It doesn’t matter if it’s GOF or not - it's dangerous

An email from NIH's Senior Program Investigations Officer with some very revealing questions and 'answers'

Chairman Rand Paul has been collecting and releasing documents as part of the ongoing (what is it now, 6 bloody years?) investigation into SARS-2 origins. You can find that here. It is “largely accepted” that SARS-2 is of natural origin and that it “most likely” arose through natural zoonotic evolution (either in an animal host or during early human transmission) and thus not a laboratory construct, thanks to this paper published on March 17, 2020 in Nature Medicine.

---

The latest release by Chairman Rand Paul is entitled: NEW: Investigation Records Released and therein on pages 2-4, you will find an email from NIH Senior Program Investigations Officer Ashley Sanders with the subject “FW: Grant Questions - FBI Inquiry - 1-R01AI110964-01 - 2-R01AI110964-06”. The importance rating was “normal”. It is on the subject matter of an NIH grant/award.

OMA/DPI received questions related to an FBI inquiry for the following NIAID grants: 1R01AI110964-01 through 2R01AI110964-06. The agent is requesting clarification on the details of the study, where certain aspects were performed, and whether seamless cloning techniques were used. I note you were the last official to sign off on 4/27/20.¹

Let’s see what normal looks like.

This email was sent on May 22, 2020 to David A. Miller at the FBI, Erik Stemmy at NIH/NIAID and Mike Shannon at NIH/OD from Ashley Sanders at NIH/OD. Erik provided the responses to the initial questions posed by the FBI officer.

N.B. Stemmy and Daszak carved out an exemption to a pause on gain-of-function research according to The Intercept and the U.S. Right to Know.

Ashley Sanders references document SF 424 AI110964-06 (received date 11/05/2018).

She writes:

Both SF 424s seem to be describing “gain of function” experiments.

On page 192 of SF 424 AI110964-06 (received date 11/05/2018) under P3CO Research, it indicates they are conducting gain of function of SARSr-CoV.

Come again? The received day is May 11, 2018, and “it indicates they are conducting gain of function of SARSr-CoV”? Sounds like The Intercept was right.

The [sneaky] answer:

The funding pause on gain-of-function (GoF) experiments was in place from 2014-2017, and explicitly involved work reasonably anticipated to enhance the transmissibility or pathogenicity of influenza, MERS-CoV, or SARS-CoV. As such, that policy would not have applied to SARS-related coronaviruses (SARSr-CoV). The replacement policy, Potential Pandemic Pathogen Care and Oversight (P3CO), requires additional review and oversight of experiments that are anticipated to increase a potential pandemic pathogen’s transmissibility or pathogenicity in humans. The viruses created under this award are chimeric bat viruses, which generally would not be anticipated to cause enhanced disease or transmission in people. When evaluating experiments for potential GoF or P3CO we determine the likelihood of altering one of these attributes compared to the wild-type or circulating viral strain.

You see that little “r” indicative of “related” coronaviruses in SARSr-CoV that got a free pass? Well guess what? The “related” part simply reflects a genetic relationship, and it applies equally to both natural and laboratory-made versions that meet the criteria of this relationship. So a virus created with recombinant technology that is genetically closely related to SARS-CoV (ie: that falls within the established genetic boundaries of the species) would still be classified as a SARSr-CoV (Severe acute respiratory syndrome-related coronavirus).

See what they did there?

Question from Ashley

Were they also conducting Seemless Cloining and Assembly? From a review of their experimental details, it looks as if they were generating recombinant DNA of the viruses using WIV1 as the backbone but I could not determine if they were using Seemless Cloning techniques.

Seamless Cloning is a molecular biology technique used to join DNA fragments together without leaving any extra unwanted sequences (“scars” or “seams”) at the junctions. It’s referred to as no-see-um (Yount et al. 2002). Ralph Baric describes using it in this paper published in 2005.

No-see-um means the ligation product of DNA recombination lacks the engineered restriction site: good way to hide evidence of engineering, in fact.

Answer:

I’m not aware of them using seamless cloning. Standard techniques for investigators in this field are to create cDNA molecular clones, which are then expressed in cull culture.

And how do you create those molecular clones? Using recombinant DNA technology and no-see-um stitching!

The use of this technique is described here in a paper entitled: “Efficient Reverse Genetic Systems for Rapid Genetic Manipulation of Emergent and Preemergent Infectious Coronaviruses” published in 2017 by Baric and friends.

In this paper, they cut up (partitioned) the SARS-related coronavirus genome into multiple cDNA fragments. Then they re-joined them using type IIS restriction enzymes (ie: BsaI, SapI, BglI). Because type IIS enzymes cut outside their recognition sites, the recognition sequences are excised during ligation, producing a completely seamless (scarless) full-length infectious clone with no residual engineered restriction sites.

This is the same method used in earlier collaborative chimeric SARSr-CoV work (ie: the 2015 Menachery/Baric/Shi Zhengli Nature Medicine paper that created SHC014 spike chimeras on a SARS backbone). Peter Daszak (EcoHealth Alliance) collaborated with Baric on grant proposals and projects that relied on these reverse-genetics systems (most notably the DEFUSE proposal), but the actual molecular assembly work was performed in Baric’s lab (or by labs using his platform).

Oh and then there’s this.

BsaXI is commonly classified and listed as a type IIS enzyme.

Next question from Ashley:

I could not determine exactly where they were conducting these gain of function experiments. I know UNC-Chapel Hill has done these but within the document, I could not determine where this was occurring. Do you know where this was occurring?

Yeah, do you know where this is occurring?

Answer:

GoF experiments were not conducted as part of this award. While chimeric viruses were created via this award, they would not be considered GoF since the results did not confer attributes that were not already exhibited by the wild type versions of the viruses. For example, expressing the spike protein of SARS-CoV in the WIV-1 backbone did not increase the pathogenicity of WIV-1 beyond that of SARS-CoV. This award did not support work to manipulate CoV genomes or create chimeric viruses at Wuhan Institute of Virology; such work was performed at UNC-CH.

But what about if those so-called “wild type versions of the viruses” were ALSO engineered? Or what if the spike protein was engineered (it was) and embedded in the WIV-1 backbone rendering the product more infectious due to the say, insertion of a furin cleavage site at the S1/S2 border?

And the work was done at UNC-CH. Got it.

Next question from Ashley:

It then appears that the recombinant DNA of the virus was then injected into humanized mice. However, again I couldn’t determine where this occurred or was to occur. On page 187 of the same SF 424 under “Vertebrate Animals” it indicates that work with vertebrate animals will be conducted at Wuhan University at the School of Medicine and UNC – CH. Then under “Laboratory Mice” it states that lab mice will be sourced commercially by Wuhan Center for Animal Experiment at Wuhan University and that humanized mice will be bread at University of Wuhan and UNC – CH. Furthermore, the mice will be inoculated with the virus.

Yeah, was it UNC-CH or China or both?

Answer:

Recombinant viral cDNA is not directly injected into humanized mice. Rather, these molecular clones are used to grow virus in culture to create an inoculum used in infectivity studies. Both WIV and UNC-CH performed studies with molecular clones, which included infecting mice with the resulting recombinant viruses.

And yes, they both infected mice with the new viruses they made.

Next question from Ashley:

Under “UNC Facilities where selected agents to be used” it continues with all mouse studies at UNC-CH will be performed….” However, on page 200 of the same SF 424 there is a letter from UNC – CH stating “The work to be performed by UNC-CH does not include animal and/or human research subjects.” I know that was a lot of information but where exactly was the experimentation of injecting the humanized mice with the recombinant DNA occurring?

Yeah were was that again? UNC-CH or China, or both? And the work performed by UNC-CH does not include human research subjects? Geez, I hope not.

Answer:

I believe this to be a typographical error on the part of UNC’s business office. The application describes animal work at UNC. Budget justifications and the consortium agreement also include references to this work. I believe UNC mean to say that the work “… does not include human research subjects.”

Ohhh! They meant to say does “not” include human research subjects. Got it. But what a weird way to turn

The work to be performed by UNC-CH does not include animal and/or human research subjects.

into

The work to be performed by UNC-CH does not include human research subjects.

Weird. Why say that at all?

Ashley continues:

On page 119 of AI110964-01 under “C3d) Humanized mouse in vivo infection experiments” it states that humanized mouse in vivo experiments in humanized mice was occurring at the Wuhan Institute of Virology. I did not see the location where these gain of function experiments were being done but if the injections were occurring at WIV then was the recombinant DNA also generated at WIV? Was Seemless Cloning also being done?

Yeah, was the no-see-um hide the evidence ball being used for these “non-GOF” experiments?

Answer:

This is not GoF work. The humanized mouse experiments described in C3d refer to work with wild type viruses isolated from wild bats. Refer to the section stating: “We will passage isolated bat-CoVs in permissive cells twice…” This is a standard virological technique to create an inoculum to infect animals. In no portion of C3d do they describe any manipulation of isolated virus, therefore this does not describe any kind of gain-of-function studies, nor does it involve the creation of any recombinant DNA or viruses. Characterization of naturally occurring viruses was explicitly excluded from the GoF policy. Further, the USG P3CO Policy Guidance states that “Wild-type pathogens that are circulating in or have been recovered from nature are not enhanced PPPs, regardless of their pandemic potential.”

This person should listen to this:

Movi

And finally Ashley asks:

Finally, in the RPPR (6/1/2017-5/31/2018) on page 28 under “In Vivo Infection of Human ACE2 Expressing Mice with SARSr-Cov S Protein variants”, it appears to be showing the results from their gain of function experiments as described in the SF 424. This is more of less the same as the question above, but do you know where this occurred?

Once again, US soil or China or both?

Answer:

These are not the results of GoF experiments. The figure you reference (Fig 35) shows weight loss and lung viral titers of chimeric viruses with bat CoV (wild type WIV-1, SHC014, WIV16, and 4231) spike proteins expressed on the WIV-1 Released by Chairman Rand Paul backbone. Weight loss and viral titers were comparable across all chimeras when compared to the wild type (Fig 35a, red series; Fig 35b small box pattern). There are no statistical differences reported, so the chimeric viruses have not gained any function/attribute they did not already exhibit.

Yes they were. Semantics don’t cut it. And again, where did it occur?

No answer.

The “This is not GOF” statement repetition kind of reminds me of the 111 invocations of the fifth by a certain someone.

This isn’t good enough. It’s just not.

1. https://www.paul.senate.gov/wp-content/uploads/2026/07/Reading-Room-FINAL.pdf