Journal of Phamaceutl Anas 1 (2020) 102-108 Contents lists 2 ‘at ScienceDirect Journal of Pharmaceutical Analysis a’ homepage: www-c! vier.com/locate/ipa os Review paper Molecular immune pathogenesis and diagnosis of COVID-19 Xiaowei Li’, Manman Geng", Yizhao Peng", Liesu Meng", Shemin Lu” a *berment of Bache end kar Bosh ofc Mea Sen Nen avo Uninet Mah ce Cent Xe, Sha arene Zp key of vnc nl ens ee Dane May of atin Shama 7208 China ARTICLE INFO ABSTRACT ‘ate sore Coronavirus disease 2019 (COVID-19) Is a kind of viral pneurnonia which is caused by severe acute Received 24 February 2020 respiratory syndrome coronavirus 2 (SARS-CoV 2). The emergence of SARS-CoV-2 has been marked 3s coved i revs fn the third introduction ofa higly pathogenic corenavinis into the human popwlation after the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coro- ‘virus (MERS-CoV) in the twenty-first century. In this minirevew, we provide a brief itreduction ofthe seneral Features of SARS-COV-2 and discuss curtent knowlege of molecular immune pathoxenesis, Aiagnosis and treatment of COVID-1 on the base ofthe present understanding of SARS-CoV and MERS- ‘Accepted 1 March 2020 coool Cov infects whch muy be hell tng ol is ad pote tape ges er ones combating the SAS CV tenon Seay ‘7020 xan anton Unies. rdctio nd osting by Fier RY. This an open ces ate wesc Under €CBYNND hen i: treasonous censored 0) Mosse 1. introduction announced that COMID-19 was liste a the Public Health Emer ‘Novel coronavirus-induced pneumonia, which was named as coronavirus disease 2019 (COVID-19) by the WHO on the February 11,2020, has rapidly increased in epidemic seale [1]-On the same day, the international virus classification commission announced that the navel coronavirus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 is not the frst severe respiratory disease outbreak caused by the coronavirus, Just in the past two decades, coronaviruses have caused three epidemic diseases, namely, COVID-19, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) [2] At pre- sent, the cases of COVID-19 have been found in many countries around the world (3]- According to the latest data. up to the March 1, 2020, the number of confirmed cases in China reached 79.968, of ‘which 2.873 were dead, and 41,681 were cured. In addition to China, the number of confirmed cases in other countries also reached 7,041, of which 105 were dead, and 459 were cured. On the 3st of January 2020, the World Health Organization (WHO) Peer review under respostty af fan Jaotong Une + Coresponding suthor at Department of iacherstry and Molecular Boley, School of fase Medial Scones, a futong Unieraty Heath Seence Center mal addres menghesaPajteducn (L Meng), Inhemindtuedicn rs eps: 01016pka202000.001 gency of International Concern (PHEIC), meaning that it may pose "sks to multiple countries and requires a coordinated international response. The review tries to explain the molecular immune pathogenesis and diagnosis of COVID-19 and provide a reference for the prevention and drug development of SARS-CoY-2 infection, based on the recent research progress of SARS-CoV-2 and the knowledge from researches on SARS-CoV and MERS-CoV. 2, Virology of SARS-CoV-2 Coronaviruses are enveloped viruses with a positive sense single-stranded RNA genome (26-32 kb) [4]. Four coronavirus srenera (2. 7, 8) have been identified so far. with human coro naviruses (HCoVs) detected in the z coronavirus (HCOV-229E and 'NUB3) and § coronavirus (MERS-CoV, SARS-CoV, HCoV-OC43 and HCOV-HKU1) genera [5] In late December 2019, patients present- ing with cough, fever, and dyspnea with acute respiratory distress syndrome (ARDS) due to an unidentified microbial infection were reported in Wuhan, China. Virus genome sequencing of five pa- tients with pneumonia hospitalized from December 18. to December 29,2019, revealed the presence of a previously unknown B-CoV strain in all of them [6]. This isolated novel [COV shows 88% Jdentity to the sequence of two bat-derived severe acute respira- tory syndromes (SARS)-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, and about 50% identity tothe sequence of MERS- dant 7790 2020 Xn tong University, Podaction an hosing by lever BY, This san open aces ate wer the CCHY-NC-ND licens (hi ljoetvermmons ‘racensesy nn.2X Wet Jamal of Parmaceua Anat 102020) 102-108 1 CoV [6]. The novel f-CoV was then named “SARS-CoV-2" by the clearly depicted in Fig. 1A. International Virus Classification Commission, The phylogenetic The genome of SARS-CoV-2 is similar to that of typical CoV’ and tee of SARS-like coronaviruses complete genome Sequences is contains at least ten open reading frames (ORFs). The first ORFS A Human Bi Pangotin Micivet w eRNA Fig. 1 The phylogeaec te of SARS-ke coronaviruses compete genome Sequences and genome Of SARS-COV, MERS CO a SARSCOV- 2. (A) hs plyogery shows evlson of ARS He reoronndrses chang apes For man (4” 20). (2 22)cet (3) and pagatia (2 6) Te phylogertc ree of Complete genome seucnces of coronaire was oiamed and naj with Nextsra (pthc kee), (8) Coronas form erloped ed sphere ares of 10-160 movin ameter. They contin apstvesense gl sanded RNA (SNA) genome of 26.32 Kins Im SARS CV, MERS CV andl SARS-CV-2 the 9 ‘erminal twothis ofthe genome ORF ences polyprotein which form the ral reas tansrpase complex. The othe ORFs onthe one-third fhe genome ence our ‘mam stoctral proteins: spike (9), enelope (0), nacecapsi (N) and mebrane (Mtns, 5 well 2 sevetlacessry prt.ws XU ea ourna of Pharmacia nay 10 (2020) 102-108 (ORFIajb), about two-thirds of viral RNA, are translated into two large polyproteins. in SARS-CoV and MERS-CoV, two polyproteins, ppia and pplab, are processed into 16 non-structural proteins {nspt-nsp16), which form the viral replicase transcriptase complex. (7), Those nsps rearrange membranes originating from the rough endoplasmic reticulum (RER) into double-membrane vesicles where viral replication and transcription occur [8.9]. The other ORFs of SARS-CoV-2 on the one-third of the genome encode four ‘main structural proteins: spike (S), envelope (E), nucleocapsid (N) and membrane (M) proteins, a well as several accessory proteins with unknown functions which do not participate in viral replica- tion (Fig. 1B) Several groups of scientists in China have all discovered that, SARS-COV-2. just like SARS-COV, requires the angiotensin- converting enzyme 2 (ACE2) [1] a5/a receptor to enter cells [10] ‘The binding of the virus with host cell receptors is a significant determinant for the pathogenesis of infection. SARS-CoV most likely originated in bats [11Jand adapted to non-bat ACE2 variants as it crossed species to infect humans [12]. Dipeptidyl peptidase 4 (PPA, also known as CD26) was identified asa functional receptor for MERS-CoV, because the receptor-binding St domain of the [MERS-CaV spike protein was copurifed with DPP4 specifically fram lysates of susceptible Huh-7 cells [13]. MERS-CoV can bind DPP4 from multiple species, which promotes the transmission to humans and other species, and infection of cells from a large number of species [i], A better understanding of the relative effects of re- ceptor binding and protease action will help predict whether spe- cific zoonotic coronaviruses infect humans and the possibility of adaptation. 3. Pathogenesis of COVID-19 Patients with COVID-19 show clinical manifestations including, fever, nonproductive cough, dyspnea, myalgia, fatigue, normal ot decreased leukocyte counts, and radiographic evidence af pneu- ‘monia [15], which are similar to the symptoms of SARS-CoV and [MERS-CoV infections [16]. Hence, although the pathogenesis of COVID-19 is poorly understood, the similar mechanisms of SARS- CoV and MERS-CoY still can give us a lot of information on the pathogenesis of SARS-CoV-2 infection to facilitate our recognition ‘of COVID-19. 34. Coronavirus entry and replication Coronavirus § protein has been reported as a significant deter- rinant of virus entry into host cells [2) The envelope spike _slycoprotein binds to its cellular receptor. ACE2 for SARS-CoV [10] and SARS-CoV-2 [17]. Cb209Ua C-type lectin, also called L-SIGN) for SARS-CoV [18], DPP¢ for MERS-CoV [13] The entry of SARS-CoV into cells was intially identified to be accomplished by direct, ‘membrane fusion between the virus and plasma membrane [19] Belouzard etal. [20] found thata critical proteolytic cleavage event occurred at SARS-CoV $ protein at position (S2") mediated the membrane fusion and vial infectivity. MERS-CoV also has evolved an abnormal two-step furin activation for membrane fusion [21] Besides membrane fusion, the clathrin-dependent and -indepen- dent endocytosis mediated SARS-CoV entry too [22,23]. After the virus enters the cells, the viral RNA genome is released into the cytoplasm and is translated into two polyproteins and structural proteins, after which the viral genome begins to replicate [5]. The newly formed envelope glycoproteins are inserted into the mem brane of the endoplasmic reticulum or Golgi. and the nucleocapsid is formed by the combination of genomic RNA and nucleocapsid protein. Then, viral particles germinate into the endoplasmic reticulum-Golgi intermediate compartment (ERIC). At last, the vesicles containing the virus particles then fuse with the plasma ‘membrane to release the virus [2 32. Antigen presentation in coronavirus infection While the virus enters the cells, its antigen will be presented 10, the antigen presentation cells(APC), which is a central part of the body's anti-viral immunity. Antigenic peptides are presented by ‘major histocompatibility complex (MHC; or human leukocyte an- tigen (HLA) in humans) and then recognized by virus-specific cytotoxic T lymphocytes (CTLs). Hence, the understanding of anti- gen presentation of SARS-CoV-2 will help our comprehension of COVID-19 pathogenesis. Unfortunately, there is still lack of any report about it, and we can only get some information from pre- vious researches on SARS-CoV and MERS-CoV. The antigen pre- sentation of SARS-CoV mainly depends on MHC 1 molecules [24], but MHC Il also contributes to its presentation. Previous research shows numerous HLA polymorphisms correlate to the susceptiil- ity of SARS-CoV, such as HLA-B°4601, HLA-B°0703, HLA-DR 'B1*1202 [25] and HLA-Cw*0801 [25], whereas the HLA-DRO3O1, HLA-Cw1502 and HLA-A*0201 alleles are related to the protec” tion from SARS infection [27] In MERS-CoV infection, MHC 1 ‘molecules, such as HLA-DRBI“I1:01 and HLA-DQBI*02:0, are associated with the susceptibility to MERS-CoV infection [28]. Be- Sides, gene polymorphisms of MBL (mannose-binding. lectin) associated with antigen presentation are related tothe risk of SARS- CoV infection {25}. These researches will provide valuable clues for the prevention, treatment, and mechanism of COVID-19. 33, Humoral and cellular immunity Antigen presentation subsequently stimulates the body's hu- ‘moral and cellular immunity, which are mediated by virus-specific Band Tells Similar to common acute vial infections, the antibody profile against SARS-CoV virus has a typical pattern of IgM and IgG production. The SARS-specific IgM antibodies disappear at the end of week 12, while the IgG antibody can last for along time, which indicates IgG antibody may mainly play a protective role [20]. and the SARS-specific IgG antibodies primarily are S-specific and N- specific antibodies 2]. Comparing to humoral responses, there are ‘more researches on the cellular immunity of coronavirus. The latest report shows the number of CD4" and CDS" Tells in the periph- eral blood of SARS-CoV-2-infected patients significantly is reduced, whereas its status is excessive activation, a5 evidenced by high proportions of HLA-DR (CD4 3.472) and CD38 (CD8 39.4%) double- positive fractions [31], Similarly, the acute phase response in pa- tients with SARS-CoV is associated with severe decrease of CD4! T and CD8* T cells. Even if there is no antigen, CD4” and CD8" ‘memory T cells can persist for four years in a part of SARS-CoV recovered individuals and can perform T cell proliferation, DTH response and production of 1FN=y [32]. Six years after SARS-CoV infection, specific T-cell memory responses to the SARS-CoV S Peptide library could still be identified in 14 of 23 recovered SARS patients [23]-The specific CDB' Tells also show a similar effect on -MERS-CoV clearance in mice [34]. These findings may provide valuable information for the rational design of vaccines against SARS-CoV-2. 34. Cytokine storm in COVID-19 ‘The report in Lancer shows ARDS is the main death cause of CCOVID-19, OF the 41 SARS-CoV-2-infected patients admitted in the early stages of the outbreak, six died from ARDS [15]. ARDS is the common immunopathological event for SARS-COV-2, SARS-CoV and MERS-CoV infections [31], One of the main mechanisms forX tec Jamal of Pharmaceutical nats 102020) 102-108 105 [ARDS is the cytokine storm, the deadly uncontrolled systemic in- flammatory response resulting from the release of large amounts of pro-inflammatory cytokines (IPN-2 IPN-y, 1-1, IL-6, I-12, 1L-18, 1L-33, TNF-a, TGF), etc) and chemokines (CC12, CCL3, CCLS, (CKCLB, CXCLS, CACLIO, etc.) by immune effector cells in SARS-COV infection [1535-37] Similar to those with SARS-CoV, individuals ‘ith severe MERS-CoV infection show elevated! levels of IL-6, FN-1, and CLS, CXCLB, CXCL-10 in serum compared to those with the ‘mild-moderate disease [38]. The cytokine storm will tigger a vi lent attack by the immune system to the body. cause ARDS and ‘multiple organ failure, and finally lead to death in severe cases of SSARS-CoV-2 infection just like what occurs in SARS-CoV and MERS- CoV infection [31] 3.5, Coronavirus immune evasion ‘To better survive in host cells, SARS-CoV and MERS-CoV use ‘multiple strategies to avoid immune responses. The evolutionarily, conserved microbial structures called pathogen-associated molec- ular patterns (PAMPs) can be recognized by pattern recognition receptors (PRRs). However, SARS-CoV and MERS-CoV can induce the production of double-membrane vesicles that lack PRRs and then replicate in these vesicles, thereby avoiding the host detection of their dsRNA [39]. IFN-I(IFN-2 and IFN-f) has a protective effect fon SARS-CoV and MERS-CoV infection, but the 17N-1 pathway is inhibited in infected mice [40,41]. Accessory protein 4a of MERS- CoV may block the induction of IFN at the level of MDAS activa tion through direct interaction with double-stranded RNA [22]. Besides, ORF4a, ORF4b, ORFS, ancl membrane proteins of MERS- CoV inhibit nuclear transport of IFN regulatory factor 3 (IRF) and activation of IFN promoter [43]. The antigen presentation can also be affected by the coronavirus. For example, gene expression related to antigen presentation is down-regulated after MERS-CoV infection [4]. Therefore, destroying the immune evasion of SARS- CoV-2 isimperative in its treatment and specific drug development. 4. Diagnosis of COVID-19 Clinical diagnosis of COVID-19 is mainly based on epidemt logical history, clinical manifestations and some auxiliary exami- nations, such as nucleic acid detection, CT” scan, immune ‘identification technology (Point-of-care Testing (POCT) of lgMjlg, enzyme-linked immunosorbent assay (ELISA)) and blood culture. However. the clinical symptoms and signs of patients infected with SARS.CoV-2 are highly atypical, including respiratory symptoms, ‘ough, fever. dyspnea, and viral pneumonia. Therefore, auxiliary ‘examinations are necessary for the diagnosis of COVID-19, ust as the epidemiological history. 4. Nucleic acd detection technology ‘The two commonly used nucleic acid detection technologies for ‘SARS-COV-2 are real-time quantitative polymerase chain reaction (RI-gPCR) and high-throughput sequencing. The authoritative ‘identification method for SARS-CoV-2 is virus blood culture and high-throughput sequencing of the whole genome [1]. However, the application of high-throughput sequencing technology in clinical diagnosis is limited because of its equipment dependency and high cost. So KT-gPCR is the most common, effective and straightforward method for detecting pathogenic viruses in res ratory secretions and blood [45] ‘After the outbreak of SARS-CoV-2 in China, many companies, soon launched RT-qPCR test kits for clinical diagnosis. The Chinese Center for Disease Control and Prevention (China CDC) recom- ‘mend the use of specific primers and probes in the ORFIab and N ‘gone regions for SARS-CoV-2 detection by RI-qPCR. "The patient is lefined as having a laboratory-confirmed infection when both targets are positive _(ttp://ivdechinaed-njkyj2/202001) 120200121_211337html). Chu et a. [46] described two t-step RT- QPCR assays (TaqMan-based fluorescence signal) to detect two ifferent regions (ORF and N)of the vital genome separately. The negative control samples were all confirmed as negative ones, while samples from two SARS-CoV-2 infected patients were confirmed as positive ones in respiratory specimens by this :method. Another study showed that the positive rate of SARS-CoV- 2 was 91.7% (11/12) in the patients’ self-collected saliva by using RT- {QPCR (non-probes SYBR based fluorescence signal}, which suggests that saliva is a promising non-invasive specimen for the diagnosis, ‘monitoring, and infection control of patients with SARS-CoV-2 infection [47]. RI-gPCR detection also showed high sensitivity and specificity for SARS-CoV and MERS-CoV infection [8]. How- ‘ever five patients with negative results of RT-qPCR for SARS-COV=2 ‘may present with positive chest CT findings, and repeated swab tests (RI-gPCR) eventually confirmed that all patients were infec- ted by SARS-CoV-2 [49]. The detection of SARS-CoV using RT-qPCR can only achieve a sensitivity of 50%-79%, depending on the pro- tocol used the sample type and number of clinical specimens collected [50]. Thus, itis essential to improve the detection rate of RI-qPCR for SARS-CoV-2 infection. Hesides, KI-qPCR has some cther shortcomings, including certain biological safety hazards brought by the retention and operation of patient samples, cumbersome nucleic acid detection operations, and long waiting time for results. 42. C¥scans and other diagnostic methods For the diagnosis of COVID-19, although RT-qPCR is specif, ts false-negative rate cannot be ignored because of the severe con- sequences of missed diagnosis. So many clinicians proposed CT scans should be one necessary auniliary diagnostic method because it is more sensitive. For individuals with a high clinical suspicion of SARS-CoV-2 infection with negative RT-PCR screening, a combi: nation of repeated RT-qPCR tests and chest CT scan may be helpful Especially the high-resolution CT (HRCT) for the chest is essential {or early diagnosis and evaluation of disease severity of patients ‘with SARS-CoV-2 [51]. Several studies have analyzed chest ages of patients infected with SARS-CoV-2 [52.53]. The typical CT images show bilateral pulmonary parenchymal ground-glass and consolidative pulmonary opacities. sometimes with a rounded ‘morphology and a peripheral lung distribution. Lung involvement with a peripheral predominance was also seen in patients with SARS-CoV and MERS-CoV infections, and the chest CTshowed that disease progressed with ground-glass opacities and consolidation ‘which is similar to that of SARS-CoV-2 infection [54,55] According, to those findings, CT scans have a great clinical diagnostic value for CCOVID-19. especially in the high prevalence area of SARS-COV-2 infection. However, CT scans also have some shortcomings, such as indistinguishability from other viral pneumonia and the hys- teresis of abnormal CT imaging Given the shortcomings of the currently used nucleic acid detection and CT scans for the diagnosis of COVID-19, clinical lab- oratories should apply some immunological detection kits that target viral antigens or antibodies as soon as possible. Currently, POCT of lgM/IgG and ELISA kits for SARS-CoV-2 have been devel- ‘oped and pre-tested by some companies and have shown higher detection rates than nucleic acid detection, but there is still no product or published article. The sensitivity of SARS-CoV N-based JgG ELISA (94.7%) is significantly higher than that of SARS-CoV S- based IgG ELISA (58.9%) [48], but the sensitivity of SARS-CoV-2 IgG IgM remains to be studied. Hence, developing other sensitive andws XU ea ourna of Pharmacia nay 10 (2020) 102-108 specific auxiliary methods is necessary and urgent for the diagnosis of COVID-19. 5. Current treatment strategies for COVID-19 Just like SARS-CoV and MERS-CoV [2,56], there is currently no Clinically proven specific antiviral agent available for SARS-CoV-2 infection. The supportive treatment, including oxygen therapy. conservation fluid management, and the use of broad-spectrum antibiotics to cover secondary bacterial infection, remains to be the most important management strategy [15]. According to the research on molecular mechanisms of coronavirus infection [57] and the genomic organization of SARS-CoV-2 [6], there are several potential therapeutic targets to reputpose the existing. antiviral agents or develop effective interventions against this novel SL Viral targeted inhibitors Remdesivir, an adenosine analogue that can target the RNA- dependent RNA polymerase and block viral RNA synthesis, has been a promising antiviral drug against a wide array of RNA viruses (including SARS/MERS-CoV) infections in cultured cells [38], mice [59] and nonhuman primate models [60,61]. The Washington Department of Health administrated remdesivir intravenously first and found that remdesivir might have potential protection from SARS-CoV-2 infection [52]. Then remdesivir and chloroquine have been demonstrated to inhibit SARS-CoV-2 effectively in vitro [63] Hence, other nucleoside analogues, such as favipiravr, ribavirin and galidesivir [56,54], may be potentially clinically applicable against SARS-COV-2. Chymotrypsin-like (3C-like protease, 3CLpro) and papain-like protease (PLP) are non-structural proteins, which have an essential function for coronaviral replication and can inhibit the hhost innate immune responses [65]. So 3CLpro inhibitors, such as cinanserin [65] and flavonoids [67], and PLP inhibitors, such as siarylheptanoids [68], are other attractive choices to fight against SARS-CoV-2. ACE2 mediates SARS-CoV-2 entry into the cell as a functional receptor of coronaviruses. So blocking the binding of S protein with ACE2 is also a meaningful strategy against SARS-CoV-2 infection [10} 52. Antibody and plasma therapy It has also been reported that there are many convalescent pa- ‘dents donating plasma against SARS-CoV-2, just as SARS-CoV [63] and MERS-CoV [70] trials. It has preliminary acquired favorable results in acute, severe SARS-CoV-2 patients. Moreover, the gen eration of recombinant human monoclonal antibody (mAb) is a fairly straightforward path to neutralize SARS-CoV. CR3022, a SARS coronavirus-specific human monocional antibody, can bind potently with the receptor-binding domain(RBD) of SARS-CoV-2 and has the potential to be developed as candidate therapeutics (of SARS-CoV-2 infections [71] Other monoclonal antibodies SARS-CoV, such a5 m396, CR3OM, could be an alter- native for the treatment of SARS-CoV-2 [72]. 53, Vaccines Effective SARS-CoV-2 vaccines are essential for reducing disease severity, viral shedding and transmission, thus helping to control the coronavirus outbreaks. There are several vaccination strategies Against SARS-CoV, MERS-CoV tested in animals, including live- attenuated virus, viral vectors, inactivated virus, subunit vaccines, recombinant DNA, and proteins vaccines [73]. These studies are in progress, but it requires months to years to develop the vaccines for SARS-CoV-2. Currently, there may be many promising targets for SARS-CoV-2,, but more laboratory and clinical evidence still should be explored! ‘The WHO is working with Chinese scientists to launch more than 80 clinical trials on potential treatments for SARS-CoV-2. Tradli- tional Chinese medicine seems to have some effects in the sup- portive treatments, Some new pharmaceutical drugs, including HIV rugs and stem cells, were testified in those clinical trials. 6. Conclusion In conclusion, the occurrence and development of SARS-CoV-2, depend on the interaction between the virus and the individual's immune system, Viral factors include virus type, mutation, viral load, viral titer, and viability of the virus in vitro The individaa's immune system factors include genetics (such as HLA genes). age. ‘gender, nutritional status, neurocndocrine-immune regulation, and physical status. These factors all contribute to whether an indi- vidual is infected with the virus, the duration and severity of the disease, and the reinfection, In the eatly stages of the epidemic, accurate diagnosis helps control the spread of the disease. It is imperative to develop new, safe, accurate, fast and simple new technologies for detecting SARS-CoV-2. Of course, physicians will intentionally intervene in the two factors to make them develop {nto a direction beneficial to human health, which can help patients recover as soon as possible. However, it must not be considered that ‘medical intervention can achieve a 100% curative effect. Conflicts of interest ‘The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (project No.81970029) and Fundamental Research Funds for the Central Universities of China (The Emer- gency Projects on COVID-19. project No.xzy032020042). We Sincerely thank the individuals who contributed to this work including Xudong Yang, Wenhua Zhu, Jing Xu, Fumeng, Huang. ‘Muhammad Saadig Khan, iaiun Ren, and Xipeng Wang. Appendix A. Supplementary data Supplementary data to this article can be found online at Intps:/ideiorg/10:1016)}jpha2020.03.001 References [1] P.2ou, XL Yang. XG. Wang etal A pacumonia outbreak associated with 2 Se,coan of pte ben Nate (0D, a [2] Ede Wit N. van Doremale,D Falarao, eta. SARS and MER: recent in. Figs int emer coromavranes Nat Rew Mra M4 (2016) 525534 tps fdorg/1010s8hnrmao201681. [5] JT Wa, K Leung GM. Leung Noweasting and ocean the potent do ‘esti an Internationa spread of te Z019-NC0V outbreak ginny ‘Wuhan. China: a modeling sty. Lance (2020), ipso orl 101016) Sotao'ersacs00608 [4] 5.5 G Wong W. Shi et al. Epidemiology, Genetic recombination, and pulhogenesi af cononavine Teens Mical 2 (2016) 490-302, ie ftoiorgft010t6p.m20\e0so0. 5 Periman,. Nelnd Coronavirues post SARS: update om replication puthogensiy NSt Rev. Nica 7 (2009) 439450, pfs OTS) fms. [6] WL, 2hao, 5 Lt a Genomic charateriatin and epidemiology of 2019 ‘ove coronavus! plication for vines ong and recapor bind Lance (2000) htpsfdoion10.1016/30140 673620}30051 8. 171 AB Fen, Perma, Coronaniuser an overview of thse replication ad Bathogenesi Methods Ma Bol 1282 (2015) 1-25 epee) 10 1007, 0X tec Jamal of Pharmaceutical nats 102020) 102-108 1 7-1-4939 2028.74, [8] PS. Alastes, Te molecular biology of coronaviruses, Adv Vis Res 65 (2006) 15-202, ts ove 1016)S0065 152705 )85009 [9] K Knoop. Mike. SH.Worm. eal, SARS-coronavnas repaton ss Suporte by a reculovesicuar network of ried endopasne reteaTom, PL Bo. 6 (2008), ns do.g/10137 Journal pa lOo0225 «226. [HO] WA) More, N. Vase, 3 Angotnsn-convertingenayne 282 Fintona ecpter forthe SARS corona Nature 425 (2003) 450454 ‘ngpso\re 10.103natre0218. [11] 29. Ge Jango olton and characterization of bat SARS oni that ses the AED recep, Nature 503 (2013) 535-538, ips Atouorgr 0 10s8)nturet2 711 [02] Chinese SaRs molecular Epdeology Consortum, Chinese Molecular evo Tuton ofthe SARS coronavirus during the course of the SARS epidemic In Ching, Seenee 303" (2004) Hos Naa9, tps 101126) Scene 050002. 103] VS fa) H Mou SL Smits tal, Dipeptiy peptidase is a ncional re poe or te emerging” human earouavnar!MC, Natute 495.2013) 251-254 hnsfdtore)10.1038natre 2005. 14) A hark | Zhan, MKS of Rep variation and suscepiiy to Middle as respiratory synarome cornaviu infection, Vito 88 (2014) 1853-4961, tps foouog/10.1128)V.00181- 1 [a5] Cuan, ¥. Wane et a, Clnea! features of paint nected wish 2019 ‘vel coronavirus in Wuhan China, Lancet (2020), hntps:fdtore 10 1016), Solan‘sran 070183, [16] 15 Pins, ¥ Guan, KY. Yue, Severe acute respiratory syndrome Nat Med. 10 (2000 SBSH, tpfdtre 10.1038) 1 (07) Ew znao, Ya, al A new coronars assocated with human ss Dratory eas fn China Nature (2020, hts foione/ 0 1038/41580-020 [U8] SA. eters, SM. Tose Gili oss, ta, CD2OBL (SIGN 2 receptor for Tr (2000) 157481575, hip duong 11073/onas 040381210, [19] 6 Simons, 1D. Reeve, A} Rennekamp, eta, Characterisation of severe ute "rspiatorysypdromesbacsed. coonsvnis”(GAKSCOV) spike lfycoprotenemedtted wal entry. Proc. Natl Acad. ScL USA. 107 (2004), ‘240-4245, ps oo.og/10.1073)pnssos06446101 (ao) S"Balouzard, VC Chu, GR Whitaker, Acuvaton of ube SARS coronavirus ‘pike protein vs aque prteoyi esvaye at two ding its, Proc Na Read's USA 108 (2008) 5871-3876, hpsjfioore/10 1073 rs. 0808524106 (21) FAM, Gi Whitaker, os clear of Mula ast respratory syndrome foronavtus ale two-step, fun medated actvabon of the spike proceln, Poca Aad SSA abl) s1¢- Toa pst 107) [22] HL Wang Yang Ki, tl, SARS coronavis entry into hos cll hough 4 novel dathrn and caveolse independent endocyac pathway Cl Res 18 (2008) 290-204, tps cu. 1038(.2008 1, (25 1M, WaT Obs Naas tal, Tony of ACED: 2 pepkidase the ‘enirargtemsia system, SARS fa purine for amin set [Bansprters Pharmacal Ther. 128 (2010) 119-128, prior 10.1016), J pharmiers 201008005 (24) 1 LP. Wik F. Ga, etal, Novel immunodominant pepe presentation fitategy: a feted {HA-A-2402 etrced etenie Tymphioryte eptope Stabe by miracham hyarogen bonds rom severe atte respiratory s— rome eoronavras muceocapst protein. 1 Virol 84 (2010) 11849-11857, faggot org/ 101 2Vio 4610 [25] NC Keio, 5. yan, K- Kashiwase tal, ASxiton of human leukocyte amese popolaton, Hum Imunol. 70 (2009) 527-53, Nps chore {L1Ot6nnnn.208.05.06, (26) VAL Chen, S¥- Lang VP. sh, eal Epidemiological and genetic cores of Seseze sete respiratory syrdiome cavonevits infection the hota with {he aghestnonacomul nection a in Tatan in 202} Cl Micra, (one) 350-365, ips oouorg/10.1 128.2. 350-35 2008, [271 SF. Wang. KML Chen M Chen, etal. Homan-eukoyteentzen ass 1 Cw 1502 and das lf DRO senuypes are assocated wi estas Lo severe dete respiratory syndrome’ (SARS) Inetion, Viral Inne. 24 (2011) {21-426 fps fog) 101089201 LD (2s) At jer aay, oan tl: Aston of unas kako ee Infection, Ann. Thorac. Med, 11 (2016) 211-213, hlpsfdowore 10103) teins 756 [29] 207 WP Chong ¥. Zh a Functional poymoypisms of the C2 ae [MBL penes cumulatively inerease suscep fo severe acute respiratory ‘yore constr eso infect 71 (2015) 101108, is sn) {ot016)int2015a0.006 [BO] GL. chen, ARG. Profle of specie antibodies to the SARS-asocited oonavinus, gh Med. 249 (2005) 308-09, tpn /dtore 10.106) Neynan0307913450520, [51] 22%, Sh ¥ Wang, Patologia findings of COID-19asocated with fete fespiratory diss syrome, Lancet Resp. Med. (2020) ips ator roneys213-260020)30076X (52) ¥¥-Fan Zang, Lt eal, Caraceration of SARS cov-specilememary Tells fom revered incivduals years after infection, Arch Viol 1 (2000) 1003-1009, ps: doiarg/10.1007/:00705-009-0400 6 [93] Feng ¥ Quan 21, tal Lack of peripheral merenyB cel responses in ecowered patents wah severe sete rnprstory syndrome! a year alo {psy | imunol 186 (2011) 7204-7268, bps fan 104049) ‘hununotososeso, {94} Zao, KL, Woblfrd-Lenane tal; Rapid generation of « mouse model for hiviie Fos respiratory symrome, Proc Natl ead Sa, USA 111 (2048) 4570-4075, np doo) IOUS pan 1323279111 155] AE Willams, RC. Chambers, The mercunal nature of neutrophils: stil an eign ARDS? Arn hyata Lang Call Met Physi” 305 (2044) 1217-1230, hs: dovord.1122)jluneo0311 2013 [265] R Ghannappanavar. Perma, Pathogenic human coronavrs infections: ‘auses ad consequences of kine stm and unsunopathaag. Sra Immunopathol 39 (2017) 529-538, hep |ioiory/ (1007/0281 O17 [57] MJ Cameron LF Bermejo-Marti, A. Danese a, Human immanopath aves of severe acute respratory syndrome (SARS), Vins Res 133 (208) Toa, poor 10 10T a) ste 2007 2201 [B81 CK Min. 5. Cheon, NY. Ha eta. Comparative and kinsc analysis of viral trod spot of daca severity, So Rep. 6 (2016) 25989, psd] O10 sep25550, [50] Ef Sayer, ¥. van der Meo.) Zevenhoven-Dobbe ta, Ulrastucure and ‘gin of membrane vesces associated with the severe acute respiratory Sydrome coronavirus epics comple, Virol 80 (208) 5927 580, itp footont011vin250T-08 [ao] R Ghannappansvar, AR. Teh, K. Way, al. Dysegulated type 1 intsferon 4d inflammatory menocte-marophage response cause thal paeumonis in SARS-CoV infected ce Cel Hest Microbe 19 (2016) 181—183. ‘vor/ 0 10love 201001007. [1] RChannappanavar. Fer.) Zheng et al IFN response timing relative 10 ‘oes epltation deternines MES Groner fnctoe autores | Cn Ives 150 G10) S625 3538, foouore 101722865, [12] D Niemeyer, Zinger, D. Math, el, Male Eat repeater sym ronan scensory protein as fpe iterteron antago Vir 87 (2015) 12489-12495, nes oon) 12RD TA (as Yvan zhang. Heng etal, Te structural and accessory proteins ML ORF “4, OWF ab, and ORF 5 of Middle Eat respzatoty synurume coronavirus (AtERS-Cov} re poten interferon antagonist, Protea Cll 4 (2013) 951-9 tp: /souog to007/s13258 015 306.5 [4] V0. Menachery, Schafer, KE Burnum Jonson, tl. MERS-CoV and HSN lnuenza vis antagonize angen resotation by altering the epigenetic ndseape. Proc Nat. Aad Sau USA 115 (2018) EIOL2 £1024, ps sotorg) 0 1073}pnae 700038115. [a5] VAI Corman 0 Lan ML Kater, tl, Detection of 2019 novel eoronvis (2o19-ncov) by realtime RT-PCR. Euro Surv 25 (2020), ipso.) ‘7/369. 7o1 75202025 3200004 DAW. cha, ¥- Pan. SAIS. Chen. eal Molecular diagnosis of a novel coro ‘vu (2019.90) coming a outcak of prow, Ch Cher (2020) ‘tp fdourg/101095fchocher haa. KK To, OF. Tsang. Chik-Van Vip. et al. Consistent derecion of 2019 novel oronavrus in salva, Cn nee Ds. (2020) psf. 1005 oe. 8] PC Woo, SK tay Wong al, Dire semstiies of severe ate respiratory syndrome (SAS cornsvis spike poypeptie enzymetnked Immunosorbent asay (ELSA) and SARS coronas ucleocaptd. poten ISA fr seodagnoss of SARS cronawus pocumon Cl MDL 3 (2005) 054-3058 ps donor 10.1 125M 43.7 20541058.2005, 9] SeXee, 2 Zhong, W- zhao, tal, Chet CT for peal 2019-nCaV pocumoni: Felatonship to negative RT-PCR testing. Ratiolgy (2020, 200383, (dior 01 agree 2nan2 00343 (50) W.c Yam, KHL Chan, LL Poo, eal Evaluation of reverse uansrption PCR {says for rapid Gagnoisof svere sete respiratory syndrome sso ‘rath novel coronaire. Cin Mri (2003) 4521-4524 ps) ioeg/ io 2sjm an 1021 5242005, 1511 ¥- Pan H. Gam S. Zhou, et al ntl CT findings and vemporal changes in Dtents withthe novel cavonavius poeumani (2010-ncaV a study of 83 patients in Waa, China, Eu Rac (2020 ps0 1007/0053 ts) Hot eg ol fcr esas apg Radiology (2020), 200269, tps: doiorg/10.1148)/ratiol 2020200265. [53] MoChung, A Berner, May ea, CT maging features of 2019 novel coronas (2010-2) Radhlgy (2520), 200730, ure 118) ‘adi 2020200220. [54] GC Oo4 PL Hon, NL Maller, et al, Severe acute respiratory syome: ‘mpora lung changes al thinsecton CT 30 patients: Radley 2902008) 336" eps [dourg 10138) 2303050855 [5] AMC alah RA Aad Lc Jamaal, Mile Et espatry syne oronavius (MERS-CoV) infection: chest CT findings. JR Ar. Roenteene Sos 2014) 782—77, hap: dotong 221 S/ATRI 30D 1X 2umla Jf Chan EL Achar, etal. Cornaviiss ~ drug discovery and ‘erapeuie opaons Nat ev Drug Deco. 15 (2016) 327-347, Nps sdvorg/ 10 1038/2015 37 [57] DA Groneberg Rigen P Zabel, Molecular mechanisms severe acute a6) (56)ws X tea Journal of harmaceten Anas 10 (2020) 02-108 respiratory syndrome (SARS), Resi. Res. 6 (2005) 8 tp: diorg/10.1196) Nason ok 158) RAL, Jordan, A Arve, a, 655754 ad ts parent naceside slog Init le, Paeuo and Paramore, Re. 7 (2017) 43395, 55) Ghion/i0 0se/sepa335% |s9) TP. Sheahan, AC Sins SI Leis et al, Comparative therapeutic cacy of rene sd cmbnation lps, ona, astern ela sat IER Ca¥, Nat Comeman 11"(2020) 22, Pp dong) 10 10381467 O19. 13940-6 [oo] Ede Wt Feldman, Cronin, a, Prophylactic and therapeutic ee ‘ua (633724) eauneat inthe Yesus hacague Dede! of MERS-COV ice fon, Proc. Natl "Acad Sc. USA. (2020) hips door 101075), ppoasaszz0ss117 [6 MK Uo. F Feldmann, IM. Gary. al Remdesivir (655734) protects Afcan sreen moneys fr Nip viru challnge S65 Teams, Med 1 (2019, fraps fdtong 0.26 )scaranstned al, [e2} REL Hote Detor 5. unaguset a Frst case of 2019 nove coronvins tn ie Unite States. NEagl Med. (2020) tp jduory 101056) [NeMos2001191 Jes KEWang Ca, Zhang al, Remdesiie and cloogeine ectvely inhi the recenly emerged novel ovonevits (2079000) vito Cel Res (Got) eps ar org 101038o41422 02002820 Isa) Ee Carey, New muccosde antogues forthe Weatment of hemorrhage Fever vrs infections. Chem. Asan 14 (2018) 3862-3908, eso taYanz/sia20 90084 165} ¥: Chen Li, D. Guo, Emerging coronaviruses: ge Yon, and pathogenesin J. Med. Veo 92 (2020) 41 atooz)me2568, [oo] L hes. © Ga. X too, et al, Cinanserin fan inti oF the 3C-ke proteinase of severe acute respiratory syndrome coronavirus and strongly Feces vies repbcaion in vitro. J iol 79 (2005) 7035-7103, hips | org aaa 11 7095-7102005, [62] So. 8. kim DH. Shine al, nhiton of SARS-CoV 3 protease by avo. ros Ey. nb Med, cere 35 (2020) 145-154, hus ors] 1 tosoj47s63562018.1650480. | 1% Pare Heong J. Kim, etal, Diarylhetanois from Alms japonce ‘Ghint papesrate rote of severe ante epatary yadnnae roy "us Bol: Pharm Bull 35 (2012) 2036-2082. por 101 28/09BB12- [69] | Mai-Jeakins M,Saavedra-Campos, JX Bale, et al, The eilectiveness of “aman pasa ae hypermmase mmanoin for heen oF Severe acute Fespratory Incons of wl ecology: 3 systematic review and ‘exploratory met-arass, Ife. Dis 211 (2015) 80-80, host iGiosnymtntasoe. KL Koenig lentiy-soletetform: a medi too for inal detection and management of Mile East Respratory Syndrome patents nthe emergency ‘Goartment, wesc} Emerg. Meds T6013) 619-624 hast) {ase iesiem 2018727915, [71 20a C1 Huan ta, Potent bing of 2019 nove eoronavius se protein by 2 SARS coonavirs-speciic human montonal anid. Emerg Nerd.” nee (2020) 382385 hepsi 0 1080) 172] L Zhang, ¥. Lu, Poel interventions for nove coronavirus in China: 2 Systematic review} Ale. Vir (220) ht svar) 2m (73) RL Graham EF. Donaldson, RS: Bac A decade after SARS: stele for ‘ontaing emerging coronsarses, Nat Rev. Mira 1 (2013) 836-848, fttpe/ tar 18 esas. 179