibitors cause a local conformational change and subsequently block access for substrate binding to the catalytic site (lee et al., 2015) . the cov genome encodes two overlapping polyproteins designated as pp1a and pp1ab (mukherjee et al., 2008) . the polyproteins are cleaved by viral proteases to yield the 16 mature nsps mentioned above, and assemble into the replication-transcription complex that is essential for viral replication. m pro is the key enzyme involved in this proteolysis process (ziebuhr et al., 2000) , and is responsible for cleavage at 11 sites on the polyproteins (ziebuhr, 2004) . owing to its dominant and indispensable role in polyprotein processing, m pro has thus emerged as an important target for anti-cov drug design. many of the inhibitors identified to date, such as peptidomimetic (ghosh et al., 2007) , tg-0205221 (yang et al., 2006) , ketones (shao et al., 2008) , and pyrimidines (ramajayam et al., 2010) , have previously been reviewed in detail (mukherjee et al., 2011; zhao et al., 2013a; zhao et al., 2013b; adedeji and sarafianos, 2014) . therefore, we here focus only on the most recent progress in inhibitor and drug discovery. in the last two years, many research groups have exerted continuous effort on the development of inhibitors targeting cov m pro s (figure 2a ). chen et al. and zhou et al. reported a new class of isatin derivatives as m pro inhibitors zhou et al., 2006) . based on their results, liu et al. started to investigate the potential of using isatin derivatives to design more efficient inhibitors against cov m pro s (liu et al., 2014) . in these studies, a series of isatin derivatives were designed, synthesized, and assessed by in vitro enzymatic assays with a fluorogenic substrate peptide. among them, the 5-sulfonyl isatin derivatives with a carboxamide group substituted by a series of sulfonamide groups showed inhibition against m pro at the micromolar range. assisted by a structure-based lead compound design, the authors effectively optimized the 5-sulfonyl isatin derivatives and obtained an optimal compound named 8k 1 (ic 50 = 1.04 μmol/l) ( figure 2b ). by comparisons of different substituted groups, they reached the conclusion that 5-sulfonyl isatin coupled with methyl at the n-1 position could dramatically promote inhibitory activity, which could facilitate further lead compound development. akaji's group reported a novel inhibitor with a decahydroisoquinolin scaffold that could target sars-cov m pro (shimamoto et al., 2015) ( figure 2c ). previous studies showed that the hydrophobic interactions at the s2 site are important for substrate/inhibitor binding. this new inhibitor was designed by connecting the substrate-based inhibitor's cyclohexyl group, located at the p2 site, with the α-nitrogen atom of the p2 position on the main chain. this approach could maintain the hydrophobic interactions at the s1 and s2 sites, which kept the imidazole and aldehyde groups in the required configuration for respective binding. the authors further synthesized a series of decahydroisoquinolin derivatives, all of which showed moderate but clear inhibitory activities against sars-cov m pro . x-ray crystallographic studies revealed that the decahydroisoquinolin scaffold was inserted into a large s2 pocket, which occupied most of the pocket to hold the terminal aldehyde tightly inside the active site cleft, resulting in the observed inhibitory activity. previously, our group investigated the conservation of cov m pro s across species. structural analyses revealed that the substrate-binding pockets of various cov m pro s are highly conserved, which led to the concept of "widespectrum inhibitors" for targeting all covs. through a structure-based drug design, we have identified a lead compound named n3 with potent inhibitory activity against all m pro s tested ( figure 2d) . recently, the wong group analyzed the substrate specificity of m pro from the p5 to p3' sites among several covs, and found that all m pro s have similar substrate specificity. this finding facilitated the design of a broad-spectrum peptidomimetic inhibitor against various cov m pro s. they identified a new peptidyl compound, cbz-tsavlq-cn, by coupling a nitrile warhead and a carboxybenzyl (cbz) group to the c-terminus and n-terminus of a peptide, respectively (chuck et al., 2014) . enzymatic assays revealed that it could inhibit m pro s from six different species of covs. the solved crystal structure of the enzyme in complex with this inhibitor indicated that the n-terminal cbz protective group could improve the interaction between the inhibitor and the protease. kim et al. also designed a series of peptidyl compounds targeting feline coronaviruses (fcov) and feline caliciviruses (fcv) (kim et al., 2015) . they reported that a compound named npi52 displayed inhibitory activity against fcov and fcv strains in cell culture ( figure 2e ). this inhibitory effect was further confirmed in a mouse model based on liver histopathology analysis. in addition, we have designed n3 and zinc ion as dual inhibitors against feline infectious peritonitis virus (fipv) m pro . the biochemical data showed that n3 and zn 2+ could serve as an irreversible inhibitor and a noncompetitive inhibitor, respectively. the solved crystal structure of fipv m pro in complex with these two inhibitors demonstrated their potential for synergistic activity to achieve an enhanced inhibitory effect . these findings provide a new approach in the design of anti-cov drugs. on the other hand, gaining a better understanding of the cleavage mechanism of cov m pro s could help to provide new directions for the design of inhibitors. dimerization is an essential step in m pro maturation for subsequent catalysis. shi et al. reported the mechanism of cov m pro for controlling the dimer-monomer switch (shi et al., 2008) . specifically, mutation of the residue arg298 at the dimer interface was shown to disturb the dimerization of m pro (paasche et al., 2014) , which could be reverted via a substrate-induced conformational change. moreover, structural studies revealed that the establishment of a new intermolecular interaction required domain iii composed of a globular cluster of five helices (anand et al., 2003) to alter its orientation. this suggests that m pro should be able to tolerate large conformational changes without loss of enzymatic activity. this further implies that intramolecular communication and regulation exists within the m pro structure. thus, better understanding of the underlying mechanism might aid in structure-based anti-cov drug design. paasche et al. reported substrate binding-induced zwitterion formation at the active site of sars-cov m pro (paasche et al., 2014) . they discovered that the cys -/his + zwitterionic state is essential for efficient proteolytic activity, which is fostered by substrate binding rather than by inhibitor binding, and enhances its substrate specificity. the free-energy calculations delineated that substrate binding could promote the proton transfer reaction, which reduces the energy between neutral and zwitterionic states. this implies that development of antiviral agents with ability of the substrate to trigger the zwitterionic state may be an alternative approach for drug design. in the wake of outbreaks of sars and mers, covs have emerged as life-threatening pathogens for humans. their zoonotic origin also sends a warning of the potential emergence of new deadly covs via interspecies 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potent sars coronavirus 3cl protease inhibitor isolation of a novel coronavirus from a man with pneumonia in saudi arabia drug targets for rational design against emerging coronaviruses recent developments on coronavirus main protease/3c like protease inhibitors isatin compounds as noncovalent sars coronavirus 3c-like protease inhibitors molecular biology of severe acute respiratory syndrome coronavirus the coronavirus replicase virus-encoded proteinases and proteolytic processing in the nidovirales the authors declare that they have no conflict of interest. this article does not contain any studies with human or animal subjects performed by any of the authors. key: cord-033952-b9fxqnni authors: lu, deng-hui; jiang, hong; lian, jian-qi title: hantavirus infection during pregnancy date: 2020-10-19 journal: virol sin doi: 10.1007/s12250-020-00300-8 sha: doc_id: 33952 cord_uid: b9fxqnni hantavirus infection is a global health challenge, causing widespread public concern. in recent years, cases of hantavirus infection in pregnant women have been reported in many countries. the infected pregnant women and their fetuses appear to have more severe clinical symptoms and worse clinical outcomes. hence, to study the prevalence of hantavirus infection in pregnant women, this study will focus on the epidemiological distribution of the virus, different virus species penetrating the placental barrier, and factors affecting the incidence and clinical outcome of the infection in pregnant women and their fetuses. in addition, this review will also discuss the diagnostic tools and treatments for pregnant patients and provide an overview of the relevant future research. hantaviruses are negative-sense, single-stranded, enveloped rna viruses belonging to the family hantaviridae. since the first hantavirus was isolated in 1978, many countries and regions have reported a series of hantavirus infections continuously . hantaviruses cause two clinical syndromes in humans; designated hemorrhagic fever with renal syndrome (hfrs), and hantavirus cardiopulmonary syndrome (hps). hfrs mostly occur in eurasian countries, caused by hantaan virus (htnv), seoul virus (seov), puumala virus (puuv) and dobrava virus (dobv) (avsic-zupanc et al. 2019) . the clinical features of hfrs are severe systemic manifestations, including fever, hemorrhage and acute renal failure. hps is mainly a group of syndromes with respiratory system involvement, caused by sin nombre virus (snv), andes virus, and other viruses. the leading cause of death in hps is acute progressive non-cardiogenic pulmonary edema and respiratory failure . hantaviruses are zoonotic viruses that can be carried by small rodents. approximately 20,000 cases of hantavirusrelated diseases occur worldwide each year (jiang et al. 2017) . furthermore, there is a clear indication that the incidence rate increases every year (jiang et al. 2017; watson et al. 2014 ). the mortality rates reported are 12% for hfrs and 60% for hps (zhang et al. 2010; jonsson et al. 2010) . pregnant women are afflicted by inhalation of host secretions and excretions carrying the virus, or by exposure to rodent carriers (pedrosa and cardoso 2011) . however, the cases of women infected with hantavirus during pregnancy are rarely reported even in areas where hantaviruses are concentrated, which poses certain difficulties for subsequent research and clinical management. here, in order to contribute to the diagnosis, treatment, and prognosis prediction, we reviewed past cases of hantavirus infections in pregnant women. the rodent host distribution, and it is influenced by factors such as the climate, environment, and food availability, which can make up a unique rodent host-hantavirus system. the rodent hosts of principal disease-causing hantaviruses are apodemus agrarius (htnv) in asia, myodes glareolus (puuv) in europe, and peromyscus maniculatus (snv) in the americas (watson et al. 2014; kariwa et al. 2007; vaheri et al. 2013 ). cases of pregnant women infected by htnv and seov hantaviruses are more than that infected by other hantavirus species (fig. 1, table 1 ). this is related to the high incidence rate and large population in the areas where these two virus species are primarily distributed. china and south korea are the top two hantavirus infection areas (kariwa 2017) , and this is consistent with the distribution of their rodent hosts, which is also in line with the final findings of this review. hantavirus infection with different species during pregnancy also has different results for pregnant women. as observed from our statistical review, the mortality rate of pregnant women infected with htnv/seov was 10.5% (27/256) (table 1), and that of those infected with snv was 25.0% (2/8) (table 1) . from the data we collected, the mortality rate of pregnant women infected with htnv was the sequelae mainly include menstrual disorders, amenorrhea, no milk secretion after delivery, sexual dysfunction, hair loss, autonomic dysfunction, and chronic renal insufficiency (hofmann et al. 2012; ji et al. 2017; howard et al. 1999; partanen et al. 1990; silberberg et al. 1993; prebensen 1997; tiilikainen and jouppila 1989; jing and jing 1994; wang et al. 1992; sha et al. 2000; mace et al. 2013; dai 2004; liu et al. 2013; georges et al. 2008; schneider et al. 2009; gilson et al. 1994; murthy et al. 2016; wang 2014; zhang 1995; duan et al. 1996; kim and choi 2006; zheng 1985; cui 2005; liu et al. 2017; todorovic et al. 2010; chen 2008; xie 1994; hao 1997; ying 1984; gao 2013; chen 1994; lu et al. 2018; li 2019; ma et al. 2003; latus et al. 2013 ). not significantly different from that of the general population, while that of those infected with snv was lower than that of the general population (zhang et al. 2010; jonsson et al. 2010) . we speculate that the sample size may primarily limit this. moreover, after being infected by a hantavirus, the residual sequelae, which mainly consist of menstrual disorders, amenorrhea, no milk secretion after delivery, sexual dysfunction, hair loss, autonomic dysfunction and chronic renal insufficiency, were only seen with htnv/seov infections (table 1) . furthermore, no deaths have been reported in pregnant women infected with puuv or dobv (hofmann et al. 2012; ji et al. 2017; partanen et al. 1993 ). however, puuv and dobv cannot be ruled out due to the limited number of cases ( table 1) . the clinical outcome for fetuses is also significantly different. since the death of the mother causes the death of the fetus in most cases, this review will only discuss the cases where the mother survived the hantavirus infection. according to the available data, fetal deaths were only reported in htnv/seov and snv infections ( table 2 ). the mortality rate of fetuses delivered by pregnant women with htnv/seov infections was 31.8% (71/223) (table 2), and that of those delivered by pregnant women with snv was 33.3% (2/6) ( table 2) . also, several sequelae, including congenital heart disease, necrotizing enteritis, restricted growth and development, as well as hydrocephalus, troubled these babies ( table 2 ). the incidence rate of sequelae in the babies, whose mothers had been infected with htnv/seov and snv was 3.6% (8/223) and 16.7% (1/6), respectively (table 2) . there were no reports of fetal malformations in pregnant women with a hantavirus infection, though many vertically transmitted viral infections are likely to cause fetal malformations (seferovic et al. 2018; silasi et al. 2015) . currently, there is no evidence that puuv, dobv and snv can be transmitted through the placenta (hofmann et al. 2012; partanen et al. 1993; howard et al. 1999) . partanen et al. (1990) presented a case, where a 29 yearold woman at 17 weeks gestation had suffered from acute abdominal pain with high fever and myalgia. as a result, she had been diagnosed with puuv infection. after treatment, the woman had recovered within 4 weeks and delivered a healthy boy naturally. besides, the postoperative serological testing showed no evidence of placental transmission. however, few cases of fetal death caused by puuv infection were also reported (partanen et al. 1993; silberberg et al. 1993; prebensen 1997; tiilikainen and jouppila 1989) . hofmann et al. (2012) conducted an immunological and molecular analysis of hantaviruses in the cord blood of four pregnant women, who had been infected with puuv or dobv. the results also showed no evidence of virus transmission through the placenta. howard et al. (1999) reviewed five cases of hps during pregnancy, and as expected, there was no evidence of vertical transmission, though snv infections mostly had severe consequences, even death, in pregnant women and fetuses. therefore, it could be speculated that htnv and seov pass through the placental barrier. liu et al. (1987) suggested that there is a phenomenon of vertical transmission between the mother and the baby. nonetheless, no evidence directly proves it. lee (1989) detected the presence of igm antibody against htnv in the blood of fetuses, whose mothers had a htnv infection, confirming the possibility of placental transmission. kim et al. (1978) studied a pregnant woman, who had had a miscarriage due to hypotension after seov infection. after the autopsy of the fetus, researchers found bleeding throughout the body, including lungs, kidneys, and adrenal glands. this is in line with the symptoms of seov infection, but unfortunately, they did not perform further serological testing of the fetus the sequelae mainly include congenital heart disease, necrotizing enteritis, restricted growth and development, and hydrocephalus (hofmann et al. 2012; ji et al. 2017; howard et al. 1999; partanen et al. 1990; silberberg et al. 1993; prebensen 1997; tiilikainen and jouppila 1989; jing and jing 1994; wang et al. 1992; sha et al. 2000; mace et al. 2013; schneider et al. 2009; gilson et al. 1994; wang 2014; zhang 1995; duan et al. 1996; kim and choi 2006; todorovic et al. 2010; chen 2008; xie 1994; hao 1997; ying 1984; chen 1994; lu et al. 2018; li 2019; ma et al. 2003; latus et al. 2013 ). (kim et al. 1978) . in addition jing pt and jing h (1994) , isolated hantaviruses from fetal brain tissues of an aborted fetus. unexpectedly, the results indicated that hantaviruses could pass not only the placental barrier but also the bloodbrain barrier. however, it is a pity that the study failed to identify the species. instead, it was based on the epidemiological analysis of the target area, which indicated that the virus was most likely seov. most of the abortion and stillbirth cases in patients with hfrs occur when they have a fever, so further research is needed to clarify the specific mechanism (chen 1994; wang et al. 1992; sha et al. 2000) . the clinical outcomes of pregnant women with a hantavirus infection are also related to their age. the mortality rate of women aged 30 or older and infected with hantaviruses during pregnancy is 45.5% (5/11) (table 3) , while for women under 30, it is only 2.5% (1/40) (table 3) . hjertqvist et al. (2010) found, in a study with 5,282 patients, that there is a significant correlation between mortality and the age of the hantavirus-infected patients. the mortality rates of pregnant women and others increase with age, and the trend remains consistent. the incidence of hantavirus infection in pregnant women varies in different gestational periods. by reviewing the literature, we conducted a statistical analysis of the gestational age of women with a hantavirus infection during pregnancy. among the reported cases of hantavirus-infected pregnant women, the incidence rate in the first trimester (\ 13 weeks) was 7.7% (4/52) ( table 3 ), whereas that in the second trimester (c 13 weeks, \ 28 weeks) and the third trimester (c 28 weeks) was 92.3% (48/52) (table 3) . coincidentally, this phenomenon was also found in other placenta-transmissible viruses. zhao (2017) conducted a controlled observation, including 288 cases of hpv-infected pregnant women at different stages of pregnancy, and found that the incidence of viral infection was the highest at the third stage. moreover, having researched epstein-barr virus infection in pregnant women at different stages of pregnancy, ming (2017) also reached the same conclusion. different mode of delivery may exert various influences on the transmission of the virus from mother to child. lee (1989) detected hantaviruses in the serum of a vaginally delivered fetus. the fetus died within 12 h of delivery. however, there were no similar cases reported in fetuses born via cesarean section. early diagnosis is vital for pregnant women with a hantavirus infection since it leads to early and more effective treatment, as well as a better clinical outcome. accurate diagnosis usually relies on clinical manifestations and laboratory tests. exploring the history of exposure in the epidemic area is also essential for a precise diagnosis. the typical clinical manifestations of hfrs are fever, hemorrhage, hyperemia, hypotensive shock and kidney damage accompanied by clinical symptoms, such as hematuria, proteinuria, and disseminated intravascular coagulation (dic) (lazzerini et al. 2017 ). similar to the general population, the typical clinical course of hfrs in pregnant women is divided into the following: fever period (3-7 days), hypotensive period (hours to 2 days), oliguria (3-7 days), polyuria (several days to weeks), and recovery period (2-3 months) (vaheri et al. 2013; latus et al. 2015; connolly-andersen et al. 2014) . the clinical symptoms of hps include flu-like symptoms in the prodromal stage (5 days), subsequent acute respiratory distress syndrome (ards), bilateral diffuse interstitial pulmonary edema, respiratory failure, as well as non-cardiogenic shock (duchin et al. 1994; khan et al. 1996; hallin et al. 1996; macneil et al. 2011) . molecular, immunochemical, and serological examination contribute to routine laboratory tests (zou et al. 2016) . igm/igg antibodies against the gn and gc of hantaviruses are often used to aid the diagnosis (hedman et al. 1991 ). however, early diagnosis is not that simple and straightforward, although diagnostic criteria are clear. misdiagnosis often happens because of inexperienced nonspecialist clinicians, a limited number of cases, and atypical clinical manifestations. misdiagnosis rate of pregnant patients with hfrs infection is as high as 50% according to several reports (xia 2009; yun 2007) . patients with hfrs that have mild or atypical presentations are most likely to be misdiagnosed, especially in the early period (xia 2009; yun 2007) . pregnant women with a hantavirus infection are often misdiagnosed with acute fatty liver of pregnancy (aflp), hemolysis, and elevated liver enzymes and low platelets (hellp), as they have similar clinical manifestations (mace et al. 2013; haram et al. 2009 ). thus, accurate differential diagnosis is vital. a substantial reduction in non-selective proteinuria in a short period is conspicuous for hfrs, but rare for aflp and hellp (clement et al. 2013) . currently, there is no safe and effective treatment for pregnant women with hemorrhagic fever. recently, new virologica sinica (chen 1994) . furthermore, continuous renal replacement therapy (crrt) plays a leading role, especially if hantavirus-infected pregnant patients display high blood volume and pulmonary edema (ji et al. 2017) . last but not the least, glucocorticoids have strong anti-inflammatory effects and are widely used in patients with stubborn and unresolved renal failure. however, the question that needs to be studied further is whether glucocorticoids can improve the health of the patients (kruger et al. 2015; martinuč bergoč et al. 2013) . sequelae: it refers to the remaining symptoms in pregnant women infected with hantavirus after their condition improves, which are mentioned in tables 1 and 2. cs cesarean section, vd vaginal delivery. in areas with high hantavirus prevalence, vaccination of women before pregnancy can effectively alleviate hantavirus infections (dai 2004; liu et al. 2013 ). however, for those who are already pregnant, the safety and effectiveness of vaccination have not been insightfully studied (dai 2004; liu et al. 2013) . although there are not many cases of hantavirus infection reported in pregnant women, even in the endemic areas, clinicians are expected to thoroughly consider severe consequences when they receive patients with similar symptoms. specific immune status of pregnant women might increase their susceptibility to hantavirus infection. pregnant women have unique immunology and physiology, and their immune system can develop a particular immunosuppressive state by suppressing cellular immunity to tolerate fetal antigens of paternal origin (beigi 2017; jamieson et al. 2006) . however, maintenance of this specific immune state during pregnancy has not been thoroughly explored. gaunt and ramin (2001) believe that this immunosuppressive state might be closely related to factors such as the absence of mhc-i antigens, the presence of unique hla surface molecules, and nonspecific reduction of systemic immunoreactivity. moreover, the blocking antibody, expressions of complement regulatory proteins, and reduced immunoreactivity might contribute to the formation of this unique immune state. these changes are likely to influence a systemic immune response to infections and lead to increased susceptibility to hantavirus infection. after being infected with hantavirus, pregnant women and their fetuses seem to have worse clinical symptoms and outcomes (sha et al. 2000; liu et al. 2003) nevertheless, currently, no theory can entirely explain the mechanism behind this phenomenon. in similar research of other viruses, this phenomenon is attributed to the unique immunological and physiological characteristics of pregnant women (beigi 2017; beigh 2012) . however, patients' immune response to a hantavirus infection, such as cytokine storms, is likely to harm the body (schönrich et al. 2008) . pregnant women are in a specific state of immunosuppression, and their immune damage is much smaller than that of other people, which is inconsistent with the findings of this literature review. therefore, it can be speculated that the cause for this difference might come from two aspects. primarily, pregnancy increases the organ burden of patients with a hantavirus infection, worsens the health, and increases the risk of death (guimarães et al. 2019) . additionally, the flow dynamics of patients might change during pregnancy. consequently, the synthesis of clotting factors increases, and the placenta synthesizes a large number of thrombogenic substances (avsic-zupanc et al. 2019). early and timely diagnosis, comprehensive symptomatic treatment based on liquid therapy, and accurate judgments improve the patient's life (dai 2004) . moreover, the cooperation between emergency room physicians, infectious disease specialists, and obstetricians is also helpful for timely 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review and distribution analysis in china acknowledgements we would like to thank editage (www.editage. cn) for their assistance with english language editing and jiamin wang from beijing foreign studies university, for improving the english writing. this work was supported by the national scientific research program of china: new technology and project on intervention and elimination of cytokine storm and secondary infection in acute severe respiratory infectious diseases (2017zx10204401-002-005). key: cord-322166-ajolu2rh authors: yin, jingchuan; liu, shi; zhu, ying title: an overview of the highly pathogenic h5n1 influenza virus date: 2013-01-16 journal: virol sin doi: 10.1007/s12250-013-3294-9 sha: doc_id: 322166 cord_uid: ajolu2rh since the first human case of h5n1 avian influenza virus infection was reported in 1997, this highly pathogenic virus has infected hundreds of people around the world and resulted in many deaths. the ability of h5n1 to cross species boundaries, and the presence of polymorphisms that enhance virulence, present challenges to developing clear strategies to prevent the pandemic spread of this highly pathogenic avian influenza (hpai) virus. this review summarizes the current understanding of, and recent research on, the avian influenza h5n1 virus, including transmission, virulence, pathogenesis, clinical characteristics, treatment and prevention. influenza virus belongs to the family orthomyxoviridae (nelson m i, et al., 2007) , which contains three different genera of influenza virus: type a, type b, and type c (shaw m, et al., 1992) . influenza viruses are enveloped rna viruses with a genome of seven or eight single-stranded negative-sense rnas consisting of from 890 to 2,341 nucleotides (noda t, et al., 2006) . avian influenza virus (aiv) is a type a virus that has eight gene segments encoding 11-12 proteins: hemagglutinin (ha), neuraminidase (na), matrix proteins (m1 and m2), non-structural proteins (ns1 and ns2), nucleocapsid proteins (np), and three polymerases, polymerase basic 1 (pb1), polymerase basic 2 (pb2) and polymerase acidic (pa) proteins. another protein, n40, which is encoded by the pb2 segment, has been identified recently (medina r a, et al., 2011; webster r g, et al., 1992; wise h m, et al., 2009) . some h5n1 viruses also express the pro-apoptotic protein pb1-f2, which is encoded by the pb1 segment (chen w, et al., 2001; medina r a, et al., 2011) (table 1) . the type a influenza viruses are divided into subtypes by two envelope proteins: hemagglutinin (ha) and neuraminidase (na). to date, 16 ha subtypes (h1 to h16) and nine na subtypes (n1 to n9) have been detected in birds (webster r g, et al., 1992) . many species of shorebirds and other waterfowl (e.g., wild geese, ducks) are natural reservoirs of aiv (webster r g, et al., 1992) . most aiv subtypes appear non-virulent to birds, since infected birds generally show no clinical symptoms of infection (de jong m d, et al., 2006; webster r g, et al., 1992) . although the majority of the known avian influenza a virus subtypes have only been isolated from wild birds, they have spread among marine mammals, including seals and whales, swine, dogs, cats, and horses (loeffelholz m j, 2010; suarez d l, et al., 2000) . currently, five aiv subtypes [h1n1, h3n2, h2n2, h1n2 and the highly pathogenic h5n1] replicate efficiently in human cells, resulting in greater potential for an aiv pandemic among humans (loeffelholz m j, 2010) . outbreaks of highly pathogenic avian influenza (hpai) h5n1 virus infections were first recorded in poultry in guangdong, china in 1996. it is now suspected that this virus contained only a few ha gene mutations that were not present in the ha of a low pandemic avian influenza 4 non-structure protein 1 involved in the post-transcription , host antiviral response antagonist ns2 (nep) nuclear export protein which regulates the virus transcription and replication pb1 core subunit of rna-dependent rna polymerase involved in rna transcription and replication; contains the polymerase activity pb2 component of the rna-dependent rna polymerase involved in rna transcription; has the temperature-dependent replication competence; recognizes capped cellular mrna n40 unknown pb1-f2 induction of apoptosis; promotion of secondary bacterial infection pa component of the rna-dependent rna polymerase involved in rna replication; possesses an endonuclease activity m1 membrane matrix protein m2 membrane ion channel protein the table lists all 12 proteins encoded by influenza a virus genome: the receptor-binding protein hemagglutinin (ha), neuraminidase (na), nucleoprotein (np), non-structural protein 1 (ns1), nuclear export protein (nep, also called ns2), matrix protein (m1), ion channel m2, the components of the rna-dependent rna polymerase complex (pb1, pb2, and pa), and the newly identified n40 protein. in addition, some viruses express the pro-apoptotic protein pb1-f2. (lpai) h5n1 virus isolated from geese (medina r a, et al., 2011; mukhtar m m, et al., 2007) . in march 1997, the previously isolated hpai h5n1 outbreaks exploded into a massive outbreak in chickens in hong kong (centers for disease control, et al., 1997) . in contrast to 1996, the h5n1 outbreak among the hong kong poultry markets spread to humans, with the isolation of an h5n1 influenza virus strain from a 3-year-old boy in hong kong (subbarao k, et al., 1998) . the patient died of severe pneumonia complicated by acute respiratory distress syndrome and reye's syndrome (subbarao k, et al., 1998) . in 1997, a total of 18 human cases were reported, and of those, 6 were fatal. in 1998, a different avian h5n1 strain emerged (li k s, et al., 2004; yuen k y, et al., 1998) . after a few years' silence, in february 2003, h5n1 infections were once again diagnosed among humans (li k s, et al., 2004) . the occurrence of frequent outbreaks of the hpai h5n1 virus among poultry since it emerged in 1996 is a cause of concern. in may 2005, more than 6,000 infected wild birds on lake qinghai in western china were infected and, during their seasonal migrations, spread the h5n1 virus westward to europe and africa in the years following (lei f, et al., 2011; liu j, et al., 2005; peiris j s, et al., 2007) . a new h5 sub-lineage, which had an increased binding affinity for human receptors, was isolated in egypt in 2008 (watanabe y, et al., 2012) . studies showed that the dominant haemagglutinin clade of the h5n1 virus has changed from clade 0 to clade 2.3， since it emerged in 1996, apparently as a result of antigenic shift (pfeiffer d u, et al., 2011) . although in the h5n1 infections that occurred in egypt, the morbidity was not as high as for seasonal influenza strains, the mortality rate of approximately 60% was substantially higher than for seasonal influenza (peiris j s, et al., 2010) . as of december 17, 2012, according to the world health organization (who), 610 confirmed cases of human h5n1 infection have been reported since 2003. these infections have been reported from 15 different countries and resulted in 360 deaths. these statistics demonstrate that a global h5n1 pandemic would have devastating effects, and that this virus represents a serious public health threat. most h5n1 influenza viruses were isolated during the winter months (peiris j s, et al., 2007; suarez d l, 2010) . the natural spread of the h5n1 virus results, in part, from the migration of wild birds, especially long-distance migratory birds (ndung'u t, et al., 2006) . the risk of hpai h5n1 infection in wild birds, and the risk of transmission between wild birds and domestic poultry may be similar at various times of the year (reperant l a, et al., 2011) . another major factor in transmission is the local commercial poultry industry. there, large numbers of infected and uninfected birds are housed together, (abdelwhab e m, et al., 2011; ndung'u t, et al., 2006) . wider spread of the virus among birds, from birds to mammals (including humans), and subsequent spread among humans requires adaptations that favor virus transmission rates (gutierrez r a, et al., 2011; vandegrift k j, et al., 2010) . most human cases seem to be the result of exposure to sick poultry or the butchering of birds. however, a number of confirmed human cases show that h5n1 can spread by close contact with an infected patient (e.g., spread to caretakers of an infected family member) (ungchusak k, et al., 2005; van kerkhove m d, et al., 2011) . although rare, human-to-human transmission of the h5n1 virus has been found in several household groups in tribal communities (van kerkhove m d, et al., 2011) . consuming food products from infected poultry is not likely to cause a human infection, but there is evidence that cats have become infected by eating raw meat from infected chickens, and that cats could, in turn, transmit the virus to their owners or to other household mammals, such as hamsters (abdelwhab e m, et al., 2011; thiry e, et al., 2007) . other studies performed since the emergence of hpai h5n1 show that the virus can be transmitted through contact with contaminated water and sewage, a risk factor that must be guarded against (beigel j h, et al., 2005; van kerkhove m d, et al., 2011) . hence, interactions of current hosts and their environment influence the ability of h5n1 influenza viruses to spread to new and different hosts (lowen a c, et al., 2007; park a w, et al., 2007) . genetic characterization of the 1997 h5n1 strain showed that it is a multiple reassortant virus from different progenitors. the ha gene of the 1997 h5n1 strain is derived from the a/goose/guangdong/1/96 strain, which was found to have close genomic relationships with h3n8 and h7n1 strains, as well as with h5n3 strains (li k s, et al., 2004; mukhtar m m, et al., 2007 ). an h6n1 virus contributed the na and some of the internal genes of the 1997 h5n1, and the h9n2 virus contributed the remaining internal genes (cauthen a n, et al., 2000; guan y, et al., 1999; hoffmann e, et al., 2000) . the hpai h5n1 virus became lethal in poultry, mammals, and human beings soon after the outbreak among wild birds. recent data suggest that there are more than 15 h5n1 genotypes distributed globally, with a newly emerged genotype, named genotype p (watanabe y, et al., 2012) . among all the circulating genotypes in china, genotype v became the dominant h5n1 representative in 2005, rather than genotype b and genotype z (duan l, et al., 2008) . inchoate studies indicate that ha is the major protein involved in aiv binding to its receptor on the cell surface (suksatu a, et al., 2009 ). six of the 16 known influenza a ha serotypes (h1, h2, h3, h5, h7, and h9) have been isolated from humans. it is widely accepted that α-2,6-linked sialic acids (α-2,6-sa) are the primary ha receptors in the upper respiratory tract of humans. in contrast, α-2,3-linked sialic acids (α-2,3-sa) that can be detected in duck intestinal epithelium are abundantly distributed on the cells of birds (van riel d, et al., 2007) . although infection is infrequent, current research shows that h5n1 viruses do infect humans. various studies have described mechanisms to account for this phenomenon. cells in the lower respiratory tract of humans, such as type ii pneumocytes, alveolar macrophages and the non-ciliated cuboidal epithelial cells of terminal bronchioles express α-2,3-sa moieties (nicholls j m, et al., 2007; van riel d, et al., 2006) . additionally, amino acid mutations in the ha receptor-binding site have been identified that may change or broaden attachment behavior and alter the inflammatory response of the host or the virulence of the virus (ayoratalavera g, et al., 2009; gao y, et al., 2009; manz b, et al., 2010; ramos i, et al., 2011; stevens j, et al., 2006; suksatu a, et al., 2009) . for example, mutation of the g225d and e190d receptors in the h5n1 virus ha protein greatly decreases its binding affinity for α-2,3-sa without changing its affinity for α-2,6-sa (stevens j, et al., 2006) . these data indicate that changes in ha dictate the receptor-binding specificity for h5n1 viruses (medina r a, et al., 2011; stevens j, et al., 2006) . further, the relative expression of ha and na in the virus envelope can be disrupted by mutations in ha, which changes the replication efficiency of the virus (chutinimitkul s, et al., 2010) . since pigs and some birds have both α-2,3-sa and α-2,6-sa receptors (and some have a third receptor), co-infection of these animals could serve as an incubator to generate novel reassortant viruses with increased ability to spread from animals to humans and humans to humans (gambaryan a s, et al., 2008; ito t, et al., 1998; kimble b, et al., 2010; li c, et al., 2010; wan h, et al., 2006) . the ha protein has also been suspected of inducing autophagy of epithelial lung cells, which would enable the virus to spread to other tissues and cause 6 further damage (manz b, et al., 2010; sun y, et al., 2012; tolnay a e, et al., 2010) . other mechanisms that could enable avian-to-human transmission of h5n1 viruses include the existence of currently unknown novel binding sites on ha that recognize the topology of sialylated pentasaccharides found on human cells (chutinimitkul s, et al., 2010; medina r a, et al., 2011; nicholls j m, et al., 2007) . no single viral protein by itself ensures successful infection and robust replication within a host; instead, the overall constellation of genes reprograms host cells for efficient propagation and spread. hpai h5n1 is no exception, as several of the viral non-structural genes have been reported to be important for replication and pathogenesis. it has been confirmed that the ns gene contributes to viral pathogenesis in cell cultures and animal models (imai h, et al., 2010) . the ns1 protein inhibits nuclear export of mrnas to the cytoplasm and binds to the cellular protein phosphatidylinositol-3-kinase (pi3k) (hale b g, et al., 2010; robb n c, et al., 2012) . avian viruses that have the hpai h5n1 ns1 gene efficiently block innate immune responses, including reduced interferon (ifn) and inducible phosphorylation of signal transducer and activator of transcription (stat) proteins (jia d, et al., 2010; seo s h, et al., 2002) . disrupted cytokine and chemokine expression, especially ifns and tnf, and decreased antiviral activity of innate immune response mediators, such as protein kinase resource and retinoic acid-inducible gene product i (rig-i), directly lead to increased virulence of the virus (haye k, et al., 2009; jackson d, et al., 2008; lipatov a s, et al., 2005; munir m, et al., 2011) . further, it has been suggested that the pdz domain binding motif, comprised of the four c-terminal residues of ns1, may influence pathogenicity through an ifn-independent pathway (jackson d, et al., 2008) . mutations of a single residue or short sequence of ns1 have enabled the virus to spread across species with increased virulence to the host (jackson d, et al., 2008; li w, et al., 2010; long j x, et al., 2008; zhu q, et al., 2008) . a deletion at position 80-84 or 191-195 , and an aspartic acid to glutamic acid substitution at ns1 residue 92 (d92e), also enhance the virulence of h5n1 by influencing the virus titers and the expression of pro-inflammatory cytokines (e.g., ifns and tnf-α) (li w, et al., 2010; lipatov a s, et al., 2005; zhu q, et al., 2008) . the e92 residue was found in viruses that were isolated during the 1997 h5n1 outbreak (lipatov a s, et al., 2005) . this confirmed the presumption that ns1 residue e92 of the hpai h5n1 may be crucial for pathogenicity (medina r a, et al., 2011; seo s h, et al., 2002) . furthermore, the existence of other ns1 gene alleles exhibiting versatile abilities to inhibit the ifn responses underscores the importance of ns1 for h5n1 pathogenesis (munir m, et al., 2011) . other aiv proteins, such as pa, pb1, pb2, m1, and m2 can also alter virus virulence and transmission, increasing the pandemic potential for this virus. (gao y, et al., 2009; guan y, et al., 2002; tafforeau l, et al., 2011; thanh t t, et al., 2008; wasilenko j l, et al., 2008; zamarin d, et al., 2006) . a study has shown that pb1-f2 interacts with two mitochondrial proteins, adenine nucleotide translocator 3 and voltage-dependent anion channel 1, which induces apoptosis (zamarin d, et al., 2005) . silencing of pb1-f2 protein expression by short interfering rna diminished aiv pathogenicity and mortality in mice without affecting viral replication kinetics in cell culture (zamarin d, et al., 2006) . another study showed that mutation of pb1-f2 residue n66s delayed activation of type i ifn pathway genes (conenello g m, et al., 2011) . similar studies have confirmed that mutations at single residues in multiple aiv proteins were associated with increased (or decreased) virulence. for example, residue k627 in pb2 has been detected in human hpai h5n1 viruses and shown to be a host range determinant, but a d701n mutation of pb2 can also enable aiv to infect mammalian cells (de jong m d, et al., 2006; gabriel g, et al., 2005; subbarao e k, et al., 1993) . in addition, some poultry-derived h5n1 viruses can cross the species barrier and replicate in mammalian cells without additional mutations, indicating that mammal-adapted aivs may exist in nature (suksatu a, et al., 2009) . the malleable nature of influenza virus, and the complex biological characteristics of the h5n1 avian virus pose a challenge for the development of vaccines and antiviral drugs needed to counter pandemic spread of this influenza virus (guan y, et al., 2002) . the time from h5n1 virus exposure to onset of symptoms ranges from one to seven days. the majority of reported cases have been in infants and young children (beigel j h, et al., 2005) . the time from onset of illness to death can be as short as few days to as long as one month, most likely reflecting different environmental conditions and characteristics of individual virus strains 7 (cattoli g, et al., 2009) . human h5n1 infection is characterized by high pharyngeal virus loads and presence of viral rna in the feces and blood (de jong m d, et al., 2006; wang h, et al., 2009) . the distribution of virus isolates and clinical features of infection vary considerably among host species (gao p, et al., 1999; kuiken t, et al., 2010) . for example, although h5n1 virus can be found in the brain of experimentally infected mice, it is more likely to infect the heart and lungs of cynomolgus macaques (rimmelzwaan g f, et al., 2001) . in humans, the clinical spectrum of h5n1 infection ranges from asymptomatic to severe or fatal disease. despite some features shared with seasonal influenza viruses, such as high fever (> 38°c), headache, myalgia, and cough, a large number of patients also have severe respiratory involvement including dyspnea and pneumonia, as well as manifestations of acute respiratory distress syndrome (beigel j h, et al., 2005; cameron m j, et al.; hui d s, 2008) . hospital records indicate that rather than upper respiratory tract symptoms, patients with h5n1 are more likely to develop lower respiratory tract symptoms within days of admission (hui d s, 2008) . respiratory epithelial cells, especially the type ii pneumocytes, are key target cells of the aiv in the human lung in humans, and respond by abundant release of inflammatory chemokines and cytokines (chan r w, et al., 2010; peiris j s, et al., 2010; snelgrove r j, et al., 2011; wang j, et al., 2009; yu w c, et al., 2011) . a similar preference for type ii pneumocytes was observed in animal models, including the cat, mouse, and ferret (van riel d, et al., 2006) . alveolar epithelium cells amplify the h5n1 virus-induced cytokine cascade, which is an important component of h5n1 pathogenesis (snelgrove r j, et al., 2011) . one of the primary clinical outcomes is acute lung injury (ali), presumed to be a consequence of autophagic cell death within the alveolar epithelium. severe ali appears to be major cause of fatality (sun y, et al., 2012; who, 2008) . numerous studies indicate that h5n1 is more pathogenic than seasonal influenza. tests in mice and primates showed more severe lung inflammation and higher virus yields following infection with the h5n1 virus than with seasonal virus, or a reassortant 1918 h1n1 virus (baskin c r, et al., 2009; sun y, et al., 2012) . however, similar levels of apoptosis were seen in a549 cells, with undetectable differences in virus yield, indicating that higher virus load does not necessarily result in more severe disease (sun y, et al., 2012) . chest radiographs revealed that although multifocal broncho-pneumonia in the parahilar of both lungs can been seen predominantly when infected with pneumococcus, h5n1 pneumonia presented with bilateral interstitial infiltration (loeffelholz m j, 2010). central nervous system (cns) manifestations are infrequent with seasonal influenza virus, but been observed in h5n1-virus-infected patients (de jong m d, et al., 2005) . other studies showed that h5n1 infection of the cns can induce parkinsonian symptoms as well as substantia nigra pars compacta dopaminergic neuron degeneration (jang h, et al., 2009) . animal studies show a similar spectrum of cns disease and encephalopathy after infection with hpai h5n1 virus, with high levels of pro-inflammatory cytokines including tnf-α, ifns, il-1 and il-6 (thiry e, et al., 2007; tolnay a e, et al., 2010) . in the mouse model, fatal h5n1 infections were associated with the appearance of cd3+ t cells and tunel+ cells in the cns, as well as the absence of perineuronal nets in the brain (bissel s j, et al., 2012). nevertheless, ifn levels, especially ifn-γ, were not as high in the cns as in the lungs (bissel s j, et al., 2012). the mechanism of h5n1 access of the cns is not fully understood, but one hypothesis is that the e223k mutation of ha facilitates virus spread to the brain and binding to neurons (manz b, et al., 2010) . in contrast with seasonal influenza strains, a large proportion of h5n1-infected patients (both children and adults) have gastrointestinal symptoms such as watery diarrhea, vomiting, and abdominal pain, which occur rarely in adults with human seasonal influenza (kandun i n, et al., 2008; wang h, et al., 2009 ). the relative increase in frequency of pneumonia and gastrointestinal symptoms is an important feature of avian influenza, and may help to distinguish avian from seasonal influenza in the clinic. multi-organ failure involving the kidneys, liver, and many other non-respiratory organs has been common (thanh t t, et al., 2008) . fatal cases of h5n1 during pregnancy, have also been reported (shu y, et al., 2006) . compared with h9 and h7 virus strains, conjunctivitis has not been prominent in h5n1-infected patients (de jong m d, et al., 2006; hui d s, 2008) . only rare h5n1 cases are associated with secondary bacterial infections, which are commonly observed with h1n1 infections however, secondary bacterial infections were observed in h5n1 cases with hypercytokinemia (gill j r, et al., 2010; peiris j s, et al., 2009; peiris m, 2006) . prominent lymphopenia, and thrombocytopenia have also been observed in h5n1-infected patients (loeffelholz m j, 2010) . laboratory testing has frequently revealed 8 elevated alanine aminotransferase, aspartate aminotransferase, creatinine kinase, and lactate dehydrogenase in h5n1 infection (de jong m d, et al., 2006) . increased levels of lactate dehydrogenase and creatinine phosphokinase, prolonged prothrombin time, and activated partial thromboplastin time, as well as hypo-albuminemia, are also common (hui d s, 2008) . in severe cases, insufficient expression of perforin in cd8+ t cells and hyperproduction of ifn-γ has occurred, leading to potential hyper-cytokinemia and hemophagocytosis (hsieh s m, et al., 2006) . finally, production of pulmonary surfactant protein d was found to be down-regulated in h5n1 infections (kongchanagul a, et al., 2011) . in humans, the h5n1 virus triggers a massive pro-inflammatory cytokine and chemokine response that contributes to systemic tissue damage and morbidity (de jong m d, et al., 2006; loeffelholz m j, 2010; suksatu a, et al., 2009; szretter k j, et al., 2007) . data show that those who died from h5n1 infection had significantly higher levels of cytokine and chemokine expression than survivors. ifn-αifn-β, ifn-λ1, tnf-α, and rantes were the primary cytokines induced by the h5n1 virus in macrophages, accompanied by up-regulation of irf3, the key transcription factor for ifn gene regulation (lee d c, et al., 2005; lee d c, et al., 2012) . further, up-regulation of cyclooxygenase 2 (cox-2), followed by il-32, inducible nitric oxide synthase and prostaglandin e2 production and accumulation were have been observed in cell cultures and in patients with h5n1 infections (li w, et al., 2010; li w, et al., 2009; li w, et al., 2008) . the stimulation of cox-2, an inducer of il-27 and ifn-λ1, acts as an important mediator of the inflammatory response during influenza virus infection (fang j, et al., 2011; liu l, et al., 2012) . although results of previous studies using different model systems (both animals and cultured cells) did not completely agree, they did lead to a consensus that h5n1 virus induces a 'cytokine storm' (yu w c, et al., 2011) . thus, patients with h5n1 disease typically present with a hyper-induced systemic inflammatory response syndrome and a higher and a more prolonged viral load in respiratory specimens than other seasonal influenza virus like human h1n1 viruses (chan m c, et al., 2005; kuiken t, et al., 2010; lee d c, et al., 2012; peiris j s, et al., 2010; sandbulte m r, et al., 2008) . the outcomes of this imbalance are always associated with fulminant viral pneumonia followed by acute respiratory distress syndrome, multi-organ failure, and/or often death (de jong m d, et al., 2006; szretter k j, et al., 2007) . most pro-inflammatory cytokines and chemokines reach higher levels in the serum of h5n1-infected individuals than comparable samples from seasonal human influenza virus-infected individuals (lee s m, et al., 2009 ). in h5n1-infected primary human macrophages, many genes showed at least a 1.5-fold greater response than the response observed in seasonal h1n1-infected cells at 6 hours post-infection. of those genes, tnf, ccl4l1, ifit2, and pmaip1 were also up-regulated to a greater degree at 3 hours post-infection (lee s m, et al., 2009) . the negative effector of fas (nef) and tnf-α seem to be unique to h5n1 infection (lee s m, et al., 2009) . despite the increased expression of ifn-α and ifn-β, an up-regulation of il-29, which was not detected during h1n1 infections, has also been observed following h5n1 infection, leading to enhanced signaling through the jak-stat pathway (lee s m, et al., 2009) . further, avian h5n1 infection triggered a delayed onset of caspaseand poly (adp-ribose) polymerase (parp)-mediated apoptotic pathways, resulting in a decreased percentage of cell death and expression level of the parp fragment than that observed with the h1n1 virus (mok c k, et al., 2007) . similar results have also been seen in previous studies using other types of human cells and animal models. much stronger natural killer (nk) cell activation, followed by up-regulation of cd69 and cd107a, significantly enhanced expression of ifn-γ production, and down-regulation of nkp46 have also been observed in h5n1 infection (du n, et al., 2010) . h5n1 virus can also hyper-induce pro-inflammatory cytokines including tnf-α, with enhanced tnf-related apoptosis-inducing ligand-induced apoptosis in jurkat t cells (lee d c, et al., 2005; mok c k, et al., 2007) . however, in contrast with findings from human macrophage studies, differences between h5n1 and h1n1 induced secretion of pro-inflammatory cytokines tnf-α and ip-10 were less pronounced in human plasmacytoid dendritic cells, although h5n1 also showed higher expression of ifn-α than did h1n1 and h3n2 viruses (sandbulte m r, et al., 2008) . despite significant diversity in the induction profile of pro-inflammatory cytokines and chemokines in h5n1 as compared with seasonal h1n1, the magnitude of the pro-inflammatory response triggered by h5n1 is also much stronger than that triggered by other human influenza viruses (bel m, et al., 2011; sandbulte m r, et al., 2008; tolnay a e, et al., 2010; zeng h, et al., 2007) . the cytokine storm caused by the induction (or suppression) of gene expression and signaling pathways 9 illustrates the extreme and serious consequences of h5n1 infection. therefore, identifying these pathways will help to find effective methods of fighting this highly pathogenic virus. although high virus titers were often detected in many tissues and blood during h5n1 infections, throat swabs and lower respiratory samples are the preferred and recommended specimen for virus detection. h5n1 virus titers are higher in pharyngeal and bronchial alveolar lavages and tracheal aspirates than in nasal epithelial cells or aspirates (beigel j h, et al., 2005; de jong m d, et al., 2006; loeffelholz m j, 2011; wang h, et al., 2009; who, 2008; yu w c, et al., 2011) . patient samples should be stored carefully (e.g., kept on ice) before testing and it is recommended to use the specimens as soon as possible (loeffelholz m j, 2011) . diagnosis of h5n1 influenza infection requires a systematic analysis. a history of recent of travel and personal contacts as well as clinical symptoms and many other factors should be considered (loeffelholz m j, 2011). many rapid diagnostic techniques can detect seasonal influenza in clinical specimens, but the rapid antigen tests designed to detect seasonal strains of influenza are less effective for h5n1 (beigel j h, et al., 2005) . in addition, since the clinical presentation of numerous illnesses may resemble influenza, and the many different influenza virus share a number of clinical features, diagnosis of a particular subtype can only be accurately identified by laboratory tests (shaw m, et al., 1992) . sensitive and specific tests are needed, but remain to be developed. at this time, laboratory diagnosis for h5n1 includes virus culture, rapid antigen detection, and viral nucleic acid amplification methods in addition to serology (loeffelholz m j, 2011). all these methods have advantages and disadvantages. for effective surveillance, the proper time to apply a particular diagnostic methods or combination of methods must be carefully considered, particularly at the pre-pandemic period of h5n1 spread. viral nucleic acid amplification, for example reversetranscription polymerase chain reaction (rt-pcr), is highly sensitive. the human h5n1 virus outbreaks in hong kong were the first time that the rt-pcr method was used for specific detection of the h5 gene segment. it proved to be a valuable diagnostic tool when used in association with identification of clinical features (yuen k y, et al., 1998) . the more recently developed real-time rt-pcr (rrt-pcr) is more efficient and accurate than the standard rt-pcr method (das a, et al., 2006) . other assays such as nucleic acid sequence-based amplification, taqman, and fluorescence resonance energy transfer have also been studied. these molecular assays are highly specific for h5n1, and can be used to detect h5n1 from a wide range of hosts (chantratita w, et al., 2008) . the disadvantage of all nucleic acid-based methods is the need for sequence information for specific genes such as ha and na, as well as np. the existence of several distinct sub-lineages and the high mutability of h5n1 viruses may then lead to increasing difficulties in identifying the virus in patient isolates (cattoli g, et al., 2009; loeffelholz m j, 2010) . hemagglutination inhibition and micro-neutralization are sensitive serological assays for virus detection, and are considered the gold standards for detection of anti-h5-specific antibodies in humans (noah d l, et al., 2009; ohnishi k, et al., 2012; stelzer-braid s, et al., 2008) . these assays are essential to providing a retrospective diagnosis to avoid potential false-negatives and are essential for epidemiological investigations. the major disadvantage of these methods is that paired specimens and h5-specific reagents for the subtypespecific antibodies are always not widely available and/or are difficult to detect (ohnishi k, et al., 2012) . as such, results from these assays may only serve as additional, but not necessarily preferential, methods for laboratory diagnosis. other methods such as rapid antigen detection tests (e.g., immunofluorescence) are simple and rapid, but suffer from a lack of sensitivity (clementi n, et al., 2011) . the rapid antigen test usually fails to distinguish between type a and type b viruses, and cannot distinguish among aiv subtypes (clementi n, et al., 2011) . two classes of antiviral drugs that target influenza virus m2 or na are commonly used for prophylaxis and treatment of influenza virus infection (sambhara s, et al., 2010) . both amantadine and rimantadine block the ion channel formed by the m2 protein, thus inhibiting viral entry and replication (couch r b, 2000; sambhara s, et al., 2010) . the second class of antiviral drug, including zanamivir and oseltamivir, acts on na to prevent virus release from infected cells (de jong m d, et al., 2006) . however, there appear to be several virus clades that are resistant to these drugs (le q m, et al., 2005) . the emergence of oseltamivir-or amantadine-resistant strains 10 restricts the ability to prevent influenza infections and treat patients effectively (cheung c l, et al., 2006; de jong m d, et al., 2005; le q m, et al., 2005; malaisree m, et al., 2009) . following analysis of the three-dimensional structure of na from oseltamivir-resistant influenza strains, six additional drug analogs have been proposed to fight h5n1 (du q s, et al., 2007) . including inhibitors of inflammation, such as the combination of celecoxib or mesalazine to zanamivir treatment has been shown to greatly reduce cell death and host tissue damage with a similar viral load compared to zanamivir alone (zheng b j, et al., 2008) . in addition to antiviral drugs that target influenza virus m2 or na, various molecules with pharmacological activity, such as isopentenyl pyrophosphate and aminobisphosphonates like pamidronate (pam), were able to activate and expand human γδ t cells (qin g, et al., 2009; tu w, et al., 2011) , suggesting activation of γδ t-cell-based immunotherapy as an alternative strategy for treating influenza a infection . recent studies have also identified several additional candidate therapeutic agents, such as a rig-i pathway activator and ifns, that inhibit influenza viral replication, das181 and its analogs that down-regulate receptor expression on the surface of the host cell, and antiviral sirnas that target viral genes that inhibit h5n1 infection in vitro and in animal models (chan r w, et al., 2009; luke j m, et al., 2011; sambhara s, et al., 2010; stewart c r, et al., 2011; szretter k j, et al., 2009) . other candidate drugs such as saponin derivatives or other active compounds that have a chacotriosyl residue and chlorogenin moiety may also serve as host cell entry inhibitors for highly pathogenic h5n1 influenza virus (ding n, et al., 2012) . development of an effective h5n1 vaccine poses a number of substantial challenges. newly emerging virus strains make the choice of vaccine strains problematic. as a result, production and deployment of vaccine are of vital importance. to date, there are at least 23 candidate vaccine viruses that can be used in clinical trials and in model systems of pandemic vaccine production (nduati r, et al., 2001) . based on specific properties of the h5n1 viruses in particular geographic areas, using one or more of these candidate viruses for vaccine production should be considered. unlike seasonal influenza virus vaccine production, using the wild-type h5n1 virus for vaccine production in eggs is not practical due to its high virulence. recent data also showed that the prototype h5n1 vaccine, which used a plasmid-based reverse genetics system, was poorly immunogenic (cheung c l, et al., 2006; harvey r, et al., 2008) . luckily, it was discovered that the addition of an adjuvant (e.g., alpo 4 or iscomatrix) to this prototype vaccine could induce a clinically significant protection in animal models (nolan t, et al., 2008; rockman s, et al., 2010) . beyond recombinant or attenuated vaccines, whole inactivated virus influenza vaccine can also provide heterosubtypic cross-protection against influenza infection (budimir n, et al., 2012) . other options that use newly established recombinant adenoviral-vector-based vaccines to express h5 ha or a polyvalent dna vaccine expressing ha antigens from h5n1 viruses have achieved protective antibody responses in animals (hoelscher m a, et al., 2006; wang s, et al., 2011) . nevertheless, the high mortality and pathogenicity of h5n1 viruses, additional development of antiviral drugs and vaccines, and novel preventive and therapeutic strategies are needed to protect the world from a potential pandemic. history witnessed three major influenza pandemics in the 20 th century: the 1918 h1n1 spanish flu, 1957 h2n2 asian flu, and 1968 collectively, these pandemics caused millions of deaths and countless more infections. the highly pathogenic influenza h5n1, despite its low morbidity (i.e., low case incidence rate) and inefficient human-to-human transmission, remains an important public health threat because of its high mortality. control efforts should be coordinated before this potentially dangerous virus becomes the next pandemic. the last 16 years have resulted in a huge influx of research into the replication and pathogenicity of h5n1, but our understanding of h5n1 remains incomplete. the recent 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optimized leader sequence elicit cross-protective antibody responses np, pb1 and pb2 viral genes contribute to altered replication of h5n1 avian influenza viruses in chickens the changing nature of avian influenza a virus (h5n1) evolution and ecology of influenza a viruses a complicated message: identification of a novel pb1-related protein translated from influenza a virus segment 2 mrna writing committee of the second world health organization consultation on clinical aspects of human infection with avian influenza a (h5n1) v. 2008. update on avian influenza a (h5n1) virus infection in humans viral replication and innate host responses in primary human alveolar epithelial cells and alveolar macrophages infected with influenza h5n1 and h1n1 viruses clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus influenza a virus pb1-f2 protein contributes to viral pathogenesis in mice influenza virus pb1-f2 protein induces cell death through mitochondrial ant3 and vdac1 highly pathogenic avian influenza h5n1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/h5n1 virus a naturally occurring deletion in its ns gene contributes to the attenuation of an h5n1 swine influenza virus in chickens key: cord-319501-a2x1hvkk authors: wong, lok-yin roy; lui, pak-yin; jin, dong-yan title: a molecular arms race between host innate antiviral response and emerging human coronaviruses date: 2016-01-15 journal: virol sin doi: 10.1007/s12250-015-3683-3 sha: doc_id: 319501 cord_uid: a2x1hvkk coronaviruses have been closely related with mankind for thousands of years. communityacquired human coronaviruses have long been recognized to cause common cold. however, zoonotic coronaviruses are now becoming more a global concern with the discovery of highly pathogenic severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) coronaviruses causing severe respiratory diseases. infections by these emerging human coronaviruses are characterized by less robust interferon production. treatment of patients with recombinant interferon regimen promises beneficial outcomes, suggesting that compromised interferon expression might contribute at least partially to the severity of disease. the mechanisms by which coronaviruses evade host innate antiviral response are under intense investigations. this review focuses on the fierce arms race between host innate antiviral immunity and emerging human coronaviruses. particularly, the host pathogen recognition receptors and the signal transduction pathways to mount an effective antiviral response against sars and mers coronavirus infection are discussed. on the other hand, the counter-measures evolved by sars and mers coronaviruses to circumvent host defense are also dissected. with a better understanding of the dynamic interaction between host and coronaviruses, it is hoped that insights on the pathogenesis of newly-identified highly pathogenic human coronaviruses and new strategies in antiviral development can be derived. [image: see text] coronaviruses (covs) are classified into four genera, namely alpha-, beta-, gamma-and deltacoronavirus, under the family of coronaviridae and the order of nidovirales (woo et al., 2012) . the first three genera were previously known as groups i, ii and iii, respectively (lau et al., 2006; zhong et al., 2012) . covs have been shown to infect many different hosts including bats, birds, dogs, mice and human (woo et al., 2009; de groot et al., 2013) . the infections are commonly zoonotic in nature (chan et al., 2013) . in the past 50 years, several human covs (hcovs) were identified. hcov-229e and hcov-oc43, belonging to alpha-and betacoronavirus respectively, were the first two hcovs identified in the mid-1960s (tyrrell and bynoe, 1965; hamre and procknow, 1966; mclntosh et al., 1967) . healthy individuals infected with either hcov-oc43 or hcov-229e develop illnesses within the range of typical common colds with good prognosis (bradburne et al., 1967) . since the identification of these two hcovs, extensive studies were conducted to understand their pathogenicity. however, almost all studies showed that hcov-oc43 and hcov-229e caused mild illnesses with high titers of neutralizing antibodies (bradburne et al., 1967) . the idea of hcov being a relatively weak respiratory diseasecausing agent was therefore presented to the field. this idea was generally accepted until the outbreak of sars in 2003. sars-cov was the first hcov identified to cause acute respiratory distress syndrome (ar-ds) (cheng et al., 2007; graham et al., 2013) . according to world health organization (who), a total of 8096 cases from 29 countries were reported with a case mortality rate of 9.6%. the sars outbreak changed the landscape of cov studies entirely and marked the new era of combating infectious diseases. tremendous efforts have been put into understanding sars-cov pathogenicity, opening a new page of cov biology. despite advances in infection control and quarantine measures in the past decade, another hcov causing ards was identified in saudi arabia as a novel lineage c betacoronavirus in september 2012 (zaki et al., 2012) . the newly identified hcov was later named mers-cov. up to october 2015, 1611 laboratory-confirmed cases were reported to who with 575 related deaths in 26 countries, including a recent outbreak involving 186 cases and 37 deaths in south korea. mers-cov is closely related phylogenetically to two bat covs, hku4 and hku5, shedding light on the possible zoonotic reservoir of mers-cov (zaki et al., 2012; . together with hcov-hku1 identified in 2005 (woo et al., 2005) and hcov-nl63 discovered in 2004 (fouchier et al., 2004; van der hoek et al., 2004) , six hcovs have been documented up to date. these 6 hcovs present diseases with a range of clinical severity from typical common cold in hcov-oc43, hcov-229e, hcov-hku1 and hcov-nl63 to ards in sars-cov and mers-cov. why these covs show dramatically different pathogenicity in human is an important but unanswered question in the field. one model to explain this difference is based on adaptation and host immunity. according to this model, bats are reservoir of various covs. bat covs constantly emerge in human via intermediate hosts such as civets and dromedaries. exposure of immunologically naïve human populations to these covs commonly causes severe diseases plausibly due to aberrant activation of innate immunity and lack of immune memory. when some covs become better adapted in human by acquiring the ability to transmit from human to human readily, pandemics could arise. meanwhile, as they become fully adapted, the covs might only cause mild diseases in human. existing evidence supports the origin of hcov-oc43, hcov-229e, hcov-hku1 and hcov-nl63 from bats and other animals (woo et al., 2009; huynh et al., 2012; corman et al., 2015) . adaptation and virus-host interaction are also known to be major determinants in cov pathogenesis (pepin et al., 2010; chan et al., 2013) . it will therefore be of great interest to see whether emerging human covs might be particularly capable of evading innate antiviral response while activating pathological inflammation. in other words, we need to determine whether the more severe clinical presentations might be accounted for by the specific interaction between host and emerging human covs, namely sars-cov and mers-cov. in this review, the host innate antiviral response to cov infection is particularly focused. in addition, the viral strategies adopted by sars-cov and mers-cov to subvert innate immunity are also summarized to provide inspiring insights that may explain the discrepancies in virulence (figure 1 ). covs are polycistronic positive-sense single-stranded rna (ssrna) viruses with genomes of about 30 kb in size. the 5′ most two-thirds of cov genome encodes polyprotein 1a (pp1a) and pp1ab replicase polyproteins, which are further cleaved by viral proteases to yield nonstructural proteins (nsps), while the 3′ end of the genome encodes structural and lineage-specific proteins (durai et al., 2015) . the cov life cycle begins with the binding to cellular receptor followed by membrane fusion as well as viral rna and protein synthesis in the cytoplasm. the pp1a and pp1ab polyproteins are co-translationally processed resulting in the formation of the replicase complex. a set of nested subgenomic mrnas and genomic rna, which possess both the same 3′ end and a common 5′ leader sequence derived from the 5′ end of the genome, is then transcribed. normally, only the 5′ end of each mrna is translated. virion assembly is achieved by budding into intracellular membranes and virion release is accomplished through the secretory pathway (cheng et al., 2007; durai et al., 2015) . the coronaviral spike (s) protein is responsible for binding to specific host receptor on cell surface and fusing viral envelope with lipid membrane of host upon infection (bosch et al., 2003; rota et al., 2003; chen et al., 2013) . hcov-nl63 and sars-cov from α-and β-genera respectively recognize angiotensin-converting enzyme 2 (ace2) (li et al., 2003; pyrc et al., 2007; frieman et al., 2008; chen et al., 2013) while mers-cov infects cells through another cell surface enzyme dipetidyl peptidase 4 (dpp4) (chen et al., 2013; raj et al., 2013) . aminopeptidase n (apn) has also been found to be recognized by some α-genus covs like hcov-229e (yeager et al., 1992) . cell surface receptor binding dictates species-specific viral entry as well as tropism. this also confines the direction of cellular antiviral response. we and others have shown the ability of cov s proteins to activate unfolded protein response and endoplasmic reticulum stress fung et al., 2014; siu et al., 2014b) . the activity of s might also be functionally related to coronaviral perturbation of innate antivir-al response including ifn and cytokine production. pattern recognition receptors (prrs) constitute an indispensable part of the host innate immune defense mechan-ism by the detection of foreign, non-self patterns from invading microbes distinct from host. these pathogenassociated molecular patterns (pamps) are usually biomolecules derived from the surface or generated during the life cycle of the microbes. the detection of pamps by host prrs activates innate immune response including the expression of type i ifns and cytokines for clearcycle are exposed to host innate immune sensors, rig-i/mda5 in cytoplasm or toll-like receptors tlr3/7/8 in endosome. activation of these immune sensors initiates a downstream signaling cascade that leads to ifn-β gene expression. rig-i/mda5 conveys signal through a mitochondrial adaptor mavs while tlr signals through trif/myd88. both pathways share the common traf adaptor to activate transcription factors. traf3 serves as an adaptor which activates tank × tbk1/ikkε complex for irf3 phosphorylation and subsequent dimerization, while traf6 is responsible for the activation of ikk complex which phosphorylates the canonical inhibitor of nf-κb (iκb). activated transcription factors are translocated into the nucleus to drive ifn-β expression. (right) ifn-β are secreted into extracellular space and bound to its cognate receptors ifnar to activate downstream jak-stat signaling. receptor-associated tyrosine kinases jak1 and tyk2 are brought to juxtaposition for self-phosphorylation and activation. stats are recruited to and phosphorylated by the tyrosine kinases. phosphorylated stat1/2 with irf9 forms a ternary complex isgf3 which translocates into the nucleus and binds to isre in the promoter region upstream of isg genes. isg genes are expressed consequently to establish an antiviral state in cells. oas is an example of isg which produces 2′, 5′-oligoadenylate (2′, 5′-a) upon detection of dsrna and activates rnase l to cleave viral rna to yield more rlr ligand as a positive-feedback mechanism of ifn production. the cov-encoded proteins shown in red are known to intervene the host innate immune signaling at various action points as evasion mechanisms to sustain viral replication and propagation. the action points at which viral proteins function marked with a question mark (?) represent controversial and inconclusive findings in the field or molecular mechanisms not well studied. mhv: mouse hepatitis virus. arms race between innate immunity and human coronaviruses ance of invading microbes. during cov infection, retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) and toll-like receptors (tlrs) are believed to bear pivotal importance in stimulating host type i ifn induction. it is therefore essential to review the sensing mechanism of the prrs to understand viral evasion mechanisms and provide insights on the development of potential viral antagonists. after viral entry, cov genomes are exposed in the cytoplasm for expression of viral proteins, providing an opportunity for viral rna sensing by host. rlrs are ubiquitously expressed cytoplasmic rna helicases of dexd/h box family responsible for sensing doublestranded rna (dsrna) (yoneyama et al., 2005) . three types of rlrs have been identified up to now, including rig-i, melanoma differentiation-associated gene 5 (mda5) and laboratory of genetics and physiology 2 (lgp2) (loo and gale, 2011) . rig-i and mda5 consist of n-terminal caspase activation and recruitment domain (card) in two tandem copies, a central dexd/h box helicase domain and a c-terminal domain (ctd) (yoneyama et al., 2004 (yoneyama et al., , 2005 . the n-terminal cards are the effector domain of rlrs to mediate downstream transduction, which is held by the ctd when unstimulated (jiang et al., 2011; kowalinski et al., 2011; luo et al., 2011) . however, in the presence of residual amount of cytoplasmic dsrna, rlrs bind to dsrna through the central dexd/h box helicase domain and ctd with atp, causing a conformational change that exposes the n-terminal cards for signal transduction (yoneyama et al., 2004; jiang et al., 2011). lgp2 lacking the n-terminal cards is thought to act as co-factor that augments the function of rig-i and mda5 (satoh et al., 2010; bruns et al., 2014) . exposure of cards leads to oligomerization of rig-i or mda5 to form filamentous structure (berke et al., 2012; peisley et al., 2013; wu et al., 2013) . the card filament recruits and further initiates similar filamentous structure formation of card on mavs, an adaptor protein which further recruits downstream effectors tumor necrosis factor receptor-associated factor 3 (traf3), tank-binding kinase 1 (tbk1) and iκb kinase ε (ikkε) (loo and gale, 2011; wu et al., 2014) . tbk1 and ikkε form a complex of activated protein kinase for phosphorylation and activation of not only mavs adaptor , but also irf3 transcription factor (loo and gale, 2011) . activated irf3 are phosphorylated, dimerized and eventually translocated to the nucleus. on the other hand, traf2/6 is also recruited to mavs for nf-κb activation. specifically, canonical nf-κb inhibitor iκb is phosphorylated and then degraded through proteasomes in a ubiquitinationdependent fashion (loo and gale, 2011) . iκb degrada-tion exposes nuclear localization signal on nf-κb dimer for nuclear translocation. activated irf3 and nf-κb together with other transcription factors including c-jun assemble the enhanceosome that binds to ifn-β promoter for ifn-β expression (ford et al., 2010; loo and gale, 2011) . infection with mouse hepatitis virus induces rig-i expression. in addition, the activation of type i ifn production by this cov in oligodendrocytes requires both rig-i and mda5 . thus, rlrs might play an important role in the sensing of cov infection. several critical questions concerning rlr recognition of covs merit further investigations. first, the role of rlrs in cov sensing should be studied in rlr-null and cov-susceptible cells and animals. when necessary cr-ispr/cas9 technology might be used to disrupt rlr genes in target cells (hsu et al., 2014; yuen et al., 2015) . second, the cov pamps recognized by rlrs should be identified and characterized. particularly, it will be of interest to see whether and how common and highly structured regions in coronaviral genome, such as the aforementioned 5′ leader sequence, might be recognized by rlrs. for example, a polyuridine motif in the 3′ untranslated region of hepatitis c virus genome and the panhandle structure in rna viruses such as influenza a virus have previously been shown to be rig-i agonists (saito et al., 2008; weber et al., 2013; kell et al., 2015; liu et al., 2015b) . in addition, possible involvement of viral proteins such as nucleocapsid (n) in this recognition as in the case of other rna viruses (saito et al., 2008; weber et al., 2013) should also be clarified. finally, comparative analysis of sars-cov, mers-cov and other hcovs for their ability to activate rlrs will shed light on whether rlr activation would be a critical determinant in cov virulence. covs have been observed to infect host cells through more than one pathway. while cov entry by the fusion of viral envelope and host membrane has been described, the endosomal pathway is still considered the classical entry pathway for covs. in this pathway the activation of s protein cleavage by cathepsin l and transmembrane serine protease tmprss2 occurs in the absence of cell surface proteases in certain cell types (shirato et al., 2013; burkard et al., 2014) . in this regard, tlr family may play an essential role in sensing cov infection through the endosomal pathway. tlr family was identified as another prr homologous to drosophila toll receptor (boehme and compton, 2004) , sensing various pamps within the endosome which leads to induction of cytokines and ifns. in human, each of the 11 tlrs is known to specifically recognize a particular pamp and preferentially resides in either plasma or endosomal membrane. the cellular localization of tlrs defines their functions in detecting different pamps. for example, tlrs critically involved in viral nucleic acid sensing, including tlr3 for dsrna, tlr7 and tlr8 for ssrna, and tlr9 for unmethylated cpg island of dsdna viruses, are mainly localized in endosomal membrane while other members having a role in sensing other biomolecules derived from microbial surface components localized to plasma membrane of infected cells (xagorari and chlichlia, 2008; kawai and akira, 2010) . tlr family members being type 1 transmembrane proteins share a similar structure with a single transmembrane domain. tlr specificity is determined by the ectodomain made up of various number of leucine-rich repeats (lrrs) that bind the corresponding pamp directly (boehme and compton, 2004) . signal transduction begins with ligand binding to lrrs in the ectodomain, thus recruiting cytosolic adaptor protein myd88 with cytoplasmic toll/il-1 receptor (tir) domain by homotypic tir-tir domain interaction (xagorari and chlichlia, 2008) . the tlr-myd88 complex then recruits and activates interleukin 1r-associated kinase (irak) by phosphorylation. the activated irak then in turn associates with traf6 and activates a series of downstream effectors leading to the activation of a range of cytokines and ifn-stimulated genes (isgs), while activation of type i ifn expression by tlr3 is independent of myd88 but dependent on trif (boehme and compton, 2004; xagorari and chlichlia, 2008) . tlr pathway is significantly involved in the suppression of cov replication and induction of type i ifn expression. mice deficient of either tlr3 or tlr4 were more prone to sars-cov pathogenesis (mazaleuskaya et al., 2012; totura et al., 2015) . notably, disruption of either myd88 or trif arm of the tlr signaling pathway causes lethal sars-cov disease, indicating the importance of both arms in host innate immunity against sars-cov (totura et al., 2015) . full characterization of the role of tlrs in host innate antiviral response against sars-cov and mers-cov versus other hcovs will not only provide new knowledge about how tlr activation might impact cov pathogenesis, but might also identify new strategies for antiviral and vaccine development. for example, synthetic tlr agonists could potentially serve as antivirals and vaccine adjuvants in the prevention and control of covs. innate antiviral response is the first line of defense against cov infection. type i ifns are important antiviral and immunomodulatory agents. type i ifns function by binding to ifn-α receptor-1 (ifnar-1) and ifnar-2 receptor complex, thus activating janus family tyrosine kinase (jak), leading to the phosphorylation of signal transducer and activator of transcription (stat), a family of transcription factors regulating the expression of isgs. activated stat and irf9 form ifn-stimulated gene factor 3 (isgf3), stimulating expression of isgs by binding to ifn-stimulated response element (isre) in promoters of isgs (levy et al., 2001; samuel, 2001) . viral induction of isgs was abrogated in stat1 -/mice infected with sars-cov. the viral infection could not be cleared resulting in severe disease, extensive lung injury and 100% mortality zornetzer et al., 2010) . this indicates the importance of stat1 in sars-cov pathogenesis. isgs are the workhorses of the innate antiviral response with diverse functions including direct antiviral activities and regulation of adaptive immune system (schneider et al., 2014) . for example, ifn-inducible gene p53 evokes apoptosis in virus-infected cells (takaoka et al., 2003) . ifn-inducible protein kinase pkr, 2′, 5′-oligoadenylate synthetase (oas) and rnase l are important modulators involved in dsrna sensing, viral gene expression and replication. they act sequentially to trigger viral rna degradation and suppression of viral activities (samuel, 2001) . other isgs encoding antiviral effectors such as mx proteins, cholesterol-25-hydrolse, ifitm proteins, trim proteins, viperin, tetherin, cgamp synthase and sting could also be highly relevant to cov infection (schneider et al., 2014; schoggins et al., 2014; ma et al., 2015a; ma et al., 2015b) . inflammatory responses triggered by inflammatory cytokines like tumor necrosis factor α (tnf-α) and ifn-γ are also found to be ifn-dependent (samuel, 2001) . ifns do not only exert antiviral effects through activation of innate immunity but also act as modulators of adaptive immunity. adaptive immune response is activated by increased level of ifns. the levels of major histocompatibility complex (mhc) proteins class i and ii are found up-regulated by ifns. this facilitates efficient antigen presentation and hence cellular immune response to cov infection (samuel, 1991 (samuel, , 2001 ivashkiv and donlin, 2014) . in addition, the roles of non-conventional isgs including micrornas, long non-coding rnas and alternatively spliced isoforms have been increasingly recognized in recent years (schneider et al., 2014) . it will be of importance to determine whether sars-cov and mers-cov might be unique in isg activation as suggested in a recent study, which demonstrated that mers-cov induces repressive histone modifications to down-regulate specific subsets of isgs (menanchery et al., 2014b) . in relation to this, two areas concerning isg activation by covs might require more attention and research efforts. first, unbiased and large-scale screening of antiviral isgs using rna interference or crispr/cas9 technology might be carried out to identify key cellular factors that restrict sars-cov and mers-cov replication and infection. second, small-molecule compounds that activate antiviral isgs could be identified and tested for inhibition of sars-cov and mers-cov replication and infection. for example, establishing the significance of cgas and sting in cov infection might lead to the development of cyclic dinucleotides such as c-di-gmp and cgamp as novel anti-cov agents. covs have been reported to directly or indirectly suppress ifn production and signaling pathways by a subset of viral proteins via various mechanisms. in many cases, infected patients have shown diminished levels of type i ifns. this is especially true for sars and mers patients with severe diseases (faure et al., 2014) . it was also shown that sars-cov and mers-cov were capable of evading type i ifn production and signaling to different extents in cultured cells (kindler et al., 2013) . when the deficiency in type i ifn production in cov-infected cells was remedied by ifn-α treatment, cov replication was inhibited (falzarano et al., 2013) . combination of ifn-α with other antiviral drugs further improves the survival of infected patients (omrani et al., 2014) . this evidence suggests an essential role of type i ifns in the antiviral effect against cov infection. covs have evolved strategies to counter host antiviral response by antagonizing type i ifn production and signaling. cov proteins have been characterized to exhibit innate immunosuppressive effects in cellular models. below we will discuss them in three categories: structural, lineagespecific and non-structural proteins (nsps) (de groot et al., 2013) . nsps of covs are involved in the assembly of the replicase complex for viral rna synthesis (sevajol et al., 2014) . certain nsps have also been reported to possess innate immunosuppressive effect that facilitates viral replication and propagation, although these proteins per se are not required for viral life cycle (narayanan et al., 2008b; lokugamage et al., 2015) . nsps of different covs are more or less evolutionarily conserved suggesting their functional significance, with the exception of nsp1 and nsp2, which are thought to contribute to virulence of certain covs (neuman et al., 2014) . four structural proteins are found in covs, namely s, membrane (m), envelope (e) and n proteins. structural proteins contribute the architecture for virion assembly. accessory proteins are lineage-specific with diverse behaviors in different covs but are not essential for viral replication and propagation (de groot et al., 2013) . cov nsps have shown suppressive effects in various immune pathways including type i ifn production and signaling. sars-cov and mers-cov nsp1 proteins have been shown to selectively induce degradation of host mrna by inducing endonucleolytic cleavage while leaving viral rnas intact (huang et al., 2011; lokugamage et al., 2015) . in addition to the induction of endonucleolytic cleavage of host mrna, general inhibition of host mrna translation is achieved by binding of 40s subunit of ribosome with sars-cov nsp1 (huang et al., 2011) . particularly, sars-cov nsp1 inhibits innate immune response by translational repression of ifn mrna transcripts, hence altering ifn production and signaling (narayanan et al., 2008a; tanaka et al., 2012) . mers-cov nsp1 has also been characterized to specifically induce endonucleolytic cleavage of nuclear transcribed mrna while sparing cytoplasmic host mrna and viral rna (lokugamage et al., 2015) . this suggests a novel mechanism for evading host immune response. cov nsp3 protein has been characterized with a papain-like protease (plpro) domain for enzymatic cleavage of pp1a and pp1ab as well as a plp2 domain with deubiquitinating and deisgylating activity (clementz et al., 2010; mielech et al., 2014) . mers-cov plpro is able to antagonize ifn production induced by rig-i and mda5 as well as nf-κb activation . mers-cov plpro is catalytically more efficient (báez-santos et al., 2014) and its catalytic activity is indispensable for the suppressive effect on rig-i, mda5 and nf-κb (mielesh et al., 2014) . in contrast, sars-cov plpro does not require enzymatic activity for ifn antagonism (clementz et al., 2010) . hcov-nl63 and sars-cov plp2 transmembrane domain can also act as potent ifn antagonists to suppress ifn production induced by rig-in, a dominant active form of rig-i (clementz et al., 2010) . in another view of direct inhibition of ifn induction, nsp3 with deubiquitinating and deisgylating activity may also influence the ubiquitination and isgylation pattern and dynamics thus indirectly hindering innate immune response against cov infections (clementz et al., 2010) . for example, isgylation and ubiquitination of irf3 required for optimal activation is probably altered by plp domain of nsp3. apart from directly manipulating the signaling pathway involved in ifn production, several cov nsps were identified to act on viral rna to minimize ifn stimulation. n7-methylguanosine is the fundamental moiety of eukaryotic mrna cap structure and 2′-o-methylation on this moiety is a representative host signature to avoid prr activation as well as isg action. particularly, viral rna with this modification evades recognition by mda5 or ifit family antiviral factors (züst et al., 2011; daffis et al., 2010) . this is a common immunoevasive mechanism adopted by not only different covs but also other rna viruses. functional screening in yeasts suggested a novel function of sars-cov nsp14 as a guanine-n7-methyltransferase, the activity of which is required for viral replication and transcription (chen et al., 2009) . another nsp of sars-cov, nsp16, also possesses 2′-o-methyltransferase activity (menachery et al., 2014a; menachery et al., 2014c) . structural modeling suggested that sars-cov nsp16 associates with nsp10 in 1:1 ratio to form a complex of mature 2′-o-methyltransferase for viral cap methylation (chen et al., 2011; decroly et al., 2011) . a short peptide derived from nsp10 conserved region has been shown to be a promising nsp16 antagonist which outcompetes native nsp10 to blunt 2′-o-methyltransferase activity and restrict viral replication . plausibly, cov nsps might execute their innate immunosuppressive roles by targeting type i ifn production and signaling. further investigations are required to clarify whether and how far the sensing of cov rna and the induction of innate antiviral response are involved in the inhibitory activity of the nsp antagonists on cov replication. cov structural proteins have been shown to inhibit ifn production and signaling at multiple levels. sars-cov n protein showed inhibitory effects on ifn production induced by sendai virus and dsrna analogue poly(i:c) but no inhibition could be observed when downstream signaling molecules of tlr and rlr pathway were overexpressed. truncation mutant of n protein shows that the c-terminal domain is critical for rna-binding and ifn-antagonizing effect (lu et al., 2011) . this suggests sars-cov n may interfere with rna recognition by host immune sensors such as rig-i and mda5 thus achieving suppressive role in ifn production. other than n protein, sars-cov m protein has been characterized to potently down-regulate ifn production by impeding the formation of traf3·tank· tbk1/ikkε complex through the first transmembrane domain (siu et al., 2009 (siu et al., , 2014a . sars-cov m protein inhibits ifn production possibly through a sequestration model in which components of traf3·tank·tbk1/ikkε complex, an active complex for irf3 phosphorylation, are sequestered to specific locations in the cell (siu et al., 2009) . sars-cov m protein therefore exerts its inhibitory effects by impeding the formation of traf3·tank· tbk1/ikkε complex but not by modulating the catalytic activity of the complex. mers-cov m protein also exhibits ifn-antagonizing effects similar to its counterpart in sars-cov. in a previous study, mers-cov m is shown to impede ifn production by preventing irf3 translocation into the nucleus (yang et al., 2013) . however, the detailed mech-anism of inhibition remains unknown. recently, our group has characterized the mode of inhibition of ifn production by mers-cov m. consistently with previous report, we show that mers-cov m suppresses ifn production by preventing irf3 activation. we showed that mers-cov m interacts with traf3 which impedes the recruitment of tbk1 to traf3 complex. irf3 activation and dimerization have also been hampered as a result. the inhibitory effect is at least in part accounted for by the n-terminal transmembrane domains. despite of the similar behaviors, mers-cov m can only moderately suppress ifn expression when compared to sars-cov m. interestingly, hcov-hku1 m protein does not exert any inhibitory effects on ifn production (siu et al., 2014a) , suggesting that the ifn-antagonizing activity of structural proteins is unique to each cov but not universal. it will be of great interest to see whether this may correlate with the pathogenicity of different hcovs. eight accessory proteins have been identified in sars-cov and five are found in mers-cov (narayanan et al., 2008b) . sars-cov genome encodes orf3a, orf3b, orf6, orf7a, orf7b, orf8a, orf8b and orf9b as accessory proteins (narayanan et al., 2008b) . sars-cov orf3b and orf6 have been found to antagonize type i ifn production and signaling. particularly, sars-cov orf3b and orf6 suppress ifn-β production by perturbing irf3 activation induced by sendai virus infection. sars-cov orf3b and orf6 also suppress ifn-β-induced activation of isre in isg promoters (kopecky-bromberg et al., 2007) , although they are not able to reduce the level of phosphorylation of stat1, a transcription factor that activates isre activity once phosphorylated. however, sars-cov orf6 has been shown to inhibit stat1 translocation for isre activation (kopecky-bromberg et al., 2007) . the findings suggest a mode of inhibition of ifn-β signaling by sars-cov. ifn antagonism of accessory proteins has also been observed in another deadly hcov. mers-cov genome encodes orf3, orf4a, orf4b, orf5 and orf8b (de groot et al., 2013) . among the five accessory proteins, orf4a, orf4b and orf5 show the ability to dampen ifn production (yang et al., 2013) . suppression of ifnβ promoter-driven luciferase activity has been observed in cells transfected with orf4a, orf4b and orf5 plasmids. all these 3 accessory proteins are able to block irf3 translocation to the nucleus to activate ifn promoter (yang et al., 2013) . mers-cov orf4a shows an additional level of inhibition of innate immunity by intervening nf-κb activation. in another study, orf4a has been shown as an antagonist of ifn production by inhib-iting irf3 translocation but has no effect on ifn signaling (niemeyer et al., 2013) . our group demonstrated that mers-cov orf4a interacts with pact, a cellular dsrna-binding protein that optimally activates rig-iand mda5-induced type i ifn production, in an rnadependent manner (siu et al., 2014c) . this suggests that orf4a may compete with rig-i and mda5 for rna, rendering the inactivation of rig-i and mda5. direct interaction of orf4a with pact may also prevent interaction of pact with rig-i and mda5, thus compromising pact-dependent activation of rig-i and mda5 required for optimal induction of ifn production. although we and others have observed the ifn-antagonizing activity of mers-cov orf4b, different activity profiles and mechanisms have been suggested (yang et al., 2013; matthews et al., 2014) . one recent report suggested that orf4b directly interacts with and inhibits tbk1/ikke in the cytoplasm but might also perturb type i ifn production in the nucleus through an unknown mechanism (yang et al., 2015) . mouse hepatitis virus, another betacoronavirus closely related to hcov-oc43 and hcov-hku1, encodes a lineage-specific accessory protein named ns2 with innate immunosuppressive property (zhao et al., 2012) . biochemical assays indicate that ns2 protein has phosphodiesterase activity against 2′, 5′-a, the product of oas . thus, ns2 is a potent inhibitor of an ifn effector molecule and it might represent a new family of viral and cellular proteins with innate immunosuppressive activity gusho et al., 2014) . whether distantly related proteins in hcov-oc43 and hcov-hku1 might have similar activity remains to be determined. more importantly, it will be of interest to see whether sars-cov and mers-cov might encode proteins with similar enzymatic activity. multiple ifn antagonists have been identified and characterized in sars-cov and mers-cov. some differences between these ifn-antagonizing viral proteins and their counterparts in other covs such as the parental bat viruses of mers-cov have also been noticed (siu et al., 2014c) . existing evidence supports several important notions. first, although sars-cov and mers-cov share some features in common, they are distinct and use unique mechanisms for innate immune evasion (perlman and zhao, 2013) . second, both sars-cov and mers-cov are bat-origin covs that are well adapted in bats but newly emerge in human. this provides a golden opportunity for the study of cov-host interaction, cov adaptation as well as the arms race between host innate antiviral immunity and covs. observing how the arms race between the host and sars-cov or mers-cov might evolve when the viruses become adapted to human will be most revealing and could provide important clues as to how a balance of power in this arms race might result in attenuation with increased transmissibility. finally, studies on sars-cov and mers-cov have overturned existing concepts and derived new principles and thoughts to cov biology. particularly, mechanisms by which sars-cov and mers-cov evade innate immunity have attracted increasing attention. however, many key issues remain obscure. particularly, better in vivo evidence should be obtained to clarify whether more potent inhibition of innate ifn production and signaling by sars-cov and mers-cov is a key determinant in virulence and disease severity. covs have drawn a lot of interests in the light of the recent emergence of mers-cov. it remains to be understood whether the emerging deadly covs causing ards might ultimately be established and adapted in human resulting in significant attenuation of virulence. from the identification of the first two hcovs, hcov-229e and hcov-oc43 in the mid-1960s, we learned that hcov was able to cause only common cold. however, the outbreaks of sars and mers that have claimed hundreds of lives revealed the other extreme of cov pathogenicity and raised new questions in cov biology. so far no vaccines have been developed against sars-cov and mers-cov. infection with sars-cov and mers-cov has been accompanied with suppression of innate immune response, most notably with the suppression of type i ifn production and signaling pathways. as the first-line defense in the immune system, suppression of innate immune response by these covs has impeded the host ability to restrict infection, causing significant casualties. although many reports have shed light on the molecular mechanism by which various cov proteins antagonize type i ifn production and signaling, most of the studies were performed with overexpression experiments in cellular models. future emphasis should be put on the characterization of knock-out viruses with which the function of a particular viral gene could be studied in a more physiologically relevant context. infectious clones and replicons for sars-cov and mers-cov have been generated for this reverse genetic approach (yount et al., 2003; almazán et al., 2006 almazán et al., , 2013 almazán et al., , 2014 scobey et al., 2013) . ifn and cytokine profiles of deadly hcovs such as sars-cov and mers-cov can be compared with hcov-229e and hcov-oc43 causing mild diseases. the pivotal significance of type i ifns in innate immune activation and modulation has been discussed in this review. suppression pattern of ifn may provide insights on the high pathogenicity of deadly hcovs. the arms race between host innate antiviral response and emerging human covs might evolve after their introduc-tion and establishment in human populations, with significant impact on virulence, transmissibility and disease severity. emerging human covs remain a potential threat to global public health. new knowledge about the host-cov arms race will provide new ideas, targets and attenuated strains for the design and development of antivirals and vaccines for prevention and control of deadly 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studies of arterivirus-and coronavirus-host interactions transcriptomic analysis reveals a mechanism for a prefibrotic phenotype in stat1 knockout mice during severe acute respiratory syndrome coronavirus infection ribose 2′-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 we thank hinson cheung, kitty fung, edwin kong and sam yuen for reading the manuscript critically. coronavirus research in our laboratory was supported by hong kong health and medical research fund (13121032, 14130822 and hkm-15-m01) and hong kong research grants council (hku1/crf/11g, c7011-15r and t11-707/15-r). the authors declare that they have no conflict of interest. this article does not contain any studies with human or animal subjects performed by any of the authors. key: cord-312080-pu6m4qad authors: he, bing; chen, guomin; zeng, yi title: three-dimensional cell culture models for investigating human viruses date: 2016-10-27 journal: virol sin doi: 10.1007/s12250-016-3889-z sha: doc_id: 312080 cord_uid: pu6m4qad three-dimensional (3d) culture models are physiologically relevant, as they provide reproducible results, experimental flexibility and can be adapted for high-throughput experiments. moreover, these models bridge the gap between traditional two-dimensional (2d) monolayer cultures and animal models. 3d culture systems have significantly advanced basic cell science and tissue engineering, especially in the fields of cell biology and physiology, stem cell research, regenerative medicine, cancer research, drug discovery, and gene and protein expression studies. in addition, 3d models can provide unique insight into bacteriology, virology, parasitology and host-pathogen interactions. this review summarizes and analyzes recent progress in human virological research with 3d cell culture models. we discuss viral growth, replication, proliferation, infection, virus-host interactions and antiviral drugs in 3d culture models. infectious diseases have been major challenges to human health and survival for centuries (morens et al., 2004) . although, over the years, significant progress has been made in the prevention and control of infectious diseases, pathogenic microbes continue to pose an enormous threat to human health (heesterbeek et al., 2015) . the level of human mortality attributed to infection is 13-15 million deaths annually (who, 2013) . in recent years, newly emerging infectious agents have represented a continuing challenge, especially those causing viral diseases, such as h7n9 influenza viruses in 2013 (lam et al., 2013) , the ebola virus outbreak in 2014 (team, 2014; heymann et al., 2015) and the zika virus outbreak in 2016 (lessler et al., 2016) . therefore, studying the mechanisms of viral diseases, antiviral agents and vaccines is critical for responding to viral diseases. our current understanding of a multitude of human viral diseases and antiviral drugs is based largely on traditional 2d cell culture, in which cells are grown on glass substrates or flat plastic, such as petri-dishes, multiwell plates and cell culture flasks baker and chen, 2012; li and cui, 2014) , or on animal model systems. indeed, 2d cell cultures have contributed tremendously to the investigation of infectious disease etiology, the immune mechanisms used to defend against human viruses barrila et al., 2010) , and especially the biochemistry and molecular biology of viral replication (andrei, 2006) . however, 2d monolayer culture has significant restrictions in mimicking the physiological complexity of the in vivo microenvironment that is encountered by pathogens; this model cannot accurately reproduce the natural infection process (barrila et al., 2010) . in addition, due to the lack of suitable cell culture models, the study of the life cycle of fastidious viruses and virus-host interactions has been hampered (lindenbach et al., 2005; sainz et al., 2009a) . furthermore, animal models are costly and their use has ethical issues (page et al., 2013) . a multitude of pathogens are species specific, and the pathogenesis mechanisms cannot be captured by time-consuming animal models (griffith and swartz, 2006) . as an example, sars (severe acute respiratory syndrome) in animal models seldom progressed to lethality, however, it may cause death in people (louz et al., 2013) . to overcome these limitations associated with 2d monolayers and animal models, various types of 3d cell culture models have been developed (edmondson et al., 2014) . 3d cell models bridge the gap between 2d cell cultures and animal models by providing an in vitro cell model system mimicking more accurately the in vivo microenvironment, which contributes to the understanding of both virus-host interaction and the fundamental mechanisms of human viruses, as well as promoting the development of antiviral drugs. cells grown on 3d culture systems can differ considerably in their morphology, viability, signaling control, gene expression, differentiation, proliferation and drug sensitivity in comparison with those grown on flat 2d tissue substrates (friedl et al., 2012; edmondson et al., 2014; antoni et al., 2015) . for example, matrigel-cultured huh7 cells assemble into 3d spheroids, whereas standard 2d-cultured cells form epithelial monolayers (molina-jimenez et al., 2012) . cells cultured in a 3d matrix can adequately reproduce the function of 3d tissues and mimic cell-cell and cellmatrix interactions in vivo. 3d culture systems have been designed to allow investigation of infectious pathogens, such as human viruses, bacteria and parasites. these models have an invaluable role in virology today. this review focuses on the recent progress of human virological research with 3d cell culture models, including human viral growth, replication, proliferation, infection, viral life cycle, virus-host interaction and the development of antiviral drugs. logical science. cells grown in a 3d environment more closely represent normal cellular characteristics and biological function that the traditional 2d monolayer culture cannot provide. for example, cells cultured in a 3d matrix can adequately reproduce the function of 3d tissues and mimic cell-cell and cell-matrix interactions that affect proliferation, differentiation, morphology and a range of cellular functions in vivo, which are lost in conventional 2d conditions (mazzoleni et al., 2009) . the classic polarized patterns of signaling that guide migration in 2d models are not essential for efficient migration in 3d models . almost all cells use lamellipodia to migrate on 2d substrates, however, multiple modes of migration are observed in three dimensions, including lamellipodial, lobopodian, amoeboid migration (madsen and sahai, 2010; and collective migration (friedl and alexander, 2011) . baker and chen (2012) further discuss examples that 3d context can provide insights in adhesion, migration and polarization which cannot be provided by the traditional 2d systems. table 1 shows the differences between 2d and 3d cell cultures. strengths and weaknesses of 3d models 3d cell cultures provide a platform for cell proliferation and differentiation. in addition, some emerging 3d culture models can allow nutrients and metabolites to be transported in and out of the 3d cells (li and cui, 2014) . although current 3d systems provide unique mechanistic insights into cell-microenvironment interaction, most 3d models do not replicate all complex physiological features of real-tissue in vivo. they often lack the normal vasculature, normal transport of small molecules, host immune responses, internal tissue tensions, tissue heterogeneity and fluid flows observed in vivo. in addition, those models cannot precisely replicate biochemical composition, gradients of soluble regulatory factors and other microenvironment factors in vivo (yamada and cukierman, 2007; grinnell and petroll, 2010; baker and chen, 2012; friedl et al., 2012) . moreover, more complex architectures are found in tissues in vivo, such as orthogonally arranged collagen sheets and collagen fibril bundles. however, the most current methods to prepare 3d fibrillar matrices result in matrices in which fibrils lack any particular organization or become aligned uniaxially (grinnell and petroll, 2010) . a brief summary of the strengths and limitations of 3d culture models is presented in table 2 . currently available and typical 3d models are spontaneous cell aggregation, liquid overlay, gyratory rotation and spinner flask spheroid cultures, scaffold-based culture systems, microcarrier beads and the rotary cell culture system (kim, 2005; page et al., 2013) , as well as 3d perfusion cell culture, microfluidic 3d cell culture and 3d cell culture by magnetic levitation (souza et al., 2010; li and cui, 2014; van duinen et al., 2015) . here, we only focus on those 3d models that have been utilized to study human viruses or will be applicable for this in the future. 3d mcs culture has become a valuable tool for mimicking the biological features and functional characteristics of native tissue. mcss are cells that aggregate and undergo the process of self-assembly on an attachment surface or scaffold. during self-assembly, mono-dispersed cells form 3d microtissues (achilli et al., 2012; . the spheroid format is particularly useful in cancer research as it enables quick discovery of morphological changes in transformed cells (antoni et al., 2015) . the most commonly used model is the multicellular tumor spheroids model, which has phenotypic characteristics close to those of human tumor tissues. consequently, it has been applied extensively in reproducing the key elements of malignant tumor behavior (hamilton, 1998) . traditional and newer techniques have been investigated for spheroid production. first, the spinner flask method, which prevents cell attachment to the vessel surface and promotes cell-cell contacts via constant stirring. this method is relatively simple and produces massive spheroids, suitable for high-throughput testing, but has a high shear force and variability in cell size/number (lin and chang, 2008; breslin and o'driscoll, 2013) . second, the hanging drop method depends on gravity forces to form spheroids on inverted substrates (kelm et al., 2003) . this method is inexpensive and suitable for highthroughput testing, and the spheroid size can be con-trolled; however, it is labor intensive and mass production is limited (lin and chang, 2008) . third, liquid overlay culture, which prevents the attachment of cells to tissue culture plates and promotes spheroid formation (achilli et al., 2012) . although this method is easy to set up, rapid and makes screening easy, the size and shape of the spheroids are heterogeneous. fourth, a rotating-wall vessel (rwv) creates a microgravity that supports cells in suspension and promotes cell aggregation into spheroids. this method allows good control of the microenvironment over time (achilli et al., 2012) . in addition, pellet culture 3d scaffolds can also form mcss, especially with micro-fluidics and the magnetic cell levitation method, which were developed in recent years to create new opportunities to form spheroids (kim et al., 2013) . mcs models have given rise to many advances in basic cell science, including understanding tumor invasion and migration, and creating models for toxicology testing and drug discovery (achilli et al., 2012; vinci et al., 2015) . the development of new technologies for analyzing spheroids has led to a rapid increase in their adoption and expansion of their applications. new technologies for analyzing spheroids have led to a rapid increase in their adoption and expansion of their applications. apart from those applications mentioned above, mcs models have also been investigated as basic units for human viruses. although the study of viruses in spheroid models is still limited so far, investigating virus-cancer interactions, the mechanism of viruses causing cancer and evaluating antiviral agents relating to tumors by those models may lead to the future direction in the field of cancer. cells cultured in 3d system can represent a more physiological microenvironment. vinci et al., 2012 as compared with 2d cultures, 3d cell cultures more accurately simulate normal cell morphology, proliferation, migration, cell-cell and cell-ecm interactions. antoni et al., 2015 edmondson et al., 2014 cell culture is flexible, cost effective and controllable, as well as a high-throughput platform. nickerson et al., 2007 several 3d models can monitor and control physiological conditions: temperature, ph, oxygen concentration, metabolites and growth factors. murakami et al., 2008; li and cui., 2014; worthington et al., 2015 limitations references in vivo complex and physiological microenvironment not to be replicated. friedl et al., 2012; van duinen et al., 2015 poor reproducibility for some biomimetic scaffolds. antoni et al., 2015 some available 3d models to be more time and expensive. vinci et al., 2012 quality of imaging interfering with 3d scaffold size, material transparency and microscope depth. antoni et al., 2015 3d cell culture using rwv or radial-flow bioreactor (rfb) the rwv is an optimized suspension culture vessel for forming 3d tissue-like assemblies (tlas). the rwv is based on a rotating cylinder that is completely full with culture medium, the sedimentation of cells in the vessel is counterbalanced by the rotating fluid, creating a constant, gentle fall of cells through the medium under conditions of physiologically relevant fluid shear (hammond and hammond, 2001; nickerson et al., 2004; barrila et al., 2010) . to generate 3d cellular aggregates, cells are first cultured in 2d monolayers. when the cells have grown to an appropriate density, they are removed from the petri dishes or flasks and re-suspended in culture medium, then they are placed within porous ecmcoated microcarrier beads for attachment (barrila et al., 2010) . lastly, the cellular aggregates are harvested and analyzed. traditional cell culture and parts of some 3d models may generate high shear forces, which may injure the cells and block proper tissue-specific differentiation (unsworth and lelkes, 1998), and inadequate nutrient and oxygenation transfer give rise to cell death, posing critical obstacles to establishing functional 3d culture systems. to overcome these problems, the nasa (national aeronautics and space administration) johnson space center developed the rwv (goodwin et al., 1992) , which provides a low fluid-shear environment and minimal turbulence that promotes cell growth and randomized gravitational vectors (goodwin et al., 1993) . the fluid dynamics of rwv bioreactors allow oxygen and nutrients to diffuse across into the cell aggregates and prevent tissue constructs from necrotic cores (goodwin et al., 2015) . studies have shown that the rwv can produce 3d models and can recreate many of the fundamental facets of the real tissue in vivo, such as 3d cellular polarity, cellular differentiation and proliferation, cell-cell interaction, multicellular complexity and functionality (rhee et al., 2001; cerwinka et al., 2012; samuelson and gerber, 2013) . rwv-derived 3d models have been applied to the investigation of infectious agents (viruses, bacteria and parasites) (barrila et al., 2010) . those models can reflect the natural infection process. the inherent flexibility of this system is an ideal platform for exploring fundamental questions in virology. a multitude of research has shown that rwv-derived models utilizing human cells are a valuable tool for investigating viral growth, replication, viral infection, viral entry, the viability of virions and virus-host interaction (margolis et al., 1997; long et al., 1998; nickerson et al., 2007; straub et al., 2007; barrila et al., 2010; berto et al., 2013; goodwin et al., 2015) . the rwv provides a useful model system for studying human viruses and has the potential for use in developing and screening antiviral drugs, as well as evaluating vaccines. the other bioreactor used for studying human viruses is the rfb, consisting of a vessel, column and pc monitoring system. it was originally designed for creating artificial liver tissues allowing human liver cells to maintain their morphological characteristics and physiological functions for a relatively long period of time (kawada et al., 1998; aizaki et al., 2003) . culture conditions can automatically be controlled. temperature, ph and oxygen concentration in the conditioning vessel are continuously monitored by pc and conditioned by mass flow controller (murakami et al., 2008) . the rfb-derived 3d models have mainly been used for hepatitis c virus (hcv). however, to our knowledge, no research associated with the rfb being used for human viruses has been reported in pubmed since 2008. the organotypic epithelial raft culture is an in vitro 3d culture system, where epithelial cells are placed on top of a dermal equivalent and then cultured at the air-liquid interface to full differentiation (meyers et al., 1992; andrei, 2006; fang et al., 2006) . raft cultures are prepared using cells or tissues derived from dispersed primary keratinocytes, explanted epithelial tissue or established cell lines (andrei et al., 2010) . organotypic raft culture was originally designed to accurately mimic the in vivo morphological and physiological characteristic of the epidermis (asselineau and prunieras, 1984; meyers et al., 1992) . this system, forming a stratified and differentiated epithelium, has provided researchers a useful means to investigate epitheliotropic viruses (chow, 2015) . over the past few years, human papillomavirus (hpv)-host interactions have been demonstrated with these raft cultures similar to those observed in vivo. this system has been a paramount milestone in the study of hpv so far. 3d scaffold materials are designed to support the attachment, proliferation and differentiation of selected cell populations grown in 3d culture models. scaffold-based 3d cultures are playing an increasing role in tissue engineering. in recent years, these models have been applied to virology. several matrices have been used to investigate human viruses. among them, the use of matrigel as a 3d scaffold has shown promise for the study of several viruses. matrigel, a gelatinous protein mixture, resembles the complex extracellular environment observed for tissues in vitro and produces a thick matrix for 3d cell culture (kleinman and martin, 2005) . hcv has been investigated by this 3d model. in addition, the use of matrigelbased systems for recombinant adenoviruses and ep-stein-barr virus has begun to capture researchers' attention (fotheringham and raab-traub, 2013; wang et al., 2014) . alginate, including sodium-alginate salt and calcium-alginate, is also used for studying hcv. mebiol gel, a thermoreversible gelation polymer (tgp), is a synthetic compound that consists of thermo-responsive and hydrophilic polymer blocks. as a 3d scaffold it was proven to be susceptible to hcv replication (rajalakshmy et al., 2015) . although the scaffold/matrix-based 3d culture models used for human viruses appear to be very limited so far, these systems may be applied in virology to define new anti-virus strategies, and may also provide a potential platform for the specific design of effective individual therapy according to patient-specific strains (aly et al., 2009 ). the multicellular complexity of tissues cannot be captured by typical 3d cell culture models, these models lack vasculature, do not provide precise control over gradients and undergo medium exchange at discrete time points instead of in a continuous manner (van duinen et al., 2015) . in recent years, several novel 3d culture models have been developed for further studying human tissue pathophysiology and physiology in vitro. microfluidic 3d cell culture allows spatial control over fluids in micrometer-sized channels. this model has become a valuable tool to further increase the physiological relevance of 3d cell culture by enabling spatially controlled co-cultures, perfusion flow and spatial control over signaling gradients. van duinen et al. (2015) have reviewed the most important progress in microfluidic 3d cell culture since 2012. using hydrogels in microfluidic systems have been a recent trend, this model offers cells a more physiologically relevant 3d matrix (huang et al., 2011; chung et al., 2012) . the microfluidics 3d system will play an important role in the development of personalized medicine, especially in the field of cancer. in addition, 3d perfusion cell culture, an emerging technology, may provide a potential research avenue for commercial applications in drug discovery, regenerative medicine and tissue engineering. this model, mimicking the blood circulation in the human body, can control physiological chemostatic conditions, and create gradients of oxygen, growth factors and other biochemical signals. the 3d perfused culture model also can maintain the stability of the local microenvironment of the residing cells by continuously providing a nutrient supply and waste removal (li and cui, 2014) . souza et al. (2010) reported a novel 3d tissue culture based on magnetic cells levitation. in this method, cells bind with a magnetic iron oxide nanoparticle assembly comprising gold nanoparticles and cell-adhesive peptide sequences. by spatially controlling the magnetic field, cells are concentrated at the airliquid interface, where they aggregate to form larger 3d cultures (haisler et al., 2013) . the magnetic levitation method (mlm) and other associated techniques (cell culture, imaging and ihc) adapted for the mlm are described by haisler et al.(2013) . although there are no reports associated with studying human viruses using those models, these approaches may become valuable tools in the field of human viruses in the future. the advantages and disadvantages of different 3d culture models used for human viral growth, replication, proliferation, infection and antiviral drugs are listed in table 3 . to develop routine and advanced 3d cell culture devices, several factors must be considered, such as the cost of equipment, running cost, throughput and the simple operation. commercial development of in vitro 3d models and applications has been summarized by li and cui (2014) , including the suppliers, the core technology and 3d products. the devices, scaffolds and technical demands for different 3d cell cultures are listed in table 4 . 3d models are modular, tractable biomedical systems, which will yield great advances in our understanding of biological science. although some shortcomings have been found in the current 3d systems, 3d cell culture models hold enormous potential for the basic cell science, tissue engineering and infectious pathogens. 3d cell culture is an evolving field and requires further research for its optimization by combining a number of key areas including materials science, cell biology and bioreactor design. human papillomavirus (hpv) is small, non-enveloped viruses with a double-stranded dna genome . more than 180 types of hpv have been identified, which are classified into low-risk and high-risk hpv types (bernard et al., 2010) . the viral genome includes six early proteins e1, e2, e4, e5, e6 and e7, and the late structural proteins l1 and l2 (malik et al., 2014) . high-risk types cause cervical cancers and other anogenital carcinomas (vulvar, vaginal and anal). types 16 and 18, the two most carcinogenic hpv types, are thought to contribute to 70% of human cervical cancer (schiffman et al., 2007) . organotypic epithelial raft cultures represented a breakthrough in the study of papillomaviruses due to the strict link of hpv replication with epithelial cell differentiation (andrei et al., 2010) . so far, more than nine aspects of hpvs have been explored in these systems. hpv cannot be propagated in 2d cell monolayer cultures, so organotypic epithelial raft cultures that generate a stratified and differentiated epithelium have been applied in studying the hpv life cycle (chow, 2015) . the organotypic raft culture system has allowed the study of the en-tire differentiation-dependent life cycle of hpvs, including virion morphogenesis (mclaughlin-drubin et al., 2003; mclaughlin-drubin and meyers, 2005) . fang et al. (2006) demonstrated episomal maintenance of hpv-11 dna in n-tert cells. hpv-11 episomal dna-containbarrila et al., 2010 hjelm et al., 2010 unsworth et al., 1998 hammond and hammond, 2001 organotypic epithelial raft cultures ing cell populations grown in raft culture showed induction of a productive viral life cycle. this system has served as a faithful in vitro model for investigating propagation, infection and neutralization of hpvs, as well as producing infectious hpv virions (ozbun, 2002; mclaughlin-drubin et al., 2004; chow et al., 2009; wang et al., 2009 ). examinations of virus-host interactions have also been reported (anacker and moody, 2012) . several researchers have further used the organotypic epithelial raft cultures to study the interaction of hpv with other epitheliotropic viruses, such as herpes simplex virus (hsv) and hpvs, adeno-associated virus and hpv interaction hermonat et al., 2005) . importantly, meyers et al. (2002) demonstrated that the nonstructural genes of hpv18 functionally interact with the structural genes of hpv16, allowing the complete hpv life cycle to occur, which is the first report of the propagation of chimeric hpv by normal life cycle pathways. screening and evaluating antiviral compounds can also be carried out with raft cultures (satsuka et al., 2010) . recently, research on the relationship between tumor progression and hpv is increasing, which will hopefully lead to the development of effective treatments for hpv-associated cancer. a detailed review concerning evaluation of the efficacy of the therapeutic interventions for hpv using epithelial raft cultures has been published by andrei et al. (2010) . so far, these studies appear to be rather limited. in recent years, an increasing number of studies have addressed more specifically the role of hpv gene products and the difference in protein function between different hpv types, as well as the mechanisms of hpv carcinogenesis, especially the relationship between hpv oncoproteins (e2, e4, e5) and cervical tumors (doorbar, 2016) . the effects of hpv16 e5 deletion mutants on epithelial morphology have been reported by barbaresi et al. (2010) . mole et al. (2009) using the organotypic raft culture with epithelial cells, demonstrated that sf2/asf (splicing factor 2/alternative splicing factor) is up-regulated in response to differentiation in hpv-infected cervical epithelial raft tissue. a specific subset of sr proteins (ser-arg rich proteins) regulated by hpv16 e2, including sf2/asf, srp20 and sc35, were also overexpressed during cervical tumor progression. furthermore, organotypic raft cultures using verruciformis-derived keratinocytes could be used to reconstruct the î²-hpv life cycle and show the relationship between î²-hpv e4 expression patterns and disease severity. this finding is indicative that e4 may be a possible marker of viral expression during î²-hpv-associated skin cancer progression (borgogna et al., 2012) . a longitudinal cell culture using organotypic raft cultures has been used to investigate the immortalizing and transforming abilities of naturally occurring e6 variants in primary human foreskin keratinocytes (phfks). the observations provided insight into the mechanisms behind how phfks are immortalized and transformed into malignant tumors by the viral oncoproteins of hpv16 (richard et al., 2010) . in addition, a breakthrough in hpv chimeric genomes producing infectious virus in organotypic raft cultures was achieved. researchers constructed hpv18 chimeric genomes in which the hpv18 capsid genes were replaced with those of evolutionarily diverse pv types, including hpv45, hpv39, hpv33, hpv31, hpv11, hpv6b, hpv1a, crpv and bpv1. each of the chimeric genomes generated infectious viral particles in organotypic raft cultures (bowser et al., 2011) . human immunodeficiency virus (hiv), originally isolated from a patient with acquired immune deficiency syndrome (aids) in france in 1983 ( barre-sinoussi et al., 1983) , is a major contributor to the global burden of disease. although dramatic progress has been made in the development of novel antiviral drugs (de clercq, 2007) , an effective vaccine remains elusive despite two decades of effort (maartens et al., 2014) . the use of antiretroviral drugs has markedly reduced the mortality rate amongst aids patients. however, the effect of these drugs on oral epithelium growth and differentiation is presently unknown. a new 3d cell culture system, organotypic raft cultures of gingival keratinocytes, has been established. research demonstrated that hiv protease inhibitor amprenavir severely inhibited the growth of gingival epithelium cultured in this model. when the drug was added at day 8, amprenavir treatments altered the proliferation and differentiation of gingival keratinocytes (israr et al., 2010) . there are several other studies that have reported similar results utilizing organotypic raft cultures of gingival keratinocytes to investigate the effect of anti-hiv drugs on gingival epithelium growth and differentiation. those drugs included protease inhibitor lopinavir/ritonavir, and nucleoside reverse transcriptase inhibitors zidovudine, efavirenz and tenofovir (israr et al., 2011; mitchell et al., 2012; mitchell et al., 2014) . furthermore, balzarini et al. (2013) developed a multi-targeted drug, 6-phosphonylmethoxyethoxy-2, 4-diaminopyrimidine (pmeo-dapym), and demonstrated that this drug efficiently suppressed both hiv-1 and hsv-2 in organotypic epithelial raft cultures of primary human keratinocytes. in addition, 3d cell cultures could be developed as a potential model for studying the neuropathogenesis of hiv infection and the development of drug candidates that could effectively treat the neurological complications of hiv infection . investigating the effects of highly active antiretroviral therapy (haart) and designing multi-targeted antiviral drugs that could effectively suppress both hiv and hepatitis b virus (hbv) (or hcv) may lead to the future direction of hiv in 3d culture systems. hepatitis c virus (hcv), first identified in 1989, poses an enormous threat to public health and affects more than 170 million people around the world (choo et al., 1989; poynard et al., 2003; cox, 2015) . although the 2d cell culture model has been the standard tool for investigating hcv in cell culture, the hcv life cycle in vivo occurs in a much more complex environment compared to that in standard 2d cultures. 3d cell culture models can more closely mimic the polarized and differentiated state of hepatocytes in vivo . so far, hcv-associated research has been reported in three independent 3d cell cultures systems: 3d/rfb, 3d rwv bioreactors and the scaffold/ matrix-based 3d cultures. differentiated human hepatoma flc4 cells transfected with full-length hcv rna can produce and secrete infectious particles in the 3d rfb culture system (aizaki et al., 2003) . a similar result has been found in the rfb system following transfection of flc4 cells with a dicistronic hcv genome derived from genotype 1b, as well as in the 3d/tgp system using huh-7 cells (murakami et al., 2006) . furthermore, research has demonstrated that a long-term culture of the 3d rfb system provides a potential platform for investigating hcv dynamics, as well as examining the therapeutic effects of interferon alpha in this 3d culture model (murakami et al., 2008) . although hcv culture infection models based on the hcv jfh-1 molecule clone and huh-7 cells permit the production of virus, these recombinant hcv genomes only proliferate in sub-lines of huh-7 cells, which do not allow infection or proliferation of blood-borne hcv. a novel in vitro culture system combining 3d/tgp and immortalized human hepatocytes (hus-e/2 cells) demonstrated efficient support of the infection and replication of natural hcv (aly et al., 2009) . moreover, another system based on a 3d hollow fiber culture system and the hus-e/2 cell line was used for investigating the life cycle of blood-borne hcv, this 3d infection system allowed the reproduction of strain-dependent events reflecting virus-cell interactions and viral dynamics (aly et al., 2009) . more recently, hcv infection, replication and life cycle have been studied frequently. rwv-cultured huh-7 cells form complex, multilayered 3d aggregates, highly permitted for hcv infection, which provides a platform for studying hcv biology and the interaction between hcv infection and host cell function (sainz et al., 2009b) . cho et al. (2009) described that huh-7.5 cells cultured in a 3d peg-based hydrogel system can be efficiently infected with hcv, which was the first report of de novo infection with the virus (both replication-defective pseudovirus particles and fully infectious hcv). in addition, matrigel-embedded 3d culture of huh-7 cells or huh-7.5 cells was used as a hepatocyte-like polarized system and supported hcv infection by jfh-1 virus, as well as producing infective viral particles (molina-jimenez et al., 2012; liu et al., 2014) . researchers succeeded in reconstituting a hepatic-like structure formation from huh-7 cells using a calcium-alginate encapsulation model, which provides an opportunity for viral studies, especially for application with hcv (tran et al., 2013) . recently, mebiolgel-derived 3d culture models were shown to support cell growth as 3d spheroids for up to 63 days, this system was susceptible to hcv infection and replication; it could be implemented as an alternate for primary hepatocytes in studies such as viral isolation from patient serum (rajalakshmy et al., 2015) . so far, many facets of hcv have been extensively studied in 3d cell culture systems. however, knowledge of several dimensions of hcv are still limited: (i) hcv biology, (ii) anti-hcv therapeutics, (iii) hcv-hiv interaction and (iv) hcv-cancer interaction. more efforts must be taken to further research hcv. 3d culture systems will provide insight into the biophysical properties and viral morphogenesis of hcv particles and assessing anti-hcv compounds. this system may have advantages in studying aspects of hcv biology, such as viral assembly and budding, and also provides a system for screening antiviral drugs that inhibit the release or transmission of infectious hcv . hepatitis e virus (hev) was discovered during the soviet occupation of afghanistan in the 1980s. anti-hev therapy has been successfully used in chronic hepatitis e, the first vaccine available for clinical use is licensed in china (mihalcin et al., 2015) . at present, the life cycle of hev is still poorly under-stood, extensive study of the viral replication cycle has been hampered by the lack of efficient cell culture systems and animal models (osterman et al., 2015) . research demonstrated that hev can replicate efficiently in human hepatoblastoma plc/prf/5 cells cultured in a rwv for up to 5 months, hev nucleic acid was detected by reverse transcription-pcr in the supernatant of the infected cells in the 3d cell culture system. in contrast, that was not observed in the 2d monolayer system. complete virions were detection by electron microscopy. however, the study of hev using 3d systems is limited so far, and 3d cultures will offer a potential platform for in vitro cultivation of hev and investigation of the biology of hev, as well as the viability of hev in pig and other environmental samples (berto et al., 2013) . human herpes virus (hsv) is a significant pathogen and responsible for a variety of disorders (nicoll et al., 2012) . hsv-1 and hsv-2 are ubiquitous human pathogens. hsv-1 is normally associated with orofacial infections and encephalitis, whereas hsv-2 usually causes genital infections. more culture models have been applied to epithelial cells for researching hsv. the organotypic raft culture system, which accurately mimics the in vivo physiology of the epidermis, is a most powerful tool for studying infectious agents that infect the epithelium. the first report about the application of 3d organotypic raft culture for the study of hsv appeared in 1996 (syrjanen et al., 1996) . researchers demonstrated that the f strain of hsv-1 had the ability to produce lytic or nonproductive infection in hacat cells (immortalized skin keratinocytes) cultured in a 3d organotypic tissue culture. the cultures were infected with hsv-1 (5 pfu) 30 min after the lifting of the epithelial cells into the air-liquid interface and were collected 1 week after inoculation. these cultures were positive for hsv dna using pcr. one year later, another research study focused on utilizing the organotypic tissue culture methodology for the study of the infection, replication and spread of hsv-1 in fully stratified and differentiated human epithelial tissue (visalli et al., 1997) . a similar hsv-1 culture system has also been described by hukkanen et al. (1999) . they reported hsv-1 could infect immortalized hacat keratinocytes cultured in an organotypic raft culture system, the virus yield was highest when the inoculation took place 72h after seeding. in addition, 3d epithelial raft culture represents a novel model for the study of antiviral agents active against hsv. researchers have shown that specific compounds, such as foscarnet and cidofovir, can reduce the replication and spread of hsv in raft cultures of human keratinocytes (andrei et al., 2005) . so far, significant advances are still limited in our knowl-edge of hsv using 3d culture systems. anti-hsv may be the future research direction, especially for evaluation of the efficacy of new anti-hsv antivirals before clinical trials. varicella-zoster virus (vzv) is a neurotropic human alphaherpesvirus that causes varicella (chicken pox) and herpes zoster (shingles), establishes latency in multiple ganglionic neurons after primary infection and can reactivate to cause zoster (arvin, 1996; heininger and seward, 2006) . unfortunately, investigating vzv pathogenesis is challenging as vzv is strictly a human pathogen and infection is highly restricted in other species. in addition, few culture models are available for studying the interaction between vzv and human neurons, because the life span of terminally differentiated human neurons in culture is short (goodwin et al., 2013; zerboni et al., 2014) . the organotypic raft culture model has been applied in studying vzv. as keratinocytes are the main target cells for productive infection in vivo for vzv, characterization of viral replication in organotypic raft cultures of these cells represents a very relevant model for studying virus-host cell interactions and antiviral agents (andrei et al., 2005) . researchers have reported that they studied the action of antiviral compounds against vzv in organotypic epithelial raft cultures. the cultures were infected by vzv after 4 to 5 days of human keratinocyte differentiation and treated with serial dilutions of antiviral compounds. the antiviral effects were quantified by determining the viral dna load by real-time pcr for vzv. furthermore, this system could be very useful for the study of the interactions between viruses and the skin. the raft cultures have been used as a novel approach for investigating vzv replication in epidermal cells undergoing differentiation (andrei et al., 2005) . similar research has been described by mcguigan et al. (2007) . recently, a 3d model of normal human neural progenitor (nhnp) cells in tlas was used to investigate vzv infection. these cells could be effectively cultured for more than 6 months in 3d culture, and exhibited an expression profile similar to that of human trigeminal ganglia. vzv produced a persistent infection in nhnp cell tlas, which could be maintained for at least 3 months. this 3d culture system is likely to contribute to deciphering the establishment of vzv latency and reactivation in future studies (goodwin et al., 2015) . despite advances having been made in understanding vzv-host interactions, numerous questions relating to vzv pathogenesis remain unresolved (zerboni et al., 2014) . 3d culture systems have the potential to provide new approaches for preventing and treating vzv infections, in particular they may provide the cell system required for the generation of high-titer, cell-free attenuated virus for vaccine production. adenovirus (adv) is a mildly pathogenic human virus that propagates prolifically in epithelial cells. advs have been increasingly used as vectors for gene transfer in tumor cells or as oncolytic viruses (grill et al., 2002) . however, because of incomplete knowledge of the complex virus-cell interactions, the predicted replication selectivity has not been realized. in addition, rodent cells do not allow the complete lytic cycle of human adenovirus (alemany et al., 2000) . furthermore, specific constraints to adenovirus distribution and spread cannot be studied in cell cultures. mcss have been applied in studying the interaction between tumor and adenoviruses. grill et al. (2002) reported that replication-defective adenoviruses do not penetrate into spheroids; in addition, they described the propagation of replication-competent adenoviruses in spheroids. organotypic spheroids may provide a useful tool for studying the spread, oncolysis and distribution of adenoviruses. the adenovirus infection process was reproduced in organotypic raft cultures of primary human keratinocytes (noya et al., 2003) . adenovirus mutants have been found to replicate and promote the killing of cells expressing hpv e6 and e7 oncoproteins, which were present in an organotypic model of human stratified squamous epithelium derived from primary keratinocytes (balague et al., 2001) . more recently, research has demonstrated that adenoviruses could effectively deliver transgenes into the cultured 3d "mini-gut" organoids using adenoviral vectors that expressed fluorescent proteins. the transgene expression could be maintained for at least 10 days. these results were indicative that adenovirus vectors should be explored as effective gene delivery vehicles to introduce genetic manipulations in 3d organoids (wang et al., 2014) . norwalk virus was first discovered in 1972 (kapikian et al., 1972) and renamed as norovirus (nov) in 2002. nov is a human enteric pathogen causing epidemic foodborne gastroenteritis (tan and jiang, 2014) . the major barrier to the research and development of effective interventions for nov has been the lack of a robust and reproducible in vitro cultivation system (robinson and pfeiffer, 2014; ettayebi et al., 2016) . straub et al. (2007) first developed a 3d culture model with human embryonic intestinal epithelial cells cultured on collagen-i porous microcarrier beads, which were infected by genogroup i and ii human novs. the results showed that this model could support the natural growth of human novs. however, other investigators reported that using these same methods had not been suc-cessful. in 2011, straub et al. (2011) designed another 3d culture model using gastrointestinal epithelial cells (caco-2); the norovirus viral rna copy number was significantly increased (> 2 log 10 ) in this 3d caco-2 cell culture system. the results showed that this model supported norovirus replication. however, papafragkou et al. (2014) reported that using the 3d cell culture model with caco-2 cells was not suitable for the replication of norovirus. at present, there is still dispute about 3d culture models for studying novs. further research efforts need to be made in the future. apart from those viruses reviewed above, the use of 3d culture models for other viruses is currently receiving widespread attention. for example, 3d porcine epithelial cell cultures were designed to help understand the interaction between foot-and-mouth disease virus (fmdv) and porcine mucosal epithelial cells. the results demonstrated that fmdv replicated only transiently without any visible cytopathic effect, and infectious progeny virus could be recovered only from the apical side (dash et al., 2010) . researchers reported that a panel of clinical human rhinovirus (hrv) species c specimens, including hrv-c2, hrv-c7, hrv-c12, hrv-c15 and hrv-c29 types, were all capable of mediating productive infection in reconstituted 3d human primary upper airway epithelial tissues, and that the virions entered and exited preferentially through the apical surface. this model is considered as a potential tool for modeling the respiratory epithelium in the study of infections caused not only by hrvs, but also by other respiratory pathogens (tapparel et al., 2013) . in addition, 3d human intestinal enteroid cultures were designed as a novel pathophysiological model for studying human rotavirus infection, host restriction and pathophysiology (saxena et al., 2015) . furthermore, lam et al. (2012) demonstrated a method for measuring the impact of infection on the mechanics of a 3d model of connective tissue using human cytomegalovirus. table 5 shows some information on human virology in 3d cell cultures. 3d cell cultures are emerging technologies that can reproduce specific morphological and biochemical features of human cells similar to those found in vivo. recent progress in the imaging of 3d dynamics may promote the development of 3d cell culture. 3d culture models provide a potential tool for studying viral growth, infection, pathogenesis, and virus-host interaction. these culture systems have allowed a breakthrough in the cultivation of fastidious viral pathogens, which has advanced our understanding of the molecular and cellular mechanisms that underlie pathogenic processes in both the virus and host (barrila et al., 2010) . in addition, these models will play a key role in preclinical drug and therapeutic discovery. it is estimated that 10%-15% of human cancers worldwide are caused by seven human viruses (moore and chang, 2010) . as an example, high-risk hpv causes cervical cancer, kaposi's sarcoma herpesvirus leads to kaposi's sarcoma and hbv contributes to hepatocellular carcinoma. however, the understanding of the relationship between human viruses and tumors is still limited, especially the therapy strategies. 3d cell culture models, capturing tumor complexity in vitro, provide a potentially powerful physical tool for investigating virus-cancer interaction and evaluating antiviral agents. in addition, analyzing the mechanism of human tumor virology in 3d in vitro may be useful for predicting cancer progress. so far, although the development and testing of viral vaccine in 3d culture systems is limited, these models may be useful as an alternative means for evaluating preclinical vaccines in the future. moreover, 3d co-culture holds enormous potential for studying virus-virus interactions (such as hsv and hpv), and for developing novel products for the prevention, diagnosis and treatment of viral disease. in recent years, the development of multi-targeted drugs for suppressing two or more viruses has been receiving widespread attention. it might also be possible to use 3d models for the specific design of effective individual therapy according to patient-specific strains. most importantly, 3d culture systems may be used not only in future virological studies, but also in more tissue engineering. we expect that these novel, easily made and reliable models will make a contribution to the further understanding of biological science. advances in the formation, use and understanding of multi-cellular spheroids production and release of infectious hepatitis c virus from human liver cell cultures in the three-dimensional radial-flow bioreactor replicative adenoviruses for cancer therapy 3d cultured immortalized human hepatocytes useful to develop drugs for blood-borne hcv generation of organotypic raft cultures from primary 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vitro tumor models for cancer research and drug evaluation modeling tissue morphogenesis and cancer in 3d molecular mechanisms of varicella zoster virus pathogenesis a review of the three-dimensional cell culture technique: approaches, advantages and applications this research was supported by the national megaprojects for infectious diseases (2014zx10004002-004-001). the authors declare that they have no conflict of interests. this article does not contain any studies with human or animal subjects performed by any of the authors. key: cord-329876-4cgrjnjo authors: lei, jian; hilgenfeld, rolf title: structural and mutational analysis of the interaction between the middle-east respiratory syndrome coronavirus (mers-cov) papain-like protease and human ubiquitin date: 2016-05-30 journal: virol sin doi: 10.1007/s12250-016-3742-4 sha: doc_id: 329876 cord_uid: 4cgrjnjo the papain-like protease (pl(pro)) of middle-east respiratory syndrome coronavirus (mers-cov) has proteolytic, deubiquitinating, and deisgylating activities. the latter two are involved in the suppression of the antiviral innate immune response of the host cell. to contribute to an understanding of this process, we present here the x-ray crystal structure of a complex between mers-cov pl(pro) and human ubiquitin (ub) that is devoid of any covalent linkage between the two proteins. five regions of the pl(pro) bind to two areas of the ub. the c-terminal five residues of ub, rlrgg, are similar to the p5–p1 residues of the polyprotein substrates of the pl(pro) and are responsible for the major part of the interaction between the two macromolecules. through sitedirected mutagenesis, we demonstrate that conserved asp165 and non-conserved asp164 are important for the catalytic activities of mers-cov pl(pro). the enzyme appears not to be optimized for catalytic efficiency; thus, replacement of phe269 by tyr leads to increased peptidolytic and deubiquitinating activities. ubiquitin binding by mers-cov pl(pro) involves remarkable differences compared to the corresponding complex with sars-cov pl(pro). the structure and the mutational study help understand common and unique features of the deubiquitinating activity of mers-cov pl(pro). electronic supplementary material: supplementary material is available for this article at 10.1007/s12250-016-3742-4 and is accessible for authorized users. to date, six coronaviruses infecting humans have been characterized. infections with human coronaviruses (hcovs) 229e (hamre & procknow, 1966) , oc43 (mcintosh et al., 1967) , nl63 (van der hoek et al., 2004) , and hku1 (woo et al., 2005) cause relatively mild symptoms in most cases, whereas severe acute res-piratory syndrome coronavirus (sars-cov; ksiazek et al., 2003; kuiken et al., 2003; peiris et al., 2003) and middle-east respiratory syndrome coronavirus (mers-cov; zaki et al., 2012) are connected with severe respiratory-tract infection and, in particular in case of mers-cov, acute renal failure (eckerle et al., 2013) , leading to high case-fatality rates of ~10 and 35%, respectively. in spite of 13 years of research on sars-cov (hilgenfeld & peiris, 2013) , no approved drugs or vaccines are available for the treatment or prevention of coronavirus infection (wang et al., 2016) . this is mainly due to the fact that although these emerging viruses have devastating effects on those infected, the absolute numbers of cases (~8000 for sars, 1733 so far for mers; (http://www.who.int)) imply that the de-velopment of specific antivirals is very likely not commercially viable. on the other hand, the global risk posed by mers-cov must not be underestimated. since its discovery in september 2012, the number of mers cases reported has been rising steadily, with some intermittent peaks connected to hospital outbreaks in saudi arabia (assiri et al., 2013) . man-to-man transmission of mers-cov has also been impressively demonstrated by the recent outbreak of mers in south korea, which was traced back to a single traveller from the arab peninsula (butler, 2015) . therefore, it is imperative that academic laboratories help increase the preparedness against a possible mers-cov pandemic by characterizing antiviral drug targets and by identifying lead compounds interfering with them. in order to successfully infect humans, a virus has to meet at least two conditions: 1), it should maintain a sufficiently correct replication of its genetic material; 2), it should inhibit the host antiviral response. the papainlike protease (pl pro ) of mers-cov (or sars-cov) is involved in both of these tasks (yang et al., 2013; barretto et al, 2005) . the pl pro is a domain located in the middle part of the largest non-structural protein, nsp3, of mers-cov (or sars-cov) . it is responsible for releasing nsp1, nsp2, and nsp3 from the polyproteins 1a (pp1a) and 1ab (pp1ab), an essential step of replication (harcourt et al., 2004) . like its sars-cov counterpart, the mers-cov pl pro also has deubiquitinating (dub) and deisgylating activities in vivo as well as in vitro (yang et al., 2013; mielech et al., 2014; lei et al., 2014; baez-santos et al., 2014b) . k48-and k63-polyubiquitin poly (ub) and isg15 (interferon-stimulated gene 15)conjugated targets are usually involved in host innate immune regulation (liu et al., 2005; maringer & fernandez-sesma, 2014) . the pl pro has the ability to digest k48-and k63-linked polyub chains and to remove isg15 from isg15-linked proteins (baez-santos et al., 2014b) , thereby interrupting the signalling pathways leading to the innate immune response. thus, the pl pro can block the activation of ifn regulatory factor 3 (irf3) (yang et al., 2013) and subsequently the production of interferon β (ifnβ) (mielech et al., 2014) . interestingly, mers-cov pl pro shows a similar cleavage rate for k48-and k63-linked polyub chains, while the sars-cov enzyme prefers k48-over k63-linked chains (baez-santos et al., 2014b) . the former enzyme degrades a polyub chain by removing mono-ubs, whereas the latter cleaves di-ub units off the polyub chain (bekes et al., 2015) . we have reported the first crystal structure of the mers-cov pl pro (lei et al., 2014) . later, two other groups also described the structure of this enzyme (bailey-elkin et al., 2014; lee et al., 2015) . the structure of pl pro can be divided into two parts: a ubiquitin-like (ubl) domain and a catalytic domain with thumb, palm, and fingers subdomains. the overall fold of mers-cov pl pro is not only similar to that of sars-cov pl pro , but also to that of several human ubiquitinspecific proteases (usps) (hu et al., 2005) . in 2014, the x-ray structure of the complex of sars-cov pl pro with ubiquitin has been reported (chou et al., 2014; ratia et al., 2014) . several key residues (such as glu168 or tyr265) of sars-cov pl pro that are important for ubiquitin recognition (chou et al., 2014; ratia et al., 2014) , are not conserved in mers-cov pl pro . bailey-elkin et al. (2014) described the structure of an artificially linked, covalent complex between ubiquitin and mers-cov pl pro . here, we present the crystal structure of a noncovalent complex between the two proteins and a mutational study of the interactions involved. for these studies, we used the cys111ser active-site variant of mers-cov pl pro . the pl pro of mers-cov (strain 2c emc/2012; gen-bank no. afv09327.1) contains 320 residues, from gln1482 to asp1801 of pp1a/1ab. for simplification, gln1482 was renumbered into gln1 here. the dna plasmid coding for mers-cov pl pro was produced earlier (lei et al., 2014) . the mers-cov pl pro c111s, d164e, d164a, d165e, d165a, and f269y variants were produced using the same strategy that we described before (lei et al., 2014) . all primers for these variants are listed in supplemental table s1 . all dna plasmids coding for the altered pl pro were verified by sequencing. genes coding for wild-type (wt) mers-cov pl pro and for its variants were expressed and the corresponding proteins were purified according to our previous description (lei et al., 2014) . performed according to the procedure described for mers-cov pl pro (lei et al., 2014) . purified mers-cov pl pro (wt) and a variant that had the active-site cys111 replaced by ser (pl pro (c111s)) were both concentrated to ~24 mg/ml in 20 mmol/l tris-hcl, 150 mmol/l nacl, ph 8.8, 10 mmol/l β-mercaptoethanol (bme). human ubiquitin (bostonbiochem) was dissolved to 6 mg/ml in 20 mmol/l tris-hcl, 150 mmol/l nacl, ph 8.8. 500 μl pl pro (wt) or pl pro (c111s) were mixed with 500 μl ubiquitin (~1:1 molar ratio) at 4°c overnight. (c111s)-ub were observed under condition no. 9 of md1-01, whereas no crystal of pl pro (wt)-ub was obtained. optimized crystals of pl pro (c111s)-ub were subsequently obtained within two days under the condition: 22% w/v peg 4000, 15% v/v 2-propanol, 0.1 mol/l tri-sodium citrate ph 4.8, and 10% glycerol. 2 μl of protein and 2.5 μl of reservoir were mixed to equilibrate against 500 μl reservoir. crystals were placed in a nitrogen-gas stream (100 k). a 3.16-å dataset was collected at wavelength 0.91841 å at beamline 14.2 of bessy, berlin (mueller et al., 2012) . the program xds (kabsch, 2010) was used to process the diffraction data. the space group was found to be p6 3 , with unit-cell parameters a = b =138.14 å, c = 57.59 å, γ = 120°. diffraction data statistics are shown in table 1 . the structure of the mers-cov pl pro (c111s)-ub complex was solved by molecular replacement using molrep (vagin & teplyakov, 2010) . the program selected the mers-cov pl pro (protein data bank (pdb) entry 4p16, lei et al., 2014) as the first search model. human ubiquitin (pdb entry: 1ubq, vijaykumar et al., 1987) was used as the second search model. the model of the complex was inspected and rebuilt using coot (emsley et al., 2010) , and refined using phenix.refine (headd et al., 2012) . the refinement statistics are shown in table 1 kinetic assays of purified pl pro s all enzymatic assays were performed using a 96-well microtiter plate and the reaction buffer 20 mmol/l tris-hcl, 150 mmol/l nacl, ph 7.9, 2 mmol/l dithiothreitol (dtt). the fluorogenic substrates cbz-arg-leu-arg-gly-gly-7-amino-4-methylcoumarin (z-rlrgg-amc) (bachem), z-lrgg-amc (bostonbiochem), and ubiquitin-amc (ub-amc) (bostonbiochem) were used. the fluorescence of free amc with different concentrations (5 nmol/l-2.5 μmol/l) in reaction buffer was measured to generate a calibration curve, in order to convert the change of fluorescence intensity per unit of time, δ(afu) /s, into the amount of hydrolyzed substrate in μmol/l/s. the enzymatic cleavage reactions were run with an flx800 fluorescence spectrophotometer (biotek), to measure the increased fluorescence signal (λ ex : 360 nm; λ em : 460 nm) resulting from amc release. reactions were initiated by addition of the proteases to the reaction system. the peptide-hydrolysis kinetic assays were performed with the following conditions: 1 μmol/l mers-cov pl pro variant (d164e, d164a, d165e, f269y), or 10 μmol/l d165a, or 0.1 μmol/l sars-cov pl pro , with different concentrations (10, 20, 40, 80, 100 μmol/l) of z-rlrgg-amc or z-lrgg-amc in a final volume of 100 μl at 25 °c. the kinetic curves for the proteases and their variants with the substrates z-rlrgg-amc or z-lrgg-amc were linear and the initial velocities also increased linearly with substrate concentration. no saturation was observed. therefore, the data were fitted to the equation v/[e] tot. = k app [s] , where k app approximates k cat /k m , as described previously (barretto et al., 2005; wojdyla et al., 2010) . the deubiquitinating kinetic assays were performed under the following conditions: 0.1 μmol/l mers-cov pl pro wild-type or its variants d164e, d164a, d165e, f269y, or 0.5 μmol/l d165a, or 0.025 μmol/l sars-cov pl pro were incubated with increasing concentrations (1, 2, 4, 8 μmol/l) of ub-amc in a final volume of 50 μl, at 25 °c. although pl pro actually cleaves the isopeptide bond between the carboxyl group of the c-terminal gly in ub and the ε-amino group of lys in ubiquitinated targets in vivo, we used the hydrolysis of ub-amc here to test the deubiquitinating activity in vitro. the kinetic curves of proteases and variants with the substrate ub-amc were hyperbolic and the initial velocities were not linear over the concentration of substrate. however, saturation was still not observed within a reasonable time. only when the ratio of protein to substrate was 1:1 or larger, were we able to achieve saturation within a limited time (data not shown). as the initial velocities did not increase in a strictly linear fashion with substrate concentration, application of the equation v/[e] tot. = k app [s] to mimic k cat /k m would lead to large standard errors. we were however able to fit the data to the michaelis-menten equation using the graphpad prism program (graphpad software), even though saturation could not be reached (supplemental figure 1 ). all assays were performed in duplicates. the substrate-binding site of mers-cov pl pro features significant differences from those of the corresponding sars-cov enzyme and human ubiquitin-specific proteases (usps, such as, usp14) (hu et al., 2005; chou et al., 2014; ratia et al., 2014) . it is therefore of interest to determine the crystal structure of the complex between mers-cov pl pro and its substrate, human ubiquitin. hence, we crystallized the ubiquitin (ub) complex of a mers-cov pl pro variant that had the active-site cys111 replaced by serine (c111s) and determined the structure at 3.16 å ( figure 1a ). there is one pl pro (c111s)-ub complex per asymmetric unit. using the pdbepisa server (krissinel & henrick, 2007) , the total interface region of mers-cov pl pro (c111s)-ub was determined as 813 å 2 , close to the 915 å 2 interface of sars-cov pl pro -ubal (ubiquitin aldehyde; pdb entry: 4mm3, ratia et al., 2014) and the 999 å 2 of sars-cov pl pro (c112s)-ub (pdb: 4m0w, chou et al., 2014) , but less than the 1503 å 2 observed for usp14-ubal (pdb: 2ayo, hu et al., 2005) . the overall structure of mers-cov pl pro (c111s) is very similar to that of the substrate-free pl pro (lei et al., 2014) , with a root-mean-square deviation (rmsd) of 0.91 å for corresponding cα atoms between these two structures. nevertheless, two differences are immediately visible: 1), the zinc-finger motif has moved and closed in onto the ub, compared to the free pl pro . the zinc ion position has shifted by about 4 å and the largest deviation between the two structures is ~6 å for cys228 of the zinc-finger region ( figure 1b) ; 2) the mobile loop 271 gietavg 277 , also named "bl2 loop", is defined by clear main-chain electron density ( figure 1c ). this loop is disordered in substrate-free pl pro (lei et al., 2014; bailey-elkin et al., 2014) . compared to mers-cov pl pro , the bl2 loop 267 gnyqcg 272 is shorter by one residue in sars-cov pl pro . at the time when we deposited in the pdb the coordinates for the crystal structure of the non-covalent complex mers-cov (c111s)-ub (pdb: 4wur), bailey-elkin et al. (2014) described two crystal structures for a covalent complex mers-cov pl pro -ub, in which an alkyl bromide group introduced at the c-terminus of ub had formed a thioether with the active-site cys111 of mers-cov pl pro . these structures were in space groups p6 5 22 and p6 3 and were named "closed" and "open" pl pro -ub complexes (pdb: 4rf0 and 4rf1), respectively (bailey-elkin et al., 2014) . even though the zinc-finger motif of pl pro in the former complex (space group p6 5 22) is closer to the ub than in the p6 3 complex, both the "closed" and "open" complex show almost the same interactions between pl pro and ub (bailey-elkin et al., 2014). the differences may be caused by crystal packing. our non-covalent complex is similar to the "open" form of the covalent pl pro -ub complex (rmsd = 0.58 å for the pl pro and 0.85 å for ub, based on all cα atoms). all parts of the pl pro except for the ubiquitin-like (ubl) domain interact with ub. most interactions in-volve five surface regions of the pl pro and two regions of ub ( figure 2a ). these five regions of the pl pro are labelled by roman numbers: i, leu106-tyr112; ii, ala162-arg168; iii, cys208-val210; iv, gly248-pro250; v, phe269-tyr279. i and ii are situated in the thumb domain; iii is in the fingers domain; and iv and v are in the palm domain ( figures 1a and 2a ). in the following and in the figure labels, residues of ub are indicated in italics to distinguish them from residues of pl pro . the two interacting regions of ub are: a, arg42-gln49; b, arg72-gly76 (figure 2a ). region a of ub consists of β3, β4, and the loop between β3 and structure of mers-cov pl pro -ubiquitin complex β4 ( figure 2b ). regions ii and iii of pl pro interact with region a of ub. region b comprises the five c-terminal residues, rlrgg, and is in contact with regions i, ii, iv, and v of pl pro ( figure 2c ). the c-terminal rlrgg motif contributes the majority of the interactions with the pl pro ; the buried surface between ub region b and pl pro is 477 å 2 (out of a total of 813 å 2 ). in order to make these interactions clear, we describe here in some detail the contacts between the pl pro and regions a and b of ub. region a (arg42-gln49) of ub inserts into the space between the thumb and fingers domains of pl pro (figures 2a-b) . residues ile44, ala46, and gly47 engage in hydrophobic interactions with tyr209 and val210 of region iii of pl pro ( figure 2b ). the hydrophobic patch (ile44, ala46, and gly47) is a common interaction region utilized by ub-binding proteins (dikic et al., 2009) . in particular, the interaction of ile44 with val210 of pl pro is important for the deubiquitinase (dub) but not for the protease activity. the variant v210r shows dramatically reduced dub activity, as demonstrated by bailey-elkin et al. (2014) (according to the numbering scheme of these authors, v210 is v1691). a salt-bridge exists between region a of ub and the pl pro ( figure 2b ), namely between the side-chains of arg42 and asp165 (region ii in pl pro ). in addition, a hydrogen bond is formed between the main-chain o atom of gly47 and the main-chain amide of val210 (region iii). in the sars-cov pl pro (c112s)-ub complex (chou et al., 2014) , arg42 forms a salt-bridge with the negatively charged glu168, a residue which is replaced by the positively charged arg168 in mers-cov pl pro . consequently, the same salt-bridge cannot be formed in the mers-cov pl pro (c111s) -ub complex. instead, mers-cov pl pro has asp165 interacting with arg42. in our deubiquitinating (dub) kinetic assays, the d165a variant shows a dramatically reduced dub activity; the k cat /k m is about 78-fold decreased compared to that of the wild-type ( table 2 ). the k m value of the d165a variant is about 4-fold higher than that of the wild-type mers-cov pl pro , suggesting that this amino-acid replacement reduces the ub binding affinity. meanwhile, the d165e amino-acid replacement shows a catalytic efficiency comparable to wild-type towards ub-7-amino-4methylcoumarin (ub-amc) ( table 2 ). however, we noticed that the k m value of d165e for the dub activity is about 2-fold larger than for the wt enzyme. although glu165 can mimic asp165 here, we propose that the longer side-chain of glu may fit less perfectly compared to asp. these results indicate that the salt-bridge between asp165 and arg42 could be important for the pl pro 's dub activity, in addition to the interaction between asp165 and the p4-amide group (see below). region b of ub comprises the five c-terminal residues, rlrgg. these five residues bind to the narrow activesite channel between the thumb and palm domains of the pl pro ( figure 2c ). they mainly interact with regions i, ii, iv, and v of the protease. residues rlrgg are compatible with the pl pro cleavage motif, (r/k) (l/i) xgg (p5-p1), in the mers-cov polyproteins; their interactions with the pl pro are discussed here in terms of subsites s1 to s5. s1 and s2 subsites. pro163 (region ii of pl pro ) and the side-chains of asn109 and tyr112 (located in region i) form a space-restricted s1 site to accommodate gly76 (p1). the carbonyl oxygen atom of gly277 (region v) accepts a hydrogen bond from the amide of gly76 (figure 3) . the side-chains of tyr112 (region i) and phe269, val276 as well as tyr279 (region v), and gly277 form a restricted space for gly75 (p2). two hydrogen bonds link the main-chain at asp164 of the pl pro and gly75 (figure 3) . the s1 and s2 sites of the protease are too small to accommodate any other residue but glycine. s3 subsite. the main-chain o atom of arg74 (p3) accepts a hydrogen bond from the gly277 amide. in the complexes sars-cov pl pro (c112s)-ub or sars-cov pl pro -ubal, the main chain at arg74 forms two hydrogen bonds (chou et al., 2014; ratia et al., 2014) , namely with the amide of gly272 and with the hydroxyl group of tyr265. the former hydrogen bond is conserved in the mers-cov pl pro (c111s)-ub complex, but the latter is not. tyr265 of sars-cov is replaced by phe269 in mers-cov, which lacks the ability to form a hydrogen bond with the main-chain amide of arg74. in agreement with this difference, the dub activity of sars-cov pl pro is 2.5-fold higher than that of mers-cov pl pro in our kinetic assay; furthermore, the mers-cov pl pro f269y amino-acid replacement leads to enhancements by about 1.5-, 1.4-, and 1.9-fold of the hydrolytic activities towards carbobenzoxy-arg-leu-arg-gly-gly-7amino-4-methylcoumarin (z-rlrgg-amc), z-lrgg-amc, and ub-amc, respectively ( table 2) . the side-chain of arg74 is exposed to the solvent in our mers-cov pl pro (c111s)-ub complex. this sidechain shows remarkable variability in the interactions it makes in the different complexes. in the "open" but not in the "closed" covalent mers-cov pl pro -ub complex (bailey-elkin et al., 2014) , it donates a hydrogen bond to the main-chain carbonyl oxygen of thr1755 of the bl2 loop (corresponding to thr274 in our numbering scheme). in sars-cov pl pro -ubal (ratia et al., 2014) but not in the sars-cov pl pro (c112s)-ub complex (chou et al., 2014) , the side-chain of arg74 forms a hydrogen bond with the main-chain carbonyl oxygen of gln270. instead, arg74 is involved in a relatively weak salt-bridge with glu162 in the sars-cov pl pro (c112s)-ub complex (chou et al., 2014) . none of these interactions exist in our mers-cov pl pro -ub complex. s4 subsite. the main-chain amide of leu73 (p4) donates a hydrogen bond to the side-chain of asp165 (region ii) (figure 3 ). this hydrogen bond is conserved in all the complexes compared here. in our peptide-cleavage assay, the d165a variant shows about 16-fold and 10-fold lower activities towards substrates z-rlrgg-amc and z-lrgg-amc, respectively, indicating that asp165 is not only important for interacting with arg42 of ub (see above). the side-chain of leu73 is embedded in a hydrophobic pocket which is formed by the cβ atom of asp165, the side-chain of pro250 (region iv), phe269, as well as the cβ and cγ atoms of glu273 (region v). asp165 and pro250 are conserved in sars-cov pl pro (asp165 and pro249). phe269 is replaced by tyr265, and glu273, situated in the bl2 loop, is replaced by tyr269. however, the side-chains of all these non-conserved residues possess the ability to provide a hydrophobic environment to accommodate leu73. s5 subsite. the side-chain of arg72 (p5) is located between the pl pro thumb domain and region a of ubiquitin. it forms a salt-bridge with the side-chain of asp164 in mers-cov pl pro . in addition, the guanidinum group of arg72 may be involved in a π-π interaction with that of arg168 (figure 3 ). these interactions have also been described for the covalent complex of mers-cov pl pro with ub (bailey-elkin et al., 2014) . arg72 is not subject to strict space limitations; accordingly, this residue displays different binding patterns in the two sars-cov residues are shown in purple in the ball-and-stick style, and they are labeled in red. for clarity, the 2f o -f c electron density (blue; 1.0 σ) of the rlrgg main chain is shown. pl pro residues are displayed in green in the balland-stick style, and they are labeled in black. hydrogen bonds are displayed as black dashed lines, and the salt-bridge between r72 and d164 is depicted as two red dashed lines. structure of mers-cov pl pro -ubiquitin complex pl pro -ub complexes. in the covalent sars-cov pl pro -ubal complex (ratia et al., 2014) , arg72 forms a saltbridge with glu168. in the non-covalent sars-cov pl pro (c112s)-ub complex (chou et al., 2014) , arg72 is exposed to the solvent and does not interact with glu168 (arg42 instead forms a salt-bridge with glu168, as mentioned above). in our kinetic assay, the d164a variant of mers-cov pl pro displays a ~4.5-fold and a ~3.5-fold reduced activity, respectively, for z-rlrgg-amc and ub-amc (table 2) . for z-lrgg-amc, the activity is decreased just a little (by about ~1.5-fold), because there is no p5-arg in this substrate (table 2 ). these data demonstrate that asp164 is important for the interaction with arg72. in summary, the main-chain heteroatoms of p5-p1 form a hydrogen-bonding network with pl pro . the binding characteristics of p1, p2, and p4 are conserved in all mers-cov and sars-cov pl pro -ub complexes. however, p3-arg and p5-arg assume binding patterns that differ between the various mers-cov and sars-cov pl pro -ub complexes. viral proteins are likely to possess multiple functions, as exemplified by non-structural protein 1 (ns1) of influenza a viruses (hale et al., 2008) , the nucleocapsid (n) protein of coronaviruses (chang et al., 2014) , or the nsp14 exonuclease-guanyl-7-methyltransferase of coronaviruses (chen et al., 2009) . exhibiting dub and proteolytic activities, the papain-like proteases of coronaviruses are no exception here. although the overall folds are conserved, the enzyme activity and substrate-binding modes of cov pl pro s differ in detail. therefore, no coronavirus pl pro can be considered a general model for all its homologues (baez-santos et al., 2014b) . we have previously noticed that the oxyanion hole of the mers-cov pl pro appears to be deficient (lei et al., 2014 ; also see the discussion below). similarly, the recognition of ubiquitin by the enzyme appears to be suboptimal. thus, the main-chain amide of the p3-arg residue has no hydrogen-bonding partner on the mers-cov pl pro , because the near-by side-chain of phe269 is incapable of accepting an h-bond. the corresponding residue is tyr265 in the sars-cov pl pro , which is perfectly positioned to accept the hydrogen bond from the p3-amide. indeed, when we replaced phe269 by tyr in mers-cov pl pro , the dub activity of the enzyme increased by a factor of almost 2 and the peptidolytic activity by ~1.5. the evolution of viral enzymes is not neces-sarily driven by optimization of catalytic efficiency. this is particularly true for viral proteases that have to ensure the availability of non-structural proteins in the correct temporal order when they cleave them out of the viral polyproteins; in fact, too rapid a polyprotein processing might be counterproductive. on the other hand, a more efficient dub activity should help the virus in counteracting the innate immune response of the host cell. as the binding of the p5-p1 residues of ubiquitin and of the polyprotein cleavage sites obviously influences both the dub and proteolytic activities of the pl pro , the suboptimal catalytic efficiency that we observe may be a consequence of a compromise between the requirements of the two activities. with regard to the oxyanion hole, we previously proposed that the backbone amide of asn109 (located in a β-turn connecting β7 and α4, figure 4 ) may contribute to the stabilization of the oxyanion intermediate in pl pro catalysis, along with the main-chain amide of the active site cys111, although this may require a slight rearrangement of this β-turn (lei et al., 2014) . in our non-covalent mers-cov pl pro -ub complex, we do not see any rearrangement of the β-turn. more or less in agreement with our suggestion, bailey-elkin et al. (2014) proposed on the basis of their covalent mers-cov pl pro -ub complex structure that the main-chain amides of asn1590, asn1591, and cys1592 (corresponding to asn109, asn110, and cys111 in our numbering scheme) form the oxyanion hole. on the other hand, lee et al. (2015) argued that the side-chain of asn109 could contribute to the oxyanion hole. they found that the n109a replacement completely abolished enzyme activity. as we reported earlier (lei et al., 2014) , the side-chain amide of asn109 makes a strong hydrogen bond with the conserved gly161 (figure 4 ). any reorientation of the asn109 side-chain towards the oxyanion would require a disruption of this strong interaction; this is not very likely. in conclusion, lacking a side-chain in the proper spatial orientation and capable of donating a hydrogen bond to the oxyanion (such as trp107 in sars-cov pl pro ), the oxyanion hole of mers-cov pl pro seems to be deficient. apart from the less than optimum binding of the p3 residue, there are other differences in the way mers-cov pl pro and sars-cov pl pro recognize human ubiquitin. the formation of a salt-bridge between arg72, the p5 side-chain of ub, and asp164 is unique for mers-cov pl pro . the same interaction exists in the two covalent mers-cov pl pro -ub complexes (bailey-elkin et al., 2014) . this binding mode is very different from the glu168 -arg72 salt-bridge in the sars-cov pl pro -ubal complex (ratia et al., 2014) . glu168 is conserved in hcov-nl63 pl2 pro and replaced by asp in the hcov-229e, hcov-oc43, and hcov-hku1 pl2 pro s (for sequence alignments, see barretto et al., 2005; baez-santos et al., 2014b) , so the same type of interaction is likely to be realized in the ub complexes of these enzymes. however, the corresponding residue in mers-cov pl pro is arg168; hence, asp164 is used instead to bind arg72. asp164 is in fact unique in mers-cov. it is replaced by gly in sars-cov pl pro and the pl2 pro s of hcov nl63 and hcov 229e, by ala in hcov-oc43 pl2 pro , and by ser in hcov-hku1 pl2 pro . our kinetic results for the d164a replacement (see results) emphasize the importance of the unique asp164 residue. in our pl pro (c111s)-ub complex, the ubl domain of pl pro shows no interaction with ubiquitin. the relative orientation of the ubl domain in the substrate-bound pl pro is the same as in substrate-free pl pro . the ubl domain of mers-cov pl pro is not required for the ifn antagonism activities (baez-santos et al., 2014b), but it is required in case of sars-cov pl pro (frieman et al., 2009) . recently, mielech et al. (2015) reported that the ubl domain of mouse hepatitis virus (mhv) is an important modulator of pl pro stability and viral pathogenesis. although the ubl domain shows variable effects in different covs, the high degree of conservation of the domain throughout the family suggests that it may play a common biological role. one possible function is that the ubl might be involved in protein-protein interactions. ubiquitin-like domains are known to function as binding modules in such interactions. for example, the kinase raf contains a ubl domain for interaction with human ras (fetics et al., 2015) . also, human ubiquitin-specific protease (usp) 7 includes five ubl (1-5) domains, of which the second is bound by the herpes simplex virus-1 immediate-early protein icp0 to counteract the intrinsic antiviral response of the host cell (pfoh et al., 2015) . the bl2 loop of sars-cov pl pro is important for binding inhibitors (baez-santos et al., 2014a; lee et al., 2015) . this loop is variable in different cov pl pro s. a potent inhibitor of the sars-cov pl pro , n-[(3-fluorophenyl) methyl]-1-[(1r)-1-naphthalen-1-ylethyl] piperidine-4-carboxamide (compound 3k, ic 50 = 0.15 ± 0.01 μmol/l) was found to have no effect on the mers-cov enzyme (baez-santos et al., 2014b) . tyr269 and gln270 of sars-cov pl pro , which are important for binding this inhibitor (baez-santos et al., 2014a) , are replaced by glu273 and ala275 in the mers-cov protease. it seems that this structural difference in the bl2 loop has a remarkable impact on the effectiveness of the inhibitor. the structure of the mers-cov pl pro (c111s)-ub complex presented here will facilitate virtual screening of chemical libraries for specific anti -mers-cov pl pro inhibitors (hilgenfeld, 2014) . the di-ub site of pl pro sars-cov and mers-cov pl pro s can digest k48-and k63-linked (poly)ubiquitin chains and remove isg15 from proteins covalently linked to it (ratia et al., 2014 , baez-santos et al., 2014b . sars-cov pl pro prefers binding of k48-ub 2 and isg15 over mono-ubiquitin (ratia et al., 2014) . all evidence suggests that on the pl pro , at least two ubiquitin-binding sites (or a binding site for diubiquitin-like molecules such as isg15) exist. ratia et al. (2014) proposed hypothetic models for complexes of sars-cov pl pro with k48-ub 2 and isg15. these authors identified two major hydrophobic binding sites on the pl pro for the first ub (ub1) and the second ub (ub2). the binding site for ub1 comprises met209, pro248, and pro249. this hydrophobic patch is conserved in mers-cov pl pro , although the residues are not exactly the same. met209 of sars-cov pl pro is replaced by val210 (region iii) in mers-cov. pro248 is replaced by thr249, whereas pro249 is conserved (pro250; region v) ( figure 5 ). the hypothetic second binding site for ub2 (also named "ridge" region, ratia et al., 2014) is located to the first α helix (α2) in the thumb domain, including residues phe70, his74, and leu76 of sars-cov pl pro . according to a structural alignment of the sars-cov and mers-cov pl pro s, phe70, his74, and leu76 are changed to lys69, gly73, and val75, respectively ( figure 5 ). in sars-cov pl pro , the f70s and f70a replacements lost the affinity to k48-ub 2 and isg15 in vitro (ratia et al., 2014) ; therefore, the presence of lys69 in mers-cov pl pro instead of phe70 strongly suggests that this enzyme should bind ub2 in a different way. bailey-elkin et al. (2014) predicted asn1673 and val1674 of the fingers subdomain (corresponding to asn192 and val193 in our pl pro ) as the distal ub site of k63 di-ub, but they found that their dub activity data do not support their prediction for the ub2 binding site. the structure of the mers-cov pl pro -ub complex reported here reveals the exact ub1 binding site on the pl pro , but the ub2 binding site should be identified by crystallizing the pl pro in complex with poly-or di-ubiquitin. in summary, the crystal structure of the mers-cov pl pro -ub complex provides valuable information that helps understand the multiple functions of coronavirus papain-like proteases. mutational studies additionally highlight features of the mers-cov pl pro . the different substrate-binding patterns should be kept in mind when designing inhibitors for pl pro s of covs, even though the overall structures of these enzymes are conserved. furthermore, it would be helpful to obtain the structure of pl pro in complex with di-ub or isg15 in the future. ing sites are depicted as purple dots. the n and c termini of pl pro are marked by underlined letters. all residues related to the two ub binding sites are labeled. the catalytic triad cys111-his278-asp293 is indicated by yellow, blue, and red spheres. ksa mers-cov investigation team. 2013. hospital outbreak of middle east respiratory syndrome coronavirus xray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases catalytic function and substrate specificity of the plpro domain of nsp3 from the middle east respiratory syndrome coronavirus (mers-cov) crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity sars hcov papain-like protease is a unique lys 48 linkage-specific di-distributive deubiquitinating enzyme south korean mers outbreak spotlights lack of research the sars coronavirus nucleocapsid protein-forms and functions molprobity: all-atom structure validation for macromolecular crystallography functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase structural basis for catalysis and ubiquitin recognition by the severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-binding domains-from structures to functions identification of a novel coronavirus in patients with severe acute respiratory syndrome in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection features and development of coot allosteric effects of the oncogenic rasq61l mutant on raf-rbd severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-κb signaling the multifunctional ns1 protein of influenza a viruses a new virus isolated from the human respiratory tract identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity use of knowledge-based restraints in phenix.refine to improve macromolecular refinement at low resolution from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design from sars to mers: 10 years of research on highly pathogenic human coronaviruses structure and mechanisms of the proteasome-associated deubiquitinating enzyme usp14 inference of macromolecular assemblies from crystalline state a novel coronavirus associated with severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome inhibitor recognition specificity of mers-cov papain-like protease may differ from that of sars-cov crystal structure of the papain-like protease of merscoronavirus reveals unusual, potentially druggable active-site features immunity by ubiquitylation: a reversible process of modification message in a bottle: lessons learned from antagonism of sting signalling during rna virus infection growth in suckling-mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease mers-cov papain-like protease has delsgylating and deubiquitinating activities murine coronavirus ubiquitin-like domain is important for papain-like protease stability and viral pathogenesis facilities for macromolecular crystallography at the helmholtz-zentrum berlin coronavirus as a possible cause of severe acute respiratory syndrome crystal structure of usp7 ubiquitin-like domains with an icp0 peptide reveals a novel mechanism used by viral and cellular proteins to target usp7 structural basis for the ubiquitin-linkage specificity and deisgylating activity of sars-cov papain-like protease identification of a new human coronavirus structure of ubiquitin refined at 1.8 å resolution recent progress in the discovery of inhibitors targeting coronavirus proteases on the use of the merging r factor as a quality indicator for x-ray data papain-like protease 1 from transmissible gastroenteritis virus: crystal structure and enzymatic activity toward viral and cellular substrates characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease isolation of a novel coronavirus from a man with pneumonia in saudi arabia technical assistance by susanne zoske is gratefully acknowledged. we thank stefan anemüller for discussion. we also acknowledge access to beamline bl14.2 operated by the helmholtz-zentrum berlin at the bessy ii electron storage ring (berlin-adlershof, germany). this work was supported by the european commission through its "silver" project (contract no. health-f3-2010-260644) and by the german center for infection research (dzif). rh acknowledges support by the dfg cluster of excellence "inflammation at interfaces" (exc 306) this article does not contain any studies with human or animal subjects. both authors declare no competing interest. jl and rh designed all experiments. jl performed the experiments. jl and rh analyzed all data. jl and rh wrote the manuscript.supplementary figure and table are available on the websites of virologica sinica: www.virosin.org; link. springer.com/ journal/12250. key: cord-274785-9jgg8ukr authors: zhang, qiang; sharma, nishi r.; zheng, zhi-ming; chen, mingzhou title: viral regulation of rna granules in infected cells date: 2019-04-29 journal: virol sin doi: 10.1007/s12250-019-00122-3 sha: doc_id: 274785 cord_uid: 9jgg8ukr rna granules are cytoplasmic, microscopically visible, non-membrane ribo-nucleoprotein structures and are important posttranscriptional regulators in gene expression by controlling rna translation and stability. tia/g3bp/pabp-specific stress granules (sg) and gw182/dcp-specific rna processing bodies (pb) are two major distinguishable rna granules in somatic cells and contain various ribosomal subunits, translation factors, scaffold proteins, rna-binding proteins, rna decay enzymes and helicases to exclude mrnas from the cellular active translational pool. although sg formation is inducible due to cellular stress, pb exist physiologically in every cell. both rna granules are important components of the host antiviral defense. virus infection imposes stress on host cells and thus induces sg formation. however, both rna and dna viruses must confront the hostile environment of host innate immunity and apply various strategies to block the formation of sg and pb for their effective infection and multiplication. this review summarizes the current research development in the field and the mechanisms of how individual viruses suppress the formation of host sg and pb for virus production. while the intracellular environment and embedded cellular machinery provide the needed vital force and necessary materials for viruses to replicate after infection, these host machineries are not available to these foreign invaders at ease. in fact, viruses have to counter the multiple layers of intracellular defense to replicate and establish their dominance for their propagation. rna granules ) are dynamic non-membrane subcellular structures (ivanov et al. 2018) containing translationally silenced messenger ribonucleoproteins (mrnps), which play an important role in regulation of cellular homeostasis, rna metabolism and gene expression at the posttranscriptional level (anderson and kedersha 2009 ). stress granules (sg) and processing bodies (pb) (eulalio et al. 2007 ) are two of rna granules well characterized in yeast and mammalian cells (poblete-duran et al. 2016) and are important components of the host cell antiviral defense. sg are non-membranous, transiently assembled cytoplasmic aggregates of 48s mrnps and associated proteins (stohr et al. 2006; buchan and parker 2009) , where stalled translation preinitiation complexes (pics) repress the translation of nonessential mrnas (anderson et al. 2015) and modulate cell signaling by sequestering key signal translation proteins . thus, sg are thought to be the aggregates of stable, translationally silent mrnas . a variety of environmental stresses, including viral infection, can trigger sg formation in eukaryotic cells . in contrast, pb can exist in the absence of stress (stoecklin and kedersha 2013) , which are sites of active mrna decay (decker and parker 2012) . sg initiate global translational arrest by storing mrna (anderson and kedersha 2009 ) for exchange with either polysomes for translation or pb for degradation . rna-binding proteins tia-1 (kedersha et al. 1999; gilks et al. 2004) , g3bp (tourriere et al. 2003; matsuki et al. 2013) and pabp (ma et al. 2009; smith and gray 2010; burgess et al. 2011) are three fundamental components of sg during stress (fig. 1 ). gw182 and de-capping/deadenylating enzymes are specific components of pb , where sirna-or mirna-guided mrnas are processed and degraded (liu et al. 2005) (fig. 1) . virus infection imposes stress on host cells (mcinerney et al. 2005) and thereby induces sg formation. sg can shut off the translation of bulk mrnas (poblete-duran et al. 2016) to regulate gene expression and compartmentalization of heterologous viral rnas and proteins. at the same time, viruses must take strategies to confront these responses and maximize their own replication efficiency (white and lloyd 2012) by inhibition of sg formation and disruption of pb assembly via virally encoded factors. the process of sg formation can be artificially divided into the following steps ( fig. 2) : (1) accumulation of stalled translation initiation complexes ) in response to various types of stress; (2) the rna-binding proteins such as ras-gtpase-activating protein sh3 domain-binding protein 1 (g3bp1) and t cell-restricted intracellular antigen 1 (tia1) bind mrnas and aggregate to nucleate sg formation. self-aggregation of g3bp1 (tourriere et al. 2003) and the binding of tia1 and tiar (tia-1-related protein) to polysome-free mrnas, which exposes prion-like domains (gilks et al. 2004) , trigger mrnp aggregation. the aggregation of proteins is dynamic, and can rapidly exchange between sg and cytosol (kedersha et al. 2000 . (3) large sg aggregate from smaller foci via posttranslational modification and microtubule transport (mccormick and khaperskyy 2017) . many sg proteins undergo multiple post-translational modifications (jayabalan et al. 2016; protter and parker 2016) . for example, g3bp1 must be demethylated (tsai et al. 2016) , dephosphorylated and poly(adp)-ribosylated (leung et al. 2011) to promote sg nucleation. accordingly, sg formation also requires ongoing transport of mrnps along with an intact microtubule cytoskeleton (ivanov et al. 2003) . theoretically, viral interference with any of these important steps may modulate sg formation in cells. in fact, many viral factors can interfere with sg formation and/or function. meanwhile, sg can entrap viral rna in some cases (mccormick and khaperskyy 2017) . therefore, sg are thought to be antiviral (rozelle et al. 2014) . thus, to illustrate the relationship between sg and rna viruses would be important for us to better understand the interactions of host and viruses. up to the present, sg can be divided into two types according to their formation mode. type i sg formation depends on phosphorylation of eukaryotic translation initiation factor-2a (eif2a) by one of the eif2 kinases-doublestranded rna (dsrna)-activated protein kinase or protein kinase r (pkr) (srivastava et al. 1998; garcia et al. 2007; onomoto et al. 2012) , pkr-like er kinase (perk) (harding et al. 2000a, b) , general control non-derepressible protein 2 (gcn2) (wek et al. 1995; deng et al. 2002) or haeme-regulated inhibitor (hri) (mcewen et al. 2005) , which are activated by distinct types of stress. phosphorylated eif2a stably binds to eif2b, which prevents the recycle of eif2 and regeneration of the eif2-gtp-met-trna i met ternary complex. thus, eif2a phosphorylation blocks recognition of the initiation codon and joining of the large ribosomal subunit, resulting in accumulation of stalled 48s mrnps (jackson et al. 2010) . type ii sg formation is independent of eif2a phosphorylation, but requires eif4f complex disruption such as inhibition of eif4a rna helicase dang et al. 2006) or disruption of eif4e activity (von der haar et al. 2004; fournier et al. 2013) for recognition and binding of rna cap structure. the stress induced by nutrient, energy, oxygen or growth factor insufficiency inhibits mtor complex 1 (mtorc1), whose activity is required for the dissociation of 4ebps from eif4e (fujimura et al. 2012) and enables eif4e to form the eif4f complex, and thus blocks assembly of pre-initiation complexes (zoncu et al. 2011) . type i sg formation induced by viruses is the most and best-studied example (table 1, fig. 3a ). various rna products derived by viruses including long dsrna (rojas et al. 2010) , 5 0 -triphosphate rna (5 0 -ppp-rna) (nallagatla et al. 2007) , dsrna that is formed by the antiparallel mrna transcripts of some dna viruses (willis et al. 2011) , and human immunodeficiency virus (hiv) transactivation-response region (tar) rna hairpins (heinicke et al. 2009 ), can be recognized by pkr. the activated pkr initiates sg assembly through eif2a phosphorylation. for instance, the persistent phosphorylation of eif2a (montero et al. 2008 ) during rotavirus infection is pkr-dependent as a consequence of the accumulation of viral dsrna in the cytoplasm outside the viroplasms (virus-induced cytoplasmic inclusion bodies called viroplasms [vms]) (rojas et al. 2010) . even though eif2a is phosphorylated in rotavirusinfected cells, the formation of sg is prevented and viral proteins are efficiently translated, suggesting that the virus prevents the assembly of these structures presumably downstream of eif2a phosphorylation to allow the translation of its mrnas (mazroui et al. 2006) . very recently, dhillon and rao found that rotavirus induces formation and sequestration of remodeled sg and pb in the vms which contain the majorities of their components but selective exclusion of a few proteins (g3bp1 and zbp1 for sg, ddx6, edc4 and pan3 for pb), to promote virus replication (dhillon and rao 2018) . oceguera et al. demonstrated that viral rna of rotavirus could interact with several rna binding proteins (rbps) (xrn1, dcp1, ago2, hur) and interfere with their subcellular localization (oceguera et al. 2018 ). lindquist et al. (lindquist et al. 2011) first determined that sg induction by respiratory syncytial virus (rsv) was mediated by pkr-dependent eif2a phosphorylation. the rsv-mediated sg formation was significantly reduced in pkr-knockdown cells (lindquist et al. 2010 ). in addition, it has been shown that hepatitis c virus (hcv) strongly activates pkr via the 5 0untranslated region (utr) of its genome (toroney et al. 2010) , thereby inducing sg. ns1-mutant influenza virus a (iav) (khaperskyy et al. 2012; mok et al. 2012; ng et al. 2013 ) and c protein-deficient sendai virus (sev) (takeuchi et al. 2008) lead to significant activation of pkr and eif2a phosphorylation. besides, perk could be activated by high levels of glycoproteins produced from enveloped gcn2. type ii sg formation: rvfv attenuates mtor signaling to inhibite 4ebp phosphorylation. all above lead to the formation of stalled translation complexes to initiate the assembly of sg. viruses (chan and egan 2005) , and general control nonderepressible-2 (gcn2) could be activated by sindbis virus (sinv) genomic rna (berlanga et al. 2006) , both leading to phosphorylation of eif2a. gcn2 prevents replication of sinv in the early stages of the viral replicative cycle by blocking the synthesis of nsps from sinv rna (berlanga et al. 2006; frolova et al. 2006; gorchakov et al. 2008) . viruses also induce sg formation independent of eif2a phosphorylation (table 1 ). the most typical example is from rift valley fever virus (rvfv) (habjan et al. 2009; ikegami et al. 2009) (fig. 2) . rvfv (hopkins et al. 2015) infection attenuates akt/mtor signaling and inhibits 4ebp phosphorylation and translation of 5 0 -top mrnas, subsequently leading to an inhibition of global protein translation. 5 0 -top-containing mrnas are indeed targeted to pb, where rvfv uses these cellular mrnas for cap-snatching (hopkins et al. 2015) . this can reflect that sg may interact with pb in a process that is thought to result in the exchange of mrna cargos ). whether any virus induces sg formation to cause translation inhibition due to the destruction of eif4g or eif4a is worth exploring in the future. sg formation shuts off bulk host protein synthesis. however, all viruses depend on the host translation apparatus for their gene expression. therefore, viruses, as intracellular parasites, have to modulate the stress response pathway and sg assembly to translate their proteins for virus replication. rna viruses modulate stress response pathway at different levels of sg formation (table 1) : one is to regulate eif2a phosphorylation, and the other is to regulate the process of sg nucleation. in some cases, viral gene products can act as antagonists by targeting the virus-activated eif2a kinases such as pkr or even by directly modulating the phosphorylation of eif2a (fig. 3a) . iav ns1 (khaperskyy et al. 2012; ng et al. 2013) , middle east respiratory syndrome coronavirus (mers-cov) accessory protein 4a (rabouw et al. 2016; nakagawa et al. 2018) , and ebola virus (ebov) multifunctional protein vp35 (nelson et al. 2016; le sage et al. 2017 ) bind viral dsrna and prevent the viral dsrna from pkr binding to inhibit sg formation. inhibition of sg formation facilitates the translation of viral mrnas, leading to efficient virus replication. hcv ns5a protein (toroney et al. 2010) binds to the pkr dimerization domain to inhibit pkr activation. japanese encephalitis virus (jev) ns2a protein (tu et al. 2012 ) might similarly interact with pkr and then prevent pkr dimer formation. sev (takeuchi et al. 2008) and measles virus (mv) (okonski and samuel 2013) encode a c protein to limit the accumulation of dsrna to inhibit sg formation. it seems that a portion of rna viruses encode rna binding proteins to antagonize the activity of pkr. there are also other groups of rna viruses which directly modulate the phosphorylation of eif2a without pkr. junín virus (junv) prevents sg assembly by impairing the phosphorylation of eif2a through its nucleoprotein (n) and glycoprotein precursor (gpc) (linero et al. 2011) . however, its mechanism remains to be elucidated, although it may be similar to hcv. ruggieri and colleagues reported that hcv rapidly de-phosphorylated eif2a through protein phosphatase 1 (pp1) and its regulatory subunit gadd34 (growth arrest and dna-damage-inducible 34) (kojima et al. 2003; clavarino et al. 2012; ruggieri et al. 2012 . redistribution or sequestering sg components to the viral replication sites is another strategy used by many viruses to impair sg assembly in infected cells (fig. 3b ). zikv infection induces the redistribution of tiar to the viral rna replication sites (hou et al. 2017) ; sev trailer rna captures tiar from sg (iseni et al. 2002) ; west nile virus (wnv) and dengue virus (denv) 3 0 -end viral genome captures tia-1/tiar (li et al. 2002; emara and brinton 2007; xia et al. 2015) ; denv 3 0 -utr interacts with g3bp1, g3bp2, caprin1 and usp10 (ward et al. 2011; reineke et al. 2015) ; jev recruits g3bp and usp10 to the perinuclear region through the interaction of jev core protein with caprin-1, a sg-associated cellular factor (ward et al. 2011) . theiler murine encephalomyelitis virus (tmev) and mengovirus, a strain of emcv, express the leader (l) protein to inhibit g3bp1 aggregation (borghese and michiels 2011) . sequestration or redistribution of sg components by viruses through protein-protein and protein-rna interactions not only prevents sg assembly, but also facilitates viral genome replication. hcv-jfh1 infection redistributes several sg components, including g3bp1, ataxin-2 (atx2), and poly(a)-binding protein 1 (pabp1), to the hcv replication complex (rc) (ariumi et al. 2011; pene et al. 2015) , and co-opts g3bp1 to mediate efficient viral replication by interaction with ns5b and the 5 0 end of the hcv minus-strand rna (ariumi et al. 2011; garaigorta et al. 2012 ). (fig. 3b ). sequestration of o-linked n-acetylglucosamine (ogn) transferase (ogt), an enzyme that catalyzes the posttranslational addition of ogn to protein targets, in rsv ibs was also proposed to regulate sg nucleation and suppression of sg formation (fricke et al. 2013) (fig. 3b ). viral transcription and replication of rabv take place within negri bodies (nbs), which are ib-like structures (lahaye et al. 2009 ). rabv-induced sg are normally located closely to nbs. viral mrnas rather than viral genomic rna accumulate in the sg-like structures together with cellular mrnas were found to be specially transported from nbs to sg-like structures (nikolic et al. 2016) . vsv infection also induces formation of the sg-like structures that co-localize with viral replication proteins and rna, which are different from canonical sg (dinh et al. 2013) . sg proteins (eif4g, eif3, pabp) are selectively sequestered within ebola virus inclusion bodies and co-localize with viral rna to form inclusion body-bound granules, which are functionally and structurally different from canonical sg, probably leading to inhibit the antiviral role of sg (nelson et al. 2016) (fig. 3b) . collectively, these findings provoke more investigations on the roles of viral ibs in viral replication and resisting cellular responses. unlike rna viruses, the regulation of sg formation during infection with dna viruses is poorly understood. it was reported that human cytomegalovirus (hcmv) infection modifies the unfolded protein response (upr) and activates perk (fig. 2) , but limiting the amount of phosphorylated eif2a to maintain translation (isler et al. 2005 ). kaposi's sarcoma-associated herpesvirus (kshv) orf57 (sharma et al. 2017) interacts with pkr and pkr-activating protein (pact) (patel et al. 2000) to inhibit pkr binding dsrna and prevent pact-pkr interaction in the pkr pathway (li et al. 2006) , respectively. hcmv ptrs1 and pirs1 antagonize pkr to facilitate virus replication (ziehr et al. 2016) . the hsv-1 vhs (sciortino et al. 2013 ) and us11 protein (cassady and gross 2002) play a key role in blocking the activation of pkr. smiley and colleagues also demonstrated that infection with virion host shutoff protein (vhs)-defective herpes simplex virus 1 (hsv-1) triggers sg formation, and pkr is essential for sg formation in the absence of vhs (dauber et al. 2016) (fig. 3a) . finnen et al. previously established that herpes simplex virus 2 (hsv-2) infection impacts stress granule accumulation in response to oxidative stress (finnen et al. 2012) . they also demonstrated that disruption of sg is mediated by vhs (finnen et al. 2014) , whose endoribonuclease activity is required to disrupt sg formation (finnen et al. 2016) . hsv-2 vhs indeed have the ability to localize to sg (finnen et al. 2016) (fig. 3b ). this implies that removal of rna from sg promotes its disassembly and that intact rna is crucial for maintaining sg structure. it will be interesting to test the function of endoribonucleases in sg disassembly. vaccinia virus (vv) sequesters crucial translation initiation factors, such as g3bp1, caprin1, eif4e, pabp and eif4g (katsafanas and moss 2007; simpson-holley et al. 2011; zaborowska et al. 2012) , within cytoplasmic viral dna factories to utilize sg components for different purposes (fig. 3b) . a recent study (meng and xiang 2019) suggested that the rna granules are resulted from untranslated mrna accumulation in viral dna factories (liu and moss 2016) and tia-1 is probably not required for granule formation and antipoxviruses. instead, the granules formation is most likely driven by an array of rna-protein interactions and requires no specific sg components (sivan et al. 2018; meng and xiang 2019) . assembly of p-bodies (pb) pb were first reported in the scientific literature by bashkirov et al. in 1997, and described as ''small granules or discrete, prominent foci'' or as the cytoplasmic location of the mouse exoribonuclease mxrn1p (bashkirov et al. 1997) . like sg, pb lack outer lipid membrane and now are recognized to be the sites where non-translating mrnas accumulate for different fates including decay, storage, or returning to translation. a variety of enzymes involved in mrna deadenylation (ccr1, caf1, not1) (sheth and parker 2006) , decapping (dcp1/2, lsm1-7, edc3proteins) (ingelfinger et al. 2002; yu et al. 2005) , nonsense-mediated decay (nmd) proteins (smg5-6-7, upf1) (ingelfinger et al. 2002; durand et al. 2007) , in addition to scaffolding proteins (ge-1/hedls) (yu et al. 2005) and translation control factors (cpeb, eif4e-t) (andrei et al. 2005; wilczynska et al. 2005) , are the components of pb and used as routine markers to distinguish these granules. nonetheless, some components (apobec3g, brf1, ddx3, fast, ttp, rap55) (mcewen et al. 2005; sen and blau 2005; gallois-montbrun et al. 2007; chen et al. 2008) have also been shown to be shared by both sg and pb, suggesting a substantial linkage of these two structures and movement of mrnas between both rna granules. interestingly, among these components, pb also include rnainduced silencing complex (risc) or mirna associated argonaute (ago) proteins (also shared with sg) and the gw182 protein which provides scaffolding activities for risc to function, suggesting pb being the sites of mirna mediated translation repression. the scaffolding activity of gw182 is critical for pb and knockdown of gw182 expression disrupts pb formation (liu et al. 2005) . notably, gw182 has been shown to bind to ago2 which is critical for mirna function and pb formation (liu et al. 2005) . recent evidence indicates that gw182 can recruit up to three molecules of ago2 via its three gw motifs (glycine-tryptophan repeats) while each ago protein has a single gw182-binding site (elkayam et al. 2017) (fig. 4) . by applying fluorescence-activated particle sorting to purify pb in combination with mass spectrometry, hubstenberger et al. identified 125 proteins that are significantly associated with pb (hubstenberger et al. 2017) . by labeling several pb-localized proteins with a bira (e. coli biotin ligase) enzyme in combination with mass spectrometry after streptavidin pulldown, youn et al. identified 38 proteins in the pb (youn et al. 2018) . isgs (interferon stimulated genes) can also be found in pb during virus infection (hebner et al. 2006) . in comparison to viral regulation of sg, interaction of virus and pb was not much explored. it is an assumption that rna viruses must regulate rna decay processes/machinery to prevent degradation of virus genomes and mrnas. recently, some progress has been made to understand the relationship between pb components and some viruses in the context of viral gene expression. the data in published literatures are summarized in (table 2) . mutation induced in the pb core components to affect the viral life cycles are well studied and tabulated in an earlier review (beckham et al. 2007) . the report linking the assembly of yeast ty3 retrotransposons virus-like particles with pb presented the first link between human retrovirus and pb (checkley et al. 2010) . the later study revealed pb to be the site of anti-viral host factors apo-bec3g and apobec3f (a3g or a3f, apolipoprotein b mrna-editing enzyme catalytic polypeptide 1-like) family of cytidine de-aminases, presumably representing a component of innate immunity against hiv (wichroski et al. 2006; gallois-montbrun et al. 2007 ). in a different study, a3f was found to specifically interact with cellular signal recognition particle rna (7sl rna). efficient packaging of 7sl rna and a3f into hiv virons was mediated by the rna-binding nucleocapsid domain of hiv-1 gag (wang et al. 2007 ). the bona fide and unique dependence of viruses on pb came from the studies on plant brome mosaic virus (bmv) (beckham et al. 2007 ). this study suggested the accumulation of bmv mrnas in pb was an important step in rna replication complex assembly for bmv, and possibly for other positive-strand rna viruses. nonetheless, many rna viruses initiate the process of transcription of viral rna by the process of 'cap snatching' which involves the acquisition of capped 5 0 oligonucleotides from cellular mrnas. interestingly, pb were shown to serve as a pool of primers in the case of hantavirus while its nucleocapsid protein, which accumulates in pb, binds 5 0 caps with high affinity (mir et al. 2008) . the base-pair complementarity between a mirna and a target mrna dictates the mirna to specifically repress posttranscriptional expression of mrnas. subsequent events in this process involve relocation of rna-induced silencing complexes (riscs) together with several other rna binding proteins to form pb. in this context, hiv-1 mrna interacts with risc proteins and disrupting pb structures enhances viral production and infectivity, suggesting a role of pb against viral infection (nathans et al. 2009 ). specific mir-29a-hiv-1 mrna interaction was found to enhance viral mrna association with risc and pb proteins and regulate hiv-1 production and infectivity. the translating mrna can be stripped of ribosomes and the initiation complex can be collaps when binding to mirna-risc complex. the mrnps targeted by pb components undergo three outcomes: 1. translational inhibition; 2. pan2/3-mediated deadenylation; 3. rna decay by other associated rna decay factors (e.g., xrn1, dcp1a, ddx6, and lsm). several rna and dna viruses which inhibit pb assembly are shown. hiv nef interacts with ago2 via its glycine-tryptophan region and functions as a viral suppressor of rnai (aqil et al. 2013) . while overexpression of mov10, a component of pb and an atp-dependent 5 0 -3 0 rna helicase, inhibits hiv production (burdick et al. 2010; furtak et al. 2010) , mov10 and apobec3g localization to pb is not required for hiv virion incorporation and antiviral activity (izumi et al. 2013) . it becomes clear that mov10 inhibits virus infection by enhancing rig-i-mavs-independent ifn induction (cuevas et al. 2016 ) and stabilizing a3g from degradation (chen et al. 2017) . the anticipated evidence of viral disruption of pb also came from the study with poliovirus (pv), a plus-strand rna virus showing that pb are disrupted during pv infection in cells by 4 h post infection (dougherty et al. 2011 ). this function is attributed to viral proteinase 3c which degrades several components of pb including xrn1 and dcp1a, but not affecting others such as gw182, edc3 and edc4. rotaviruses disassemble pb by using viral rna as a sponge for rna binding proteins to redistribute several pb components, including ago2, gw182 and dcp1 pb (oceguera et al. 2018) . in fact, rotavirus disrupts pb through multiple mechanisms. the viral nsp1 protein seems to degrade pb component pan3, while relocalizing other two components (xrn1 and dcp1a) (bhowmick et al. 2015) . intriguingly, exclusion of sg and pb components from the viroplasm is important for rotavirus replication and progeny virus production (dhillon and rao 2018) . while rna viruses have evolved to co-opt or modulate the assembly of pb, this effect is rather unclear during infection by dna viruses. since most of the dna viruses replicate and assemble in the nucleus, therefore as proposed for rna viruses, accumulation of viral rnas in pb for assembly cannot be a strategy required by dna viruses. however, the close relationship of pb with translational repression reasonably provides a foundation for pb being antiviral cellular components against dna viruses. thus it is assumed that those factories suppressing mrna translation would inhibit protein production of dna viruses. to fight back, the dna viruses have to develope strategies to bypass this antagonism mediated by pb for their survival and productive infection (table 2) . adenovirus e4 11 k, the product of e4 orf3, accumulates viral late mrna transcripts and at least five proteins of pb (rck/p54/ddx6, ago2, xrn1, ge1, and lsm-1) in the e4 11 k-induced cytoplasmic aggresomes. redistribution of the pb components to the aggresomes, not to the pb, leads to inactivate or destroy these proteins. e4 11 k protein interacts with rna helicase ddx6, one of the pb proteins, for its redistribution. because pb are the sites for mrna degradation, their alteration by e4 11 k suggests a role of e4 11 k in viral late mrna accumulation (greer et al. 2011) . the role of pb in regulation of cytomegalovirus infection remains elusive. first, hcmv infection does not affect, but rather accumulates the formation of pb; second, pb formed during hcmv infection do not contain ago2; third, hcmv prevents viral ie1 mrna, a major ie gene product to encode a critical protein for viral gene expression and replication, from colocalization with pb (seto et al. 2014) . by generating a transgenic mice deficient of pb component lsm14a (or rap55), recent studies showed that lsm14a plays a critical and specific role in the induction of antiviral cytokines (ifn-b, ifn-a, and il-6) in dendritic cells (dcs). dna viruses (hsv-1 and murine herpesvirus 68) and rna virus vsv trigger this induction, but sendai virus lacks such an effect (anderson and kedersha 2009; . lsm14a deficiency specifically downregulates mita/sting (stimulator of interferon genes) level in dcs by impairing its nuclear mrna precursor processing. in contrast to its role in mrna decay, this study revealed a role of lsm14 in nuclear mrna precursor processing and cell-specific regulatory mechanism of antiviral immune responses . kshv kaposin b, a latent protein linked with cancer progression, induces pb dispersion (corcoran et al. 2015) . kaposin b activates the stress-responsive kinase mk2 in endothelial cells (ecs) to selectively block the decay of au-rich mrnas (are-mrnas) which encode pro-inflammatory cytokines and angiogenic factors and to reprogram ecs through post-transcriptional control of ec gene expression and secretion. kshv orf57 protein inhibits the formation of pb during lytic infection by disrupting the essential interaction of ago2 with gw182 (unpublished data). these data provide the first evidence that a tumor virus rna-binding protein orf57 antagonizes the rna regulatory pathway of host antiviral defenses during lytic infection. sg are highly dynamic structures , which constantly exchange their components to regulate gene expression and are thought to be antiviral. sg composition appears to vary according to the inducing stimulus (table 3) . it's clear that sg assembly/disassembly is a tightly regulated process which accompanies rearrangements of rna and proteins . although significant advances have been made to understand how viruses regulate sg formation, our current knowledge is not suffucient to fully elucidate the machanism how sg are regulated in living cells. further works are needed to address the following questions: first, is there any pathway to be a target for antiviral drug development? second, do sg function as platforms that potentiate virus recognition? third, is any unexplored pathway leading to sg formation which could be visualized by fluorescence in situ hybridization techniques-including single molecule rna tracking methods in combination with super-resolution microscopy? using viruses as a research tool will definitely teach us how the host fights virus infections and how the viruses get away from its host resistance. pb affect viral infections in multiple ways. thus, it is difficult to generalize a common viral strategy in a particular virus group to interact with the components of pb. the noticed evidence is that viruses in the same family may show extremely distant behavior when they come to interact with pb (table 2 ). more studies on virus interactions with pb will be required to characterize the pb to be proviral or antiviral in a context-dependent manner. other key questions in the field for future studies are: (1) to understand the mechanisms that regulate pb formation in cells. viral manipulation of pb may provide a better platform to understand this regulation; (2) to determine which viral rna species preferentially travel through these rna granules and which ones do not? (3) to identify the rna elements dictating viral rna to escape from sg and pb. thus, discovery of virus regulations of pb assembly represents a new paradigm of virus-host interactions. (1ziasc010357 to zmz). conflict of interest the authors declare that they have no conflict of interest. animal and human rights statement this article does not contain any studies with human or animal subjects performed by any of the authors. open access this article is distributed under the terms of the creative commons attribution 4.0 international license (http://creative commons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. abrahamyan lg, chatel-chaix l, ajamian l, milev mp, monette a, clement jf, song r, lehmann m, desgroseillers l, laughrea novel staufen1 ribonucleoproteins prevent formation of stress granules but favour encapsidation of hiv-1 genomic rna stress granules: the tao of rna triage rna granules: post-transcriptional and epigenetic modulators of gene expression stress granules, p-bodies and cancer a role for eif4e and eif4e-transporter in targeting mrnps to mammalian processing bodies the hiv-1 nef protein binds argonaute-2 and functions as a viral suppressor of rna interference hepatitis c virus hijacks p-body and stress granule components around lipid droplets a mouse cytoplasmic exoribonuclease (mxrn1p) with preference for g4 tetraplex substrates interactions between brome mosaic virus rnas and cytoplasmic processing bodies antiviral effect of the mammalian translation initiation factor 2alpha kinase gcn2 against rna viruses rotavirus disrupts cytoplasmic p bodies during infection rna-mediated sequestration of the rna helicase eif4a by pateamine a inhibits translation initiation the leader protein of cardioviruses inhibits stress granule assembly eukaryotic stress granules: the ins and outs of translation p bodyassociated protein mov10 inhibits hiv-1 replication at multiple stages nuclear relocalisation of cytoplasmic poly(a)-binding proteins pabp1 and pabp4 in response to uv irradiation reveals mrna-dependent export of metazoan pabps the herpes simplex virus type 1 u(s)11 protein interacts with protein kinase r in infected cells and requires a 30-amino-acid sequence adjacent to a kinase substrate domain p-body components lsm1, gw182, ddx3, ddx6 and xrn1 are recruited to wnv replication sites and positively regulate viral replication hepatitis c virus envelope proteins regulate chop via induction of the unfolded protein response p-body components are required for ty1 retrotransposition during assembly of retrotransposition-competent viruslike particles multiple pathways differentially regulate global oxidative stress responses in fission yeast moloney leukemia virus 10 (mov10) inhibits the degradation of apobec3g through interference with the vifmediated ubiquitin-proteasome pathway protein phosphatase 1 subunit ppp1r15a/gadd34 regulates cytokine production in polyinosinic:polycytidylic acidstimulated dendritic cells viral activation of mk2-hsp27-p115rhogef-rhoa signaling axis causes cytoskeletal rearrangements, p-body disruption and are-mrna stabilization mov10 provides antiviral activity against rna viruses by enhancing rig-i-mavsindependent ifn induction eukaryotic initiation factor 2alpha-independent pathway of stress granule induction by the natural product pateamine a the herpes simplex virus virion host shutoff protein enhances translation of viral true late mrnas independently of suppressing protein kinase r and stress granule formation p-bodies and stress granules: possible roles in the control of translation and mrna degradation activation of gcn2 in uv-irradiated cells inhibits translation rotavirus induces formation of remodeled stress granules and p bodies and their sequestration in viroplasms to promote progeny virus production induction of stress granule-like structures in vesicular stomatitis virus-infected cells poliovirus-mediated disruption of cytoplasmic processing bodies inhibition of nonsensemediated mrna decay (nmd) by a new chemical molecule reveals the dynamic of nmd factors in p-bodies multivalent recruitment of human argonaute by gw182 interaction of tia-1/tiar with west nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly p-body formation is a consequence, not the cause, of rnamediated gene silencing herpes simplex virus 2 infection impacts stress granule accumulation the herpes simplex virus 2 virion-associated ribonuclease vhs interferes with stress granule formation herpes simplex virus 2 virion host shutoff endoribonuclease activity is required to disrupt stress granule formation inactivation of the mtorc1-eukaryotic translation initiation factor 4e pathway alters stress granule formation and ogt sequestration into viral inclusion bodies in cells infected with human respiratory syncytial virus suppresses mk2 activities and stress granule assembly formation of nsp3-specific protein complexes during sindbis virus replication selenite targets eif4e-binding protein-1 to inhibit translation initiation and induce the assembly of non-canonical stress granules production of a dominant-negative fragment due to g3bp1 cleavage contributes to the disruption of mitochondria-associated protective stress granules during cvb3 infection perturbation of the p-body component mov10 inhibits hiv-1 infectivity antiviral protein apobec3g localizes to ribonucleoprotein complexes found in p bodies and stress granules hepatitis c virus (hcv) induces formation of stress granules whose proteins regulate hcv rna replication and virus assembly and egress the dsrna protein kinase pkr: virus and cell control stress granule assembly is mediated by prion-like aggregation of tia-1 different types of nsp3-containing protein complexes in sindbis virusinfected cells the adenovirus e4 11 k protein binds and relocalizes the cytoplasmic p-body component ddx6 to aggresomes nss protein of rift valley fever virus induces the specific degradation of the double-stranded rna-dependent protein kinase regulated translation initiation controls stress-induced gene expression in mammalian cells perk is essential for translational regulation and cell survival during the unfolded protein response human papillomaviruses target the double-stranded rna protein kinase pathway rna dimerization promotes pkr dimerization and activation protein expression redirects vesicular stomatitis virus rna synthesis to cytoplasmic inclusions inclusion bodies are a site of ebolavirus replication virusinduced translational arrest through 4ebp1/2-dependent decay of 5 0 -top mrnas restricts viral infection zika virus hijacks stress granule proteins and modulates the host stress response inclusion bodies of human parainfluenza virus type 3 inhibit antiviral stress granule formation by shielding viral rnas p-body purification reveals the condensation of repressed mrna regulons feline calicivirus infection disrupts assembly of cytoplasmic stress granules and induces g3bp1 cleavage rift valley fever virus nss protein promotes posttranscriptional downregulation of protein kinase pkr and inhibits eif2alpha phosphorylation the human lsm1-7 proteins colocalize with the mrna-degrading enzymes dcp1/2 and xrnl in distinct cytoplasmic foci sendai virus trailer rna binds tiar, a cellular protein involved in virus-induced apoptosis human cytomegalovirus infection activates and regulates the unfolded protein response disruption of microtubules inhibits cytoplasmic ribonucleoprotein stress granule formation stress granules and processing bodies in translational control mov10 and apobec3g localization to processing bodies is not required for virion incorporation and antiviral activity the mechanism of eukaryotic translation initiation and principles of its regulation atpase-modulated stress granules contain a diverse proteome and substructure neddylation promotes stress granule assembly colocalization of transcription and translation within cytoplasmic poxvirus factories coordinates viral expression and subjugates host functions stress granules: sites of mrna triage that regulate mrna stability and translatability rnabinding proteins tia-1 and tiar link the phosphorylation of eif-2 alpha to the assembly of mammalian stress granules dynamic shuttling of tia-1 accompanies the recruitment of mrna to mammalian stress granules stress granules and processing bodies are dynamically linked sites of mrnp remodeling real-time and quantitative imaging of mammalian stress granules and processing bodies stress granules and cell signaling: more than just a passing phase? g3bp-caprin1-usp10 complexes mediate stress granule condensation and associate with 40s subunits influenza a virus inhibits cytoplasmic stress granule formation modulation of stress granules and p bodies during dicistrovirus infection the function of gadd34 is a recovery from a shutoff of protein synthesis induced by er stress: elucidation by gadd34-deficient mice functional characterization of negri bodies (nbs) in rabies virus-infected cells: evidence that nbs are sites of viral transcription and replication ebola virus vp35 blocks stress granule assembly poly(adp-ribose) regulates stress responses and microrna activity in the cytoplasm cell proteins tia-1 and tiar interact with the 3 0 stem-loop of the west nile virus complementary minus-strand rna and facilitate virus replication molecular basis for pkr activation by pact or dsrna respiratory syncytial virus induces host rna stress granules to facilitate viral replication activation of protein kinase r is required for induction of stress granules by respiratory syncytial virus but dispensable for viral replication junin virus infection impairs stress-granule formation in vero cells treated with arsenite via inhibition of eif2alpha phosphorylation opposing roles of double-stranded rna effector pathways and viral defense proteins revealed with crispr-cas9 knockout cell lines and vaccinia virus mutants microrna-dependent localization of targeted mrnas to mammalian p-bodies lsm14a plays a critical role in antiviral immune responses by regulating mita level in a cell-specific manner expression of poly(a)-binding protein is upregulated during recovery from heat shock in hela cells both g3bp1 and g3bp2 contribute to stress granule formation inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation translation inhibition and stress granules in the antiviral immune response heme-regulated inhibitor kinase-mediated phosphorylation of eukaryotic translation initiation factor 2 inhibits translation, induces stress granule formation, and mediates survival upon arsenite exposure importance of eif2alpha phosphorylation and stress granule assembly in alphavirus translation regulation rna granules associated with samd9-mediated poxvirus restriction are similar to antiviral granules in composition but do not require tia1 for poxvirus restriction storage of cellular 5 0 mrna caps in p bodies for viral capsnatching the ns1 protein of influenza a virus interacts with cellular processing bodies and stress granules through rna-associated protein 55 (rap55) during virus infection rotavirus infection induces the phosphorylation of eif2alpha but prevents the formation of stress granules inhibition of stress granule formation by middle east respiratory syndrome coronavirus 4a accessory protein facilitates viral translation, leading to efficient virus replication 0 -triphosphate-dependent activation of pkr by rnas with short stem-loops cellular microrna and p bodies modulate host-hiv-1 interactions ebola virus does not induce stress granule formation during infection and sequesters stress granule proteins within viral inclusions encephalomyocarditis virus disrupts stress granules, the critical platform for triggering antiviral innate immune responses rabies virus infection induces the formation of stress granules closely connected to the viral factories rotavirus rnas sponge host cell rna binding proteins and interfere with their subcellular localization stress granule formation induced by measles virus is protein kinase pkr dependent and impaired by rna adenosine deaminase adar1 critical role of an antiviral stress granule containing rig-i and pkr in viral detection and innate immunity mechanistic insights into mammalian stress granule dynamics pact, a stressmodulated cellular activator of interferon-induced doublestranded rna-activated protein kinase, pkr dynamic interaction of stress granules, ddx3x, and ikk-alpha mediates multiple functions in hepatitis c virus infection who regulates whom? an overview of rna granules and viral infections principles and properties of stress granules middle east respiratory coronavirus accessory protein 4a inhibits pkrmediated antiviral stress responses hiv-1 gag co-opts a cellular complex containing ddx6, a helicase that facilitates capsid assembly stress granules regulate double-stranded rnadependent protein kinase activation through a complex containing g3bp1 and caprin1 functional organization of cytoplasmic inclusion bodies in cells infected by respiratory syncytial virus protein kinase r is responsible for the phosphorylation of eif2alpha in rotavirus infection activation of stress response pathways promotes formation of antiviral granules and restricts virus replication dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection the virion host shutoff rnase plays a key role in blocking the activation of protein kinase r in cells infected with herpes simplex virus 1 argonaute 2/risc resides in sites of mammalian mrna decay known as cytoplasmic bodies processing bodies accumulate in human cytomegalovirus-infected cells and do not affect viral replication at high multiplicity of infection kshv inhibits stress granule formation by viral orf57 blocking pkr activation targeting of aberrant mrnas to cytoplasmic processing bodies an rna pseudoknot is required for production of yellow fever virus subgenomic rna by the host nuclease xrn1 formation of antiviral cytoplasmic granules during orthopoxvirus infection human host range restriction of the vaccinia virus c7/k1 double deletion mutant is mediated by an atypical mode of translation inhibition poly(a)-binding protein (pabp): a common viral target sindbis virus usurps the cellular hur protein to stabilize its transcripts and promote productive infections in mammalian and mosquito cells phosphorylation of eukaryotic translation initiation factor 2 mediates apoptosis in response to activation of the double-stranded rna-dependent protein kinase relationship of gw/p-bodies with stress granules zbp1 regulates mrna stability during cellular stress sendai virus c protein plays a role in restricting pkr activation by limiting the generation of intracellular double-stranded rna rna granules: the good, the bad and the ugly regulation of pkr by hcv ires rna: importance of domain ii and ns5a the rasgap-associated endoribonuclease g3bp assembles stress granules arginine demethylation of g3bp1 promotes stress granule assembly blocking double-stranded rna-activated protein kinase pkr by japanese encephalitis virus nonstructural protein 2a foot-and-mouth disease virus leader protease cleaves g3bp1 and g3bp2 and inhibits stress granule formation the mrna cap-binding protein eif4e in post-transcriptional gene expression 7sl rna mediates virion packaging of the antiviral cytidine deaminase apobec3g quantitative mass spectrometry of denv-2 rna-interacting proteins reveals that the dead-box rna helicase ddx6 binds the db1 and db2 3 0 utr structures the histidyl-trna synthetaserelated sequence in the eif-2 alpha protein kinase gcn2 interacts with trna and is required for activation in response to starvation for different amino acids distinct stages in stress granule assembly and disassembly regulation of stress granules in virus systems inhibition of cytoplasmic mrna stress granule formation by a viral proteinase human retroviral host restriction factors apobec3g and apobec3f localize to mrna processing bodies the translational regulator cpeb1 provides a link between dcp1 bodies and stress granules viral double-stranded rnas from vaccinia virus early or intermediate gene transcripts possess pkr activating function, resulting in nf-kappab activation, when the k1 protein is absent or mutated dengue virus infection induces formation of g3bp1 granules in human lung epithelial cells picornavirus 2a protease regulates stress granule formation to facilitate viral translation foot-and-mouth disease virus counteracts on internal ribosome entry site suppression by g3bp1 and inhibits g3bp1-mediated stress granule assembly via post-translational mechanisms high-density proximity mapping reveals the subcellular organization of mrna-associated granules and bodies ge-1 is a central component of the mammalian cytoplasmic mrna processing body recruitment of host translation initiation factor eif4g by the vaccinia virus ssdna-binding protein i3 human cytomegalovirus ptrs1 and pirs1 antagonize protein kinase r to facilitate virus replication mtor: from growth signal integration to cancer, diabetes and ageing acknowledgements this work was supported by grants from the china natural science foundation (81825015 and 31630086), the natural science foundation of hubei province innovation group (2017cfa022), and intramural research program of nci/nih 