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

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Abstract
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.
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DOI: 10.13140/RG.2.2.31358.13129/1
Is considering a genetic-manipulation origin for SARS-CoV-2 a conspiracy
theory that must be censored?
Rossana Segreto1# and Yuri Deigin2
1Department of Microbiology, University of Innsbruck, Austria.
2Youthereum Genetics Inc., Toronto, ON Canada.
#Correspondence should be addressed to Rossana.Segreto@uibk.ac.at
RS and YD contributed equally to the manuscript.
Abstract
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 the ability to cross species and tissue barriers. Chimeric viruses can
be the product of natural recombination or genetic manipulation. 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
market.1 Unfortunately, the market was quickly closed and sanitized before enough animal samples
could have been collected; the few market samples that did get collected exhibit only human-
adapted SARS-CoV-2 and no traces of zoonotic predecessor strains.2
The closest relatives to SARS-CoV-2 are bat and pangolin
coronaviruses
Zhou and colleagues3 from the Wuhan Institute of Virology (WIV) first identified and characterized
the new coronavirus (CoV), later 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 (MN996532), 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 colleagues3 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 found4 and it has
not been published before the outbreak of SARS-CoV-2.
The RdRp of RaTG13 has 100% nucleotide identity with the sequence BtCoV/4991 (KP876546)
identified by Ge and colleagues5 in a Rhinolophus affinis bat in the Yunnan province in 2013, as
RaTG13. The original sample was collected in a mine colonized by bats near Tongguanzhen, Mojiang,
Yunnan. The WIV researchers were invited to investigate the mine after 6 miners contracted severe
pneumonia in 20126, and 3 of the miners had died.7 The miners were tasked with clearing out bat
droppings in the mine, and the severity of their pneumonia correlated with the duration of exposure
to the mine.8 Some of the miners’ samples subsequently underwent testing at WIV, where IgG
antibodies against SARS were identified in 4 of the samples.9 Considering that only about 5300
people were infected in mainland China during the SARS outbreak of 2002-2004, most of whom
resided in Guandong, the odds of 4 miners in Yunnan retaining antibodies from the 2002-2004 SARS
outbreak are quite low. On the other hand, it is possible that the SARS antibody test administered to
the miners cross-reacted with a novel SARS-like bat virus that the miners had acquired at the mine.
Ge and colleagues5 had identified a number of CoVs in the mine, but based on the phylogenetic
analysis, BtCoV/4991 was the only SARS-related strain, clearly separated from all known alpha- and
beta-CoVs at that time. Ge et al. also amplified spike genes of collected CoVs and made them
available upon request. BtCoV/4991 differentiates from other bat CoVs also in the phylogenetic
analysis carried out by Wang and colleagues in 2019.10 Chen and colleagues11 identified BtCoV/4991
as the closest sequence to SARS-CoV-2 because RaTG13 had not yet been published at that time.
BtCoV/4991 and RaTG13 have been recently confirmed to be two different coding names of the
same strain by their original authors at WIV, as they registered the two strains as one entry in the
Database of Bat-associated Viruses (DBatVir).12
The second non-human RdRp sequence closest to BtCoV/4991 (91.89% nucleotide identity) is the
CoV sequence MP789 (MT084071) isolated in 2019 in a Malaysian pangolin (Manis javanica) from
the Guangdong province, China.13 The envelope protein of MP789 has 100% aminoacidic identity
with the corresponding protein in RaTG13, in bat-SL-CoVZXC21 (MG772934.1), in bat-SL-CoVZC45
(MG772933.1) and in some SARS-CoV-2 isolates (e.g. YP_009724392).14 The envelope protein of CoVs
is involved in critical aspects of the viral life, as viral entry, replication and pathogenesis.15
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.16, 17 Some of these viruses can directly infect humans18, 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.19 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 might naturally
occur.20, 21 These experiments with gain of function have raised biosafety concerns and controversy
among researchers and the public. One of the main arguments in favour of gain of function studies is
the need to be prepared with an arsenal of drugs and vaccines for the next pandemic. By contrast,
one of the main arguments against them is that the next pandemic could be caused by those
experiments, due to the risk of lab leakage.22, 23, 24
In recent years, the field of corona-virology had been focused on pan-coronavirus therapies and
vaccines, as evident from research conducted in the past five years,25, 26, 27, 28 as well as from media
reports.29 Synthetically generating diverse panels of potential pre-emergent coronaviruses was
declared as a goal of active grants for EcoHealth Alliance which funded some of such research at
WIV.30
Key difference between SARS-CoV-2 and its closest relative RaTG13
SARS-CoV-2 differs from its closest relative RaTG13 by a 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 b31 and similar to that of Middle
East Respiratory Syndrome Coronavirus (MERS-CoV).32 Host protease processing plays a pivotal role
as a species and tissue barrier. Engineering of the cleavage sites of CoV spike proteins modifies virus
tropism and virulence.33 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 systemic
infection in the body.34 Cell-cultured SARS-CoV-2 that was missing the above-mentioned cleavage site
caused attenuated symptoms in infected hamsters,35 and mutagenesis studies have confirmed that
the polybasic furin site is essential for SARS-CoV-2’s ability to infect human lung cells.36
The polybasic furin site in CoV2 was created by a 12-nt insert TCCTCGGCGGGC coding for a PRRA amino
acid sequence at the S1/S2 junction (Fig. 1). Interestingly, the two joint arginines are coded by two
CGGCGG codons, which are quite rare for these viruses: only 5% of arginines are coded by CGG in CoV2 or
RaTG13, and CGGCGG in the new insert is the only doubled instance of this codon in CoV2. The CGGCGG
insert includes a FauI restriction site, of which there are six instances in CoV2 and four instances in
RaTG13 (and 2 in MP789). The serendipitous location of the FauI site could allow using restriction
fragment length polymorphism (RFLP) techniques37 for cloning38 or screening for mutations,39 as the new
furin site is prone to deletions in vitro.40, 41
Fig. 1 Nucleotide sequence of the S protein at the S1/S2 junction in SARS-CoV-2 (NC045512.2),
showing the furin cleavage site (in blue) that includes a FauI enzyme restriction site.
A study by Zhou et al.42 recently reported the discovery of a novel CoV strain RmYN02, which the authors
claim exhibits natural PAA amino acid insertions at the S1/S2 cleavage site where SARS-CoV-2 has the
PRRA insertion. However, upon close examination of the underlying nucleotide sequence of RmYN02 in
comparison with its closest ancestors ZC45 and ZXC21, no insertions are apparent, just nucleotide
mutations (Fig. 2).
Fig. 2 Alignment of nucleotide and amino acid sequences of the S protein from RaTG13 (MN996532)
and RmYN02 at the S1/S2 junction site. No insertions of nucleotides possibly evolving in a furin
cleavage site can be observed (in blue).
Therefore, SARS-CoV-2 remains unique among its beta coronavirus relatives not only due to a polybasic
furin site at the S1/S2 junction, but also due to the four amino acid insert PRRA which had created it (Fig.
3).
Fig. 3 Alignment of nucleotide and amino acid sequences of the S protein from RaTG13 (MN996532),
MP789 (MT084071) and SARS-CoV-2 (NC045512.2) at the S1/S2 site. The common nucleotides and
amino acids are given in black, SARS-CoV-2 unique nucleotides and amino acids in red, RaTG13
unique nucleotides and amino acids in green and common nucleotides and amino acids in SARS-CoV-
2 and RaTG13 that differ in M789 in blue. The codon for Serine (TCA) in RaTG13 and MP789 is split in
SARS-CoV-2 to give part of a new codon for Serine (TCT) and part of the amino acid Alanine (GCA).
Interestingly, the insertion of the furin cleavage site in SARS-CoV-2 is not in frame with the rest of
sequence, when compared with the MP789 and the RaTG13 sequences. The insertion causes a split
in the original codon for Serine (TCA) in MP789 or RaTG13 to give part of a new codon for Serine
(TCT) and part of the amino acid Alanine (GCA) in SARS-CoV-2 (Fig. 3).
SARS-CoV-2 seems to merge some exclusive features of SARS-CoV together with those typical for
MERS-CoV. A recent study has identified the MERS-CoV transmembrane dipeptidyl peptidase 4
receptor (DDP4) as a candidate binding target or coreceptor of SARS-CoV-2.43
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.44, 45, 46 SARS-CoV-2 and RaTG13 mostly diverge because of the RBD of their spike protein.4
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-2.32 ACE2 sequence similarity is higher between humans and pangolins than
between humans and bats. Intriguingly, the Spike protein of SARS-CoV-2 has a higher predicted
binding affinity to human ACE2 receptor than to that of pangolins.47 Before the SARS-CoV-2 outbreak,
pangolins were the only mammals other than bats documented to carry and be infected by SARS-
CoV-2 related CoV.13, 45 Recombination events between the RBD of CoV from pangolins and RaTG13-
like backbone could have produced SARS-CoV-2 as a chimeric strain. For such recombination to
occur, the two viruses must have infected the same cell in the same organism simultaneously.32
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 human,48 a possible synthetic origin by laboratory
engineering of SARS-CoV-2 is a reasonable hypothesis. For Andersen and colleagues,49 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 hACE2 could not have been predicted by models based on the RBD
of SARS-CoV. Based on the structural analysis conducted by Wan and colleagues,50 SARS-CoV-2 has
the potential to recognize hACE2 more efficiently than the SARS-CoV which emerged in 2002.
Moreover, generation of CoV chimeric strains has recently demonstrated that bat CoV spikes can
bind to the hACE2 receptor with more plasticity than previously predicted.16 All amino acids in the
RBD have been extensively analysed and new models to predict ACE2 affinity are available.51 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 synthesized by combining a backbone similar to RaTG13 with the RBD of CoV similar to the one
recently isolated from pangolins13, 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 colleagues49 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.52 Experiments on proteolytic cleavage of CoV spike proteins have
been recently suggested as future key studies to study virus transmissibility in different hosts. 51 The
pangolin from which MP789 was isolated was co-infected by several viruses13, among others the
Herpes Virus that is characterized by a furin cleavage site. In a context of an evolutionary study, this
observation might have suggested the idea of this insertion into SARS-CoV-2.
Andersen and colleagues49 also state, based on the work of Almazan and colleagues53 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
publication.
Andersen and colleagues49 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 colleagues54 and resulted
in increased pathogenicity. Concerning the predicted O-linked glycans around the newly inserted
polybasic site, it should be noted that this prediction was not confirmed by Cryo-EM inquiry into the
SARS-CoV-2 spike glycoprotein.55 Nevertheless, while it is true that O-linked glycans are much more
likely to arise under immune selection, they could be added in the lab through site-directed
mutagenesis56 or arise in the course of in vivo experiments, for example, in BLT-L mice that have
human lung implants and autologous human immune system57 or in mice expressing human ACE2
receptor.58 To overcome problems of bat CoV isolation, experiments based on direct inoculation of
bat CoV in suckling rats have been carried out.59 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.
Conclusion
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. New technologies based on synthetic genetics
platforms even allow the reconstruction of viruses based on their genomic sequence, without the
need of a natural isolate.60
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”.61
Acknowledgements
We thank Prof. Allan Krill (NTNU) for proof reading the manuscript and the valuable comments; Prof.
Heribert Insam (Head of the Department of Microbiology; University of Innsbruck) for his support
and Dr. Lawrence Sellin for all the valuable information. A special thanks goes to Dr. Fernando
Castro-Chavez (New York Medical College) and to René Bergelt for their support.
Conflicts of Interest Statement
RS and YD do not have any conflicts of interest.
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