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On the SARS-CoV-2 Virus Laboratory Origins Hypothesis: What Fauci and Collins Knew, and When They Knew It
And how the "Proximal Origins" paper distorted their shared understanding, preventing the world from early comprehension of the virological and immunological landscape...
Prelude: I do not pretend to reproduce the full 72 pages of the emails here. I have selected what I think are the most important parts. Read them all here.
Freelance journalist Jimmy Tobias is a hero. His FOIA fight yielded emails between former NIAID Director Anthony Fauci, Francis Collins, Kristian Anderson (who started the email thread), Jeremy Farrar, and Marion Koopsman.
Wikipedia: Sir Jeremy James Farrar OBE FRCP FRS FMedSci (born 1 September 1961) is a British medical researcher and director of the Wellcome Trust since 2013.
It’s one thing to publish a paper based on data and to have made an error in the data analysis. It’s a whole different story to gin up a perspective piece based on no data supportive of, or ruling out, causality, and to represent one’s conclusion as ‘firm’ when the data are, in fact, equivocal - or wrong - on the matter. And then’s an entirely different matter to freely discuss how the SARS-CoV-2 sequence data clearly show that serial passage in humanized ACE-2 mice was a plausible - and likely - means to given SAR-CoV-2 its uncanny ability to infected human ACE-2 expressing cells, only then to turn around in public and say the evolution of that capacity was evidence AGAINST the possibility of serial passage using humanized ACE-2 mice and mispresent the literature on whether such studies had been done in the past, or indeed ongoing leading up the discovery of the SARS-CoV-2 virus in humans.
The authors of the paper also failed to mention the serious - and repeated - and long-standing biosecurity issues at the Wuhan Institute for Virology (WIV), despite communications brought forward by Tobias showing they were all well aware of the lax standards of biosecurity in place at WIV.
In the abysmally bad “Proximal origins” paper, the authors represented laboratory evolution of the virus via serial passage - the infection of one animal another, or one cell line after another - as unlikely given the sequence similarity of part of the SARS-CoV-2 spike protein to the pangolin sequence that miraculously appeared in the databases on 2020-01-22. Importantly, the pangolin has since been ruled out as an intermediate host related to the origins of SARS-CoV-2.
The emails, however, show that there was a range of willingness to accept laboratory origin and that the group was waiting for the publication of the pangolin sequences so they might have a footing to use to reject serial passage.
Fauci also first thought of going to the FBI if the concerns were warranted - but in the end, the group wanted to defer to Tedros of the World Health Organization.
First, let’s look at a summary of an analysis by Mike Farzan (edited only to correct OCR ambiguity). Remember, Fauci is a recipient and participant in the email thread:
Farrar to Fauci:
From Mike Farzan (discoverer of SARS receptor):
1. The RBD didn't look 'engineered' to him - as in, no human would have selected the individual mutations and cloned them into the RBD (I think we all agree)
2. Tissue culture passage can often lead to gain of basic sites - including furin cleavage sites (this is stuff they have seen with human coronaviruses)
3. He is bothered by the furin site and has a hard time explaining that as an event outside the lab (though, there are possible ways in nature, but highly unlikely)
4. Instead of directed engineering, changes in the RBD and acquisition of furin site would be highly compatible with the idea of continued passage of virus in tissue culture
5. Acquisition of furin site would likely destabilize the virus, but would make it disseminate to new tissues
So given above, a likely explanation could be something as simple as passaging SARS-like CoVs in tissue culture on human cell lines (under BSL-2) for an extended period time, accidentally creating a virus that would be primed for rapid transmission between humans via gain of furin site (from tissue culture) and adaptation to human ACE2 receptor via repeated passage.
All of this brings it back to a simple conversation about how this virus might have gained a furin site (but with a stretch and series of coincidences you can find a way to explain others - although very odd all together (sic)) and there are ways in which that could occur both in nature and in the lab. Nothing seems to
specifically suggest whether this virus was most likely to be "adapted", "evolved", or maybe even "engineered". So I think it becomes a question of how do you put all this together, whether you believe in this series of coincidences, what you know of the lab in Wuhan, how much could be in nature - accidental release or natural event? I am 70:30 or 60:40.
And “From Bob” (Robert F. Garry, PhD) (via Farrar to Fauci):
“…I aligned nCoV with the 96% bat CoV sequenced at WIV. Except for the RBD the S proteins are essentially identical at the amino acid level - well all but the perfect insertion of 12 nucleotides that adds the furin site. 52 is over its whole length essentially identical. I really can't think of a plausible natural scenario where you get from the bat virus or one very similar to it to nCoV where you insert exactly 4 amino acids 12 nucleotide (sic) that all have to be added at the exact same time to gain this function- that and you don't change any other amino acid in 52? I just can't figure out how this gets accomplished in nature. Do the alignment of the spikes at the amino acid level - it's stunning. Of course in the lab it would be easy to generate the perfect 12 base insert that you wanted.
Another scenario is that the progenitor of nCoV was a bat virus with the perfect furin cleavage site generated over evolutionary time. In this scenario RaTG13 the wiv virus was generated by a perfect deletion of 12 nucleotides while essentially not changing any other S2 amino acid. Even more implausible imo.
That is the big if.
You were doing gain of function research you would NOT use an existing clone of sars or mersv. These viruses are already human pathogens. What you would do is clone a bat virus they had not yet emerged, Maybe then pass it in human cells for-a while to lock in the rbs, then you reclone and put in the mutations you are interested - one of the first a polybasic cleavage site.”
The theory of the origin of the has gathered considerable momentum not in social media, but increasingly among some scientists, in mainstream media, and among politicians.
The aim of this was to bring a neutral, respected, scientific group together to look at the data and in a neutral, considered way provide an opinion and we hoped to focus the discussion on the science, not on any conspiracy or other theory and to lay down a respected statement to frame whatever debate goes on - before that debate gets out of hand with potentially hugely damaging ramifications.
With the additional information on the pangolin virus, information not available even 24 hours ago, I think the argument is even clearer.
My preference is that a carefully considered piece of science, early in the public domain, will help mitigate more polarised debate. If not, that debate will increasingly happen and science will be reacting to it. Not a good position to be in.
A lot of good discussion here, so I just wanted to add a couple of things for context that I think are important - and why what we're considering is far from "another conspiracy theory", but rather is taking a valid scientific approach to a question that is increasingly being asked by the public, media, scientists, and politicians (e.g., I have been contacted by Science, NYT, and many other news outlets over the last couple of days about this exact question).
To Ron's question, passage of SARS, like CoVs have been ongoing for several years, and more specifically in Wuhan under BSL-2 conditions -see references 12-15 in the document for a few examples. The fact that Wuhan became the epicenter of the ongoing epidemic caused by nCoV is likely an unfortunate coincidence, but it raises questions that would be wrong to dismiss out of hand. Our main work over the last couple of weeks has been focused on trying to disprove any type of lab theory, but we are at a crossroad where the scientific evidence isn't conclusive enough to say that we have high confidence in any of the three main theories considered. Like Eddie - and I believe Bob, Andrew, and everybody on this email as well - I am very hopeful that the viruses from pangolins will help provide the missing pieces. For now, giving the lab theory serious consideration has been highly effective at countering many of the circulating conspiracy theories, including HIV recombinants, bioengineering, etc. - here's just one example: https://www.factcheck.org/2020/02/baseless-conspiracy-theories-claim-new-coronaviru.s was-bioengineered/.
As to publishing this document in a journal, I am currently not in favor of doing so. I believe that publishing something that is open-ended could backfire at this stage. I think it's important that we try to gather additional evidence - including waiting on the pangolin virus sequences and further scrutinize the furin cleavage site and O-linked glycans - before publishing. That way we can (hopefully) come out with some strong conclusive statements that are based on the best data we have access to. I don't think we are there yet.
I just got off the phone with Kristian Anderson and he related to me his concern about the Furine site mutation in the spike protein of the currently circulating 2019-nCoV. I told him that as soon as possible he and Eddie Holmes should get a group of evolutionary biologists together to examine carefully the data to determine if his concerns are validated. He should do this very quickly and if everyone agrees with this concern, they should report it to the appropriate authorities. I would imagine that in the USA this would be the FBI and in the UK it would be MIS. It would be important to quickly get confirmation of the cause of his concern by experts in the field of coronaviruses and evolutionary biology.
In the meantime, I will alert my US. Government official colleagues of my conversation with you and Kristian and determine what further investigation they recommend. Let us stay in touch.
As reported, Fauci never went to the FBI. Instead, the group went to the WHO in an attempt to turf the hot potato over to Tedros. One can speculate that Fauci understood that Tedros, answering to the CCP, might tap into a plan of action to create data that could make it appear as if the SARS-CoV-2 virus came from an animal intermediate, or at least minimize the impact of the clear evidence that natural zoonotic transfer was the least likely explanation.
I want to go to the main focus on the group on the O-glycans, starting with Kristian’s reluctance to publish the paper he was being pushed to work on:
8 Feb 2020 10:15PM
“As to publishing this document in a journal, I am currently not in favor of doing so. I believe that publishing something that is open-ended could backfire at this stage. I think it's important that we try to gather additional evidence - including waiting on the pangolin virus sequences and further scrutinize the furin cleavage site and O-linked glycans - before publishing. That way we can (hopefully) come out with some strong conclusive statements that are based on the best data we have access to. I don't think we are there yet.”
Earlier discussion included some back-and-forth question and commentary including the now-disgraced former NIH Director Francis Collins:
8 Feb 2020
Would serial passage in an animal in the laboratory give the same result as prolonged adaptation in animals in the wild? Or is there something fundamentally different in what happens when you serially passage versus natural animal adaption? This is not my specific area of expertise and so I do not know.
8 Feb 2020 1:15AM
From Bob, via Farrar
"I'd say the existence of the glycans is pretty strong evidence of evolution in the presence of an immune system. l don't think it is random chance since the glycans appear in other betacoronaviruses that "evolve" a furin site, eg MHV and HKUl. MHV and HKUl also simultaneously evolve a variable and sometimes large patch of 0-linked glycans at the top of the prefusion (virion) form of the spike. Seems pretty clear this is immune based selection all around to me.
Yes serial passage in animals would do the same thing. There are a couple passage of HSNl in chicken papers - the furin site appears in steps."
We also then see this remarkable exchange between Fauci, Collins, and Farrar, pointing clearly to a lack of confidence in WIV biosafety by Farrar - which is not further addressed in the email exchange by either Fauci or Collins:
Fauci: “?? Serial passage in ACE2-transgenic mice”
Collins: “Surely that wouldn't be done in a BSL-2 lab?”
Farrar: “Wild West”
February 5, 2020 (time obscured)
Francis and Tony Couple of things:
I spoke again with WHO this morning. I believe they have listened and acted. Let me know if you agree
At the WHO meeting next week they will set up the Group that will "look at the origins and evolution of 2019n-CoV"
They have asked for names to sit on that Group - please do send any names
We can have a call this week with a core group of that to frame the work of the Group including - if you could join?
I think this puts it under the umbrella of WHO, with action this week and into next
With names to be put forward into the Group from us and pressure on this group from your and our teams next week.
The team will update the draft today and I will forward immediately-they will add further comments on the glycans
Does that sound reasonable to you? Jeremy
''Engineered'' probably not.
Remains a very real possibility of accidental lab passage in animals to give glycans. Will forward immediately or if you want to give Eddie a ring.
Eddie would be 60:40 lab side. I remain 50:50...
Yes, I'd be interested in the proposal of accidental lab passage in animals (which ones?). Francis
Wed, 5 Feb 2020 06:57
I asked Eddie about the addition of the glycans and where these could be added accidentally by passage in lab animals or of course in the wild-the reply from Bob and Kristian
?Kristianthat's correct about everything he said for the P residue. It's what's shifted me to thinking that the insert of the furin site is the result of cell culture passage [or less likely intense transmission in a nonbat host]. Really need to see the data from Ron about generating the furin cleavage site on in vitro passage. Really!
CoV come with or without a furin site. CoV without a furin site are said to be non-cleaved and rely on endosomal proteases like cathepsin for entry. However if you infect a virus like SARS in culture in the presense of exogenous protease like trypsin its lOOX more effective at entering because the spike gets cleaved and it can enter at the cell surface.
You have to infect flu viruses (the ones without the multibasic cleavage site) in the presence of trypsin, and include trypsin in the overlay if you want to get virus spread aka plaques.
This also contributes to the pathogenicity of - well - highly pathogenic flu virus- different tissues have different proteases and are able to "activate" flu to different extents - if the flu v has a furin cleavage site it has a lot more choices and canmore easil go systemic.
This is an excellent review on CoV fusion - deals with all the complexities: https://www.ncb,.nlm.nih.gov/pmc/articles/PMC33973S9/
Bottom line- I think that if you put selection pressure on a Cov without a furin cleavage site in cell culture you could well generate a furin cleavage site after a number of passages (but let's see the data Ron!). It will infect a lot better if it can effectively fuse at the cell surface and doesn't have to rely on endosomal cleavage and receptor mediated endocytosis..?
How did it end up?
Well, the paper pooh-poohed the laboratory origin concept, even though the scientists were clearly aware the other variation should have accumulated elsewhere in the viral sequence that seems to be missing.
The final published paper included language that introduced unnecessary ambiguity regarding the O-linked glycans:
“The function of the predicted O-linked glycans is unclear, but they could create a ‘mucin-like domain’ that shields epitopes or key residues on the SARS-CoV-2 spike protein... Several viruses utilize mucin-like domains as glycan shields involved immunoevasion... Although prediction of O-linked glycosylation is robust, experimental studies are needed to determine if these sites are used in SARS-CoV-2.”
The paper also points to the Chinese’s then-newly reported Pangolin sequence as evidence sufficient to rule out laboratory serial passage origin of the SARS-CoV-2 virus, sequences published on 2020-01-22; most other scientists studying the sequences have shown that the rest of the pangolin SARS-CoV-2 sequence is too distant from the SARS-CoV-2 virus, and the similarity may be the result of convergent evolution (of the virus to ACE2 receptors (see, for example, THIS STUDY and references therein).
Near the end of the email, a message prophecies the demotion of the idea of serial passage/lab leak from an hypothesis to just a comment addressing the issue in the discussion - not because it was not valid to address the question at the level of a full-blown hypothesis, but because what people might do with the fact that scientists were actually interested in it as a scientifically plausible question:
9 Feb, 2020
Marion Koopsman (Dutch Virologist)
Wow took off from e-mail for a day,
As mentioned to Jeremy, I would not be in favour of publishing something specific on the lab escape hypothesis, because I agree (with Kristian) that this could backfire. Yes, there is speculation in the public domain, triggered by several papers, including the rubbish ones. By zooming in on a specific finding that is NOT in the public domain as far as l know, I think this will generate its own conspiracy theories.
So if published, I would suggest zooming out a bit for starters, describing that one of the key challenges is where this virus came from, discuss some of the (wild) guesses out there, and then argue step by step what the challenges are in inferring this, from sequence data, where you do not know exactly what the pool is that you are sampling from, so end up interpreting the needle drawn out of a haystack, Here, the many pieces of the discussion that passed by these last few days can be included, like rates of evolution and dating of possible origins; examples of cleavage site acquisition from other viruses, recombination in coronavirus evolutionary history, possible abrupt changes in spillover events, ability to confirm or disproof things in vitro. etc
And I would leave "lab escape" for the discussion, because putting that in the public domain as a hypothesis in my view will be read as "see, they also thought so"
Science done like this is not the science I recognize, do and teach.
Recap: What Key Details Did the Final Paper Leave Out?
The “Proximal Origins” paper also buries the key information studies had been conducted that pointed to the likely evolution of O-glycans in serial passage studies - and that Wuhan was studying humanized ACE-2 mice.
“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.”
After misrepresenting the scientific literature the author cited and learned about in the literature with each other via email,and misrepresenting the facts of the humanized mice experiments that had been ongoing at WIV, the authors turfed cell-culture serial passage, saying (unbelievably) it was unlikely because it would have required serial passage in animals:
“Finally, the generation of the predicted O-linked glycans is also unlikely to have occurred due to cell-culture passage, as such features suggest the involvement of an immune system.”
Marion Koopman’s involvement is not made apparent even with an acknowledgement.
What Could Have Happened?
Knowledge that the SARS-CoV-2 virus had new capabilities compared to other coronaviruses would have helped researchers focus on therapies and treatments that might interfere with the furin cleavage/O-glycan 1,2-punch.
For example, one you’ve never heard of:
Kifunensine is an alkaloid compound, originally isolated from the actinomycete Kitasporia kifunensis, that exhibits potent and selective inhibition of class I ER- mannosidases. Kifunensine is used to suppress Endoplasmic Reticulum-Associated Degradation (ERAD) via the inhibition of endoplasmic reticulum-associated mannosidase activity.
Now, I’m not saying people should run out and get some Kitasporia fungi, but the specific mechanism of action of kifunensine might be mimicked by some compounds researched by Universities funded by the NIH - by Collins - instead of wheeling and dealing with distortions of reality.
What’s Happened Since?
Francis Collins has resigned. Fauci has resigned.
Kristian Anderson has defended his change in position in the NYTimes as due to the fact that other coronaviruses also have furin cleavage sites… contrary to the paper which reports it has not been seen in betacoronaviruses.
But that does not rule out acquisition in SARS-CoV-2 by serial passage followed by lab escape.
Further, the furin cleavage site was not the main reason why scientists thought serial passage was plausible given their own, independent analyses of the sequence data.
For his part, “Bob” was given the senior author position on the paper.