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manipulate CoV genomes or create iuldn't jJicates 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 cation >E2 Expressing Mice with on experiments as described this occurred? ters of 35a, red ained 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. 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? 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." RPPR (AI110964-05) 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? 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 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.