Slack / Private Message Drop — page 23

[2020-05-12 21:12:48] [Eddie Holmes] There's so many I've lost track! [2020-05-12 21:13:30] [Eddie Holmes] Bob/Kristian - can you give me your latest thinking about whether any lab manipulation would involve cell culture or live animal passage? Can we exclude the former? [2020-05-12 21:15:13] [Kristian Andersen] Yeah, either cell culture or animal passage. I find animal passage to be unlikely, since those are complicated experiments. I'm starting to worry about cell culture again because of some of the things we're noticing in the cell culture sequencing experiments (plus all their historical work of exactly that type of work - but we have already discussed that). [2020-05-12 21:20:07] [Eddie Holmes] What do we now think about the o-linked glycans and the low dn/ds values? Can they be used to rule anything in/out? [2020-05-12 21:22:00] [Kristian Andersen] Initially we thought the O-linked glycans could somewhat rule out lab culture (or wouldn't be consistent with), but I'm much less sure now - I'm not convinced they actually form a glycan shield and instead may help regulate the site itself. The low dN/dS value - primarily in the spike - has always been peculiar to me and a tiny bit suggestive of culturing. [2020-05-12 21:24:31] [Eddie Holmes] Thanks. Very important. [2020-05-12 21:25:59] [Eddie Holmes] My first look at the virus suggested that the pattern of distribution of sequence variation across the genome looked natural, but that may too broad brush. [2020-05-12 21:28:09] [Kristian Andersen] The three main new things that bothers me - the first two shown in this post http://virological.org/t/identification-of-a-common-deletion-in-the-spike-protein-of-sars-cov-2/451: 1. The furin site is clearly active in tissue culture. We see the virus lose it under some culturing conditions, but this effect could be very cell-type specific. What bothers me here is that the virus is clearly fiddling with this site in culture. If it can lose it, likely it can gain it? Typically SARS-like CoVs are cultured in the presence of trypsin (to allow for cleavage of S1/S2), but if trypsin isn't added, there's a *huge* selection pressure for gaining a furin site. SARS-CoV-2 can grow without trypsin - other SARS-like CoVs can't. 2. The example from bovine CoVs (post #6 on the thread on virological) is exactly a 12bp insertion seen in tissue culture creating a furin cleavage site. 3. We have now seen multiple examples of SARS-CoV-2 de novo obtaining exactly 12bp insertions in different parts of the spike, but only in tissue culture. They're not furin sites, but there seems to be some affinity specifically for 12bp insertions (in T/C) - and of course, the furin cleavage site is exactly 12bp. There could be some specific mechanism behind this. We're looking into this a little closer at the moment. [2020-05-12 21:28:47] [Kristian Andersen] > My first look at the virus suggested that the pattern of distribution of sequence variation across the genome looked natural, but that may too broad brush