Emerging Microbes & Infections

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SARS-CoV-2: no evidence of a laboratory origin

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Journal: Emerging Microbes & Infections

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Manuscript ID Draft

Manuscript Type: Commentary

Date Submitted by the

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Author:
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Complete List of Authors: Liu, Shan-Lu; The Ohio State University, Infectious Diseases Institute
Saif, Linda J.; The Ohio State University
Weiss, Susan; University of Pennsylvania
Su, Lishan; University of North Carolina at Chapel Hill
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Keywords: SARS-CoV-2, COIVD-2019, origin, evolution

Abstract:

URL: https://mc.manuscriptcentral.com/temi E-mail:[email protected]

Page 1 of 9 Emerging Microbes & Infections

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6 SARS-CoV-2: no evidence of a laboratory origin
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9 Shan-Lu Liu 1, 2,3,4, Linda J. Saif 4,5, Susan Weiss 6, and Lishan Su 7
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12 1 Center
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for Retrovirus Research, The Ohio State University,
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15 Columbus, OH 43210, USA
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17 2 Department of Veterinary Biosciences, The Ohio State University, Columbus,
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OH 43210, USA
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3 Department of Microbial Infection and Immunity, The Ohio State University,
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24 Columbus, OH 43210, USA

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4 Viruses and Emerging Pathogens Program, Infectious Diseases Institute,

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29 The Ohio State University, Columbus, OH 43210, USA
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5 Food Animal Health Research Program,
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33 Ohio Agricultural Research and Development Center, CFAES

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Department of Veterinary Preventive Medicine,
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The Ohio State University, Wooster, Ohio 44691, USA
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40 6 Department of Microbiology, Perelman School of Medicine,
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University of Pennsylvania, Philadelphia, Pennsylvania, USA
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7 Lineberger Comprehensive Cancer Center, Department of Microbiology and Immunology,
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47 University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
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52 Contact: Dr. Lishan Su, [email protected]
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54 Dr. Shan-Lu Liu, [email protected]
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60 URL: https://mc.manuscriptcentral.com/temi E-mail:[email protected]
 Emerging Microbes & Infections Page 2 of 9

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3 The emergence and outbreak of a newly discovered acute respiratory disease in
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6 Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as
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8 of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and
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10 the associated disease is now referred to as coronavirus disease discovered in 2019
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(COVID-19) (https://globalbiodefense.com/novel-coronavirus-covid-19-portal/).
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17 According to what has been reported [1, 2, 3], COVID-2019 seems to have similar
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clinical manifestations to that of the severe acute respiratory syndrome (SARS) caused
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22 by SARS-CoV. The SARS-CoV-2 genome sequence also has ~80% identity with SARS-
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24 CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96%
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identity [4, 5].

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31 Currently, there are speculations, rumors and conspiracy theories that SARS-CoV-2
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33 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was
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leaked directly from a laboratory in Wuhan where a bat CoV (RaTG13) was recently
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reported, which shared ~96% homology with the SARS-CoV-2 [4]. However, as we know,
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40 the human SARS-CoV and intermediate host palm civet SARS-like CoV shared 99.8%
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homology, with a total of 202 single-nucleotide (nt) variations (SNVs) identified across the
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45 genome; among these SNVs, 200 were in the coding sequences, and among the 128
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47 nonsynonymous mutations, 89 led to predicted radical amino-acid changes [6]. Given that
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49 there are greater than 1000 nt differences between the human SARS-CoV-2 and the bat
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52 RaTG13-CoV [4], which are distributed throughout the genome in a naturally occurring
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54 pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that
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Page 3 of 9 Emerging Microbes & Infections

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3 RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted
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6 pattern in the new viral sequences and a close relative in a wildlife species (bats) are the
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8 most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an
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10 intermediate animal host between bats and humans is needed to identify animal CoVs
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more closely related to human SARS-CoV-2. There is speculation that pangolins might
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15 carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet
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17 published (https://www.nature.com/articles/d41586-020-00364-2).
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22 Another claim in Chinese social media points to a Nature Medicine paper published in
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24 2015 [7], which reports the construction of a chimeric CoV with a bat CoV S gene
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(SHC014) in the backbone of a SARS CoV that has adapted to infect mice (MA15) and
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29 is capable of infecting human cells [8]. However, this claim lacks any scientific basis and
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31 must be discounted because of significant divergence in the genetic sequence of this
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33 construct with the new SARS-CoV-2 (>5,000 nucleotides).

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The mouse-adapted SARS virus (MA15) [9] was generated by serial passage of an
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40 infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15
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passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in
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45 aged mice (hence M15), due to six coding genetic mutations associated with mouse
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47 adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients
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49 due to the mouse adaptation.
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 Emerging Microbes & Infections Page 4 of 9

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3 When the original SARS-CoV was isolated, it was concluded that the S gene from bat-
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6 derived CoV, unlike that from human patients- or civets-derived viruses, was unable to
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8 use human ACE2 as a receptor for entry into human cells [10, 11]. Civets were proposed
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10 to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans
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[6, 12]. However, in 2013 several novel bat coronaviruses were isolated from Chinese
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15 horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from
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17 humans, civets and Chinese horseshoe bats for entry [8]. Combined with evolutionary
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evidence that the bat ACE2 gene has been positively selected at the same contact sites
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22 as the human ACE2 gene for interacting with SARS CoV [13], it was proposed that an
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24 intermediate host may not be necessary and that some bat SL-CoVs may be able to
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directly infect human hosts. To directly address this possibility, the exact S gene from bat
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29 coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the
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31 mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus
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33 could indeed efficiently use human ACE2 and replicate in primary human airway cells to
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similar titers as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate
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efficiently in young and aged mouse lungs, infection was attenuated, and less virus
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40 antigen was present in the airway epithelium as compared to SARS MA15, which causes
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lethal outcomes in aged mice [7].
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47 Due to the elevated pathogenic activity of the SL-SHC014-MA15 chimeric virus
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49 relative to the SARS-MA15 CoV in mice, such experiments with SL-SHC014-MA15
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52 chimeric virus were later restricted as gain of function (GOF) studies under the US
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54 government-mandated pause policy (https://www.nih.gov/about-nih/who-we-are/nih-
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Page 5 of 9 Emerging Microbes & Infections

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3 director/statements/nih-lifts-funding-pause-gain-function-research). The current COVID-
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6 2019 epidemic has restarted the debate over the risks of constructing such viruses that
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8 could have pandemic potential, irrespective of the finding that these bat CoVs already
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10 exist in nature. Regardless, upon careful phylogenetic analyses by multiple international
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groups [5, 14], the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15,
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15 with >6,000 nucleotide differences across the whole genome. Therefore, once again there
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chimeric SL-SHC014-MA15 virus. Finally, we note that the synthetic and chimeric panels
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22 of bat and SARS-like CoV led to the identification of remdesivir as a broad spectrum
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24 inhibitor of all group 2b SARS-like coronaviruses tested in vitro or in vivo [15, 16],
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providing critical pre-clinical data that has led to the ongoing clinical trials in China and is
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29 critical for the future development of universal vaccines for all the SARS-like
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31 coronaviruses.
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35 There are also rumors that the SARS-CoV-2 was artificially, or intentionally, made by
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37 humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv (a
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40 manuscript sharing site prior to any peer review), claiming that SARS-CoV-2 has HIV
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sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by
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44 an HIV-1 expert Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate
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that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific
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49 but random (Gao et al., EMI paper 2/12/2020 in press). Because of the many concerns
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51 raised by the international community, the authors who made the initial claim have already
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3 Evolution is stepwise and accrues mutations gradually over time, whereas synthetic
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6 constructs would typically use a known backbone and introduce logical or targeted
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8 changes instead of the randomly occurring mutations that are present in naturally isolated
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10 viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to
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support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is
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15 more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat
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explore this possibility and resolve the natural origin of SARS-CoV-2.
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Page 7 of 9 Emerging Microbes & Infections

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