bat viruses ZXC21 and ZC45--main trail pursued by Dr. Yan Li-Meng

9-12-18  by Dan Hu et alia Department of Epidemiology, College of Preventive Medicine, Third Military Medical University, Chongqing; Department of Epidemiology, Research Institute for Medicine of Nanjing Command, Nanjing

  To further understand the evolutionary relationship between SARS-CoV and its reservoirs, 334 bats were collected from Zhoushan city, Zhejiang province, China (on the coast just above Taiwan) between 2015 and 2017.  PCR amplification of the conserved coronaviral protein RdRp detected coronaviruses in 26.65% of bats belonging to this region, and this number was influenced by seasonal changes. Full genomic analyses of the two new SL-CoVs from Zhoushan (ZXC21 and ZC45) showed that their genomes were 29,732 nucleotides (nt) and 29,802 nt in length, respectively, with 13 open reading frames (ORFs).  These results revealed 81% shared nucleotide identity with human/civet SARS CoVs   https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135831/

………………………................................................……

1-28-20 by Jasper Fuk-Woo Chan of U. of Hong Kong, et alia

  Spike glycoprotein comprised of S1 and S2 subunits. ...We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs (SL-CoV ZXC21 and ZC45) and human SARS-CoV (Figure 2).  https://www.tandfonline.com/doi/full/10.1080/22221751.2020.1719902

……………………..................................................…

6-8-20 by Hong Zhou of Universities of Shandong et alia

Table 1. Sequence Identity for SARS-CoV-2 Compared with RmYN02 and Representative Beta-CoV Genomes 

https://www.cell.com/current-biology/pdf/S0960-9822(20)30662-X.pdf

………………………………………................................................................…........................................................................... 
   9) A comparison between the amino acid sequences of the Wuhan SARS-CoV-2 virus as originally described and the ZC45 and ZXC21 viruses shows a remarkable identity in all but one crucial region.  In the majority of the virus there is 95% amino acid sequence identity, but there is one crucial region where they are strikingly dissimilar, with only 69% identity.  That is the S1 region of the spike protein that harbours the RBD.  Given the very high identity in all other regions of the SARS-CoV-2 virus when compared with ZC45 and ZXC21, it is highly improbable that such a huge difference in just the S1 part of the spike protein of SARS-CoV-2 could have arisen naturally over the timespan in which they are supposed to have co-existed in nature.

  10) The other striking result of a comparison between SARS-CoV-2 and ZC45/ZXC21 relates to another component, the E protein.  The E protein is a structural protein of coronaviruses that can tolerate a large number of mutations without any negative impact on function.  This is highlighted by the fact that even after just two months after the outbreak of the COVID-19 pandemic, mutations in the E protein of SARS-CoV-2 were identified.  However, when comparing the original SARS-CoV-2 virus with the ZC45/ZXC21 bat viruses, they have a 100% identical E protein amino acid sequence.  Given the high mutation rate observed in SARS-CoV-2 (and in coronaviruses in general), and given the fact that mutations can occur anywhere in the virus genome, including in the E protein region, it makes no biological sense that the original SARS-CoV-2 virus would have a 100% identical E protein amino acid sequence to the ZC45/ZXC21 bat viruses.

  11) Both the above basic biological observations strongly indicate that the only way that SAS-CoV-2 can be so dissimilar in the S1 region of the spike protein (crucial to human infectivity), yet identical in a far less crucial component such as the E protein, is through intentional design (genetic manipulation in the lab) and not by natural mutation and selection in animal and human hosts.

  12) The above information strongly points to SARS-CoV-2 being constructed based on one or both of the two bat viruses, ZC45 and ZXC21, rather than the purported RaTG13.     https://www.gmwatch.org/en/news/latest-news/19396-evidence-that-the-sars-cov-2-virus-is-genetically-engineered

………………………..........................…...................

  One thing we haven’t mentioned so far is that ZC45 and ZXC21 are bat coronaviruses discovered, collected, and published by a military research lab of the Chinese Communist Party (CCP) (6)....
  The E protein of β coronaviruses is a structural protein that is tolerant of mutations as evidenced both in SARS and in bat coronaviruses.  However, on the amino acid level, E protein of the Wuhan coronavirus identified at the beginning of the outbreak is 100% identical to those of the suspected templates, ZC45 and ZXC21 (Figure 4).  What is striking is that after a short two-months spread of the virus in humans, the E protein is already mutating.  Sequence data obtained within the month of April indicate that mutations have occurred to four different locations (Figure 4).  Note that the E protein makes very limited interactions with host proteins and thus is not under evolutionary pressure to adapt to a new host.  Not only the E protein can tolerate mutations but also its mutational rate is held constant across different coronavirus species.  The fact that the E protein of the Wuhan coronaviruses is already mutating in the short period of human-to-human transmission is consistent with its evolutionary feature.  In stark contrast, while ZC45/ZXC21 and the Wuhan coronavirus are more distant evolutionarily, the E proteins within them are 100% identical.  In no way this could be a result of natural evolution....
  As shown in Figure 5, mutations and insertion/deletions in E proteins have been observed at multiple locations both in SARS coronaviruses and in bat coronaviruses.  This clearly indicates E protein’s tendency and permissiveness toward mutations across β coronavirus species.  What is inconsistent with this trait is the fact that ZC45/ZXC21 and the Wuhan coronavirus, while significantly distant from each other in evolution, share 100% identity in E proteins.  Again, in no way this could be a result of natural evolution.  This further supports the claim that the Wuhan coronavirus is made in a lab by following ZC45/ZXC21 as a template....

• 

  Figure 5. Sequence alignment of E proteins from Wuhan coronavirus (Wuhan-Hu-1), SARS coronaviruses (SARS_GD01, SARS_ExoN1, SARS_TW_GD1, SARS_Sino1_11), and bat coronaviruses (Bat_AP040581.1, RsSHC014, SC2018, Bat_NP_828854.1, BtRs-BetaCoV/HuB2013, BM48-31/BGR/2008). The ready-for-analysis sequences were kindly prepared by Viennah K. Erchus.      https://nerdhaspower.weebly.com/ratg13-is-fake.html
  ...................................................................................

    Unusual Features of the SARS-CoV-2 Genome Suggesting Sophisticated Laboratory Modification Rather Than Natural Evolution and Delineation of Its Probable Synthetic Route 

  Li-Meng Yan (MD, PhD)1, Shu Kang (PhD)1, Jie Guan (PhD)1, Shanchang Hu (PhD)1 1Rule of Law Society & Rule of Law Foundation, New York, NY, USA. Correspondence: team.lmyan@gmail.com   (published also at zerohedge.com on 9-14 and at youtube.com)

    Origin of SARS-CoV-2 has remained mysterious and controversial.  The natural origin theory, although widely accepted, lacks substantial support.  The alternative theory that the virus may have come from a research laboratory is, however, strictly censored on peer-reviewed scientific journals.  Nonetheless, SARS-CoV-2 shows biological characteristics that are inconsistent with a naturally occurring, zoonotic virus. …the laboratory-creation of this coronavirus is convenient and can be accomplished in approximately six months….Sars-Cov-2 /is highly efficient in binding the human ACE2 receptor (hACE2), the affinity of which is greater than that associated with the ACE2 of any other potential host2,3…

    As we have described above, if natural recombination event is responsible for the appearance of SARS- CoV-2, then the ZC45/ZXC21-like virus and a coronavirus containing a SARS-like RBM would have to recombine in the same cell by swapping the S1/RBM, which is a rare form of recombination. Furthermore, since SARS has occurred only once in human history, it would be at least equally rare for nature to produce a virus that resembles SARS in such an intelligent manner – having an RBM that differs from the SARS RBM only at a few non-essential sites (Figure 4). The possibility that this unique SARS-like coronavirus would reside in the same cell with the ZC45/ZXC21-like ancestor virus and the two viruses would recombine in the “RBM-swapping” fashion is extremely low. Importantly, this, and the other recombination event described below in section 1.3 (even more impossible to occur in nature), would both have to happen to produce a Spike as seen in SARS-CoV-2. …

    abundant literature shows that gain-of-function research, where the Spike protein of a coronavirus was specifically engineered, has repeatedly led to the successful generation of human- infecting coronaviruses from coronaviruses of non-human origin44-47 …Evidently, the technical barrier is non-existent for the WIV and other related laboratories to carry out and succeed in such Spike/RBM engineering and gain-of- function research. …

    Strikingly consistent with the RBM engineering theory, we have identified two unique restriction sites, EcoRI and BstEII, at either end of the RBM of the SARS-CoV-2 genome, respectively (Figure 5A). These two sites, which are popular choices of everyday molecular cloning, do not exist in the rest of this spike gene. This particular setting makes it extremely convenient to swap the RBM within spike, providing a quick way to test different RBMs and the corresponding Spike proteins. 

    Such EcoRI and BstEII sites do not exist in the spike genes of other β coronaviruses, which strongly indicates that they were unnatural and were specifically introduced into this spike gene of SARS-CoV-2 for the convenience of manipulating the critical RBM. Although ZC45 spike also does not have these two sites (Figure 5B), they can be introduced very easily as described in part 2 of this report. …

    In 2008 Dr. Zhengli Shi’s group swapped a SARS RBM into the Spike proteins of several SARS-like bat coronaviruses after introducing a restriction site into a codon-optimized spike gene (Figure 5C)47.  They then validated the binding of the resulted chimeric Spike proteins with hACE2.  Furthermore, in a recent publication, the RBM of SARS-CoV-2 was swapped into the receptor-binding domain (RBD) of SARS- CoV, resulting in a chimeric RBD fully functional in binding hACE2 (Figure 5C)39.  Strikingly, in both cases the manipulated RBM segments resemble almost exactly the RBM defined by the positions of the EcoRI and BstEII sites (Figure 5C).  Although cloning details are lacking in both publications39,47, it is conceivable that the actual restriction sites may vary depending on the spike gene receiving the RBM insertion as well as the convenience in introducing unique restriction site(s) in regions of interest. 

    It is noteworthy that the corresponding author of this recent publication39, Dr. Fang Li, has been an active collaborator of Dr. Zhengli Shi since 201049-53.  Dr. Li was the first person in the world to have structurally elucidated the binding between SARS-CoV RBD and hACE238 and has been the leading expert in the structural understanding of Spike-ACE2 interactions38,39,53-56.  The striking finding of EcoRI and BstEII restriction sites at either end of the SARS-CoV-2 RBM, respectively, and the fact that the same RBM region has been swapped both by Dr. Shi and by her long-term collaborator, respectively, using restriction enzyme digestion methods are unlikely a coincidence.  Rather, it is the smoking gun proving that the RBM/Spike of SARS-CoV-2 is a product of genetic manipulation. 

    Although it may be convenient to copy the exact sequence of SARS RBM, it would be too clear a sign of artificial design and manipulation. The more deceiving approach would be to change a few non- essential residues, while preserving the ones critical for binding. This design could be well-guided by the high-resolution structures (Figure 3)37,38. This way when the overall sequence of the RBM would appear to be more distinct from that of the SARS RBM, the hACE2-binding ability would be well-preserved. We believe that all of the crucial residues (residues labeled with red sticks in Figure 4, which are the same residues shown in sticks in Figure 3C) should have been “kept”.  As described earlier, while some should be direct preservation, some should have been switched to residues with similar properties, which would not disrupt hACE2-binding and may even strengthen the association further. Importantly, changes might have been made intentionally at non-essential sites, making it less like a “copy and paste” of the SARS RBM. …

    (If we) assume that this site in SARS-CoV-2 is a result of natural evolution, then only one evolutionary pathway is possible, which is that the furin-cleavage site has to be derived from a homologous recombination event. Specifically, an ancestor β coronavirus containing no furin-cleavage site would have to recombine with a closely related coronavirus that does contain a furin-cleavage site. 

    However, two facts disfavor this possibility.  First, although some coronaviruses from other groups or lineages do contain polybasic furin-cleavage sites, none of them contains the exact polybasic sequence present in SARS-CoV-2 (-PRRAR/SVA-). Second, between SARS-CoV-2 and any coronavirus containing a legitimate furin-cleavage site, the sequence identity on Spike is no more than 40%66.  Such a low level of sequence identity rules out the possibility of a successful homologous recombination ever occurring between the ancestors of these viruses. Therefore, the furin-cleavage site within the SARS-CoV-2 Spike protein is unlikely to be of natural origin and instead should be a result of laboratory modification. …

    To engineer and create a human-targeting coronavirus, they would have to pick a bat coronavirus as the template/backbone.  This can be conveniently done because many research labs have been actively collecting bat coronaviruses over the past two decades32,33,70,72,81-85. However, this template virus ideally should not be one from Dr. Zhengli Shi’s collections, considering that she is widely known to have been engaged in gain-of-function studies on coronaviruses.  Therefore ZC45 and/or ZXC21, novel bat coronaviruses discovered and owned by military laboratories33, would be suitable as the template/backbone.  It is also possible that these military laboratories had discovered other closely related viruses from the same location and kept some unpublished. …

  References: 

  1. Zhan, S.H., Deverman, B.E. & Chan, Y.A. SARS-CoV-2 is well adapted for humans. What does this mean for re-emergence? bioRxiv, https://doi.org/10.1101/2020.05.01.073262 (2020).
     
  2. Mou, H. et al. Mutations from bat ACE2 orthologs markedly enhance ACE2-Fc neutralization of SARS- CoV-2. bioRxiv, https://doi.org/10.1101/2020.06.29.178459 (2020).
     
  3. Piplani, S., Singh, P.K., Winkler, D.A. & Petrovsky, N. In silico comparison of spike protein-ACE2 binding affinities across species; significance for the possible origin of the SARS-CoV-2 virus. arXiv, arXiv:2005.06199 (2020).
     
  4. Andersen, K.G., Rambaut, A., Lipkin, W.I., Holmes, E.C. & Garry, R.F. The proximal origin of SARS- CoV-2. Nat Med 26, 450-452 (2020). 

  5. Maiti, A.K. On The Origin of SARS-CoV-2 Virus. Preprint (authorea.com), DOI: 10.22541/au.159355977.76503625 (2020).
     
  6. Lin, X. & Chen, S. Major Concerns on the Identification of Bat Coronavirus Strain RaTG13 and Quality of Related Nature Paper. Preprints, 2020060044 (2020).
     
  7. Bengston, D. All journal articles evaluating the origin or epidemiology of SARS-CoV-2 that utilize the RaTG13 bat strain genomics are potentially flawed and should be retracted. OSFPreprints, DOI: 10.31219/osf.io/wy89d (2020).
     
  8. Segreto, R. & Deigin, Y. Is considering a genetic-manipulation origin for SARS-CoV-2 a conspiracy theory that must be censored? Preprint (Researchgate) DOI: 10.13140/RG.2.2.31358.13129/1 (2020).
     
  9. Rahalkar, M.C. & Bahulikar, R.A. Understanding the Origin of ‘BatCoVRaTG13’, a Virus Closest to SARS-CoV-2. Preprints, 2020050322 (2020).
     
  10. Robinson, C. Was the COVID-19 virus genetically engineered? (https://gmwatch.org/en/news/latest- news/19383, 2020).
      
  11. Robinson, C. Another expert challenges assertions that SARS-CoV-2 was not genetically engineered. (https://gmwatch.org/en/news/latest-news/19383, 2020).
      
  12. Sørensen, B., Dalgleish, A. & Susrud, A. The Evidence which Suggests that This Is No Naturally Evolved Virus. Preprint, https://www.minervanett.no/files/2020/07/13/TheEvidenceNoNaturalEvol.pdf (2020).
      
  13. Zhang, B. SARS-CoV-2 Could Come from a Lab - A Critique of “The Proximal Origin of SARS-CoV-2” Published in Nature Medicine. (https://www.linkedin.com/pulse/sars-cov-2-could-come-from-lab- critique-proximal-origin-billy-zhang?articleId=6651628681431175168#comments- 6651628681431175168&trk=public_profile_article_view, 2020).
      
  14. Sirotkin, K. & Sirotkin, D. Might SARS‐CoV‐2 Have Arisen via Serial Passage through an Animal Host or Cell Culture? BioEssays, https://doi.org/10.1002/bies.202000091 (2020).
      
  15. Seyran, M. et al. Questions concerning the proximal origin of SARS-CoV-2. J Med Virol (2020).
      
  16. China Honors Ian Lipkin. (https://www.publichealth.columbia.edu/public-health-now/news/china-honors-ian-lipkin, 2020).
      
  17. Holmes, E. Academic CV .
      (https://www.sydney.edu.au/AcademicProfiles/profile/resource?urlid=edward.holmes&type=cv, 2020).
      
  18. Zhou, P. et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature (2020).
      
  19. Rahalkar, M. & Bahulikar, R. The Abnormal Nature of the Fecal Swab Sample used for NGS Analysis of  RaTG13 Genome Sequence Imposes a Question on the Correctness of the RaTG13 Sequence. Preprints.org, 2020080205 (2020).
      
  20. Singla, M., Ahmad, S., Gupta, C. & Sethi, T. De-novo Assembly of RaTG13 Genome Reveals
      Inconsistencies Further Obscuring SARS-CoV-2 Origins. Preprints, 2020080595 (doi:
      10.20944/preprints202008.0595.v1) (2020).
      
  21. Zhang, D. Anomalies in BatCoV/RaTG13 sequencing and provenance. Preprint (zenodo.org),
      https://zenodo.org/record/3987503#.Xz9GzC-z3GI (2020).
      
  22. Robinson, C. Journals censor lab origin theory for SARS-CoV-2.
      (https://www.gmwatch.org/en/news/latest-news/19475-journals-censor-lab-origin-theory-for-sars-cov-2, 2020).
      
  23. Scientific evidence and logic behind the claim that the Wuhan coronavirus is man-made.
      https://nerdhaspower.weebly.com (2020).
      
  24. Zhang, Y. et al. The ORF8 Protein of SARS-CoV-2 Mediates Immune Evasion through Potently Downregulating MHC-I. bioRxiv, https://doi.org/10.1101/2020.05.24.111823 (2020).
      
  25. Muth, D. et al. Attenuation of replication by a 29 nucleotide deletion in SARS-coronavirus acquired during the early stages of human-to-human transmission. Sci Rep 8, 15177 (2018).
      
  26. Schoeman, D. & Fielding, B.C. Coronavirus envelope protein: current knowledge. Virol J 16, 69 (2019).
      
  27. Lam, T.T. et al. Identifying SARS-CoV-2-related coronaviruses in Malayan pangolins. Nature (2020).
      
  28. Liu, P. et al. Are pangolins the intermediate host of the 2019 novel coronavirus (SARS-CoV-2)? PLoS Pathog 16, e1008421 (2020).
      
  29. Xiao, K. et al. Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins. Nature (2020).
  30. Zhou, H. et al. A Novel Bat Coronavirus Closely Related to SARS-CoV-2 Contains Natural Insertions at the S1/S2 Cleavage Site of the Spike Protein. Curr Biol 30, 2196-2203 e3 (2020). 

  30. Zhang, T., Wu, Q. & Zhang, Z. Probable Pangolin Origin of SARS-CoV-2 Associated with the COVID- 19 Outbreak. Curr Biol 30, 1578 (2020).
      
  31. Yang, X.L. et al. Isolation and Characterization of a Novel Bat Coronavirus Closely Related to the Direct Progenitor of Severe Acute Respiratory Syndrome Coronavirus. J Virol 90, 3253-6 (2015).
      
  32. Hu, D. et al. Genomic characterization and infectivity of a novel SARS-like coronavirus in Chinese bats. Emerg Microbes Infect 7, 154 (2018).
      
  33. Wang, Y. Preliminary investigation of viruses carried by bats on the southeast coastal area (东南沿海地 区蝙蝠携带病毒的初步调查研究). Master Thesis (2017).
      
  34. Wu, F. et al. A new coronavirus associated with human respiratory disease in China. Nature 579, 265-269 (2020).
      
  35. Lab That First Shared Novel Coronavirus Genome Still Shut Down by Chinese Government. Global Biodefense, https://globalbiodefense.com/headlines/chinese-lab-that-first-shared-novel-coronavirus- genome-shut-down/ (2020).
      
  36. Song, W., Gui, M., Wang, X. & Xiang, Y. Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2. PLoS Pathog 14, e1007236 (2018).
      
  37. Li, F., Li, W., Farzan, M. & Harrison, S.C. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 309, 1864-8 (2005).
      
  38. Shang, J. et al. Structural basis of receptor recognition by SARS-CoV-2. Nature (2020).
      
  39. Hassanin, A. The SARS-CoV-2-like virus found in captive pangolins from Guangdong should be better sequenced. bioRxiv, https://doi.org/10.1101/2020.05.07.077016 (2020).
      
  40. Zhang, D. The Pan-SL-CoV/GD sequences may be from contamination. Preprint (zenodo.org), DOI: 10.5281/zenodo.3885333 (2020).
      
  41. Chan, Y.A. & Zhan, S.H. Single source of pangolin CoVs with a near identical Spike RBD to SARS-CoV-2. bioRxiv, https://doi.org/10.1101/2020.07.07.184374 (2020).
      
  42. Lee, J. et al. No evidence of coronaviruses or other potentially zoonotic viruses in Sunda pangolins (Manis javanica) entering the wildlife trade via Malaysia. bioRxiv,
      https://doi.org/10.1101/2020.06.19.158717 (2020).
      
  43. Becker, M.M. et al. Synthetic recombinant bat SARS-like coronavirus is infectious in cultured cells and in mice. Proc Natl Acad Sci U S A 105, 19944-9 (2008).
      
  44. Menachery, V.D. et al. A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. Nat Med 21, 1508-13 (2015).
      
  45. Menachery, V.D. et al. SARS-like WIV1-CoV poised for human emergence. Proc Natl Acad Sci USA 113, 3048-53 (2016).
      
  46. Ren, W. et al. Difference in receptor usage between severe acute respiratory syndrome (SARS)
      coronavirus and SARS-like coronavirus of bat origin. J Virol 82, 1899-907 (2008).
      
  47. Li, X. et al. Emergence of SARS-CoV-2 through Recombination and Strong Purifying Selection. bioRxiv (2020).
      
  48. Hou, Y. et al. Angiotensin-converting enzyme 2 (ACE2) proteins of different bat species confer variable susceptibility to SARS-CoV entry. Arch Virol 155, 1563-9 (2010).
      
  49. Yang, Y. et al. Two Mutations Were Critical for Bat-to-Human Transmission of Middle East Respiratory Syndrome Coronavirus. J Virol 89, 9119-23 (2015).
      
  50. Luo, C.M. et al. Discovery of Novel Bat Coronaviruses in South China That Use the Same Receptor as Middle East Respiratory Syndrome Coronavirus. J Virol 92(2018).
      
  51. Cui, J., Li, F. & Shi, Z.L. Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol 17, 181-192 (2019).
      
  52. Wan, Y. et al. Molecular Mechanism for Antibody-Dependent Enhancement of Coronavirus Entry. J Virol 94(2020).
      
  53. Li, F. Receptor recognition mechanisms of coronaviruses: a decade of structural studies. J Virol 89, 1954-64 (2015).
      
  54. Li, F. Structure, Function, and Evolution of Coronavirus Spike Proteins. Annu Rev Virol 3, 237-261 

  56. Shang, J. et al. Cell entry mechanisms of SARS-CoV-2. Proc Natl Acad Sci U S A 117, 11727-11734 (2020).
      
  57. Hoffmann, M., Kleine-Weber, H. & Pohlmann, S. A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells. Mol Cell 78, 779-784 e5 (2020).
      
  58. Coutard, B. et al. The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade. Antiviral Res 176, 104742 (2020).
      
  59. Claas, E.C. et al. Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351, 472-7 (1998).
      
  60. Watanabe, R. et al. Entry from the cell surface of severe acute respiratory syndrome coronavirus with cleaved S protein as revealed by pseudotype virus bearing cleaved S protein. J Virol 82, 11985-91 (2008).
      
  61. Belouzard, S., Chu, V.C. & Whittaker, G.R. Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites. Proc Natl Acad Sci U S A 106, 5871-6 (2009).
      
  62. Kido, H. et al. Role of host cellular proteases in the pathogenesis of influenza and influenza-induced multiple organ failure. Biochim Biophys Acta 1824, 186-94 (2012).
      
  63. Sun, X., Tse, L.V., Ferguson, A.D. & Whittaker, G.R. Modifications to the hemagglutinin cleavage site control the virulence of a neurotropic H1N1 influenza virus. J Virol 84, 8683-90 (2010).
      
  64. Cheng, J. et al. The S2 Subunit of QX-type Infectious Bronchitis Coronavirus Spike Protein Is an Essential Determinant of Neurotropism. Viruses 11(2019).
      
  65. Ito, T. et al. Generation of a highly pathogenic avian influenza A virus from an avirulent field isolate by passaging in chickens. J Virol 75, 4439-43 (2001).
      
  66. Canrong Wu, Y.Y., Yang Liu, Peng Zhang, Yali Wang, Hua Li, Qiqi Wang, Yang Xu, Mingxue Li, Mengzhu Zheng, Lixia Chen. Furin, a potential therapeutic target for COVID-19. Preprint (chinaXiv), http://www.chinaxiv.org/abs/202002.00062 (2020).
      
  67. Zeng, L.P. et al. Bat Severe Acute Respiratory Syndrome-Like Coronavirus WIV1 Encodes an Extra Accessory Protein, ORFX, Involved in Modulation of the Host Immune Response. J Virol 90, 6573-6582 (2016).
      
  68. Lau, S.Y. et al. Attenuated SARS-CoV-2 variants with deletions at the S1/S2 junction. Emerg Microbes Infect 9, 837-842 (2020).
      
  69. Liu, Z. et al. Identification of common deletions in the spike protein of SARS-CoV-2. J Virol (2020).
      
  70. Ge, X.Y. et al. Detection of alpha- and betacoronaviruses in rodents from Yunnan, China. Virol J 14, 98 (2017).
      
  71. Guan, Y. et al. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 302, 276-8 (2003).
      
  72. Ge, X.Y. et al. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature 503, 535-8 (2013).
      
  73. Lau, S.K. et al. Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc Natl Acad Sci U S A 102, 14040-5 (2005).
      
  74. Kam, Y.W. et al. Antibodies against trimeric S glycoprotein protect hamsters against SARS-CoV challenge despite their capacity to mediate FcgammaRII-dependent entry into B cells in vitro. Vaccine 25, 729-40 (2007).
      
  75. Chan, J.F. et al. Middle East respiratory syndrome coronavirus: another zoonotic betacoronavirus causing SARS-like disease. Clin Microbiol Rev 28, 465-522 (2015).
      
  76. Zhou, J., Chu, H., Chan, J.F. & Yuen, K.Y. Middle East respiratory syndrome coronavirus infection: virus-host cell interactions and implications on pathogenesis. Virol J 12, 218 (2015).
      
  77. Yeung, M.L. et al. MERS coronavirus induces apoptosis in kidney and lung by upregulating Smad7 and FGF2. Nat Microbiol 1, 16004 (2016).
      
  78. Chu, D.K.W. et al. MERS coronaviruses from camels in Africa exhibit region-dependent genetic diversity. Proc Natl Acad Sci U S A 115, 3144-3149 (2018).
      
  79. Ommeh, S. et al. Genetic Evidence of Middle East Respiratory Syndrome Coronavirus (MERS-Cov) and Widespread Seroprevalence among Camels in Kenya. Virol Sin 33, 484-492 (2018).
      
  80. Sia, S.F. et al. Pathogenesis and transmission of SARS-CoV-2 in golden hamsters. Nature (2020).
      
  81. Ren, W. et al. Full-length genome sequences of two SARS-like coronaviruses in horseshoe bats and genetic variation analysis. J Gen Virol 87, 3355-9 (2006). 24 

  82. Yuan, J. et al. Intraspecies diversity of SARS-like coronaviruses in Rhinolophus sinicus and its implications for the origin of SARS coronaviruses in humans. J Gen Virol 91, 1058-62 (2010).
      
  83. Ge, X.Y. et al. Coexistence of multiple coronaviruses in several bat colonies in an abandoned mineshaft. Virol Sin 31, 31-40 (2016).
      
  84. Hu, B. et al. Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus. PLoS Pathog 13, e1006698 (2017).
      
  85. Luo, Y. et al. Longitudinal Surveillance of Betacoronaviruses in Fruit Bats in Yunnan Province, China During 2009-2016. Virol Sin 33, 87-95 (2018).
      
  86. Kuo, L., Godeke, G.J., Raamsman, M.J., Masters, P.S. & Rottier, P.J. Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier. J Virol 74, 1393- 406 (2000).
      
  87. Drexler, J.F. et al. Genomic characterization of severe acute respiratory syndrome-related coronavirus in European bats and classification of coronaviruses based on partial RNA-dependent RNA polymerase gene sequences. J Virol 84, 11336-49 (2010).
      
  88. Agnihothram, S. et al. A mouse model for Betacoronavirus subgroup 2c using a bat coronavirus strain HKU5 variant. mBio 5, e00047-14 (2014).
      
  89. Johnson, B.A., Graham, R.L. & Menachery, V.D. Viral metagenomics, protein structure, and reverse genetics: Key strategies for investigating coronaviruses. Virology 517, 30-37 (2018).
      
  90. Wu, K., Peng, G., Wilken, M., Geraghty, R.J. & Li, F. Mechanisms of host receptor adaptation by severe acute respiratory syndrome coronavirus. J Biol Chem 287, 8904-11 (2012).
      
  91. Follis, K.E., York, J. & Nunberg, J.H. Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry. Virology 350, 358-69 (2006).
      
  92. Yount, B., Denison, M.R., Weiss, S.R. & Baric, R.S. Systematic assembly of a full-length infectious cDNA of mouse hepatitis virus strain A59. J Virol 76, 11065-78 (2002).
      
  93. Yount, B. et al. Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus. Proc Natl Acad Sci U S A 100, 12995-3000 (2003).
      
  94. Almazan, F. et al. Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis. J Virol 80, 10900-6 (2006).
      
  95. Scobey, T. et al. Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus. Proc Natl Acad Sci U S A 110, 16157-62 (2013).
      
  96. Almazan, F., Marquez-Jurado, S., Nogales, A. & Enjuanes, L. Engineering infectious cDNAs of coronavirus as bacterial artificial chromosomes. Methods Mol Biol 1282, 135-52 (2015).
      
  97. Thao, T.T.N. et al. Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform. Nature (2020).
      
  98. Oldfield, L.M. et al. Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods. Proc Natl Acad Sci U S A 114, E8885-E8894 (2017).
      
  99. Vashee, S. et al. Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination. mSphere 2(2017).
      
  100. Xie, X. et al. An Infectious cDNA Clone of SARS-CoV-2. Cell Host Microbe 27, 841-848 e3 (2020).
       
  101. Roberts, A. et al. A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice. PLoS Pathog 3, e5 (2007).
       
  102. Roberts, A. et al. Animal models and vaccines for SARS-CoV infection. Virus Res 133, 20-32 (2008).
       
  103. Takayama, K. In Vitro and Animal Models for SARS-CoV-2 research. Trends Pharmacol Sci 41, 513-517 (2020).
       
  104. Wang, Q. hACE2 Transgenic Mouse Model For Coronavirus (COVID-19) Research. The Jackson Laboratory Research Highlight, https://www.jax.org/news-and-insights/2020/february/introducing-
       mouse-model-for-corona-virus# (2020).
       
  105. Zhang, L. et al. The D614G mutation in the SARS-CoV-2 spike protein reduces S1 shedding and increases infectivity. bioRxiv, https://doi.org/10.1101/2020.06.12.148726 (2020).
       
  106. Yurkovetskiy, L. et al. Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant. bioRxiv, https://doi.org/10.1101/2020.07.04.187757 (2020).
       
  107. Korber, B. et al. Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of
       the COVID-19 Virus. Cell 182, 812-827 e19 (2020). 25 

  
  

  108. Plante, J.A. et al. Spike mutation D614G alters SARS-CoV-2 fitness and neutralization susceptibility. bioRxiv, https://doi.org/10.1101/2020.09.01.278689 (2020).
       
  109. Poon, L.L. et al. Recurrent mutations associated with isolation and passage of SARS coronavirus in cells from non-human primates. J Med Virol 76, 435-40 (2005).
       
  110. Pervushin, K. et al. Structure and inhibition of the SARS coronavirus envelope protein ion channel. PLoS Pathog 5, e1000511 (2009).
       
  111. Nieto-Torres, J.L. et al. Severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis. PLoS Pathog 10, e1004077 (2014).