Note -- this is a guest post thus the "Other Voices" category; the author(s) have asked to remain unattributed.   There have been no edits other than spacing and formatting necessary to post, and make sure the links to references display.

Many questions remain unanswered about the origin of SARS-CoV-2, and we are certainly not the only scientists that have them. There are likely benign convincing explanations to everything, but to date we have not seen them. 

Some will say: why does knowing the origin matter? It matters for several reasons. First of all, it will help us plan for the future. If this indeed was a virus that arose from close contact with wildlife and humans, this contact in the future will have to be managed. Secondly, if in the unlikely event this was perhaps escape from a lab, then lab procedures will have to be evaluated, and lab experiments with infectious possibly pandemic viruses will have to be additionally regulated. Finally, if this again was an unlikely escape from a lab, then knowing the exact type of virus we are dealing with would help us manage the current pandemic.

The story starts, we believe, with a noble goal: to prevent the world from ever having the type of pandemic we are currently experiencing, through production of a vaccine effective against all coronaviruses past and future.

Coronavirus vaccines can be difficult to make. In animals, while vaccines are sometimes successful, toxicity of the vaccine as well as incomplete immunity can happen. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284272/ .

SARS-CoV-2 (COVID19) is a lot like SARS-CoV (SARS). SARS initially hit China and East Asia in 2003, killed 774 people, infected over 8000, and scared everyone. For the past 17 years there has been an enormous effort worldwide to develop a vaccine not only against SARS but also against all coronavirus strains. As we have detailed in another post, scientists knew in 2006 that recombinant spike protein RBD vaccines to SARS didn’t protect all animals from a re-challenge from a slightly different mutated coronavirus ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7124095/pdf/978-0-387-33012-9_Chapter_101.pdf ). Young animals can gain immunity, but it can be harder to get protective immunity in older animals ( https://pubmed.ncbi.nlm.nih.gov/17194199/ ). 

Killed whole coronavirus vaccines in animal models of SARS infection demonstrated that in some animals such as ferrets, killed whole virus vaccines gave a predominant Th2 response (you want Th1) and that there was an antibody dependent enhancement (ADE) of lung toxicity in mice ( https://jvi.asm.org/content/85/23/12201 ). This led to the idea of a live attenuated coronavirus vaccine ( https://www.nature.com/articles/nrmicro3143 ) ( https://www.nature.com/articles/nm.2972 ) ( https://www.nature.com/articles/nbt.1635 ), which is a vaccine that infects and reproduces in a human, results in immunity, but does not cause severe disease.

This has been an important issue since the SARS pandemic in 2003 and was reinforced by MERS epidemic in 2012. Enormous amounts of resources and human effort have been thrown at this problem, and these resources have come from just about everyone: governments, industry, NGOs, and philanthropy. 

Various live virus attenuated recombinant vaccines have two issues among many. First, these vaccines have to be able to be grown in culture in great quantity for testing and for mass production. Second, the vaccines can mutate and revert back to severe pathogenicity once administered, especially in those with weaker innate immune systems. These dual problems have confronted vaccine developers. 

To make a recombinant attenuated live coronavirus vaccine that you can grow in culture, it should be pretty obvious that (a) you have to have it gain function to make it more viable in culture; and (b) while at the same time make it less pathogenic and less able to recombine. These are somewhat contradictory goals and can be hard to do.

Live attenuated vaccines can be hard to make without them mutating a lot in culture in ways you do not expect, not being able to grow them in culture to make lots of virus to work with, and without them reverting back to a dangerous live virus once someone (animal or human) is vaccinated, as happened in 1998 with a live attenuated poliovirus vaccination on the island of Hispaniola (Haiti and the Dominican Republic) ( https://pubmed.ncbi.nlm.nih.gov/11896235/ ).

Virologists have been making recombinant man-made coronaviruses to try to find one that is safe and will work as an attenuated vaccine for over 15 years. To do this they use “reverse genetic systems” to make the virus they want in bacteria or yeast ( https://www.pnas.org/content/100/22/12995 )( https://www.pnas.org/content/110/40/16157 ). In these reverse genetic systems, to make man-made viruses with specific functions, mutations are inserted into the DNA copies of the virus. Bacteria or yeast make lots of these DNA copies, which are added mammalian cells in culture to provide live RNA viruses in the fluid surrounding them. These fluids containing the man-made viruses can then be used to infect animals or people ( https://pubmed.ncbi.nlm.nih.gov/19036930/ ). The goals of these experiments were likely twofold: (1) to find out how viruses like SARS can jump from bats to humans, to develop countermeasures; and (2) to develop universal vaccines to protect the world in the case of a coronavirus pandemic.

Scientists have used several man-made strategies to make viruses weaker to use as possible coronavirus vaccines. These strategies included increasing the number of attenuating mutations in the virus through alteration of a necessary viral RNA proofreading enzyme called ExoN ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3518599/ ), but these types of viruses were found to revert to a more aggressive phenotype with long term culture of 250 passages ( https://mbio.asm.org/content/8/6/e01503-17.abstract ). Viruses also were mutated to not recombine with each other (possibly increasing virulence through recombination) by changing a viral RNA “leader” sequence (ACGAAC to UGGUCGC) possibly responsible for this recombination ( https://www.nature.com/articles/s42003-018-0175-7 ).  Additionally, there has been consideration to mutate viruses in a way, called “codon de-optimization” in which the RNA of the virus is changed to make the same viral proteins, but make them slower, and thus have slower virus growth ( https://jvi.asm.org/content/89/7/3523 ). This has been done for multiple viruses including influenza ( https://pubmed.ncbi.nlm.nih.gov/20543832/ ). 

As noted above, a major issue with experiments like these is that to fully examine the ways viruses jump from bats to people, and to fully develop man-made recombinant vaccines sometimes viruses are unexpectedly made that have the potential to become much more transmissible in animals and possibly in humans. These are called “gain-of-function” experiments. This appeared to happen in at least one experiment in mice published in 2015 ( https://pubmed.ncbi.nlm.nih.gov/26552008/ ). The danger of these experiments needs to be carefully weighed against the possible costs, since leaks from laboratories of viruses are not uncommon ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC416634/ ), and if a gain of function strain were to escape, we could have a pandemic similar to the one we are currently experiencing.

The US scientific community held a symposium in 2014, which led to the banning of funding of these “gain of function” experiments https://mbio.asm.org/content/5/6/e02366-14 . This ban was rescinded in 2016 https://osp.od.nih.gov/biotechnology/gain-of-function-research/ . Minutes of the meetings of the advisory body (the National Scientific Advisory Board for Biosecurity, or NSABB) to help the NIH and Secretary of Health (at the time, Sylvia Burwell) to decide to rescind the ban were heated ( https://osp.od.nih.gov/wp-content/uploads/2016/11/NSABB_January_2016_Meeting_Minutes.pdf ). The NSABB members asked for multiple safety “guardrails” for this research to proceed.

In 2013, six miners in Yunnan Province, 550 miles south of Wuhan, were cleaning out a copper mine of bat droppings. They all developed a severe pneumonia with symptoms very similar to SARS-CoV-2 pneumonia, and three died. During the workup of these miners it was determined that several of the miners developed antibodies to a SARS-like coronavirus ( https://www.documentcloud.org/documents/6981198-Analysis-of-Six-Patients-With-Unknown-Viruses.html ).

The WIV (Wuhan Institute of Virology) became involved, and in 2013-2014 isolated viruses from fecal swabs of 276 bats in the cave and found novel SARS-like coronaviruses in 138 of them. These samples were brought to Wuhan for further study. Manuscripts describing these viruses were published in 2016, but for some reason the publication left out the deaths of the miners from the cave ( https://www.pnas.org/content/100/22/12995 ) ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090819/ ).

Investigators then determined that about 9% of residents of villages around the Yunnan cave had developed antibodies to a SARS-like coronavirus, yet none of them appeared to have severe symptoms. All of these villagers described bats flying around, and none of them visited the cities where SARS was endemic in 2003, so they likely developed an asymptomatic infection ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6178078/ ). 

This experiment of nature likely became a matter of intense interest. How could these humans around the cave be exposed to bats carrying a supposedly lethal SARS-like coronavirus yet have no or minimal symptoms? The implications of the answers to this question were obvious: (a) bat to human transmission could be probed in depth, and (b) a live attenuated vaccine to coronavirus could be developed by mimicking the natural process that led to an attenuated virus that caused antibody production to a SARS-like coronavirus without symptoms.

It is entirely possible that investigators at the WIV could have been performing such experiments in coronavirus gain-of-function manipulation, trying to obtain a live attenuated virus for an attenuated vaccine, based on coronaviruses isolated from Yunnan province or elsewhere, when there was a laboratory accident/leak of virus sometime in the fall of 2019.

Prominent virologists argued in a letter to Nature Medicine in early March ( https://www.nature.com/articles/s41591-020-0820-9 ) that lab escape, while not being entirely ruled out, was unlikely. Yet there are a number of unusual features of SARS-CoV-2, as well as some unusual scientific behavior of Chinese and US investigators, that requires explanation. Hopefully there are simple and trivial explanations, and that SARS-CoV-2 can be explained as a natural experiment that mimicked very closely a series of laboratory experiments performed in the exact place (Wuhan) where the virus was initially found in humans.

These questions point to a manipulated virus, which may have escaped from a WIV lab. These questions can be answered by an independent audit of the WIV P4 laboratories where the novel coronaviruses were being studied, which would have been one of the first steps international authorities usually take. Such an independent audit has not taken place, which is highly unusual scientific behavior.

Hopefully there are simple and trivial explanations, and that SARS-CoV-2 can be explained as a natural experiment that mimicked very closely a series of experiments performed in the exact place (Wuhan) where the virus was initially found in humans.

These questions, in no particular order, are as follows:

• Why didn’t the investigators mention the 2013 deaths of the miners from a likely novel coronavirus in their 2016 paper describing the sequence of novel Yunnan coronaviruses ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090819/ )? This would have been a matter of intense interest worldwide.
  
  
• SARS-CoV-2, from its earliest isolates, appears to have optimal spike protein binding to ACE2, with very few new receptor binding domain (RBD) mutations in SARS-CoV-2 ( https://www.biorxiv.org/content/10.1101/2020.05.03.074567v2 ). Usually viruses like coronavirus adapt their spike protein over time to bind human ACE2 more tightly. Can this be explained naturally?
  
  
• A pangolin coronavirus isolated in 2019 under study at the WIV and elsewhere appears to have an RBD of its spike protein is very close to SARS-CoV-2 (97.4% amino acid homology) where the rest of this pangolin coronavirus has less homology (85-92%) ( https://www.nature.com/articles/s41586-020-2169-0 ). RaTG13 has a lot less homology in the RBD with SARS-CoV-2, yet is more than 96% identical to SARS-CoV-2 over the entire virus ( https://www.cell.com/cell/pdf/S0092-8674(20)30262-2.pdf ). Recombination to make SARS-CoV-2 from an ancestor of these two viruses would be an extremely rare one-time event. Is there a natural explanation for this?
  
  
• In 2018, experiments with a novel recombinant SARS coronavirus substituted the typical TRS transcription leader and body sequences with a novel sequence (UGGUCGC) in an attempt to further reduce recombination in animal models as a live vaccine candidate ( https://www.nature.com/articles/s42003-018-0175-7#Sec7 ). This TRS leader sequence, supposedly novel, is found starting at nucleotide 1465 in SARS-CoV-2 and could result, if utilized, in a novel viral RNA transcript that deletes part of the nsp2 protein of the ORF 1ab polyprotein. It is also found at nucleotide 1446 in RaTG13, one of the viruses found in the Yunnan cave in 2013, which has been proposed as a precursor to SARS-CoV-2. It is found in that area in no other coronavirus. This novel TRS sequence is also found in the 3’ ends of viral spike RNA transcripts of SARS-CoV. Why was this not mentioned in the 2018 paper describing the novel TRS sequence recombinant viruses? Is there an explanation for this? 
  
  
• A recombinant SARS-like coronavirus (SHC015-MA15) containing a human SARS Urbani spike protein sequence inserted into a mouse adapted SARS-like coronavirus was found to develop increased pathogenicity during infection of aged but not young mice compared to the parent MA15 strain in a 2015 publication ( https://www.nature.com/articles/nm.3985 ). The RNA sequence of this recombinant manipulated coronavirus was not deposited in Genbank until May 2020, five years later ( https://www.nature.com/articles/s41591-020-0924-2 ). This is highly unusual behavior. Is there an explanation for this?
  
  
• In the publication of SHC015-MA15 above in November 2015, the attribution of funding of Shi Zheng Li by the US NIAID was initially left out ( https://www.nature.com/articles/nm0416-446d ).  It was reinstated in the publication in 2016 in a corrigendum, perhaps after the meeting in January 2016 to possibly reinstate NIH funding for gain of function virus research. This is also unusual scientific behavior. Is there an explanation for this?
  
  
• After amino acid 604 of the spike protein of RaTG13, there is no difference between the spike of RaTG13 and SARS-CoV-2, yet other coronaviruses have multiple differences in this area ( https://www.cell.com/cell/pdf/S0092-8674(20)30262-2.pdf ).  The only difference between these two viruses is in the PRRA furin cleavage site, and there are novel nucleotide changes apparently inserted into SARS-CoV-2 that allow insertion of this furin cleavage site into RaTG13 ( https://medium.com/@yurideigin/lab-made-cov2-genealogy-through-the-lens-of-gain-of-function-research-f96dd7413748 ).  Is there a natural explanation for this?

There are quite possibly very simple and very benign explanations for all of these questions as well as others. They likely can be answered by a simple independent audit of the WIV P4 laboratories where the novel coronaviruses were being studied. This is one of the first steps international authorities usually take. Such an independent audit has not taken place, which is highly unusual scientific behavior.

The origin of the virus is extremely important in helping us determine what is going to happen going forward in the pandemic, as well as to suggest what possible therapies could have activity. 

It is therefore critical that we determine a precisely as we can the origin of the virus.

We are not the only scientists considering this.
https://onlinelibrary.wiley.com/doi/10.1002/bies.202000091 

We do not want in any way to cast aspersions on individual scientists or countries. All we want to do, as we have done from the beginning, is ask questions of the data.  We hope that there is international cooperation to put this issue to rest, hopefully with a benign conclusion. We also hope there is again widespread honest discussion about the risks and the benefits of gain of function viral research.