Needed, a transparent investigation into origins of SARS-CoV-2

Shortly after the outbreak of COVID-19 disease from inside Wuhan, China, when the virus infections had reached numerous people across the world, some people began exploring various possibilities around the origins of the current SARS-Coronavirus, also known as SARS-CoV-2. These people included individuals across diverse sectors from many countries such as Scientists, Politicians, Activists, Lawyers and of course the people from Security and Intelligence fraternity, as they are supposed to inform the policy makers.

Eventually, these efforts at uncovering the truth about the COVID-19 origins began to play out in public domain via various media outlets shaping the public discourse around the subject. Multiple theories began emerging and these theories can be broadly grouped into three categories, namely 1) Natural Origin Theory, 2) Laboratory Origin Theory and/ or 3) Bioweapon Theory.

The theorists advocating natural origin of SARS-CoV-2 contend that it is a result of a bat coronavirus (bat-CoV) undergoing zoonotic transfer after a recombination event inside the Pangolins with another coronavirus. The Pangolins in question were reported to have been smuggled into China from Malaysia and were sold in the Sea Food Market in Wuhan. Therefore, according to Natural Origin Theory, current pandemic is supposed to have emerged from the Wuhan Sea Food Market.

The Lab Origin theorists suggest that a genetically engineered virus from the laboratory escaped and led to the current pandemic. They say that this virus was produced through ‘Gain of Function’ research or experiments and according to most of them, it escaped from the BSL-4 laboratory in Wuhan, China, also known as Wuhan Institute of Virology (WIV).

The Bioweapon theory is basically a version of Lab Origin theory wherein theorists indicate towards the use of ‘gain of function’ technology by scientists at WIV to create a highly pathogenic virus at the behest of People’s Liberation Army (PLA). It is this virus that is supposed to have accidently leaked according to some of the Bioweapon theorists while others have asserted that Chinese Communist Party (CCP) leadership decided to release this virus to wreak social and economic havoc at a Global Level.

It is important to examine the scientific information that underpins these theories,  so as to enable an informed public discussion on the subject of COVID-19 origins. So, in the next section we will begin by discussing the basics of this subject to be able to make sense of what is written in the scientific publications that have either been cited or produced by proponents of theories discussed above.

Also important is to review the conduct of Chinese Government during the initial days of the current pandemic. All this would easily suffice to establish that China must be held accountable for the adversities that are being currently experienced across the world because of this pandemic.

BASICS

Coronavirus are a group of viruses that infect mammals and birds to cause respiratory diseases. They can be classified as Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus. The SARS-CoV-2, SARS-CoV-1 – coronavirus responsible for 2002–2004 SARS outbreak -and MERS-CoV virus come under the category of Betacoronavirus.

The Betacoronaviruses can be further categorized in terms of lineages – A, B, C and D, so SARS coronaviruses are lineage B Betacoronaviruses while MERS coronaviruses belong to lineage C.

Now viruses are either made of DNAs or RNAs. They can be double stranded or single stranded and single stranded RNAs can further be classified as positive and negative. So, the SARS-CoV-2 virus is a positive single stranded RNA.

These DNAs or RNAs are basically chains or a sequence of nucleotides that encode all the components of an organism. Each nucleotide consists of a molecule known as Nitrogenous Base. The RNA is made up of nucleotides, each having different nitrogenous base and there are four different types of nitrogenous bases – Adenine (A), Uracil (U), Cytosine (C) and Guanine (G).

Simply speaking, we can refer to nitrogenous bases as A, U, C and G and they are basically alphabets (or codes) that go on to form words (or codons). Every word is formed from a combination of any three out of these four alphabets and this way we can make 64 words. Each word or a codon codes for an amino acid which is the building block of protein and as such there are 20 amino acids. So, a single amino acid can be coded by more than one codon or word.

The RNA strand or viral genome can be represented as sequence of A, U, C and G which is known as nucleotide sequence. Another way of representing them is, as a sequence of amino acids. These two different types of sequences serve different purposes in studying the virus.

STRUCTURE OF CORONAVIRUS

Every virus has a genome which is either a DNA or RNA but never both unlike all other organisms. They have a protein coat called ‘Capsid’ which are proteins arranged in a symmetrical manner and form a protective shell for the nucleic acid. In case of SARS-CoV-2, this symmetry is helical.

Diagram: Coronavirus Structure

The SARS-CoV-2 is an enveloped virus which has a lipo-protein envelope around the capsid. This envelope is constituted by lipids that are derived from the host cell and proteins that are synthesized using the host cell machinery according to the codons inside the RNA Genome. Now there is certain protein that projects outward from this envelope and it is known as Spike protein (S-Protein).

The spike protein (S-protein) in coronavirus has two sections – 1) a receptor binding domain (RBD) and 2) a second section which is made of sequences that facilitate the fusion of virus cell envelope and the host cell membrane.

So, the RBD binds to something called as ACE-2 receptor of the host cell, after which there is an enzyme in the host cell called ‘protease’ which breaks the protein and this enzyme cleaves (split or sever) the S-protein and the fusion sequences of the second section get exposed and virus envelope fuses with the host cell membrane. Different coronaviruses have different S-proteins and they are severed by different host cell protease.

Also, the S-proteins in some coronaviruses get split during their entry into the host cell while others during their exit from the host cell so that they are ready to enter the next host cell. The site at which this cleavage happens is called ‘cleavage site’.

NATURAL ORIGIN THEORY

RATG13

On 3^rd February, 2020 a paper authored by Zheng-Li Shi was published in the Nature that identified a previously found bat coronavirus named RaTG13 as sharing 96% identity – the closest known so far – with the SARS-CoV-2 at a whole genomic level. The scientific publications that support the Natural origin of SARS-CoV-2 base their studies and assertions mainly on this piece of evidence.

According to the database of Global Initiative on Sharing Avian Influenza Data (GISAID), this RaTG13 virus is known to infect the horseshoe bat and it is said to have been discovered in 2013 from a mining cave near the town of Tongguan in Mojiang county, Yunnan, China. However, it has not been mentioned in any of the publications before by Zhengli Shi that were about the bat coronaviruses.

Instead, one of Shi’s previous studies that was published in 2016, highlights a bat coronavirus strain called BtCoV/4991 whose short region of RNA-Dependent RNA Polymerase (Rd-Rp) gene has shown 100% nucleotide identity with the one that belongs to RaTG13. Basically, this implies that RaTG13 and BtCoV/4991 are the same strain because Rd-RP gene is integral to replicate the virus’s genetic material inside the cell. Also, if they were different, Shi’s report should have separately identified BtCoV/4991 as also having high similarity with SARS-CoV-2 Rd-Rp. However, if they are the same strain then one can’t help but wonder, as to what is the rationale behind giving two different names to the same virus strain.

This and many other inconsistencies around the accuracy of RATG13 Genome Sequence and even it’s actual existence have been pointed out in many scientific publications. However, many of such publications exist only as pre-prints or non-peer reviewed articles because of the alleged censorship against arguments that raise objections to the information supporting the Natural Origin narrative.

Pangolins

Several scientific institutions and experts have suggested Pangolins as being the likely intermediate host for the SARS-CoV-2 after the reports emerged that Sunda Pangolins carrying coronaviruses were smuggled into China from Malaysia.

The reason behind this suggestion was that the S-protein of RATG13 virus was very different from that of SARS-CoV-2 as the amino acids in it’s Receptor Binding Domains (RBDs) differ hugely at the sequence level with that of the latter but they were similar to the RBD of Pangolin coronavirus.  However, a recently published study in Nature that was conducted by scientists in Australia, has shown results that strongly contradict this suggestion.

The scientists in Australia compared the affinity of ACE-2 receptors of human, pangolin, dog, monkey, hamster, ferret, cat, tiger, bat, civet, horse, cow, snake and mouse, towards the RBD of SARS-CoV-2. They found that human ACE-2 (h ACE-2) and Pangolin ACE-2 had highest affinity towards SARS-CoV-2 RBD but most importantly, h ACE-2 had much higher binding potential to the SARS-CoV-2 RBD than that of the Pangolin. Now this is not normal if we assume Pangolin as an intermediate host for SARS-CoV-2 because a zoonotic virus would normally have the highest affinity for it’s immediate preceding host, as it takes time to adapt to the new host.

LAB ORIGIN THEORY

ZC45 and ZXC21

On 12^th January, 2020 Eddie Holmes an Australian virologist who is also a colleague of Professor Yong-Zhen Zhang from Fudan University, Shanghai put out the genome of SARS-CoV-2 and later, a paper based on Professor Zhang’s discovery was published by Nature on 3^rd February, 2020, alongside the paper authored by Zheng-Li Shi that linked the SARS-CoV-2 with RATG13.

An interesting feature of Professor Zhang’s paper was that it did not mention RATG13 or BtCoV/4991 in it’s paper but found another bat coronavirus named bat SL-CoVZC45 (or ZC45) as sharing high sequence identity of 89.1 % with the SARS-CoV-2 at nucleotide level. The paper also discusses another bat SARS-like virus ZCX21 which is highly identical to ZC45 and suggests possible recombination events between these viruses.

Professor Zhang’s paper was picked up by many among the Scientific community and one of them is Dr. Li-Meng Yan, a Chinese virologist who as of now is probably the leading proponent of bio weapon theory. She has fled to U.S. from Hong Kong and has authored multiple papers to substantiate her assertions. Her paper provides some more information corroborating the findings of Professor Zhang.

Dr. Yan’s paper explains that when SARS-CoV-2 and ZC45/ZXC21 are compared at amino acid level there is a high sequence identity observed for most of the proteins between these viruses. Among those proteins, she highlights the high sequence identity for Orf8 proteins and E protein, simultaneously, between these viruses as one of the definitive evidence that ZC45/ZXC21 is the backbone for SARS-CoV-2. According to her findings, the Orf8 proteins in these viruses are 94.2% identical while no other coronaviruses share more than 58% identity with SARS-CoV-2 on this particular protein. Also, the E proteins in these viruses share 100% sequence identity and this has been observed between the previous SARS-CoV-1 and other SARS-like viruses but none of those pairs simultaneously share over 83% identity on the Orf8 protein.

Therefore, Dr. Li Meng Yan suggests that ZC45/ZXC21 is probably the template used for creating SARS-CoV-2 in the laboratory through genetic ‘gain-of-function’ modifications.

Receptor Binding Domain (RBD)

According to Professor Zhang’s findings there were evidences to suggest that recombination events had occurred between SARS-CoV-2, SARS-CoV-1, ZC45/ZXC21. However, this recombination event was probably limited to the Spike gene and not the entire genome. So, the Receptor Binding Domain (RBD) region of the spike gene in SARS-CoV-2 was highly similar to the SARS-CoV-1 RBD, while the rest of the sequences were most closely related to ZC45 and ZXC21.

The paper also stated, “Despite these recombination events, which seem relatively common among sarbecoviruses, there is no evidence that recombination has facilitated the emergence of WHCV (SARS-CoV-2).” So, if there is no evidence of recombination behind the emergence of SARS-CoV-2 then this opens up an avenue for discussion on genetic engineering as one of the possible reasons.

Dr. Yan’s paper dwells more into the subject of Spike protein of SARS-CoV-2 and it’s similarity with SARS-Cov-1. Her findings show that the S2 functional domain of the Spike protein in SARS-CoV-2 shares a high sequence identity of 95% with that of ZC45/ZXC21 but the S1 functional domains – the RBDs – in these viruses which determine the host that the virus can infect share only 69% amino acid sequence identity. This is because the RBD of SARS-CoV-2 is highly identical to that of SARS-CoV-1.

Also, Dr. Yan’s paper further examines the bind between the h-ACE-2 and the RBDs of SARS-CoV-2 & SARS-CoV-1. Her findings show that the hACE2 bindings with both the RBDs resemble greatly however they are not exact copy paste. This is perhaps because of the difference in nucleotide sequences between the two as there can be more than one codon used to code for same amino acid. Also, some of the amino acid residues in the RBD of SARS-CoV-1 that are non-essential for the RBD-hACE2 binding are absent in SARS-CoV-2 RBD Which probably helps to obscure the link between the RBDs of these viruses.

Two unique restriction sites – EcoRI and BstEII

As we discussed in the previous section that many laboratory origin theorists contend that the SARS-CoV-2 RBD is actually the customized SARS-CoV-1 RBD which is swapped with the ZC45/ZXC21 RBD.

So, Li Meng Yan in her paper provides information to substantiate this contention. Her paper has emphasized on the presence of Restriction Sites on either end of the SARS-CoV-2 RBD that are absent in the ZC45/ZXC21 viruses. The Restriction Sites are those segments of nucleotide sequences that can be cut by Restriction Enzymes, such as EcoRI and BstEII, which happen to be popular choices for everyday molecular cloning, as suggested by Dr. Yan. A particular Restriction Enzyme will only cut a specific sequence of nucleotide bases which means that a Restriction site is unique to a Restriction Enzyme.

Dr. Yan’s paper states that SARS-CoV-2 RBM has a restriction site on one of it’s end that can be cut by EcoRI and on the other end there is another restriction site that can be cut by BstEII. Now, the ZC45/ZXC21 viruses originally do not have these restriction sites so Dr. Yan suggests that they could be easily introduced on either end of their RBM by deleting and inserting certain nucleotide bases through a method known as Site Directed Metagenesis. Then they could have been cut by EcoRI and BstEII so as to swap the original RBM with the customized SARS-Cov-1 RBD.

However, this can only be true if ZC45/ZXC21 are indeed the backbone of the current SARS-CoV-2 virus because the RATG13 virus which is currently accepted backbone for SARS-CoV-2 does have these restriction sites. Also, the restriction sites can be naturally present in virus genomes. Which is why Dr. Yan also states, “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.”

Furin like Cleavage Site

Back in the year 2020, Dr. Francis Boyle, an American Human Rights Lawyer, gave an interview to Geopolitics & Empire where he said that the SARS-CoV-2 is a bioweapon that leaked from the BS4L facility in Wuhan, China. He based his claims on a paper authored by a group of scientists from France and Canada which was published by Elsevier.

Dr. Boyle is known to have drafted the US domestic implementing legislation for the biological weapons convention, known as the Biological Weapons Anti-Terrorism Act of 1989. He has served as a counsel to governments and has advocated for various organisations in the areas of human rights, war crimes and genocide, nuclear policy, and biowarfare.

This paper that was cited by Dr. Boyle highlighted the presence of peculiar furin-like cleavage site in the SARS-CoV-2 that was absent in the other SARS or SARS-like CoVs. This furin like cleavage site is found present in the spike protein of SARS-CoV-2 virus.

As discussed before, once the RBD binds to the receptor of a host cell, the protease in host cell cleaves the S-protein and the fusion sequences of the second section get exposed and virus envelope fuses with the host cell membrane. Now according to what is usually observed, SARS-CoV-1 belongs to the category of coronaviruses whose S-protein gets split during entry into the host cell and MERS-CoV belongs to the second category whose S-protein gets split during exit from the host cell so that it is ready to enter the next host cell.

Therefore, the cutting of S-protein is a very decisively important factor when it comes to cross species transmission of virus because even if the RBD in the first section of S-Protein is able to bind to a receptor of host cell, if the S-protein is not already cleaved or if a protease required to split it is not present in the host cell, the virus genome cannot enter inside it.

The S-protein of SARS-CoV-1 is reported to be cleaved by the protease called TMPRSS2 present in human epithelial cells during it’s entry. The research presented in the paper cited by Dr. Boyle shows that S-protein of SARS-CoV-2 contains cleavage site for furin protease. ‘Furin’ is a protease found abundantly in respiratory tract of humans and furin cleavage site is found generally in the S-protein of MERS-CoV which, as mentioned before is known to be cleaved during the virus exit from the host cell. Which is why the presence of a furin like cleavage site in the S-protein of SARS-CoV-2 indicates towards the possibility of cleavage occurring during virus exit which is in contrast to what is generally known and therefore ‘peculiar’.

However, the paper does not confirm if this cleavage site actually gets severed or not therefore it is possible that the S-protein of SARS-CoV-2 gets cut during virus entry, just as normally observed. Otherwise, it is also possible that there are two cleavage sites in the S-protein of SARS-CoV-2 that can be cleaved by two different protease in the cell – TMPRSS2 and Furin – during virus entry and exit respectively. Also important is that the paper does not confirm or deny if this Furin-like cleavage site is the only cleavage site present.

Now how does the presence of furin-like cleavage site allude to the possibility of laboratory origin? Interestingly, this furin like cleavage site is not known to be present inside any of the bat-CoVs – RATG13, ZC45/ZXC21 – that have been suggested as possible backbones for the present SARS-CoV-2 virus by different theorists. This indicates towards the occurrence of an insertion of this furin-like cleavage site into the bat CoV, which allowed it to infect humans. Now such an insertion can either occur due to it’s recombination with another virus that possessed that site inside an intermediate host or they can be inserted into the backbone virus inside lab.

Variations around the S-protein cleavage site are known to play a role in cellular tropism and pathogenesis. The researchers in the paper say, “This furin-like cleavage site, is supposed to be cleaved during virus egress (exit) for S-protein “priming” and may provide a gain-of-function to the 2019-nCoV for efficient spreading in the human population compared to other lineage b betacoronaviruses”. So, Dr. Boyle while citing this paper, specifically emphasized on the use of phrase ‘Gain of Function’ in this sentence when he made his assertion that this is a “smoking gun” evidence because “GOF technology is DNA genetic engineering which has no other legitimate scientific purpose except make biological weapons and can be carried out safely only in BSL-4 or a BSL-3 facility”.

Now, GOF is a result of mutations. Mutations are classified on the basis of their impact on the function of virus or a protein inside it. The GOF mutations have incremental effects on this function and therefore they are also known as activating mutations. These mutations can either occur naturally or they can be carried out in laboratories. There is a discipline of research in virology known as Gain of Function research or studies which involves increasing the ability of a virus to cause disease. While this increased ability of the virus can indeed be used as a ‘bio-weapon’, as suggested by Dr. Boyle and therefore he is not completely inaccurate but it is not true that that there is “no other legitimate scientific purpose” behind GOF research other than making bioweapon.

GOF research are conducted to study the human-pathogen interactions which can enable appraisal of the pandemic potential of emerging infectious agents. Also, GOF studies are required for vaccine development. However, there is a consensus within the Scientific community that GOF research involve grave biosecurity risks if they are not carried out properly.

RISKY EXPERIMENTS, LABORATORY SAFETY AND GENERAL OPAQUENESS

In April, 2020 there was a report in Washington Post by Josh Rogin which claimed to have obtained two U.S. Diplomatic Cables sent from the U.S. Embassy in China to State Department. In these cables, the U.S. Diplomats in China voiced their concerns about the safety and management issues in BSL-4 lab facility of WIV. One of those cables also emphasized on the lab’s work on bat coronaviruses and their potential human transmission which posed a risk of new SARS-like pandemic. According to those cables, as reported in Washington Post, in their visit to the BSL-4 lab facility, the U.S. officials found serious shortage of appropriately trained technicians and investigators needed to safely operate that high-containment laboratory.

Besides, there have been other previously reported instances of such concerns raised by researchers and scientists about works in which Shi Zengli was involved.

In 2015, U.S. and Chinese scientists which included Shi Zengli, published a paper about a chimeric virus that they developed by substituting or swapping a gene that encoded spike glycoprotein of SARS like virus obtained from bats, with a gene inside the mouse adopted SARS-CoV backbone. The ‘mouse adopted SARS-CoV’, means the SARS-CoV was able to replicate in mice after series of passages from mouse to mouse. The scientists found that the chimeric virus produced by them was able to replicate efficiently in primary human airway cells which they described as surprising.

This paper drew criticism from experts such as Richard H. Ebright and Simon Wain-Hobson. Elbright, an esteemed American micro-biologist who is also known for his work on biosecurity said, “The only impact of this work is the creation, in a lab, of a new, non-natural risk” and Wain-Hobson, virologist at the Pasteur Institute in Paris described this study as the creation of a novel virus that grew remarkably well in human cells and if it escaped, nobody could predict the trajectory.

Also, there have been multiple known instances of laboratory leaks of SARS-viruses in different countries before, which includes China itself. The other known countries are Singapore and Taiwan. Therefore, laboratory leak theory cannot be discounted. It is also important to consider that governments in Singapore and Taiwan had addressed their occurrences of laboratory leak with complete transparency and co-operation with the International Agencies such as WHO. The Chinese government institutions, however, failed to do so.

Even this time in case of the current COVID-19 pandemic as reported by the media outlet Caixin Global, it was discovered as early as December, 2019 that there is an outbreak of a viral disease which has pandemic potential but China tried to conceal it from rest of the world and covered it up by prohibiting further tests and public sharing of test results, etc. This kept the measures such as restrictions on travel or public gathering from implementing. Besides, in 2002-03, the SARS epidemic had originated in China and it spread from China to 37 countries and even then China had not disclosed about the SARS disease until it was too late.

CONCLUSION

The above presented information makes it clear that there is a need for a thorough investigation into the origins of the current SARS-CoV-2 virus and it must be conducted in an unbiased manner. For that to happen, the International Community must exert pressure over the Chinese government to cooperate with absolute transparency. This pressure will have to be exerted by governments through international organisations and agencies that are comprised of them. However, this is not possible unless the public mandate forces their governments to do that which is necessary and this in turn is dependent on the quality of public discourse on this subject. It is therefore important that this debate about the COVID-19 origins, within the scientific community is brought to masses. This article is an attempt towards that end.

Tanmay Kadam is an engineer-turned-geopolitical practitioner.