Tracing The Origin Question Of Coronavirus And Associated Controversies

More than 3.36 million people have succumbed to Covid-19, a highly infectious disease caused by SARS-CoV-2 (SARS2), that first surfaced in Wuhan, China in December 2019.

SARS2 is a novel coronavirus belonging to the beta-coronavirus genus and is the third lethal virus (SARS-CoV and MERS-CoV being the other two) of this genus to have crossed the species barrier and was identified as the primary cause of severe respiratory distress and pneumonia — Covid-19.

In addition to the three outbreaks by newly emerged highly pathogenic coronaviruses, four other low-pathogenicity coronaviruses — HCoV-OC43, HCoV-HKU1, HCoV-NL63 and HCoV-229E — have crossed over to human beings and are endemic in nature.

While SARS-CoV (hereinafter called SARS1) which caused severe acute respiratory syndrome (SARS) in 2003 and MERS-CoV, the reason for Middle East Respiratory Syndrome (MERS) in 2012 — did not go past epidemic stage; Covid-19 caused by SARS2 is in the seventeenth month of global pandemic and continues to endanger global health and disrupt businesses globally.

For the sake of easy reading, this essay is divided into two sections. Section one deals with the molecular structure of virus and how it works, and Section two is for various hypothesis regarding its origin and related controversies.

Section One — Virus Structure And How It Works

Molecular Structure Of SARS-CoV-2

SARS2 is an enveloped, positive sense single strand RNA virus of 29.9 kilobase length and 65–125 nanometer diameter with crown-like spikes protruding on the outer surface. Like other two lethal coronaviruses, SARS2 also contains four structural proteins — spike (S) protein, nucleocapsid (N) protein, membrane ‘matrix’ (M) protein, and envelope (E) proteins and several accessory proteins.

Inside the core of SARS2, sits a single strand genetic material called RNA genome. This genetic material is like a blueprint that encodes everything virus needs to replicate inside the host cell.

Bound to the RNA inside capsid are N-proteins that allow the virus to hijack human cells and turn them into virus factories. N-protein provides the packing for genome to help it protect from host cells and viral infections.

A hard protective shell formed by M-proteins forms a protective layer and it is then followed by viral envelope made from twin layer of lipids. This layer protects the genetic material while providing anchor structures needed to infect a cell.

Embedded on the viral envelope are E-proteins, whose role is to help in the assembly of new virus particles formed after host cell is infected by the virus.

Last comes crown like projections protruding from the coronavirus surface known as spike proteins or S-proteins. In common language, they are the keys that help virus open the door of the host cell. S-proteins play a pivotal role in initiating the viral infection in a two-stage process.

First, it helps SARS2 virus in identifying and binding to the host cell through a receptor known as angiotensin converting enzyme 2 (hACE2). Simultaneously, it helps in the fusion of viral and hosts cell membranes and enable virus to penetrate the host cell.

This 3D video beautifully explains the SARS2 cell structure described above.

Viral Replication Process Inside Host Cell

After S-proteins complete the fusion of viral and cell membrane, SARS2 virus enters the host cell, via a process called endocytosis. Upon entry, genome packing opens up first and the genetic material or RNA genome is released into the cell.

Using the genetic information encoded in the RNA genome, the virus starts the replication process and uses ribosomes — also known as protein production machines — to make proteins required for new viruses.

The next stage of replication involves hijacking cell machinery to manufacture a new viral machine called replicase-transcriptase complex or RTC.

The RTC then helps in manufacturing of viral genome, four viral structure proteins and other proteins found in the viral envelop. All the viral components are then assembled into new virus and released as new coronavirus outside the cell, leaving the host cell to die its natural death.

Acute Respiratory Distress Syndrome (ARDS) Caused By SARS2

As we know that SARS2 virus uses hACE2 receptor to bind itself to the target host cell, it is the location of the receptor that decides the trajectory of Covid-19 disease. hACE2 receptors are found on the respiratory tract cell inside lungs and upper esophagus, stratified epithelial cells, myocardial cells, kidney proximal tubule cells and bladder urothelial cells etc.

So when virus uses its natural affinity for hACE2 receptor of these cells to finds its host, the severely infected patients face respiratory problems such as pneumonia, acute respiratory distress syndrome as also disorders of heart, kidneys, and the digestive tract.

After SARS2 coronavirus infects human body, it first enters human respiratory system through the air the infected person breathes in and settles in the space between the pulmonary alveoli and surrounding blood vessels known as capillaries.

The entry of a hitherto unknown virus activates the immune system and overwhelms it, which in turn causes inflammation of the bronchioles and air sacs also known as alveoli.

As infection increase, air sacs are filled with fluids (situation is known as pneumonia) and deprives human organs of oxygen needed to function. This inflammation of the air sacs, when it becomes acute, causes respiratory failure or ARDS.

Section 2 — Origin Hypothesis And Related Controversies

More than 16.5 months, 164 million infections, 3.4 million deaths, millions of jobs losses, trillions of dollars of economic losses since SARS2 first hit Wuhan, China on 31 December 2019 — we remain clueless about the origin of the virus that caused it all.

With every passing day, the mystery on the origin of this virus continues to deepen and the deafening silence of the global leaders is not helping the cause either.

Thankfully, 17 renowned scientists have appealed for the investigation into the origins of Covid-19; igniting hopes of millions of global citizens searching for answers of their continued suffering and pain.

Several theories have been propounded on the mysterious origins of SARS2, ever since it first showed up. Let me revisit each of them for an improved and easy understanding of all such theories.

Zoonotic Origin Of SARS2

Within days of Wuhan being hit by a wave of mysterious fever, pneumonia and respiratory illness, Chinese researchers and virologists quickly zeroed in on a novel coronavirus as the causative agent.

Full length genome of this virus was decoded from the swabs of the affected patients and was uploaded on GISAID databases on 10 January 2020 by Shi Zhengli and her team of virologists of Wuhan Institute of Virology (WIV).

Once SARS2 genome was sequenced, a frantic search to explore the origins of the novel coronavirus began.

As majority of initially infected patients had direct connection with Huanan seafood market in Wuhan, virologists and researchers veered around the theory of natural emergence.

Simultaneously, Shi Zhengli team, after studying the SARS2 genome sequences, found out that genome code of SARS2 was 79.6 per cent and 96.2 per cent identical to genome of SARS1 and RaTG13 respectively.

Interestingly, global virologists came to know about RaTG13 for the first time in seven years after it was first collected from a horseshoe bat fecal sample by Shi Zhengli in 2013 from a mine in Yunnan province.

To date, RaTG13 with 96.2 per cent genomic match is closest known coronavirus to SARS2.

All these findings led Shi Zhengli team to claim that novel coronavirus belongs to the same family as SARS1, and hinted at the possibility that SARS2 evolved naturally, either directly from bats to humans or possibly through intermedite species.

All these findings were published by Shi Zhengli in Nature on 3 February 2020 and this became the foundational paper for zoonotic origin theory.

Premature Death Of Wuhan Wet Market And Pangolin Theory

The Wuhan wet market theory suffered its first jolt after Chinese authorities failed to find any SARS2 positive frozen animals in samples drawn from the market.

Also, viral genome copies of the virus found on the doors knobs/surface/sewer etc, in that market was very low.

Besides, no bats were found traded in the seafood market at that time as bats go into hibernation during winters.

Lastly, Wuhan is not known to have any bat species that carries any of the coronaviruses closely related to SARS2. All these factors were enough to disprove Wuhan wet market as a possible source of spillover.

Next hypothesis in support of natural emergence was that of Pangolin.

For months, Pangolin, one of the most trafficked exotic animals, was touted as the intermediate host species in the zoonotic transmission chain that helped virus jump from bat to humans.

And the reason in support thereof was the coronavirus found in pangolin seized by Chinese custom authorities in 2019.

As per studies, it was found to have 90 per cent genetic similarity with SARS2; albeit much lesser than RaTG13, which is 96.2 per cent identical with SARS2.

Interestingly, while comparing spike protein sequences of SARS2, RaTG13 and Pangolin strain of coronavirus, researchers in both China and Hong Kong found out that receptor binding motif (RBM) of pangolin coronavirus strain was 97.4 per cent identical to RBM region of SARS2.

However, pangolin theory was laid to rest by this paper published by Peter Daszak for the fact that no coronavirus infections/traces were found in pangolin population upstream.

SARS2 Jumped Directly From Bats To Humans

Another extension of natural emergence theory is a recent study that SARS2 jumped directly from bats to humans without needing any intermediate species.

As per the proponents of this theory, the natural evolution process amongst bats, from whom SARS2 has emerged, led to creation of a generalist virus with higher pathogenicity for human beings.

They further cited findings that SARS2 has infected cats, minks and other species — albeit to a lesser degree than humans — in support of their claim.

The other argument given in support of the “direct bat-human jump” claim was the stable and highly optimised nature of SARS2 even after 17 months of its first emergence.

In other words, SARS2 genome has not undergone any evolutionary changes and the genome of currently infected patients can be traced back to the first virus genome extracted from patient “0” of PLA Hospital in Wuhan.

As against this, SARS1 and MERS underwent significant evolutionary changes after jumping to humans so as to adapt to its new biological host before achieving any stability.

However, this theory was questioned by the other researchers on the basis of the findings that SARS2 (that hasn’t changed/adapted since it first infected humans) did not show matching affinity for the bats.

This may be due to the fact that SARS2 has changed genetically so much that it can no longer infect any bat species cell culture, thus rendering direct bat-humans transmission hypothesis highly improbable.

Also, the fact that horseshoe bat species (Rhinolophus affinis) which was used for drawing sample of RaTG13- genetically closest relative of SARS2 — is not native to Wuhan and was instead found 1,900 km away in Monjiang, Yunnan is enough to disprove direct transmission hypothesis.

More so, no outbreak was reported in those areas before Wuhan. However, an exception to this does exist and has been explained in greater detail under subheading — Curious Story of RaTG13 — below.

Evidently, even after more than 16 months, virologists and scientists have not been able to identify the species of the host reservoir or the intermediate species.

Hence, suffice to say that zoonotic transmission trail of SARS2 found on patient “0” of Wuhan outbreak remains incomplete thus eluding us of any conclusive evidence in support of the proximal origin of SARS2.

As compared to SARS2, the zoonotic transmission trail for SARS1 (batsàcivetsà humans) and MERS-CoV {batsàcamelsàhumans) was fully established within four months and nine months of outbreak respectively.

However, the research into the source of origin is continuing and Chinese researchers via this new preprint of 8 March 2021 mentioned a new coronavirus — RpYN06 — having 94.5 per cent identical gene as SARS2 but with a much depleted spike protein.

Natural Emergence: Controversies And Unanswered Questions

1. Curious Story Of RaTG13

RaTG13 shot to fame via Shi Zhengli paper in February 2020 but its curious story goes back to 2012 when six miners from Mojiang mine in Yunnan contracted Covid-19 like illness, leaving three of them dead. The swab samples of these miners were sent by Kunming hospital to the Wuhan Institute of Virology (WIV) for analysis.

It was found that the root cause of the mysterious illness was a SARS (SARS2 was not known then) like virus from horseshoe bats.

Interestingly, the miners’ deaths were not reported to World Health Organization (WHO) nor an attempt was made to investigate the causes of their deaths. Eight years after those deaths, Shi Zhengli, in her interview to Scientific American brushed aside the deaths/infection of miners as fungal infection.

Following the 2012 incident, a team led by Shi Zhengli from WIV went to the site of the infection four times between 2012 and 2013 for sample collection from six bat species found inside the mine. No data is available about visits thereafter and according to some news reports this mine was closed in 2013.

WIV detected 150 alpha-coronaviruses and two beta-coronaviruses in the samples; of the two beta-coronaviruses, one was SARS family virus and other was new coronavirus species.

This February 2016 (the findings of 2016 paper about number of coronaviruses detected were revised in November 2020 here) by Shi Zhengli about Monjiang expeditions gave details of all the coronaviruses including RaBtCov/4991.

But full genome sample of RaBtCov/4991 was not shared with the researchers until Shi Zhengli published her paper on 3 February 2020.

Alongside Shi Zhengli’s foundational paper, a team of researchers from Wuhan University published their own paper comparing genome samples of two patients of 2019 Wuhan outbreak with partial genome sample of RaBtCov/4991.

Note how they used old nomenclature as against newly rechristened RaTG13 used by Shi Zhengli.

Later in an interview to Science magazine, Shi Zhengli explained that RaBt/4991 and RaTG13 are one and the same and she switched to RaTG13 only after her team sequenced the entire virus.

She further claimed that they had never isolated RaTG13 from the sample; and entire sample of RaTG13 was used by the time full genome sequencing was completed following improvement in NGS sequencing technology, and technology in her lab in 2018.

However, a full genome sequence was published only in February 2020. In scientific parlance, any arbitrary switching of tags without disclosing reasons therefor as also non publishing of any crucial information is hair-raising incident but for reasons best known to scientific community, every attempt to ask questions was shot down as conspiracy.

Another noteworthy fact is that her lab removed access to pathogen database Shi Zhengli used to maintain for the project 2013FY113500. If all that wasn’t enough to raise doubts, Shi Zhengli in November 2019 wrote an addendum to her Nature paper of 3 February 2019 and identified Monjiang mine as the site of 2012 incident.

Surprisingly, the number of alpha-coronaviruses and beta-coronaviruses (as compared to what she disclosed in her February 2016 paper) went up from 150 to 284 and two to nine respectively.

In short, it took Shi Zhengli four years and a pandemic to revisit the data of coronaviruses detected in 2016. Bizarre and hair-raising to say the least.

Here it is important to mention that while RaTG13 and SARS2 share 96.2 per cent identical genome, the similarity between the two ends just at that as 4 per cent genomic mismatch in the field of evolutionary biology translates to 51 years of evolutionary gap.

The curious story of RaTG13 holds lot of importance in finding answers to the origin question of SARS2.

Most importantly, the hypothesis that RaTG13/similar coronavirus from these mines or even infected samples sent to WIV by Kunming Hospital for research purpose might have undergone experiments at the WIV including the "gain of function" experiments and evolved into SARS2.

Was RaTG13 used as a backbone for gain of function research involving S-proteins?

Why a mysterious illness of six miners from a site where so many coronaviruses were detected, not studied in detail and reported to WHO?

Did researchers from Shi Zhengli teams catch infection during their trips/or in lab while working on the coronaviruses detected from the mine samples? And, answers for all these important questions can only be found with one person — Shi Zhengli.

2. Conflict Of Interest Of Lead Proponents Of Natural Emergence Theory

Wuhan Institute of Virology, headed by Shi Zhengli, is the leading global centre in the field of coronavirus research. As soon as Wuhan was hit by SARS2, first needle of suspicion began pointing at WIV and its role came under scrutiny.

On 19 February 2020, the Lancet published a statement by 27 public health scientists from eight countries, extending full support to scientists, public health professionals, and medical professionals of China combatting Covid-19.

Expressing their solidarity with Chinese researchers and lauding their efforts in combatting Covid-19, they condemned all conspiracy theories suggesting that Covid-19 does not have a natural origin.

One name amongst the group of 27 was that of Peter Daszak. As the head of EcoHealth Alliance, New York-based non-profit, working in the area of pandemic prevention, Daszak was the principal collaborator of WIV and Shi Zhengli and was responsible for channelising US grants to WIV for research and surveillance of bat coronaviruses in China to prevent future pandemics.

Another conflict of interest stems from the fact that Daszak is a key member of both committees commissioned by WHO and Lancet for examining the evidence for determining origin of the SARS2 virus.

This statement was followedby an article “The proximal origin of SARS-CoV-2” and published by Nature on 17 March 2020, on behalf of Kristian Andersen of Scripps Research Translational Institute and his associates.

Arguing in support of natural origin of SARS2, this article claimed that SARS2 is not a laboratory construct or a purposefully manipulated virus.

His main contention in support of his claim was that SARS2 binds perfectly to ACE2 receptor of the target cell; but when checked with computational analysis, it can’t be termed as best fit and therefore the virus must have arisen by natural selection and not by laboratory manipulation.

To say that as binding is not perfect, it has to be natural only is an absurd argument. Besides, in reality, study of amino acid receptor binding region showed that SARS2 is 95 per cent optimised for binding to ACE2 — perfect by all means.

On the issue of presence of polybasic cleavage site at S1/S2 junction, a scientist of Anderson’s repute made another bizarre argument that it is likely that some of the viruses may have that. Without going into further detail, let me share this blog on point by point rebuttal of Anderson paper.

Thanks to their reputation and clout amongst scientific community, both didn’t face much questioning until this 14 May 2021 letter by 17 leading scientists including Ralph Baric — the leading coronavirus researcher — demanding investigation into the origins of SARS2.

This letter represents the watershed moment in the origin debate and is expected to help assign equal scientific weightage to all conflicting theories denied thus far.

3. Gain Of Function Research At WIV

Gain of function (GOF) research is about using genetic tools and processes like serial passaging on pathogens to increase their ability to cause disease by enhancing their viral properties (like transmissibility, virulence, immunogenicity and host tropism), so as to define the fundamental nature of human-pathogen interaction and gain knowledge about how close a virus is to make a jump to humans.

The argument in favour of GOF is that it allows an assessment of the pandemic potential of emerging pathogens, help public authorities increase community surveillance and health preparedness efforts and make vaccines available in advance so as to deal with the potential outbreak.

Shi Zhengli has been at the forefront of carrying out GOF experiments at her lab in Wuhan.

In fact, Anthony Fauci headed National Institute of Allergy and Infectious Diseases sanctioned Shi Zhengli/WIV grants worth $3.748 million via Peter Daszak run Eco Health Alliance for GOF research on coronaviruses from 2014 onwards.

Interestingly, these grants were issued even while US had imposed moratorium on GOF research between October 2014 and February 2017.

Just to prove how GOF research can help create chimera, collaborative work between two leading coronavirus researchers — Ralph Baric and Shi Zhengli — holds great value.

As detailed in this 2015 paper, both went on to create a novel coronavirus using SARS-CoV as a backbone and replacing its spike protein with another known coronavirus — SHCO14-CoV — for testing experiments on lab culture of human cells/humanised mice in-vivo and in-vitro.

Shi Zhengli followed it up with this 2017 paper detailing how her team of WIV lab virologists created eight chimera viruses using a chimera — WIV1 developed and isolated in 2013 from SARS, as a backbone and after transplanting it with receptor-binding domain (RBD) of different types of coronaviruses.

In fact, stated aim of US funding of GOF research by Shi Zhengli and her lab was to ensure that jump of a coronavirus from humans can be predicted for faster remedial action.

Unfortunately, currently raging pandemic has already exposed claims of GOF proponents as the supposed remedial action took long and by the time it came, pandemic was already out of control with 3.5 million lives lost.

Who knows, in their bid to go ahead of nature, these experiments alone exposed humanity to greater risks, much earlier and differently, that what nature might have planned.

4. Presence Of Furin Site And Higher Affinity of SARS2 To Human ACE2 Receptor

Higher infectivity of SARS2 is best explained by very high affinity of its spike (S) protein for hACE2 receptor of the target host cell as also the presence of a polybasic furin site at the junction of two halves (S1 and S2) of S protein.

Just to explain it in simple terms, if hACE2 receptor is taken as a lock, then S1 subunit containing RBD is its key, that not only helps it to identify hACE2 but also help it to bind with each other tightly.

The tighter the bind, lesser will be the need for extra viruses and higher will be infectivity.

Additionally, for spike protein to get activated, it is essential that both the halves (S1 and S2) must separate from one another.

This separation is carried out by an enzyme called furin. Furin, an enzyme readily available in our body, acts as a cleaver (cutter) and it helps cut S1 and S2 at a site of their junction called as furin cleaver site (FCS).

Later, S2 helps viral membrane fuse with cell membrane to allow virus to penetrate the cell and take over its protein production machinery using viral genome code for production of viral proteins and other components for replication.

It is the presence of FCS, which makes SARS2 unique from other coronaviruses as most coronaviruses don’t contain this site.

Acquiring a furin site in nature is possible by recombination but only between two beta-coronaviruses at clade level (meaning of same ancestry).

As of now, no coronavirus of similar clade as SARS2 is known to possess FCS.

Also, given the nature of SARS2 furin cleavage site, made by insertion of four amino acids (PRRA) at same place, mutation based acquisition is completely ruled out.

That leaves us with the GOF experiments as one plausible reason behind emergence of this site.

However, anything can be said conclusively only if Chinese authorities allow open inspection of WIV lab records.

Another crucial feature of SARS2 is 10-20 times higher affinity of its RBD to hACE2 as compared to that in SARS.

Additionally the RBD present inside S1 sub-unit is placed in a lying down (or hidden position) thus helping it evade immune system.

It is this higher affinity coupled with ability to evade immune system, that helps SARS2 spike bind tightly with hACE2 receptor of the host cell. This near perfect structure to optimise spike protein binding with hACE2 receptor can best be achieved by GOF experiment only.

As we reach the end of this analysis, we can safely conclude that the argument in support of the natural emergence theory of SARS2 is not backed by solid hard conclusive evidence.

Even after 17 months, zoonotic transmission chain leading up to humans from the reservoir host of the virus is still incomplete.

Those opposed to natural emergence hypothesis cite absence of proven zoonotic trail as well as the fully optimised/stable nature of the virus as a reason to claim that SARS2 escaped from the lab.

It is a strange coincidence that the city of outbreak also happens to be the city which houses Wuhan Institute of Virology (WIV) lab — home to largest number of coronaviruses globally, and a leading centre for gain of function experiments.

We should also remain mindful of the fact that lab of WIV acquired BSL-4 level in 2018 only but it has been dealing with coronaviruses and dangerous GOF experiments ever since SARS outbreak of 2003.

Additionally, Wuhan Center for Disease Prevention and Control also has a BSL-2 level lab dealing with coronaviruses.

History is replete with examples of lab leak incidents in world’s most safe and secure labs.

Hence the lab leak theory becomes relevant as it is a real possibility that researchers from these two labs might have contacted a SARS2 like coronavirus during their sample collecting trips or in their lab.

There is also a possibility that the pathogen they may have been working on, jumped out.

US State Department also alluded to this possibility in this factsheet published on 15 January 2021.

Here it is important to mention this paper by two Chinese researchers — Botao Xiao and Lei Xiao — whose preprint titled “The Possible Origins of 2019-nCoV coronavirus” of 6 February 2020 talked about lab leak possibility. Both the researchers have since removed their paper and deactivated their accounts.

The origin of virus question is a question of science and hence every hypothesis needs to be tested scientifically only. It will help global community fight this pandemic effectively and aid in avoiding similar situations in future.

Political and scientific compulsions aside, those 3.4 million (and counting) lives lost to this virus, deserve to know the source of the virus that killed them and so do their loved ones left behind by them.

Initial sign of cracks in the omerta code amongst researchers and virologists is clearly visible but real key to origin question lies with Chinese authorities and Wuhan labs.