Reading Room Production — page 34

ated aring for investigators in this field are to create cDNA I know UNC-Chapel Hill you know where this was he iuldn't jicates Then ent at mice these molecular clones are used to grow id UNC-CH performed studies with molecular e "mation but tion describes animal work at work. I believe UNC mean to say C3a and C3b) although it does not use that 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. 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. 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. 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? 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. 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. 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. 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? 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." SF 424 AI110964-01 (received date 06/05/2013) In this SF 424 Aim 3 seems indicative of gain of function experimentation (C3a and C3b) although it does not use that exact term. The receipt date of this application was June of 2013, and the term "gain-of-function" was not widely used before the USG funding pause was announced in 2014. C3a and C3b describe work using pseudovirus assays, which would not have been considered GoF as they do not involve creating full replicating viruses.