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> First and foremost the central claim is that 5 potential restriction binding sites versus 2 means that SARS-CoV2 is non natural. That does not necessarily follow. Just as SARS-CoV2 is unusually infectious and damaging to humans it could just happen to have an additional 3 restriction binding sites. So there is nothing inconsistent with natural selection of viral characteristics, only a comparison between wild and lab viruses.

No, you've completely misunderstood the analysis. The number of restriction sites is not what is important. It's the location of the sites, and the spacing between them. This is suspicious, and has a high degree of variability, as is shown in Figure 3c. They also generated 100,000 random mutations to RaTG13 and BANAL52, and found that only ~1.2% and 0.1% of these, respectively, had restriction maps as deviant as the one found in SARS CoV2 (Figure 4).

The spacing here alone is suspicious, but couple with the number of synonymous (silent) mutations, and you're looking at an outcome extremely unlikely to be found in nature.

https://www.biorxiv.org/content/10.1101/2022.10.18.512756v1....

We've seen horizontal gene transfer in fish, was it aliens or natural selection?

Extremely unlikely events happen when an extreme amount of attempts are made, and natural selection amplifies them if they increase fitness

Your reply after reading the explanation is like you seeing a farm of branded cows and wondering if it was due to extreme natural selection that caused that.

From the layperson article:

> In wild viruses, these cutting/pasting sites are randomly distributed because there's no evolutionary pressure for the virus to be thusly cut and pasted in nature. In infectious clones, however, the humans behind the screen tend to modify restriction sites in a regular way. For any given restriction enzyme or set of enzymes, the set of all cutting sites is called the “restriction map”, and looking at these restriction maps helps us see the fingerprint of infectious clones.

> It turns out, the sticky ends produced by BsaI/BsmBI digestion of SARS-CoV-2 are all unique, non-palindromic, and all contain at least one A or T - all criteria either required or recommended for in vitro genome assembly.

Cows don't randomly develop brands on the order of 0.1% to 1% probability. There isn't a great analogy for this and your example, as well as the hyena one in another comment, are way overstating the case.

The explanation given is based on some premises that I'm not qualified to assess, and others I am.

One of these premises is that their work properly models reality, there seems to be a lot of well informed doubt by subject-matter experts.

Another is that an event with a probability of 0.1-1% is exceptionally rare, its occurrence thus being most likely artificial, and with that I disagree, by looking at endless counter-examples nature provides.

Is the fact that humans found some optimization a proof that any occurrence of it is man-made? I believe most people would say it isn't.

> One of these premises is that their work properly models reality, there seems to be a lot of well informed doubt by subject-matter experts.

The model is fine. There's no more "well informed doubt" than for any other paper. You can certainly debate the details of what they did, but none of this debate is substantial enough to invalidate the work.

What you're seeing is a group of people who have largely pre-judged the outcome, inventing reasons to reject an experiment that disagrees with their prior conclusions. This always happens, in any scientific domain. Nonetheless, there are also a large number of well-informed people who see this as an interesting result. If you don't listen to both groups, you will be misled.

> They also generated 100,000 random mutations to RaTG13 and BANAL52, and found that only ~1.2% and 0.1% of these

This could also be selection pressure - right? Ie imagine 99,000 yield viruses that are non viable… you’d see the same behavior of rare traits being common.

It was an in silico experiment.

But regarding your broader question: there's no reason to believe that thesse viruses experience any selective pressure for the number or location of cutting sites of the particular enzymes being investigated here. They're bacterial enzymes, entirely unrelated to coronaviruses.

Your summary of the paper is unfair. From one of the author's blog:

> Recap: BsaI/BsmBI are particularly useful restriction enzymes to use if you wanted to study a bunch of chimeric coronaviruses like the close relatives of SARS-CoV-2. The SARS-CoV-2 BsaI/BsmBI cutting sites look regularly-spaced (ish). The maximum fragment length is in the bottom percentile of all CoVs digestions in the idealized fragment-number range, the bottom 0.07% for all type IIS digestions within the idealized range, and the number of fragments is also in the idealized range. The SARS-CoV-2 BsaI/BsmBI restriction map looks a lot more like known pre-COVID infectious clones than a wild coronaviruses. All sticky ends are unique & meet other nice criteria for good assembly. All mutations separating these sites from close relatives are silent, and there’s a significantly higher rate per nucleotide of silent mutations within BsaI/BsmBI recognition sites than the rest of the viral genome.

> The odds of meeting any one of these criteria vary, from 1%-0.07% of having such a small maximum fragment length to 1/250 to 1/100 million odds of having such high concentration of silent mutations within BsaI/BsmBI recognition sites. The odds of meeting every single one of these criteria are even smaller. Much smaller.

https://alexwasburne.substack.com/p/a-synthetic-origin-of-sa...

So it's not just "5 vs 2" sites, it's the spacing of the sites, the fact that the site mutations are silent, and the fact that the "sticky ends" are unique.

This paper should move your needle toward "synthetic origin". Similar to how some other papers published in the last few months (the wet market paper among them) should have moved your needle toward "natural origin".

> That argument points out that genetic drift is well characterized and the presence of two closely related SARS-CoV2 variants cultured from the wet market is extremely strong evidence that is where the virus initially appeared.

I assume you're referring to Pekar et al. here? The two lineages are literally just two SNPs apart, so it's near-impossible to distinguish whether they arose from two separate introductions, or just from two super-spreading events after cryptic evolution in humans from a single earlier introduction. Pekar builds an epidemiological model that purports to find that evolution in humans is p ~ 0.5% unlikely; but that result is highly sensitive to the assumptions in that model, most notably their choice of a scale-free infection network (and thus power-law distribution of number of other people each patient infects). Robustness to that infection network isn't studied.

The author of this endonuclease fingerprint preprint also has a preprint on Pekar's model,

https://www.biorxiv.org/content/10.1101/2022.10.10.511625v1

Note that I'm criticizing Pekar here, not endorsing the endonuclease preprint. I don't have a great sense of the correct Bonferroni correction (to borrow Prof. Balloux's framing) to apply to the latter's probabilities.

Asserting that understanding the origins of a virus helps control its adverse impacts is completely bog-standard fare in the literature. Finding it suspicious in this case smacks of an isolated demand for rigor and unfamiliarity with the field. Even a cursory look at papers making a case for a zoonotic origin is likely to reveal statements to the same effect, here's just two for example:

https://www.sciencedirect.com/science/article/pii/S009286742...

>Failure to comprehensively investigate the zoonotic origin through collaborative and carefully coordinated studies would leave the world vulnerable to future pandemics arising from the same human activities that have repeatedly put us on a collision course with novel viruses.

https://www.nature.com/articles/s41591-020-0820-9

> Detailed understanding of how an animal virus jumped species boundaries to infect humans so productively will help in the prevention of future zoonotic events. For example, if SARS-CoV-2 pre-adapted in another animal species, then there is the risk of future re-emergence events.

Right - it should never really be suspicious for a research paper to include text rationalizing why it's a good idea to invest in researching that topic. That's just basic self advocacy.

Their claims around Type IIS assembly are also suspect. eg in Golden Gate assembly, you choose Type IIS that reach over and cut, so the restriction site is absent from the final assembled product.

"Additionally, because the final product does not have a Type IIS restriction enzyme recognition site, the correctly-ligated product cannot be cut again by the restriction enzyme, meaning the reaction is essentially irreversible"

https://en.wikipedia.org/wiki/Golden_Gate_Cloning ----

The choice of focusing on a particular RE pair also smells of p-hacking. Their claim that BsaI/BsmBI makes for easy mixing/matching genomes doesn't make sense in this day and age, when you can use other techniques to make hybrids more effectively (eg, you are not restricted to the natural location of those restriction enzyme sites)

The argument is about the negative space of the RE. Regardless of what the article says, To do golden gate/Gibson/etc. best practice is to cut the template first (with RE) then assemble against the open ends, so to do this you must ablate the existing re sites. The alternative is to linearize by round the horn pcr. At 33kb, it's not impossible but why bother with the pain when it's much easier to snip.

Their description of the assembly strategy as "Golden Gate" seems like incorrect terminology. The WIV has at least published papers using BsaI and BsmBI, though.

https://twitter.com/jbkinney/status/1583267221047869441

https://twitter.com/jbkinney/status/1583248052969562112

https://journals.plos.org/plospathogens/article?id=10.1371/j...

EDIT: Kinney also asserts here that they left the sites in a final assembled genome (i.e., the genome of a replication-competent virus). I'm still trying to figure out if that's true, though.

Just because there are more modern techniques doesn't mean there haven't been older ones used, or techniques used incorrectly.

>Thirdly, this paper emphasizes the strong impact of the COVID pandemic and asserts that understanding the origins of the virus would necessarily aid in preventing future pandemics.

I strongly disagree. If of natural origin, there are a plethora of simple controls that could be implemented. Control doesn't necessarily need to be perfect. Some simple controls could be restrictions or bans on Commercial trade or transport of high-risk animals. If not of natural origin, it obviously indicates that BSL4 controls are inadequate or inconsistently applied. A simple but perhaps costly solution might be to not certify bsl4 Laboratories in dense Urban settings.

I generally agree, but would note that the WIV worked with novel natural or synthetic bat-origin viruses at BSL-2 or -3, mostly not BSL-4. From an interview with Dr. Shi:

> A: The coronavirus research in our laboratory is conducted in BSL-2 or BSL-3 laboratories. [...]

https://web.archive.org/web/20210727042832/https://www.scien...

I believe the research on the bat viruses was being done under BSL-2 conditions, which basically means normal lab practices, an autoclave for waste, and some of the work needs to be done in a biosafety cabinet. Of course, that assumes the procedures are strictly followed.

BSL-2 biosafety cabinets only have laminar flow hoods. They're effectively open air and depend upon proper protocols being followed.

Typically you're more concerned about what's getting into your BSL-2 hood than out.

"the central claim is that 5 potential restriction binding sites versus 2 means that SARS-CoV2 is non natural"

That claim is not in the paper at all. The argument is based on the length of longest fragment and could apply to any number of restriction binding sites.

"the evidence for the wet market origin is trivialized."

It's a statistical analysis of viral genomes. The wet market evidence is unrelated.

Your third point is arguing in favor of ignorance and defeatism. "there is no clear and obvious way of systematically reducing risk" because we do not understand the origin. If we did, there may very well be; e.g. restrict trade in civet cats. Simple actions like this have controlled pandemics in the past.

I really don't see how saying that the pandemic was a bad thing and that there is value in understanding its origin is "a dangerous level of sloppiness".

Well considering that's not what the OP said at all I guess it's ok you don't see that.

> third criticisms...show a dangerous level of sloppiness

and the second criticism?

>These second and third criticisms are not direct against the evidence and logic presented, but show a dangerous level of sloppiness in the research

OP _literally_ said that.

Neither the "second [or] third criticisms" were "saying that the pandemic was a bad thing and that there is value in understanding its origin".

Just because "show a dangerous level of sloppiness in the research" was used in both sentences does not mean they are saying the same thing at all.

The OP's second and third criticisms were:

"the evidence for the wet market origin is trivialized."

and

"Thirdly, this paper emphasizes the strong impact of the COVID pandemic and asserts that understanding the origins of the virus would necessarily aid in preventing future pandemics. This does not clearly follow."

While "saying that the pandemic was a bad thing and that there is value in understanding its origin" might approach a summary of the third criticism it doesn't address the second - more serious - criticism at all.

The accusation of sloppiness is based on two claims, and you're only responding to a bastardization of one of them.

You're suggesting that the species jump is likely to have happened in the wet market, but also claiming that there's no clear & obvious way to reduce risk in the future? That's inconsistent, isn't it?

This comment appears more like slanted analysis from someone with an agenda than a critical thinking scientist genuinely interested in the truth.

It’s amazing how long this farce has gone on. People who had every incentive to lie lied. That’s all.

>Thirdly, this paper emphasizes the strong impact of the COVID pandemic and asserts that understanding the origins of the virus would necessarily aid in preventing future pandemics. This does not clearly follow. Especially if the virus had natural selection origins there is no clear and obvious way of systematically reducing risk. Simply living or traveling where host populations like bats live could be enough to generate exposures and it is not simple to clear people off of rural habitations.

Exterminating all the bats is an option. It would be our first purposeful extinction and would no doubt cause many problems. We need to at least weigh up the possibility. Then we can run the risk benefit analysis.

> first purposeful extinction

Smallpox? And extinction of Guinea worm is in process.

Fair point

People could just try and stop eating them first instead, that's my humble idea.