COVID origins: Why unfunded DEFUSE is the smoking gun.

Opponents of the lab leak hypothesis often dismiss the 2018 DEFUSE proposal by saying, "But it wasn't funded!" This misses the point entirely: My thesis: SARS2 origin is most likely a lab leak precisely because its two most unique features – the furin cleavage site and the missing N370 glycan – align with the parts of DEFUSE that were NOT funded by US agencies.

Latest SARS2 preprint provides additional evidence that not only was the novel FCS a key factor in its pandemic potential, but the way the FCS was acquired — via a 4 aa insertion — was also important. Re: "why would a genetic engineer use an insertion to create an FCS?"

1/

https://twitter.com/TheMenacheryLab/status/1899800371272290622

I've hypothesized previously that the answers to "why use an insertion" and "why use a leading proline" lie in MERS, whose S1/S2 loop is 4 AAs longer than that of SARS-like CoVs, and also has the leading proline, making it more exposed to proteases:

https://x.com/ydeigin/status/1782490749889552740

Increasing neuroinvasiveness could indeed have been the research goal of a genetic engineer opting to use a “suboptimal” or “non-canonical” furin cleavage site like the one found in SARS2. And Baric did say that MHV and FIPV inspired the FCS interest of DEFUSE. Thread below:

https://twitter.com/breakfast_dogs/status/1900331687768137900

Somewhat counterintuitively, in a human coronavirus, OC43, when it has the less efficient (“suboptimal”) RRSR furin cleavage site instead of the canonical RRSRR one, it is more lethal and more *neurovirulent* when tested in a mouse model:

journals.plos.org/plospathogens/…

Excellent analysis of SARS2 cryptic lineages from persistent human infections (those nightmare scenarios happen when the virus colonizes your gut and remains there for months or years). Some implications for Covid origins there that I’d like to address in the thread below.

https://twitter.com/solidevidence/status/1872953895280169172

First an aside on extrapolating insertions found in SARS2 circulating in humans to the likelihood of insertions occurring (and getting fixed) in a SARS2 progenitor circulating in an intermediate host — SARS2 has now been circulating for years in hundreds of millions of humans, while in the wild it could not have had even a sliver of such a reservoir of non-bat hosts (and we all agree the FCS insertion could not have arisen in bats because it is detrimental to their preferred enteric tropism).

🧵 Covid Origins: Lab leak critics claim the coincidence of a novel coronavirus emerging near the Wuhan Institute of Virology is dwarfed by the “counter-coincidence” of it first being noticed at a wildlife market. But the market was actually one of the most likely places for a lab-leaked virus to get noticed.

Here’s a clip from my podcast with @robertwrighter and a deep dive in the thread below: Basically, if we imagine the map of Wuhan as a “probability landscape”, where each location has an inherent probability that a roaming lab-leaked virus would get noticed if it got there, the Huanan seafood market would dominate that landscape like Mount Everest.

Some critics of the Covid lab leak hypothesis say the SARS2 furin cleavage site is unlikely to have been engineered because in previous cases of creating novel FCSes in coronaviruses virologists have never inserted an FCS but rather created them in place via point mutations. But! A close collaborator of Zhengli Shi and the Wuhan Institute of Virology, Shibo Jiang, in 2013 published a paper creating a furin cleavage site in a non-CoV synthetic vector via a 12-nt insertion (note the CGG codon for the leading arginine):

🚀 More amazing partial reprogramming news! This time from @davidasinclair — he mentioned some unpublished animal results for:

- *reversing* Alzheimer’s symptoms
- hearing loss
- ALS
- glaucoma (anticipating clinical trials in 2025!)
- rejuvenating skin, kidneys and liver Great to hear that the FDA is ok with Tet-inducible (via rtTA3) partial reprogramming gene therapies, and that Life Bio is so close to the clinic. They presented encouraging results in monkeys in an eye stroke model (NIAON); I didn’t know they also tried a glaucoma monkey model.

🧵 I wrote a new Medium article about how Ralph Baric's January 2024 testimony provides new insights into the origins of COVID-19. Check out the article here:

yurideigin.medium.com/why-ralph-bari… 2/
Highlights:

- Ralph Baric confirmed that DEFUSE proposed inserting novel furin cleavage sites into live viruses, inspired by feline coronaviruses
- SARS-CoV-2’s furin cleavage sites is identical to the one found in several lethal feline coronavirus strains
- Ralph Baric strongly believes WIV had unpublished viruses and viral reverse genetics systems
- Most plausible Covid origin is the result of research on identifying new SARS-like viruses and developing broad vaccines against them

I was curious to hear Peter Dazsak mention at the @COVIDSelect hearing that prior to the Covid outbreak, he actually met with Dr. (Yi-Gang) Tong who was working on a SARS-like CoV found in pangolin samples. In his Feb 2020 paper, Dr. Tong mentions that they isolated this virus long before the outbreak and routinely cultured it at BSL2. More info in the thread below.

PS: Daszak erroneously claiming in 2020
that WIV didn’t have live bats goes to show that he could well be unaware of what other research relevant to Covid origins WIV was engaged in. While Dr. Tong's lab is in Beijing, he did collaborate with WIV and EcoHealth previously, e.g. in 2018 on a SADS-CoV paper titled "Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin":

pubmed.ncbi.nlm.nih.gov/29618817/

4 years after my first Medium article on Covid origins, I wrote another one. In it, I make the case that SARS-CoV-2 is precisely the virus WIV was hunting for in 2019.



Below is a thread on the key points:yurideigin.medium.com/sars-cov-2-is-… 2/

In 2018, WIV started looking for SARS-like viruses that were 10–25% different from SARS1 in their spike but could still enter human cells, and escape SARS1-based antibodies. SARS2 fits those criteria like a glove.

In our debate on Covid origins, Peter @tgof137 kept insisting that the DEFUSE proposal was only interested in viruses that are only within a 95% similarity to (i.e. at most 5% different from) SARS1. I thought I fully explained that Peter was mistaken and the judges have agreed with me (see the clip below) but Peter keeps repeating that claim (e.g. in the Astral Codex writeup).

Now, with the FOIA of the DEFUSE drafts we now have clear evidence that even strains with up to a 25% difference from SARS1 were of interest.

🚨 Moreover, the 2019 EcoHealth grant renewal letter for their joint grant with WIV actually said that they would PRIORITIZE strains that had between 10% and 25% difference from SARS1 in their spike gene.

Details in the thread below. This is the relevant excerpt from the DEFUSE FOIA:

Skeptics of epigenetic rejuvenation via partial reprogramming often point to low efficiency of full reprogramming in support of their skepticism.

Indeed, under standard conditions in vitro, only a small proportion of cells forced to express Yamanaka factors end up finishing the journey to pluripotency. This has led skeptics to further suggest that the rejuvenation we observe in reprogrammed cells is a manifestation of selection of apriori healthier cells by the reprogramming process rather than bona fide rejuvenation.

However, several observations mentioned in the attached video argue against this. First observation is that essentially 100% of starting cells can be reprogrammed to iPSC by Yamanaka factors if their H3K36 repression is transiently ablated in the early stages of reprogramming. And secondly, the earliest stages of reprogramming actually open up chromatin in all cells, even in the ones that don’t end up transitioning to pluripotency.

I think this has implications for both safety and efficacy of epigenetic rejuvenation by partial reprogramming, as its goal is actually to avoid a change in cell identity while at the same time giving cells a quick epigenetic jolt in the hopes of resuscitating them back to a healthier state. If we now observe that essentially all cells expressing Yamanaka factors get that jolt in the first days of partial reprogramming, that’s quite encouraging.

So the video below has excerpts from interviews with Konrad Hochedlinger and Ken Zaret from this year’s Cold Spring Harbor’s Cell State Conversions Meeting, as well as from Ken Zaret’s excellent CSHL keynote there. I’ll post links to the original videos in tweets below. Bonus: Juan Carlos Izpisua Belmonte asks two very insightful questions:

(Dr. Belmonte is one of the pioneers of partial reprogramming as he led the group at Salk that published the seminal Ocampo et al. 2016 paper. He is now at Altos Labs)

Here’s another example of virologists trying to downplay the importance of DEFUSE using bad arguments. I’ve already addressed objections built on treating DEFUSE as gospel or dismissing its importance because it wasn’t funded but here I want to address two more arguments: “DEFUSE proposed inserting an FCS only in pseudoviruses” and “DEFUSE only talked about cleavage sites in S2 and the novel SARS2 furin cleavage site separating S1 and S2 doesn’t count”:

https://twitter.com/ydeigin/status/1731399158479675632

In actuality, DEFUSE clearly stated that pseudovirus work was meant as a first, pre-screening step which could then lead to chimeric virus testing and ultimately to testing using the full backbone of the original virus. Here I’ve highlighted the relevant DEFUSE excerpts:

I was quite shocked to discover that Yaroslav Hunka — the Nazi veteran invited to the House of Commons — was actually previously honored (along with several dozen other Waffen SS veterans) by the Ukrainian Canadian Congress in 2007. Notably, the Ukrainian ambassador to Canada was a special guest at the ceremony and gave a celebratory speech:








Previously, in 2003, Yaroslav Hunka represented the very same Ukrainian Canadian Congress at the 8th Ukrainian World Congress in Kiev. Since 1989 he frequently visited Ukraine and even ran for Verkhovna Rada (Parliament) in 1994.

In 2004 he was made an honorary citizen of Berezhany; war criminals Stepan Bandera and Roman Shukhevych were given the same honor in 2011:

1/ A key weakness of the Huanan market zoonosis hypothesis is that all 16 earliest Wuhan patients with link to the market had lineage B of SARS2 which is phylogenetically placed later in the ancestry tree than lineage A.
2/ Lineage A initially represented 33% of the Wuhan early cases but quickly went extinct as lineage B seemed to have a fitness/growth advantage as evidenced by early epidemiological data.

1/

Besides the furin cleavage site (FCS), SARS2 has another unique feature mentioned in DEFUSE not yet seen in any natural SARS-like viruses – an ablated N-linked glycan at position N370. This glycan was ablated via a T372A amino acid mutation that came about via a double nucleotide mutation of the original ACT codon into GCA (the latter, incidentally, is the same codon as the one coding for alanine – out of 4 possible alanine codons – in the PRRA insertion which has created an FCS in SARS2).

Importantly, the T327A mutation greatly increases SARS2 infectivity in human lung cells but, just like an FCS, this kind of a mutation seems to have selective pressure AGAINST it in ancestral bat viruses.

DEFUSE’s interest in N-linked glycans stems from a very curious observation about SARS1 whose bat progenitor seems to have temporarily lost two of its N-linked glycans in civet SARS1 progenitors before re-acquiring them, and this led virologists to hypothesize that those glycans could be relevant for host switching. This is described in DEFUSE in a somewhat convoluted way:

“N-linked glycosylation: Some glycosylation events regulate SARS-CoV particle binding DC-SIGN/L-SIGN, alternative receptors for SARS-CoV entry into macrophages or monocytes [76,77]. Mutations that introduced two new N-linked glycosylation sites may have been involved in the emergence of human SARS-CoV from civet and raccoon dogs [77]. While the sites are absent from civet and raccoon dog strains and clade 2 SARSr-CoV, they are present in WIV1, WIV16 and SHC014, supporting a potential role for these sites in host jumping. To evaluate this, we will sequentially introduce clade 2 disrupting residues of SARS-CoV and SHC014 and evaluate virus growth in Vero cells, nonpermissive cells ectopically expressing DC-SIGN, and in human monocytes and macrophages anticipating reduced virus growth efficiency. We will introduce the clade I mutations that result in N-linked glycosylation in rs4237 RBD deletion repaired strains, evaluating virus growth efficiency in HAE, Vero cells, or nonpermissive cells +/- ectopic DC-SIGN expression [77]. In vivo, we will evaluate pathogenesis in transgenic hACE2 mice.”

The [77] paper cited in DEFUSE is a 2007 work by Han et al. titled “Specific Asparagine-Linked Glycosylation Sites Are Critical for DC-SIGN- and L-SIGN-Mediated Severe Acute Respiratory Syndrome Coronavirus Entry”. It looked at the 5 civet progenitor strains of SARS1 and showed that initially those strains did not have glycans around positions N227 and N699 but then eventually acquired them in civet progenitors and kept in human SARS1.

2/

What the 2007 paper did not know at the time that the DEFUSE authors pointed out is that the bat progenitor strains like WIV1/Rs3367 or SHC014 also have glycans at those positions. This is what likely made the DEFUSE authors interested in the host jumping potential of these glycans and potentially genetically modifying them to further study their role:

🧵Ben Hu being named as one of the 3 WIV staffers sick with Covid-like symptoms in Oct/Nov 2019 is an interesting 🧩 to the Covid origins saga. He connects several threads together: DEFUSE, WIV sampling in Mengla County (which borders Laos), RmYN02 with its proto-FCS etc. --> He even has a possible connection to the mysterious pre-pandemic pangolin coronavirus (that has a near-identical RBD to SARS2) reported in mid-2019 by Guandong’s GIABR.

Ok, the 2014 West African Ebola outbreak is also looking suspicious. Until 2014, the outbreaks of that (Zaire) Ebola species were confined to Central Africa, over 5000 km away.

Here’s a detailed article about that outbreak by @samhusseini and @BioSRP.

independentsciencenews.org/health/did-wes… One aspect of the story I found interesting is that while some virologists like Bob Garry have denied that the Kenema lab (rumored to have possibly caused the Ebola outbreak in West Africa) was studying Ebola before the outbreak, I found the following study which reported… twitter.com/i/web/status/1…

1/n
The Wuhan CDC is back in focus after reports that it was the reason behind DOE changing its Covid origin assessment to a possible lab leak. Really curious what data led to that conclusion, hope they'll declassify it! For now, let me share some observations about Wuhan CDC: 2/n
WHCDC was, of course, an early lab leak suspect, as outlined in a Feb 2020 preprint by Xiao & Xiao where they pointed to Junhua Tian (JHT)’s interest in bat ticks, as well as his statements about having to quarantine after being attacked by bats:
web.archive.org/web/2020021414…

At the protein level the BANAL-52 spike is ~99% identical to the pre-FCS SARS2 spike, and its RBD (responsible for binding to the ACE2 receptor) differs by just 1 aa. Other BANAL genes are 98-99% identical to SARS2. So BANAL-52+FCS itself would do quite well at human spillover.

https://twitter.com/ydeigin/status/1631785943916060672

Of course a lab in Wuhan could have had a BANAL-like bat strain even closer to SARS2 than strains found in Laos in 2020. The point is, all it takes for a BANAL-like virus to become human-transmissible is an FCS, as it opens up respiratory tropism. Some papers:

Yes, WIV (with funding from NIAID and USAID via EcoHealth) was creating SARS-like chimeras and using them to infect cell cultures with live viruses — all in merely BSL2 conditions.

https://twitter.com/R_H_Ebright/status/1629543155807965184

Here is the 2017 paper in which they created 8 SARS-like chimeras:

ncbi.nlm.nih.gov/pmc/articles/P…