About a month ago I recorded a video examining the scientific evidence of the lab-leak hypothesis for the origin of the SARS-CoV-2 virus that causes COVID-19. Since then, a heated exchange between Senator Rand Paul and Dr. Anthony Fauci has transpired in which the issue of whether the United States, through the National Institutes of Health (NIH), has funded so-called gain-of-function research in the Wuhan Institute of Virology (WIV). I was planning on making another video on this subject (and probably will at some point), but after some discussion with a commenter on my earlier video, some more philosophical questions have emerged that I am going to discuss here a bit.

Here is my video going through evidence for the lab-leak hypothesis:

The paper in question (in the Paul-Fauci confrontation) can be found here:
https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006698

For some background, what the paper’s authors did (or at least what is relevant to the discussion) is to take the backbone of a coronavirus variant known as WIV1 and switch out its wild type Spike protein for the Spike proteins of other natural variants. From the paper in question (words in {red} are mine; hyperlinks added by me):

In the current study, we successfully cultured an additional novel SARSr-CoV {SARS related-COV} Rs4874 from a single {bat} fecal sample using an optimized protocol and Vero E6 cells [17]. Its S {Spike} protein shared 99.9% aa {amino acid} sequence identity with that of previously isolated WIV16 and it was identical to WIV16 in RBD {receptor binding domain – the part that binds to the ACE2 receptor}. Using the reverse genetics technique we previously developed for WIV1 [23], we constructed a group of infectious bacterial artificial chromosome (BAC) {a type of plasmid that allows for conjugation} clones with the backbone of WIV1 and variants of S {Spike protein} genes from 8 different bat SARSr-CoVs. Only the infectious clones for Rs4231 and Rs7327 led to cytopathic effects {causes damage to the cells i.e. caused illness} in Vero E6 cells after transfection {adding the virus to the cells} (S7 Fig). The other six strains with deletions in the RBD region, Rf4075, Rs4081, Rs4085, Rs4235, As6526 and Rp3 (S1 Fig) failed to be rescued {the virus was dead i.e. unable to infect the cells}, as no cytopathic effects was observed and viral replication cannot be detected by immunofluorescence assay in Vero E6 cells (S7 Fig). In contrast, when Vero E6 cells were respectively infected with the two successfully rescued chimeric SARSr-CoVs, WIV1-Rs4231S and WIV1-Rs7327S, and the newly isolated Rs4874, efficient virus replication was detected in all infections (Fig 7). To assess whether the three novel SARSr-CoVs can use human ACE2 as a cellular entry receptor, we conducted virus infectivity studies using HeLa cells with or without the expression of human ACE2. All viruses replicated efficiently in the human ACE2-expressing cells. The results were further confirmed by quantification of viral RNA using real-time RT-PCR (Fig 8)

So, the question is: does this experiment constitute gain-of-function research? In order to assess this, we first need to look at what the technique they were using is. It is known as recombinant DNA techniques. Essentially, it is taking DNA from two or more sources and bringing it together into a single organism. It is a standard and extremely common technique used in biochemistry, microbiology, cell biology, etc. I have used such techniques in my own research: inserting A. baumanii beta-lactamase enzymes into plasmids in order to be expressed in E. coli and inserting human CDK8 and MED13 genes into plasmids in order to be expressed in E. coli. Recombinant DNA is also used in the biomedical industry to make things like insulin. The point is, it is a standard laboratory technique.

Here is how the conversation in the comments of my lab-leak hypothesis video has gone:

Interlocutor

Here is the Paper Rand Paul and Fauci are arguing gain of function. https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006698 Fauci is listed in the funding section. This sentence seems to say gain of function: Recombinant viruses with the S gene of the novel bat SARSr-CoVs and the backbone of the infectious clone of SARSr-CoV WIV1 were constructed using the reverse genetic system described previously Is this paper actually gain of function? Or is fauci right that it is not?

This sentence seems like gain of function too. In this study, we con- firmed the use of human ACE2 as receptor of two novel SARSr-CoVs by using chimeric viruses with the WIV1 backbone replaced with the S gene of the newly identified SARSr- CoVs. Rs7327’s S protein

Me

I actually just started looking into this yesterday in preparation for a video I am going to make on the subject. Spoiler alert for that video:

From what I can discern so far (as I continue down the rabbit hole of papers) is that what the paper is talking about is not gain-of-function research, but standard recombindant DNA techniques used in all genetics, biochemistry, cell biology, microbiology etc. laboratories. They are inserting the naturally discovered Spike protein sequences into a naturally occurring coronavirus backbone (WIV1) which is known to be able to infect human cells. The purpose is to measure changes in infectivity of WIV1 given different Spike proteins.

Why this is not gain-of-function research is because the scientists are not creating new functions that had not existed before. They are testing functions that already existed in the wild.

This is standard recombinant DNA research, where a particular function (in this case the Spike protein) is looked at in a controlled context (in this case the WIV1 backbone) in order to isolate the particular function (the Spike protein) to further characterize and understand the function.

Gain-of-function research is creating brand new functions that had not existed before. Since this is only looking at the already existing function of naturally occurring Spike proteins, it is not gain-of-function research.

Interlocutor (posted at same time as my next response)

You explanation is why Rand Paul also had the definition of gain of function as defined by Fauci: Gain-of-function (GOF) research involves experimentation that aims or is expected to (and/or, perhaps, actually does) increase the transmissibility and/or virulence of pathogens. This is quickly turning into the “I did not have sexual relations with that woman” where it does not have any common sense. Your definition would mean combing AIDS with corona viruses would not be considered gain of function because both are natural even though nearly impossible to occur in nature.

As an aside, my interlocutor is quoting (bolded by me) the definition of gain-of-function research given here:
https://osp.od.nih.gov/wp-content/uploads/2013/06/Gain-of-Function_Research_Ethical_Analysis.pdf

Me (posted At same time as my interlocutor’s previous response)

I should add that this is somewhat of a gray area insofar as it would be possible to take already existing functions from multiple (two or more) sources to create something with a new function. There are two ways one could go about doing this. The first would be introducing a foreign gene, such as adding bioluminescence to some bacteria that does not naturally have bioluminescence. The second more relevant way is doing what this paper is talking about, which is switching out one naturally occurring Spike protein for a different naturally occurring Spike protein. One could certainly see that if, for instance, the WIV1 backbone itself was not conducive to infecting humans, and some arbitrary Spike protein that was not conducive to infecting humans, were both recombined to make something that was conducive to infecting humans, that this would constitute a gain of function.

A few issues with characterizing this as identical to the sort of gain-of-function research I spoke of in this video:

1) recombinant DNA techniques are not only standard procedure in all biology labs, but are essential to such research; banning recombinant DNA techniques altogether would halt all biological and biomedical research completely, as well as removing certain life-saving techniques (vaccines, insulin, antibiotics, etc.).

2) what the scientists in the paper were doing would not be able to produce SARS-CoV-2; the furin cleavage site insertion was not done using the methods in the paper (whether through natural recombination events or through directed evolution via multiple passages).

3) the viral backbone (WIV1) was already able to infect human cells (i.e. able to bind to human ACE2 receptor, which is why it was used as the backbone), so the experiments were not introducing a new function (invectivity) to the chimeras they were producing.

Me (in direct response to my interlocutor’s previous response)

You are certainly right that this comes down to an issue of definitions. What are the necessary and sufficient conditions that must obtain in order to classify an experiment as “gain-of-function”? It is certainly something I will have to consider before making my next video on this topic.

It is this response of mine that has me wondering: what exactly are the necessary and sufficient conditions to call something “gain-of-function” research? Certainly a case could be made that any and all recombinant DNA experiments are gain-of-function in a broad sense. And I can certainly see how the experiment done in the paper in question could be construed as gain-of-function in an even narrower sense in that recombining the Spike protein into the WIV1 backbone could possibly have made a more virulent strain that could then have infected one of the researchers and thereby escaped the lab. But it still does not reach the level of gain-of-function research in a narrower sense of attempting to generate novel viruses that did not (or maybe even could not) have come about by natural processes.

Interestingly, I found the following in a workshop summary available on the NCBI (part of the NIH) website:

To answer these questions, virologists use gain- and loss-of-function experiments to understand the genetic makeup of viruses and the specifics of virus-host interaction. For instance, researchers now have advanced molecular technologies, such as reverse genetics, which allow them to produce de novo recombinant viruses from cloned cDNA, and deep sequencing that are critical for studying how viruses escape the host immune system and antiviral controls.

This is exactly what the researchers in the paper in question did. Thus, in addition to the exact type of research being pointed out here when talking about what gain-of-function research is, the paper also seems to satisfy the definition given above by my interlocutor (“Gain-of-function (GOF) research involves experimentation that aims or is expected to (and/or, perhaps, actually does) increase the transmissibility and/or virulence of pathogens”).

I guess a question we can then ask ourselves is whether this is a necessary and sufficient definition of gain-of-function research. The issue is how broadly or narrowly we want to define it. If we define it too broadly, we are going to encroach into vital, yet exceedingly low risk standard lab techniques. If we define it too narrowly, we might leave out certain types of research that is of potentially higher risk of causing another outbreak.

The other salient issue, of course, is whether the United States was complicit in any gain-of-function research at the Wuhan Institute of Virology (and further, whether this makes the United States complicit in the origin or spread of COVID-19). Obviously a working definition of gain-of-function research is required to confidently make that determination. You can look at the grant that funded the research online: The NIH grant to EcoHealth Alliance (who worked in collaboration with the Wuhan Institute of Virology):

https://grantome.com/grant/NIH/R01-AI110964-06

https://taggs.hhs.gov/Detail/RecipDetail?arg_RecipId=ZfeVER2uWwKllnTv6X4AxA%3D%3D

https://www.usaspending.gov/award/ASST_NON_R01AI110964_7529

I think an issue I have is that this has become a highly politicized discussion. How any particular individual will want to define gain-of-function research is likely going to be influenced by which side of the argument the individual already accepts (i.e. motivated reasoning). Those who already think it is not gain-of-function research, or perhaps just people who like Anthony Fauci or hate Rand Paul, are going to define it in such a way that the definition does not cover the research in question. Those who already think it is gain-of-function research, or perhaps those who hate Anthony Fauci or like Rand Paul, are going to define it in such a way that the definition does cover the research in question. My own position, likely to raise the ire of all sides, is that I am withholding judgement. I am ambivalent about both Anthony Fauci and Rand Paul and am not uncomfortable with ambiguity as definitions are made and agreed upon and while risk assessments are carried out as to what is an acceptable amount of risk and which sorts of experiments pose an acceptable risk. These important tasks, however, are likely not to be decided in the absurd theater that is congress or on social media.

More references for the debate on gain-of-function research:
https://osp.od.nih.gov/biotechnology/gain-of-function-research/

EDIT

While looking through the National Science Advisory Board for Biosecurity report on gain-of-function experiments, one part of it stuck out to me as a way of distinguishing between gain-of-function experiments with acceptably low risk and gain-of-function experiments with unacceptably high risk:

Research proposals involving GOF research of concern entail significant  potential risks and should receive an additional, multidisciplinary review, prior to determining  whether they are acceptable for funding. If funded, such projects should be subject to ongoing  oversight at the federal and institutional levels. As part of this recommendation, the NSABB has proposed a conceptual approach for guiding funding  decisions about GOFROC. First, the NSABB identified the attributes of GOFROC, which is research  that could generate a pathogen that is: 1) highly transmissible and likely capable of wide and  uncontrollable spread in human populations; and 2) highly virulent and likely to cause significant  morbidity and/or mortality in humans. Next, the NSABB identified a set of principles that should guide funding decisions for GOFROC. Only research that is determined to be in line with these principles should be funded. Additional risk mitigation measures may be required for certain research studies to be deemed acceptable for funding.

Essentially, what came to my mind is that we could have four tiers of gain-of-function research:

1. Acceptable Low Risk: recombinant DNA experiments in which the altered gene must be manually “switched on” (e.g. the lac operon and IPTG) in order to be expressed and/or expression of the gene does not confer any new traits to the host organism (e.g. expressing a human CDK8 protein in E. coli does not alter the physiology of the E. coli in any way)
2. Acceptable Medium Risk: recombinant DNA experiments that do alter the host organism’s genome but is unlikely to significantly increase the danger of that organism (e.g. giving E. coli a gene for beta-lacatamse (antibacterial resistance), but the E. coli is not itself dangerous and so having that resistance does not make it any more dangerous)
3. Acceptable High Risk: recombinant DNA experiments that may increase the danger posed by the resulting chimeric organism, but the risk can be acceptably mitigated by proper laboratory biosafety measures and/or the benefits of performing the experiment outweigh the risks (e.g. we are in the middle of a deadly pandemic and only by doing such an experiment do we have hope of finding a way to combat the pandemic)
4. Unacceptable Risk: recombinant DNA experiments where under no circumstances do the benefits outweigh the risks (the potential danger posed by the resultant chimeric organism is too great for any level of biosafety measures and/or there is no foreseeable benefit to such an experiment)

This 4-tier outline is something just off the top of my head while continuing to research this issue, but something along these lines must be constructed if we want to both continue enjoying the benefits of modern science and medicine while also not exposing ourselves to undo risk. I have a feeling (or, perhaps, a misguided hope) that somewhere in all these long, boring papers seen here there is a more concrete and comprehensive list of criteria than I have yet to find.

EDIT 2

My interlocutor in the comment section of my lab-leak hypothesis video introduced me to the following video which I think is very informative on this issue:

EDIT 3

I made a video about gain-of-function research and how it relates to the paper discussed in the confrontation between Dr. Fauci and Senator Rand Paul: