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Yuri Deigin

@ydeigin

Apr 11

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:

🚨 But this is an even more important piece of evidence as this is the 2019 EcoHealth NIH grant renewal application for their joint grant with WIV that has been ongoing since 2014. In it we see that by 2019 their interest has shifted to focusing on novel SARS-like strains that are 10-25% different from SARS1 in their spike.

It is also noteworthy that they are talking of anticipating finding another 100-200 novel CoVs by sampling 5000 bats.

ecohealthalliance.org/wp-content/upl…

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Yuri Deigin

@ydeigin

Dec 13, 2023

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)

Source interview 1 (Konrad Hochedlinger):

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Yuri Deigin

@ydeigin

Dec 4, 2023

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

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:

Moreover, the quote from DEFUSE about creating novel “human-specific” cleavage sites in SARS-like viruses is found in the “Testing Synthetic Modifications” section that clearly means work with chimeric viruses:

“We will synthesize [SARS-like quasispecies] with novel combinations of mutations to determine the effects of specific genetic traits and the jump potential of future and unknown recombinants.
…
We will analyze all SARSr-CoV S gene sequences for appropriately conserved proteolytic cleavage sites in S2 and for the presence of potential furin cleavage sites. SARSr-CoV [spikes] with mismatches in proteolytic cleavage sites can be activated by exogenous trypsin or cathepsin L. Where clear mismatches occur, we will introduce appropriate human-specific cleavage sites and evaluate growth potential in Vero cells and [human epithelial airway] cultures.”

The mention of pseudoviruses in the sentence that follows refers to something different — ablating existing rather than creating novel cleavage sites:

“In SARS-CoV, we will ablate several of these sites based on pseudotyped particle studies and evaluate the impact of select SARSr-CoV S changes on virus replication and pathogenesis.”

To be honest, I’m not even sure what that sentence refers to, as it seems to be talking about SARS1 rather than novel SARS-like (SARSr-CoV, i.e. SARS-related) CoVs. Moreover, SARS1 doesn’t have even a single FCS to ablate, let alone several, which makes that sentence even more puzzling. I actually think it might have gotten misplaced from the N-linked glycans section which follows right after and talks about civet SARS1 strains having some ablated glycans that were present in human SARS1, and in that context ablating “several of these sites” in SARS1 actually makes sense.

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Yuri Deigin

@ydeigin

Sep 28, 2023

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:

Given Christia Freeland’s close ties with the Ukrainian Canadian Congress, I can’t help but wonder whether she knew Yaroslav Hunka personally and/or was well aware of his invitation to the House.

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Yuri Deigin

@ydeigin

Aug 27, 2023

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.

3/ So lineage A is two mutations closer to ancestral bat viruses than lineage B and most likely lineage B evolved from it before eventually outcompeting it into oblivion. However, lineage A itself is not at the root of the SARS2 ancestry tree because several phylogenetically earlier genomes are known, i.e. ones that have even fewer mutations than lineage A when compared to bat viruses like RaTG13 or BANAL-52.

One such mutation is C18060T (i.e. bat viruses have a T nucleotide in their genomes [actually, a U as they are RNA viruses but let’s speak DNA here] in position 18060, while the reference human SARS2 genome, Wuhan-Hu-1, has a C nucleotide), and several investigators of SARS2 phylogeny (e.g. @jbloom_lab or Kumar et al. 2021) think that it is likely that the earliest ancestor of all human SARS2 viruses had that mutation.

Such an ancestor is sometimes called proCoV2, and it is basically lineage A with the C18060T mutation (so, in total, it is 3 mutations away from Huanan market’s lineage B: C8782T, C18060T, and T28144C).

Here is the Kumar et al. paper:

Read 8 tweets

Yuri Deigin

@ydeigin

Aug 24, 2023

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:

3/ Circling back to the DEFUSE proposal, the N370 glycan in SARS2 is the same glycan as N357 in SARS1 which was implicated as being important for DC-SIGN binding in 2006:

ncbi.nlm.nih.gov/pmc/articles/P…

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