Thread by @VBruttel

The DEFUSE proposal has IMO always been a precise blueprint for how SARS2 came into existence.
Now @emilyakopp and @USRightToKnow used the FOIA to get access to draft documents that contain further damning details. a 🧵.
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I explain in detail how a DEFUSE-like research project would explain all the oddities that cumulatively prove a lab origin of SARS2 in this academic talk:

The new draft documents can be found here:

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usrtk.org/wp-content/upl…

DEFUSE was not funded, but researchers sometimes start projects before the funding is approved, and very similar research projects at the WIV were funded by US and Chinese institutions. Making synthetic viruses is also very cheap, about $6000 are enough according to R. Baric.
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These are key oddities about the SARS2 outbreak, and how a DEFUSE-like gain of function project would explain pretty much all of them.
LOCATION
viruses closely related to SARS2 are not found around Wuhan, but 1500km further south in Yunnan or Lao.
WIV collected samples there.
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TRACELESS INTRODUCTION
Other related viruses such as SARS👇 or MERS jumped several times at various locations from intermediate species into humans, they left a trail. Transportation from Lao or S China to Wuhan in a sealed container would explain why there is no trail.
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TIMING
Wildlife trade or markets in China did not change much over the last decades, a market origin could have happened decades earlier. In contrast, DEFUSE-like projects at the WIV were started in or just before 2019.
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HIGH INFECTIVITY
The COVID pandemic spread extremely fast also in countries with excellent nutrition and healthcare. The virus seemed to be perfectly adapted to humans. The receptor binding domain (RBD) interacts with the human receptor ACE2 and is important here
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One would expect this domain to be adapted to an intermediary host, and then to slowly adapt to the human receptor over several jumps. However, the SARS2 RBD seemed to bind better to human receptors than to all suspected intermediary host receptors

8/x nature.com/articles/s4159…

The RBD in SARS2 is ~99% identical to a RBD of a pangolin coronavirus, while the rest of SARS2 (except for the FCS) is ~98-99% identical to RaTG13, a virus the WIV had.
SARS2 is highly similar to RBD chimeric viruses that were made by DEFUSE virologists.
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The DEFUSE drafts show that they planned to use “3D protein modeling” to prioritize the RBDs that “interact with human ACE2 molecules” and “Through recombination ... the correct RBD could be dropped into a strain w/ 25% variation (=divergence from SARS1).
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"...and allow the virus to enter human cells and replicate”. They planned to culture “full length recombinant viruses” on human airway epithelium (HAE) cultures and in mice with the human ACE2 receptor.
This would perfectly explain the optimised, inserted RBD in SARS2!
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THE FCS
A furin cleavage site enables coronaviruses to infect new hosts and to cause severe diseases, as it enables infections of neurons. Virologists including Baric and Shi introduced FCSs into all sorts of dangerous coronavirus spike proteins ().
12/x ncbi.nlm.nih.gov/pmc/articles/P…

However, before SARS2, SARS-related coronaviruses have never evolved such a FCS in nature. The FCS in SARS2 is further suspect as it contains unusual codons, looks cleanly inserted, and is "human- specific".
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Some virologists argued that only FCSs in S2 are mentioned in DEFUSE, and that the boundary of S1 and S2 (the 2 parts of the spike) would not be included here. The draft now mentions a FCS exactly at the S1/S2 boundary!!!
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GLYCOSYLATION SITES
Enzymes in host cells can add sugar residues to glycosylation sites, which enables viral spike proteins to escape from immune responses and to bind additional receptors (DC-SIGN in SARS2). DEFUSE mentioned DC-SIGN as a desired additional receptor.
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The draft mentions 2 specific sites in SARS1, N227 and N669. These correlate to N243 and N726 in SARS2. And both of these also happen to be very good glycosylation sites... (@ydeigin wrote an excellent thread on this topic )
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SYNTHETIC SPIKES
They planned to “produce codon optimized, stabilized and purified prefusion SARS-CoV glycoprotein ectodomains” in which “the chimeric S ectodomain will be linked to a C-terminal T4 fibritin trimerization domain” in a human cell line (293-F cells).
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which translates to: they wanted to make vaccines for bats based on the spike proteins. This is best done using codon-optimised plasmids. Exactly such codon-optimized plasmids (only difference: no FCS) were found in patient samples collected in China in 2019!!!
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References and further explanations:

19/xyoutube.com/embed/EuuY94ts…

SYNTHETIC VIRUS DESIGN
The ~30,000 nucleotides (“letters”) of the RNA genome of synthetic viruses are assembled as DNA from several DNA segments. These are linked using short, complementary single-strand overhangs (so called sticky ends) that are unique for each junction.
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@WashburneAlex, @tony_vandongen and I had written preprint in which we describe that the SARS2 genome contains a pattern of restriction sites typically found in synthetic, but not in related natural viruses.

21/xbiorxiv.org/content/10.110…

We postulated that SARS2 was
a) assembled from 6 fragments
b) using a restriction enzyme called BsmBI for the backbone (BsaI for RBD or FCS variants)
c) these restriction sites were introduced with abnormally many synonymous mutations
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The preprint is IMO still being censored by journals.
Virologists such as @DolkenL said our analysis was flawed (), claiming that the aim must have been to replace the entire spike (not the FCS and RBD)...

23/x idw-online.de/de/news803624

...or that similar patterns can be found in virtually any coronavirus genome if other enzymes are used (they used enzymes here that do not produce unique sticky ends and admitted in internal emails that they do not know if these can be used btw...).
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the DEFUSE draft documents show that, exactly as we had postulated, they planned to use 6 SEGMENTS to assemble synthetic viruses, to use unique endonuclease sites that do not disturb the coding sequence, and TO BUY BsmBI!!!
(= NE Bio labs order # R0580S or R0580L)
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THE MINDSET
Other comments suggest that project leader Peter Daszak did not really understand what exactly other scientist were planning to do, which of course could also lead to unnecessary riks.
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Daszak also seemed to be proud that they can do these experiments under “highly cost-effective” BSL2 conditions. BSL2 is cost-effective as it is also very unsafe, it does not even mandate simple face masks. 27/x

Other comments indicate that Peter Daszak tried to convince US reviewers that the high-risk work would be done at safer US institutions, but would have been fine with later doing a lot of the bioengineering in Wuhan once they got the funds.
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CONCLUSION
These documents provided further insights into what gain of function experiments virologists connected to the WIV were planning. Location, timing, the traceless introduction, the divergent RBD and inserted FCS, the glycosylation sites, restriction enzyme choices..
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and restriction site pattern, leaked synthetic spike-coding plasmids...
IMO the a lab origin of SARS-CoV-2 is proven beyond reasonable doubt. Still, please draw your own conclusions.
please consider supporting @emilyakopp, @USRightToKnow or !
thanks!
30/30biosafetynow.org