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

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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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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 Rossana.Segreto@uibk.ac.at. Tel.: +43-512-50751252.
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 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
market.1
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
publication.
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.
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.
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).
Acknowledgements
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.
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