Thread by @breakfast_dogs

The BANALity of Evil

Early sequences supporting a natural origin for SARS-CoV-2 all came from WIV and the PLA. In time, concerns about their provenance would inevitably bubble up from the realms of "conspiracy theorists". Independent validation was needed.

Enter the Bat-ANALs.

The BANAL viruses were the result of an expedition to bat caves in Laos in July 2020. Pasteur had been part of a group exploring the same caves 3 years earlier, but they never sequenced the >500 bat samples they collected. Pasteur's Marc Eloit didn't respond when I asked why not.

The 2020 field trip was intended to involve others, including a team from Vietnam, and some from Institut Pasteur Shanghai. Ultimately it appears these groups didn't participate. Two months before the expedition a PLA delegation visited the IP du Laos outpost.

h/t @tommy_cleary

The BANAL sequences have many inconsistencies that should trouble evolutionary biologists @DarrenM98230782, although these concerns aren't unique to them. They also apply to pre-pandemic SARS-like covs WIV claims to have found in a cave, which I believe are artificial chimeras.

BANAL-20-52 is the closest known bat sequence to SARS-CoV-2, after it usurped RaTG13 for the title. Unlike RaTG13 the RBM is also very similar, there's just one amino acid different (Q498H).

The RBM is the key to the ACE2 receptor lock. Different hosts (human/bats/pangolins) have different ACE2 sequences. In this case, the key seems designed to work in multiple locks. But research shows it doesn't work well with bat ACE2, unlike human.

So why is it nearly identical?

The dS/dN ratio is used to measure pressure on organisms to adapt. Between BANAL-20-52 and SARS-CoV-2 there are 9 times as many synonymous mutations as non-synonymous. This shows curiously little selection pressure, despite switching from bat enteric virus to human respiratory.

Interestingly if we compare some pairs of BANAL bat viruses there are a similar number or more, non-synonymous mutations (where an amino acid is changed), than synonymous. There is apparently more pressure for these viruses to adapt in bats, than between bats and humans.

But the stand-out feature of many BANAL comparisons are that some regions have mutated a lot, others very little - the slope of the lines changes sharply, and large regions are flat. This is characteristic of a lab made chimera, though Pasteur claims it is natural recombination.

Why do I think these are artificial, not natural recombination?

-the recombining viruses aren't very similar near the breakpoint (a prerequisite)
-in most cases recombination appears very recent
-breakpoints are exactly where a bioengineer would choose - but why would nature?

The entire RBD region (and more) of BANAL-20-247 is aa identical to RmYN02. It has the same S1|S2 that was claimed to have resemblance to SARS-CoV-2's FCS. There are questions over the finer detail of some charts e.g. there are lengthy regions (~100aa) with no mutations at all.

We can look to early sarbecovs discovered before WIV started delving into making synthetic viruses, to see what we might expect natural pairwise evolutions to look like. All of these viruses were discovered before 2013. Most early sarbecov pairs look similar to these examples:

The features these earlier covs have in common:

-no obvious signs of recombination (green line fairly straight)
-the most variable regions are the RBM and NTD (but these evolved by mutation, not recombination)
-inserts/deletions are rare and short (blue)
-S1|S2 is conserved

We can also look at sequences from outside China. These are distantly related but still have similar patterns of mutation, although there are more overall. Though some geographically distant viruses have a different S1|S2 junction sequence, it isn't due to an insert.

In 2008, speculating how SARS acquired its then unique RBD, Baric proposed it might be recombination. So he made a chimera consisting of a bat virus (a consensus similar to HKU3) with the RBD of human SARS. The SNAP comparison of before and after looks like this:

In 2011, WIV tried to emulate the feat using Rp3 as the backbone instead of Baric's HKU3 consensus. I discovered this from an FOIA to CSIRO which turned up this email from WIV scientist Huajun Zhang who was then interned at CSIRO's BSL-4 lab under Linfa Wang.

Apparently they didn't succeed on this attempt, but in 2013 they announced two new viruses which purportedly proved the bat origin of SARS: RsSHC014 and Rs3367 (or WIV1).

But the SNAP comparison resembles Baric's chimera experiment more than any known natural virus pair.

While Rs3367 has an RBM close to human SARS, RsSHC014's RBM seems to be a consensus which includes Bulgarian bat cov BM48-31, discovered in 2008 by a Christian Drosten group.

Hongkui Deng of Peking University links Drosten's team to WIV's early synthetic biology efforts.

Having successfully passed these off as natural to a receptive audience (including Baric, whose hypothesis seemed validated), WIV went on to make *many* more such chimeras, some of which they claimed were natural. This one Rs9401 (aka WIV16) is an obvious NTD chimera.

This pair of important BANAL viruses show resemblance. Although there are a few more mutations outside the substituted region, I believe it is just a higher quality fraud. WIV learned to add extra mutations in the intervening years, particularly synonymous ones.

This pair from WIV suggest a chimeric construct with both RBM and NTD replaced. This is the basic structure that was proposed in DEFUSE. WIV had been making similar chimeras for years, while claiming they found them in their unique bat cave.

Some claim the restriction enzymes BsaI and BsmBI point to DEFUSE, but WIV were making chimeras without outside help using these. In this 2017 thesis uncovered by @theseeker268 they make many infectious clones using this RGS.

@tony_vandongen points out that the BANAL sequences provide an evolutionary explanation for certain suspicious inserts in the NTD. These attracted a great deal of attention early in the pandemic when IIT scientists announced they had HIV homology.

This prompted Fauci et al to redouble efforts to quash "conspiracy theories". He called the theory "outlandish". The proximal origins conspirators were also disparaging. But there is no doubt that compared to sequences known before the pandemic these NTD inserts stood out.

I have a different theory regarding the origin of these inserts, based on homology to MERS rather than HIV. But I agree that the combination of sequence homology, structural proximity, and evolution by insertion is very suspicious.

A year and a half later, in Sep 2021, Pasteur's Banal sequences were eventually published, purportedly supporting a natural evolution of these NTD features, and further obfuscating the relationship of PLA viruses ZC45/ZXC21 to SARS-CoV-2.

In another example of evolution making special exceptions for this clade, these loops, among the most variable regions of spike, are rarely conserved in bat covs or SARS-CoV-2 variants. But this one is identical between viruses that evolved 3000km apart and in different species.

Comparing BANAL-20-52 with the bizarre RaTG13, there are over 50 synonymous mutations between their NTDs, and just one amino acid change, no adaptation due to immune pressure. But between other BANALs the opposite is true, there are more amino acid changes than silent mutations.

A baseline comparison with an early pair of sarbecovs shows there is selection pressure on the NTD compared to elsewhere in the spike. NTD is exposed to the immune system and must adapt to evade immune recognition. A typical dS/dN is 1-1.5.

The BANALs are just different.

The Banal sequences were very successful in papering over holes in the evolutionary trail. Conveniently they:

-explain the suspicious NTD inserts
-supplant RaTG13 as closest relative to SARS-CoV-2
-support RmYN02s "proto-FCS"
-assert sarbecovs evolve primarily by recombination

Before the Banals we mostly had to rely on sequence data from WIV and the PLA to support a zoonotic origin. But now there is a reputable institution from a country other than China to validate their fraud. The only feature of the genome whose evolution isn't explained is the FCS.

But with so much reliance on these sequences we should also ask if we can implicitly trust Institut Pasteur- are they truly independent of WIV/the PLA/China?

My answer is an emphatic "no".

Pasteur's engagement with China goes back a long way. They worked closely with the PLA's AMMS (including Wuchun Cao) on the epidemiology and origin of SARS.

France was intimately involved in the planning and construction of WIV's BSL-4. The project was driven by businessman Merieux, over the objections of French security services - who warned the PLA would use it to make bioweapons.

Excellent article 👇
mediapart.fr/en/journal/fra…

Pasteur was to be the international science partner of WIV in the new lab, believing they would have some oversight of operations. Institute Pasteur Shanghai, a JV with CAS, was established for this purpose. But things didn't work out so well, recently the JV was dissolved.

Christian Brechot, a former director of both Instituts Pasteur and Merieux was one of the China enthusiasts pushing to override the warnings of the security services. Despite the evidence at the time pointing to WIV and the PLA, he came out strongly against such a possibility:

I don't know how, or by who, BANAL sequences/RNA fragments/isolates were made, but I'm certain they are artificial. Some assert the Paris lab is above suspicion. The same doesn't apply to Laos, where corruption is rife. It would be easy to contaminate a sample with synthetic RNA.

And in any case, security of samples isn't assured - even in France. In 2015 Pasteur managed to "lose" more than 2000 vials of SARS samples from their BSL-3 lab in central Paris, while Brechot was in charge. This case remains unsolved.

science.org/content/articl…

As with WIV's other international collaborators - like Australia's CSIRO and Canada's CNML, there were mysterious and disconcerting incidents after they engaged too closely. At least they seem to be re-evaluating the relationship. They should do the same for the BANAL sequences.

Pasteur maintains their joint venture with HKU, while Hong Kong is rapidly subsumed into the totalitarian state. @drlimengyan1 has made allegations against people at this lab where she was employed. Increasingly they have little choice but to submit to CCP censorship and control.

It isn't only the BANAL sequences that need re-evaluating. All of the zoonotic sarbecovs on this tree are suspect. If fraudulent, the implication is there are no SARS-like viruses lurking in wildlife with potential to cause a human respiratory pandemic.

Not without human help.