Thread by @gadboit

Does the furin cleavage site in SARS-CoV-2 originate from these Pangolin samples? The samples are from 2019 but were sequenced in Guangzhou after the pandemic started, and a limited set of raw reads uploaded to ncbi here...

ncbi.nlm.nih.gov/sra/SRR1109326…

Those reads contain one that is a 49nts long, is a perfect match with SARS-CoV-2, and contains the furin cleavage site (FCS). It's effectively impossible that this is a coincidence but is it contamination that was introduced in early 2020 when the samples were sequenced, by...

which time SC2 was already quite prevalent in China, or was it actually present in the sample? @humblesci reckons it's contamination. Hassanin says we can't tell. I have looked in more detail for...


arxiv.org/abs/2108.08163
biorxiv.org/content/10.110…

evidence of contamination, not found any, and I think there is some evidence against it. First some explanation. The archive that was uploaded is a collection of "short reads"-- little bits of genome, mostly around 75nts along, picked at random out of whatever was in the...

sample. One approach to see where the reads might have come from is to match them up to reference genomes, which I have done both with some of my own code, and with a program called bowtie2. This is a bit like doing a jigsaw puzzle where you use the completed picture on the...

box as a guide. The idea here is that maybe our sample contains a mixture of pieces from two different jigsaws-- the Pangolin virus P2S, and the SC2 contamination; or perhaps P2S and some other virus; or maybe it's all P2S (but the sample contains a mixture of variants)...

If I match the reads up to a collection of related viruses using this method, I get slightly better coverage of BANAL-20-236 and BANAL-20-52 (two closely related bat viruses) than I do of Wuhan-Hu-1. So according to this measure there's no reason to think the sample is...

contaminated with SC2 rather than with some other closely related virus. But perhaps this result is confounded by the fact that BANAL-20-236 and BANAL-20-52 might be more closely related to P2S than SC2 is? In other words, there might be more jigsaw pieces that match both...

references (the possibly-contaminating virus and P2S) when one of the references is one of the BANALs, due to their higher similarity in the first place, leading to the higher coverage. But they aren't more similar. The nucleotide similarity between P2S and the two BANALs...

is 89.49% and 89.23%. It's 89.96% with SC2. Basically nothing in it, but if anything, SC2 is closer to P2S than they are. So no evidence here the contamination is with SC2 rather than something else. Another approach I tried is to look at whether the reads themselves cluster...

into two distinct groups. I defined a "group" as containing all reads that share more than some threshold minimum number of nts when aligned. If I set that threshold to anything less than 25 I get one big group. At 25, the read containing the FCS itself jumps into a second...

group. At 26 I get three groups (two of length 1 plus the rest), then with a longer threshold than that it quickly explodes into lots of groups. What I was looking for here was whether there was a robust division into two groups across a range of thresholds. This would have...

implied that two distinct viruses were contributing to the reads. However, because those two viruses are closely related (even if it is SC2 contamination, SC2 is very closely related to P2S anyway) this test doesn't prove they *aren't* two different viruses. But it confirms...

that we don't know. So far all we have established is that we don't know whether these reads are from one virus, or two or more closely-related ones, and whether there is any SC2 contamination there or not. So then I tried to look for whether, if it was SC2, we could find...

any clues as to what variant of SC2 it was. As luck would have it the reads that match WH1 *better* than they do P2S happen to contain a site containing a key mutation: C8782T. There are 16 reads there that have T...
ncbi.nlm.nih.gov/pmc/articles/P…

And none that have C. Of those 16 one aligns significantly better with SC2 than it does with P2S. So what this tells us is that *if* this is SC2 contamination then it is with a very early Lineage A variant, before the big spreading event at the wet market...

This is significant because Guangzhou, where the samples were sequenced, is 1000km away from Wuhan. Although Covid was widespread in China at that time, I would have expected human samples to have had C at 8782, not T. This counts as some evidence that the sample was not...

contaminated at the point of sampling, and that the FCS was actually present in those samples in March 2019. Interestingly there are also no reads in the set that cover the part of P2S where the FCS should be except the one that actually does contain the FCS. And that...

read does match up with P2S by a few nts at the far end. In other words, we don't actually know that P2S *itself* doesn't have the FCS. In summary, we are looking at reads from one or more variants of one or more closely related viruses and there is an FCS in there somewhere...

So what are the possible explanations here? If this really is a Pangolin virus with an FCS, very close to SC2, then that could have actually jumped into humans (natural origin). But it's not clear why that should have happened in Wuhan-- there were no Pangolins at the wet...

market. Another possibility is that some of the tissue samples from these contraband Pangolins were sent from Guangzhou to the WIV. They may have sequenced them there (and perhaps got a much larger set of reads), and discovered the FCS, which it's likely they would have...

taken some interest in. Perhaps they were doing experiments with P2S (or whatever contained the FCS) itself, or perhaps they were swapping that FCS into other viruses. An FCS that occurred naturally in related viruses would be a good choice, and this might also explain why...

it's a bit unusual (it's been described as "non-canonical", "crap", etc). If there was a subpopulation of variants with an FCS then that might become the dominant population after passaging in cells where it was an advantage...

as was found in this experiment with bovine coronaviruses. And of course it is still possible that this is just contamination with human SC2 from 2020 (although 8782T speaks against that). So we don't know...
journals.plos.org/plosone/articl…

This set of reads has been highly filtered (I suspect to whatever matched SARS-CoV-2 or looked "coronavirusy" in BLAST searches). We could tell much more if we had the complete set. I doubt we will ever get it. END.

@stevenemassey @WashburneAlex @humblesci @Daoyu15