Private channel session 158
41 messages over 4h 59m, 2020-06-11 – 2020-06-11.
A “conversation” here is an activity session — a run of messages with under 60 minutes of silence inside it. The channel had no native conversation boundaries.
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While I knew about the furin site insertion experiments, I did know realize this (that the insertion in SARS2 is exactly where they inserted it experimentally in SARS):https://twitter.com/Ayjchan/status/1266805310313967617?s=20
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Gotta say - some smoking(ish) guns starting to appear that I'm not comfortable with and Alina here is touching on alot of points we have discussed previously that were concerning (e.g., lack of selection - I should have looked into that a little closer earlier :wink: ). The furin site in SARS2 is exactly the same as it is in some of the HKU1 sequences. When I talked to Farzan way back in the day he mentioned that the one thing he'd be looking for was whether SARS2 would lose the furin site under different conditions - we now know this to be true (primarily in T/C, but there are examples in people too - intrahost).
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"exactly where they inserted it experimentally in SARS" Sure - but that site with the single R is a required cleavage site - if you're going to convert to a furin site then adding RRA at that exact position is the only thing that makes sense - or even RRAR to make it a super-optimal site like Nunberg did - what would not make sense is to stick aproline in and then to do the whole insertion out-of-frame.
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Yeah, agreed - however, this exact site already exist in other CoVs (just not in exactly this site) - not in HKU1 as Isaid, but in that Feline CoV. The part I'm really struggling with is that at the end of the day, we really don't have any hard evidence one way orthe other - and especially given some of the recent evidence, we also can't rule out that somebody actually put it inthere. That's obviously not to say that somebody _*did*_, but we can't rule it out. Our paper was pretty strong insaying "there's no way", but I have less confidence in that statement at this stage.
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What smoking guns Kristian? I don't quite see it. What recent evidence are you talking about? That Follis paper was literally the first one I looked at. Doesn't it's presence in HKU1 suggest that these events in nature a lot? Do we know what animal HKU1 comes from? I was reading Mike's paper to suggest that D614G was under selection to increase the infectivity of SARS-CoV-2, because it was directly related to the furin cleavage insertion, such that it not perfectly adapted to humans on emergence.
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What about Bill Gallaher's evidence?
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HKU1 actually does have an optimal furin site - some strains have a insert or deletion of SSS - yes some of these Shave predicted O-glycans [shared file(s): HKU-1 Spike.pdf]
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"animal HKU1 comes from" Rodents maybe - probably
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May be. Perhaps rodents are involved in SARS-CoV-2? I'm really struggling to understand why someone would "put" a furin site into a random bat virus they had not previously sequenced and published. Has the Baric group ever done such an experiment? Accidental lab escape is one thing, but why would deliberately do this to a bat virus? Then why would publish the sequence of a related bat virus that puts you in frame? There was no need to that if you were guilty.
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"Then why would publish the sequence of a related bat virus that puts you in frame?" To throw off M15 and Pompeo?
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People have put furin sites into CoVs previously - here's one example from SARS: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111780/
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There's a paper where they put it into a bat virus and shows it has much broader host range - I believe from Baric's group. I'll find it.
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At S1/S2?
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Yup - the paper above is _exactly_ where it was inserted in SARS2 - that's the part I missed previously (I have been aware of these papers all the time).
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(BUT, the inserted site _is not_ the same as in the paper - that would have been a dead giveaway)
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We're going round in circles...that's the Follis paper...the first paper we discussed and I sent you in early Feb.
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My take is that furin cleavage sites are *everywhere* in natural viruses e.g. MERS. I'm revising a paper on avianavulaviruses and they have one. To me, there is a strong natural prior.
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Yes - I have been aware of the Follis paper all along - I was _not_ aware that their insertion was exactly where it is inSARS2
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Also, the Zhou et al. paper never call it a furin cleavage site insertion and I think the alignment in that paper is the optimal one. If so, it is not a simple 'shift' but rather a complex set of indel events.
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Here's the paper from Baric - not the insertion, but showing that cleavage broadens host range (https://pubmed.ncbi.nlm.nih.gov/31801868/) - which is what furin does. This is also not a new paper - I have been aware of this all along as well, so not a smoking gun, but there are several concerns here related to tissue culture
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I don't think the Zhou et al. paper shows that this is multiple indels - to me it just looks like a region not aligning very well?
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Let me spend some time on a table with things that bothers me - because there are several.
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That would be useful. Thanks.
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Yeah, let me get it all down - including the silly stuff :wink:
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Make sure there is a column in your table that can be used for 'possible natural explanation'. For example, I don't see why adaptation in animals/culture would then produce a virus that would need not to adapt in humans themselves.
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I also think that focusing on these genetic details - which are not conclusive - is in danger of missing the bigger picture: that bats (and like other wildlife) carry a huge diversity of coronaviruses some of which are closely related toSARS-CoV-2; that coronaviruses link species boundaries all the time; that 4 other CoVs have emerged in humans inthe last 20 years; that furin cleavage sites are common in viruses and come and go all the time; that people have been warning about emerging CoVs for years. This is not a blue sky event. It just seems to me that we are going over the same arguments we went through in Feb.
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I of course agree with all of this (and still believe this is a natural virus), but the issue is that some of the new evidence certainly is peculiar - plus some additional details that have come to light, e.g., acquisition of furin site intissue culture in bovine CoV. Nothing has changed in terms of prior risk of lab accident, but the issue is that we're dealing with the fact that WIV has been doing culturing of bat CoVs for over a decade exactly to understand what it takes for these viruses toemerge in humans (in BSL-2). We describe this in the paper and say that this work is performed "all of the world", but that isn't true - it has exclusively been going on at WIV. The EgoHealth grant was also specifically funded for this type of work (and described the kind of experiments to be done in tissue culture). This research primarily involvescollecting and culturing viruses from bats in Yunnan - which is where RaTG13 is from and it seems plausible that SARS2 is also from there (no roosting bats in Wuhan in winter). A lot of the GOF work on SARS-like CoVs is also from WIV, although there are also other labs that have been doing that (e.g., Baric). None of this tells us anything new - but it means that given no data, I find both scenarios to be equally probable.Given the data we had seen at the time of our writing, I was highly skewed towards 'natural'. But with some of this new data (plus stuff I didn't know previously), I'm moving much more towards the middle (again).Suffice to say, since writing the paper I have explained to a ton of scientists (and policy makers) over and over again why this is a natural virus, and I have been pretty surprised to learn that most scientists I talk to believe there is alikely connection to the lab - people that are themselves doing GOF and culture-type of experiments, they know the risks and don't seem to think that a natural explanation is more likely than lab culture.
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Fair points. But that work isn't exclusively done at the WIV because as you say yourself the Baric lab are doing it. Istill don't quite see what the new data is. The bovine stuff? What else is there? To me, there is more evidence against lab escape than when we wrote the paper. I think the bats not roosting in winter argument is nothing because I reckon it went through an intermediate host anyway. Do you have a copy of the EgoHealth grant?
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Aim3: https://projectreporter.nih.gov/project_info_description.cfm?aid=9819304&icde=50404048&ddpara m=&ddvalue=&ddsub=&cr=1&csb=FY&cs=DESC&pball=
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> But that work isn't exclusively done at the WIV because as you say yourself the Baric lab are doing it No, that work _is_ exclusively at WIV AFAIK. Baric's lab is doing (did) gain of function research where they created reverse genetics systems to study the viruses. All of this is done in BSL-3, so much (much!) less risk of exposure. The work in Shi's lab is different - here they are taking bat CoVs and culturing them under BSL-2 in cell lines from different animals - to e.g., understand which ones can infect human cells and what type of mutations that occur inT/C may make them permissive. They probably have more than 10 papers where they describe that type of work onall kinds of SARS-like CoVs.
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Got it, thanks. That helps.
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Have the Shi lab published all the viruses they use in these culture experiments? Do they find the same mutations that appear in SARS-CoV-2?
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In our paper we argue against (a) manipulation and (b) tissue culture. Our main arguments were that manipulation ishard, but the German's came out and created a reverse genetics system of SARS2 in less than two weeks, soclearly it's not that hard. Our arguments against tissue culture mostly came down to the O-linked glycans and a'mucin-like' domain. But by now it seems clear that these don't actually form a mucin-like domain, but rather those glycans modulate the function of the furin site. All our arguments about natural viruses emerging, the RBD identicalin pangolin CoVs, etc., all still stand. The main new things that bothers me the most:1. Lack of selection (a lot of the selection is for replication in a particular host species, so yes, I would assume culture would lover selection during subsequent transmission in humans - however, immune selection will still occur).2. Gain of furin cleavage of bovine CoV in tissue culture.3. Loss of furin site in tissue culture meaning it's active.4. The little details about the furin site - e.g., that it's exactly in the spot where experiments had previously inserted it,the fact that the exact sequence exist in a cat CoV, Shi herself being sketchy with details and considering lab accident, etc. But all of this can go both ways
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Unclear to me if they published sequences of all these viruses - it's a lot and names are changing over time (e.g.,RaTG13 had a different name previously).
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And yes - of the six key mutations in the RBD, one of them occur in tissue culture for SARS (*but* it's also present inthe pangolin RBD).
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Are you saying they deliberately inserted the furin cleavage site? I see no evidence of them ever doing that before. (Idon't think RaTG13 had a different name...I think there a partial sequence from another virus that is identical. Canwe do a quick zoom?
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To me, "Loss of furin site in tissue culture meaning it's active" strongly argues AGAINST culturing as being the cause of the cleavage site insertion.
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Just making some dinner - let's Zoom in ~30mins?
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Let's say 6:30 pm time. OK?
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https://uni-sydney.zoom.us/j/98148315438
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I'm on!