PreprintPDF Available

Is considering a genetic-manipulation origin for SARS-CoV-2 a conspiracy theory that must be censored?

Preprints and early-stage research may not have been peer reviewed yet.

Abstract and Figures

Based on our experience in genetic manipulation we cannot exclude a synthetic origin of SARS-CoV-2 and we believe that this topic should not be censored. In our manuscript we suggest a possible experiment that could have originated SARS-CoV-2, known to be chimeric and characterized by a furin cleavage site, missing in other beta-coronaviruses of the same lineage. Moreover, we do a critical analysis of the paper of Andersen and colleagues published in Nature on the Proximal Origin of SARS-CoV-2. This paper is considered to prove that SARS-CoV-2 has a natural origin, but in our opinion it lacks scientific evidence. We do not want to accuse a specific research group, but raise attention of the scientific community on this topic.
Content may be subject to copyright.
Is considering a genetic-manipulation origin for SARS-CoV-2 a conspiracy
theory that must be censored?
Rossana Segreto1# (Ph.D.)
1Department of Microbiology, University of Innsbruck, Technikerstraße 25, 6020 Innsbruck (Austria).
#Correspondence should be addressed to Tel.: +43-512-50751252.
The origin of SARS-CoV-2 is still controversial. Comparative genomic analyses have shown that SARS-
CoV-2 is likely to be chimeric, most of its sequence being very close to the CoV detected from a bat,
whereas its receptor binding domain is almost identical to that of CoV obtained from pangolins. The
furin cleavage site in the spike protein of SARS-CoV-2 was previously not identified in other SARS-like
CoVs and might have conferred ability to cross species and tissue barriers. Chimeric viruses can be
the product of natural recombination or genetic manipulations. The latter could have aimed to
identify pangolins as possible intermediate hosts for bat-CoV potentially pathogenic for humans.
Theories that consider a possible artificial origin for SARS-CoV-2 are censored as they seem to
support conspiracy theories. Researchers have the responsibility to carry out a thorough analysis,
beyond any personal research interests, of all possible causes for SARS-CoV-2 emergence for
preventing this from happening in the future.
Several months have passed since the outbreak of SARS-CoV-2 in Wuhan, China, and its origin is still
controversial. The theory that the Wuhan’s Huanan Seafood Wholesale Market was the first source
for animal–human virus transmission has lost credibility. During the first phase of the epidemic in
Wuhan, several hospitalized patients with confirmed SARS-CoV-2 infections had no link with the
The closest relatives to SARS-CoV-2 are bat and pangolin coronaviruses
Zhou and colleagues2 from the Wuhan Institute of Virology (WIV) first identified and characterized
the new coronavirus (CoV), recently named SARS-CoV-2. The genomic sequences obtained from early
cases shared 79% sequence identity to the CoVs that caused Severe Acute Respiratory Syndrome
(SARS-CoV) in 2002-2003 and 96·2 % sequence identity to RaTG13, a total genomic sequence of a
CoV detected from a Rhinolophus affinis bat. This sample was collected in the Yunnan province
(China) by the same group of researchers in 2013. Zhou and colleagues2 found a short region of RNA-
dependent RNA polymerase (RdRp) in their data and then fully sequenced the original sample. This
sequence is currently the closest phylogenetic relative for SARS-CoV-2 found3 and it has not been
published before the outbreak of SARS-CoV-2.
The RdRp of RaTG13 has 100 % identity with the sequence BtCoV/4991 (KP876546) identified by Ge
and colleagues4 in a Rhinolophus affinis bat in the Yunnan province in 2013 as RaTG13. Based on the
phylogenetic analysis carried out by Ge and colleagues4, BtCoV/4991 is a novel beta-CoV, clearly
separated from all known alpha- and beta-CoVs at that time. Spike genes were amplified as well, and
made available upon request to Ge and colleagues.4 BtCoV/4991 clearly differentiates from other bat
CoVs also in the phylogenetic analysis carried out by Wang and colleagues.5 How BtCoV/4991 and
RaTG13 relate to each other remains unclear.
Chen and colleagues6 identified BtCoV/4991 as the closest sequence to SARS-CoV-2 because RaTG13
had not yet been published at that time. The second non-human RdRp sequence closest to
BtCoV/4991 (91·89%) is the CoV sequence MP789 isolated in 2019 in a Malaysian pangolin (Manis
javanica) in the Guangdong province, China (MT084071).
Bat CoVs have been studied intensely and genetically manipulated
Several studies point out that bats are reservoirs for a broad diversity of potentially pathogenic SARS-
like CoV.4,7,8 Some of these viruses can directly infect humans9, whereas others need to mutate their
spike protein in order to effectively bind to the human angiotensin 1-converting enzyme 2 (hACE2)
receptor and mediate virus entry.10 In order to evaluate the emergence potential of novel CoVs,
chimeric CoVs with Bat CoV backbones not able to infect human cells were fused to spike proteins of
CoVs compatible with human ACE2, simulating recombination events that occur naturally.7,11,12 These
experiments with gain of function have raised biosafety concerns and controversy among
researchers and the public.13,14
Key difference between SARS-CoV-2 and its closest relative RaTG13
SARS-CoV-2 differs from its closest relative RaTG13 by few key characteristics. The most striking one
is the acquisition in the spike protein of SARS-CoV-2 of a cleavage site activated by the host-cell
enzyme furin, previously not identified in other beta-CoVs of lineage b15 and similar to that of Middle
East Respiratory Syndrome Coronavirus (MERS-CoV).16 Host protease processing plays a pivotal role
as species and tissue barrier. Engineering of the cleavage sites of CoV spike proteins modifies virus
tropism and virulence.17 The ubiquitous expression of furin in different organs and tissues may have
conferred to SARS-CoV-2 the ability to infect body parts insensitive to other CoVs, leading to
systematic infection in the body.18 Cell-cultured SARS-CoV-2 that was missing the above-mentioned
cleavage site caused attenuated symptoms in infected hamsters.19
Pangolin or not pangolin, that is the question
The possibility that pangolins could be the intermediate host for SARS-CoV-2 is still under
discussion.20,21 SARS-CoV-2 and RaTG13 mostly diverge because of the RBD of their spike protein.3
Although the average genome similarity is lower compared to RaTG13, CoV isolated from pangolins
has RBDs almost identical to that of SARS-CoV-2. Indeed, pangolin CoVs and SARS-CoV-2 possess
identical amino acids at the five critical residues of the RBD, whereas RaTG13 only shares one amino
acid with SARS-CoV-2.16 ACE2 sequence similarity is higher between humans and pangolins than
between humans and bats. Before the SARS-CoV2 outbreak, pangolins were the only mammals other
than bats documented to be infected by a SARS-CoV-2 related CoV.22 Recombination events between
the RBD of CoV from pangolins and RaTG13-like backbone could have originated SARS-CoV-2 as
chimeric strain. For recombination to occur, the two viruses must have infected the same cell in the
same organism simultaneously.16
Is a lab origin for SARS-CoV-2 a baseless conspiracy theory?
Due to the broad-spectrum of research conducted over almost 20 years on bat SARS-CoV justified by
their potential to spill over from animal to human23, a possible synthetic origin by laboratory
engineering of SARS-CoV-2 is a reasonable hypothesis. Andersen and colleagues24 stated that strong
evidence that SARS-CoV-2 did not result from genetic manipulation is that the high-affinity binding of
the SARS-CoV-2 spike protein to human ACE2 could not have been predicted by models based on the
RBD of SARS-CoV. As described above, creation of chimeric viruses has been carried out over the
years with the purpose to study the potential pathogenicity of bat CoVs for humans. In this context,
SARS-CoV-2 could have been synthetized by combining a backbone similar to RaTG13 with the RBD of
CoV similar to the one recently isolated from pangolins20, because the latter is characterized by a
higher affinity with the hACE2 receptor. Such research could have aimed to identify pangolins as
possible intermediate hosts for bat-CoV potentially pathogenic for humans.
Regarding the furin cleavage site, Andersen and colleagues24 state that “The functional consequence
of the polybasic cleavage site in SARS-CoV-2 is unknown”. New studies from several groups have
lately identified this activation site as possibly enabling the virus to spread efficiently between
humans and attack multiple organs.25
Andersen and colleagues24 also state, based on the work of Almazan and colleagues26 that “the
genetic data irrefutably show that SARS-CoV-2 is not derived from any previously used virus
backbone”. In the last six years before the outbreak of SARS-CoV-2 the number of potential bat
backbones has been undeniably increased by several bat CoV screenings, last but not least bringing
RaTG13 to scientific attention in January 2020. Other possible backbones could, as well, still wait for
Andersen and colleagues24 also state that “The acquisition of both the polybasic cleavage site and
predicted O-linked glycans also argues against culture-based scenarios”. Methods for insertion of a
polybasic cleavage site in infectious bronchitis CoV are given in Cheng and colleagues27 and resulted
in increased pathogenicity. The addition of O-linked glycans typically occurs under immune selection
and could have arisen during in vivo experiments. To overcome problems of bat CoV isolation,
experiments based on direct inoculation of bat CoV in suckling rats28 have been carried out. Pangolins
or other animals with similar ACE2 conformation could have been used as experimental animals as
well. The authors also state that “Subsequent generation of a polybasic cleavage site would have
then required repeated passage in cell culture or animals with ACE2 receptors similar to those of
humans, but such work has also not previously been described.” It should not be excluded that such
experiments could have been aborted due to the SARS-CoV-2 outbreak, before a possible publication
of the results or that the results were never intended to be published.
Due to the gravity of SARS-CoV-2 impact on humanity, researchers have the responsibility to carry
out a thorough analysis, beyond any personal research interests, of all possible causes for SARS-CoV-
2 emergence. Unfortunately, theories that consider a possible artificial origin for SARS-CoV-2 are
censored by international scientific journals as they seem to support conspiracy theories. Genetic
manipulation of SARS-CoV-2 may have been carried out in any laboratory in the world with access to
the backbone sequence and the necessary equipment.
Xiao Qiang, a research scientist at the School of Information at the University of California at
Berkeley, recently stated “To understand exactly how this virus has originated is critical knowledge
for preventing this from happening in the future” (Washington Post, April 14, 2020).
I am grateful to Prof. Allan Krill (NTNU) for proof reading the manuscript and all the valuable
comments. I want to thank Prof. Heribert Insam (University of Innsbruck) for his support.
1 Huang C, Wang Y, Li X et al. Clinical features of patients infected with 2019 novel coronavirus
in Wuhan, China. Lancet 2020; 395: 497–506.
2 Zhou P, Yang X, Wang X et al. A pneumonia outbreak associated with a new coronavirus of
probable bat origin. Nature 2020; 579: 270–273.
3 Cagliani R, Forni D, Clerici M, Sironi M. Computational inference of selection underlying the
evolution of the novel coronavirus, SARS-CoV-2. J Virol. 2020; DOI: 10.1128/JVI.00411-20
4 Ge XY, Wang N, Zhang W et al. Coexistence of multiple coronaviruses in several bat colonies
in an abandoned mineshaft. Virol Sin. 2016; 31: 31–40.
5 Wang N, Luo C, Liu H et al. Characterization of a new member of alphacoronavirus with
unique genomic features in Rhinolophus bats. Viruses 2019; 11: 379.
6 Chen L, Liu W, Zhang Q et al. RNA based mNGS approach identifies a novel human
coronavirus from two individual pneumonia cases in 2019 Wuhan outbreak. Emerg Microbes Infect.
2020; 9: 313–9.
7 Hu B, Zeng L-P, Yang X-L et al. Discovery of a rich gene pool of bat SARS-related coronaviruses
provides new insights into the origin of SARS coronavirus. PLoS Pathog. 2017; 13: e1006698.
8 Fan Y, Zhao K, Shi Z-L, Zhou P. Bat coronaviruses in China. Viruses 2019; 11: 210.
9 Ge XY, Li JL, Yang XL, Chmura AA et al. Isolation and characterization of a bat SARS-like
coronavirus that uses the ACE2 receptor. Nature 2013; 503: 535.
10 Graham RL, Baric RS. Recombination, reservoirs, and the modular spike: mechanisms of
coronavirus cross-species transmission. J Virol. 2010; 84: 3134–3146.
11 Agnihothram S, Yount BL Jr., Donaldson EF et al. A mouse model for Betacoronavirus
subgroup 2c using a bat coronavirus strain HKU5 variant. mBio. 2014; 5: e00047-14.
12 Johnson BA, Graham RL, Menachery VD. Viral metagenomics, protein structure, and reverse
genetics: key strategies for investigating coronaviruses. Virology 2018; 517: 30–37.
13 Weiss S, Yitzhaki S, Shapira SC. Lessons to be learned from recent biosafety incidents in the
United States. Isr Med Assoc J. 2015; 17: 269–273.
14 Racaniello V. Moving beyond metagenomics to find the next pandemic virus. Proc Natl Acad
Sci U S A. 2016; 113: 2812–2814.
15 Coutard B, Valle C, de Lamballerie X, Canard B, Seidah NG, Decroly E. The spike glycoprotein
of the new coronavirus 2019- nCoV contains a furin-like cleavage site absent in CoV of the same
clade. Antiviral Res. 2020; 176: 104742.
16 Zhang T, Wu Q, Zhang Z. Probable pangolin origin of SARS-CoV-2 associated with the COVID-
19 outbreak. Curr Biol. 2020; 30: 1346–1351.
17 Letko M, Marzi A, Munster V. Functional assessment of cell entry and receptor usage for
SARS-CoV-2 and other lineage B betacoronaviruses. Nat Microbiol. 2020; 5: 562–569.
18 Wang Q, Qiu Y, Li JY, Zhou ZJ, Liao CH, Ge XY. A unique protease cleavage site predicted in the
spike protein of the novel pneumonia Coronavirus (2019-nCoV) potentially related to viral
transmissibility. Virol Sin. 2020. Available from:
19 Lau SY, Wang P, Mok B W-Y et al. Attenuated SARS-CoV-2 variants with deletions at the S1/S2
junction. Emerg Microbes Infect. 2020. Available from:
20 Liu P, Chen W, Chen J-P. Viral metagenomics revealed Sendai virus and coronavirus infection
of Malayan Pangolins (Manis javanica). Viruses 2019; 11: 979.
21 Li X, Zai J, Zhao Q, Nie Q, Li Y, Foley BT, Chaillon A. Evolutionary history, potential
intermediate animal host, and cross-species analyses of SARS-CoV-2. J Med Virol. 2020. Available
22 Lam TT, Shum MH, Zhu H et al. Identifying SARS-CoV-2 related coronaviruses in Malayan
pangolins. Nature 2020. Available from:
23 Wang LF, Anderson DE. Viruses in bats and potential spillover to animals and humans. Curr
Opin Virol. 2019; 34: 79–89.
24 Andersen KG, Rambaut A, Lipkin WI, Holmes, Garry RF. The proximal origin of SARS-CoV-2.
Nat Med. 2020; 26: 450–452.
25 Mallapati S. Why does the coronavirus spread so easily between people? Nature 2020; 579:
26 Almazan F, Gonzalez JM, Penzes Z. Coronavirus reverse genetic systems: Infectious clones
and replicons. Virus Res. 2014; 189: 262–270.
27 Cheng J, Zhao Y, Xu G. The S2 Subunit of QX-type infectious bronchitis coronavirus spike
protein is an essential determinant of neurotropism. Viruses 2019; 11: 10.
28 Hu D, Zhu C, Ai L et al. Genomic characterization and infectivity of a novel SARS-like
coronavirus in Chinese bats. Emerg Microbes Infect. 2018; 7: 154.
ResearchGate has not been able to resolve any citations for this publication.
Full-text available
The devastating impact of the COVID-19 pandemic caused by SARS–coronavirus 2 (SARS-CoV-2) has raised important questions about its origins and the mechanism of its transfer to humans. A further question was whether companion or commercial animals could act as SARS-CoV-2 vectors, with early data suggesting susceptibility is species specific. To better understand SARS-CoV-2 species susceptibility, we undertook an in silico structural homology modelling, protein–protein docking, and molecular dynamics simulation study of SARS-CoV-2 spike protein’s ability to bind angiotensin converting enzyme 2 (ACE2) from relevant species. Spike protein exhibited the highest binding to human (h)ACE2 of all the species tested, forming the highest number of hydrogen bonds with hACE2. Interestingly, pangolin ACE2 showed the next highest binding affinity despite having a relatively low sequence homology, whereas the affinity of monkey ACE2 was much lower despite its high sequence similarity to hACE2. These differences highlight the power of a structural versus a sequence-based approach to cross-species analyses. ACE2 species in the upper half of the predicted affinity range (monkey, hamster, dog, ferret, cat) have been shown to be permissive to SARS-CoV-2 infection, supporting a correlation between binding affinity and infection susceptibility. These findings show that the earliest known SARS-CoV-2 isolates were surprisingly well adapted to bind strongly to human ACE2, helping explain its efficient human to human respiratory transmission. This study highlights how in silico structural modelling methods can be used to rapidly generate information on novel viruses to help predict their behaviour and aid in countermeasure development.
Full-text available
The ongoing outbreak of the novel coronavirus pneumonia COVID-19 has caused great number of cases and deaths, but our understanding about the pathogen SARS-CoV-2 remains largely unclear. The attachment of the virus with the cell-surface receptor and a co-factor is the first step for the infection. Here, bioinformatics approaches combining human-virus protein interaction prediction and protein docking based on crystal structures have revealed the high affinity between human dipeptidyl peptidase 4 (DPP4) and the spike (S) receptor-binding domain of SARS-CoV-2. Intriguingly, the crucial binding residues of DPP4 are identical to those as bound to the MERS-CoV-S. Moreover, E484 insertion and adjacent substitutions should be most essential for this DPP4-binding ability acquirement of SARS-CoV-2-S compared with SARS-CoV-S. This potential utilization of DPP4 as a binding target for SARS-CoV-2 may offer novel insight into the viral pathogenesis, and help the surveillance and therapeutics strategy for meeting the challenge of COVID-19.
Full-text available
The outbreak of COVID-19 poses unprecedent challenges to global health1. The new coronavirus, SARS-CoV-2, shares high sequence identity to SARS-CoV and a bat coronavirus RaTG132. While bats may be the reservoir host for various coronaviruses3,4, whether SARS-CoV-2 has other hosts remains ambiguous. In this study, one coronavirus isolated from a Malayan pangolin showed 100%, 98.6%, 97.8% and 90.7% amino acid identity with SARS-CoV-2 in the E, M, N and S genes, respectively. In particular, the receptor-binding domain within the S protein of the Pangolin-CoV is virtually identical to that of SARS-CoV-2, with one noncritical amino acid difference. Results of comparative genomic analysis suggest that SARS-CoV-2 might have originated from the recombination of a Pangolin-CoV-like virus with a Bat-CoV-RaTG13-like virus. The Pangolin-CoV was detected in 17 of 25 Malayan pangolins analyzed. Infected pangolins showed clinical signs and histological changes, and circulating antibodies against Pangolin-CoV reacted with the S protein of SARS-CoV-2. The isolation of a coronavirus that is highly related to SARS-CoV-2 in pangolins suggests that they have the potential to act as the intermediate host of SARS-CoV-2. The newly identified coronavirus in the most-trafficked mammal could represent a future threat to public health if wildlife trade is not effectively controlled.
Full-text available
Reverse genetics has been an indispensable tool revolutionising insights into viral pathogenesis and vaccine development. Large RNA virus genomes, such as from Coronaviruses, are cumbersome to clone and manipulate in E. coli due to size and occasional instability1–3. Therefore, an alternative rapid and robust reverse genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Paramyxoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples, or synthetic DNA, and reassembled in one step in Saccharomyces cerevisiae using transformation associated recombination (TAR) cloning to maintain the genome as a yeast artificial chromosome (YAC). T7-RNA polymerase has been used to generate infectious RNA to rescue viable virus. Based on this platform we have been able to engineer and resurrect chemically-synthetized clones of the recent epidemic SARS-CoV-24 in only a week after receipt of the synthetic DNA fragments. The technical advance we describe here allows a rapidly response to emerging viruses as it enables the generation and functional characterization of evolving RNA virus variants—in real-time—during an outbreak.
Full-text available
In a side-by-side comparison of evolutionary dynamics between the 2019/2020 SARS-CoV-2 and the 2003 SARS-CoV, we were surprised to find that SARS-CoV-2 resembles SARS-CoV in the late phase of the 2003 epidemic after SARS-CoV had developed several advantageous adaptations for human transmission. Our observations suggest that by the time SARS-CoV-2 was first detected in late 2019, it was already pre-adapted to human transmission to an extent similar to late epidemic SARS-CoV. However, no precursors or parallel branches of evolution stemming from a less human-adapted SARS-CoV-2-like virus have been detected. The sudden appearance of a highly infectious SARS-CoV-2 presents a major cause for concern that should motivate stronger international efforts to identify the source and prevent near future re-emergence. Any existing pools of SARS-CoV-2 progenitors would be particularly dangerous if similarly well adapted for human transmission. To look for clues regarding intermediate hosts, we analyze recent key findings relating to how SARS-CoV-2 could have evolved and adapted for human transmission, and examine the environmental samples from the Wuhan Huanan seafood market. Importantly, the market samples are genetically identical to human SARS-CoV-2 isolates and were therefore most likely from human sources. We conclude by describing and advocating for measured and effective approaches implemented in the 2002-2004 SARS outbreaks to identify lingering population(s) of progenitor virus.
Full-text available
The emergence of SARS-CoV-2 has led to the current global coronavirus pandemic and more than one million infections since December 2019. The exact origin of SARS-CoV-2 remains elusive, but the presence of a distinct motif in the S1/S2 junction region suggests possible acquisition of cleavage site(s) in the spike protein that promoted cross-species transmission. Through plaque purification of Vero-E6 cultured SARS-CoV-2, we found a series of variants which contain 15-30-bp deletions (Del-mut) or point mutations respectively at the S1/S2 junction. Examination of the original clinical specimen from which the isolate was derived, and 26 additional SARS-CoV-2 positive clinical specimens, failed to detect this variant. Infection of hamsters shows that one of the variants (Del-mut-1) which carries deletion of 10 amino acids (30 bp) does not cause the body weight loss or more severe pathological changes in the lungs that is associated with wild type virus infection. We suggest that the unique cleavage motif promoting SARS-CoV-2 infection in humans may be under strong selective pressure, given that replication in permissive Vero-E6 cells leads to the loss of this adaptive function. It would be important to screen the prevalence of these variants in asymptomatic infected cases. The potential of the Del-mut variant as an attenuated vaccine or laboratory tool should be evaluated.
Full-text available
The Coronavirus disease (COVID-19) is a new viral infection caused by severe acute respiratory coronavirus 2 (SARS-CoV-2) that was initially reported in city of Wuhan, China and afterwards spread globally. Genomic analyses revealed that SARS-CoV-2 is phylogenetically related to severe acute respiratory syndrome-like (SARS-like) Pangolin and Bat coronavirus specific isolates. In this study we focused on two proteins of Sars-CoV-2 surface: Envelope protein and Membrane protein. Sequences from Sars-CoV-2 isolates and other closely related virus were collected from the GenBank through TBlastN searches. The retrieved sequences were multiply aligned with MAFFT. The Envelope protein is identical to the counterparts from Pangolin CoV MP798 isolate and Bat CoV isolates CoVZXC21, CoVZC45 and RaTG13. However, a substitution at position 69 where an Arg replace for Glu, and a deletion in position 70 corresponding to Gly or Cys in other Envelope proteins were found. The Membrane glycoprotein appears more variable with respect to the SARS CoV proteins than the Envelope: a heterogeneity at the N-terminal position, exposed to the virus surface, was found between Pangolin CoV MP798 isolate and Bat CoV isolates CoVZXC21, CoVZC45 and RaTG13. Mutations observed on Envelope protein are drastic and may have significant implications for conformational properties and possibly for protein-protein interactions. Mutations on Membrane protein may also be relevant because this protein cooperates with the Spike during the cell attachment and entry. Therefore, these mutations may influence interaction with host cells. The mutations that have been detected in these comparative studies may reflect functional peculiarities of the Sars-CoV-2 virus and may help explaining the epizootic origin the COVID-19 epidemic.
Full-text available
Two notable features have been identified in the SARS-CoV-2 genome: (1) the receptor binding domain of SARS-CoV-2; (2) a unique insertion of twelve nucleotide or four amino acids (PRRA) at the S1 and S2 boundary. For the first feature, the similar RBD identified in SARs-like virus from pangolin suggests the RBD in SARS-CoV-2 may already exist in animal host(s) before it transmitted into human. The left puzzle is the history and function of the insertion at S1/S2 boundary, which is uniquely identified in SARS-CoV-2. In this study, we identified two variants from the first Guangdong SARS-CoV-2 cell strain, with deletion mutations on polybasic cleavage site (PRRAR) and its flank sites. More extensive screening indicates the deletion at the flank sites of PRRAR could be detected in 3 of 68 clinical samples and half of 22 in vitro isolated viral strains. These data indicate (1) the deletion of QTQTN, at the flank of polybasic cleavage site, is likely benefit the SARS-CoV-2 replication or infection in vitro but under strong purification selection in vivo since it is rarely identified in clinical samples; (2) there could be a very efficient mechanism for deleting this region from viral genome as the variants losing 23585-23599 is commonly detected after two rounds of cell passage. The mechanistic explanation for this in vitro adaptation and in vivo purification processes (or reverse) that led to such genomic changes in SARS-CoV-2 requires further work. Nonetheless, this study has provided valuable clues to aid further investigation of spike protein function and virus evolution. The deletion mutation identified in vitro isolation should be also noted for current vaccine development.
The pandemic coronavirus SARS-CoV-2 threatens public health worldwide. The viral spike protein mediates SARS-CoV-2 entry into host cells and harbors a S1/S2 cleavage site containing multiple arginine residues (multibasic) not found in closely related animal coronaviruses. However, the role of this multibasic cleavage site in SARS-CoV-2 infection is unknown. Here, we report that the cellular protease furin cleaves the spike protein at the S1/S2 site and that cleavage is essential for S-protein-mediated cell-cell fusion and entry into human lung cells. Moreover, optimizing the S1/S2 site increased cell-cell, but not virus-cell, fusion, suggesting that the corresponding viral variants might exhibit increased cell-cell spread and potentially altered virulence. Our results suggest that acquisition of a S1/S2 multibasic cleavage site was essential for SARS-CoV-2 infection of humans and identify furin as a potential target for therapeutic intervention.