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[1]: One must remember that SARS-CoV-2 was never found in the wild and that RaTG13 does not exist as real virus but instead only as a sequence in a computer. It is a virtual virus which thus cannot leak from a laboratory. (from WP:NOLABLEAK) What exactly does that mean? RaTG13 is a real virus, isn't it? ProcrastinatingReader (talk) 19:32, 30 July 2021 (UTC)

I think what this means in context is that RaTG-13 is a reconstructed set of sequences, from pcr samples. As far as we know, no full reconstruction and rescue via reverse genetics of RaTG-13 ever took place. And there’s a good reason for that…

It’s extremely difficult to do viral rescues at all, and it’s usually done using sequences that have been OPTIMIZED for this purpose. This would be trying to do it in a completely NEW sequence that hasn’t been optimized or tested to see which conditions work best.

The actual process of rescuing a virus requires a sequence that has been “codon optimized” so that it uses the codons (for tRNA) that are most efficient for transcribing into RNA and then translating from RNA into proteins. Because you’re usually doing it in an extremely artificial environment.

For example, typically when you do this for influenza, you “co culture” two types of cells together (one from human embryonic kidney cells (HEK) and another from dog kidney cancer (MDCK)). One of these cell types is really good at getting all the dna pieces inside of it to make RNA and then the proteins to build a virus, but cannot really get infected. The other type is really good at getting infected.

For each new virus, these super specific conditions (which cell types, which media (meaning nutrient soup), which ratio of DNA pieces, which modifications to that DNA) need to be figured out.

Turns out, trying to do something artificial like this (make a virus from scratch) is much harder than you might think.

That’s why Ralph Baric’s lab used chimeras when they were investigating novel bat coronaviruses, because they’ve got it optimized for the “backbone” already! So all they’re changing is the spike protein. This means the rescue is actually feasible.

It’s also why people use “pseudo viruses” because pseudo viruses don’t need to go through this whole process, since they’re not actually functioning as viruses. They just need to be lil spheres of membrane with spike protein on them. They aren’t replicating etc, like a rescued virus would be.

Rescues are difficult because you’re taking the genome of an RNA virus, turning it into DNA, then trying to get a cell to make RNA out of that DNA, then make protein out of that RNA, then package the RNA into the protein, in a way that makes the actual factual virus (or an artificial “version” of it anyway). All in cells that normally don’t get infected with this virus. Because we don’t have good high quality robust bat cell lines.

This is Also why rescued viruses tend to be, for lack of a better term, a little janky. They are just a poor substitute for the real thing, since they weren’t “put together” the way a normal virus is. We try to guess the ratio of the different parts of the virus, the mutations that will make it easier to rescue but not make it not work at all, etc etc.

I do not know of ANY virus that replicated better as a viral rescue than it did as a wild natural isolate. Can’t think of a single one.

Doing the rescue for the first time is a Science or Cell paper in and of itself, and takes multiple years. There’s lots of viruses that most people have just decided it’s impossible for.

But if you have a good system working to rescue a very closely related virus, you can just pop in the spike protein from your new one, rescue it again, and you have a good way to run the tests on the spike you wanted to run on the actual virus. And it’s still a hell of a lot easier than trying to grow the natural isolate (which is a whole thing in and of itself, and is probably the biggest bottle neck in virus discovery).

TL;DR— it’s a real virus in nature, somewhere. In that cave where they found it, probably. But it’s never existed as a real virus in the lab, as far as we know.—Shibbolethink ^{(♔ ♕)} 20:04, 30 July 2021 (UTC) Shibbolethink ^{(♔ ♕)} 20:04, 30 July 2021 (UTC)

That's quite fascinating (and some topics there will make for some interesting late night reading material I think). Thanks! It's not directly WP-related per se, but I figured I'll finally read up on the science here. Maybe a couple follow-up questions, if you got a sec...

First, backtracking a lil bit... RaTG-13 is a reconstructed set of sequences, from pcr samples & it’s a real virus in nature, somewhere. In that cave where they found it, probably. -- so they don't actually have the virus? Maybe a bit of a stupid/elementary question, but if they took droppings from bats within which they found this virus, that thing would be a real virus right? Assuming it's still live (apparently possible for SARS-CoV-2, perhaps for similar viruses too?) why can't you just multiply it in a petri dish with some suitable host cells or something? Then you'd have a lot of normal virus, right?

Secondly, As far as we know, no full reconstruction and rescue via reverse genetics of RaTG-13 ever took place. And there’s a good reason for that… I presume this process is used to learn more about infectious diseases? Since the process seems time consuming and difficult, how do researchers identify which samples seem promising to work on?

Is there any good/quality reading material on the sentence about Ralph Baric's work you mention above? I've seen him mentioned on talk pages with links to blogs/journalism, presumably it's about the same thing, but never bothered to read those since they didn't seem of the best quality.

Does high similarity with RaTG13 indicate anything about where SARS-CoV-2 came from? I mean, would it be a reasonable conclusion that it originates in an animal in or near the cave they found RaTG13? (the area they found RpYN06 seems to be near, too)? Also, since SARS-CoV-2 spreads well in humans, does it also spread well in bats? If so, would it be a reasonable assumption that there are probably a lot of samples of SARS-CoV-2 in caves in that area (in bats or their excrements etc)? ProcrastinatingReader (talk) 11:34, 31 July 2021 (UTC)

ProcrastinatingReader, sorry for the delay, I have been formulating an explanation to this! Forthcoming--Shibbolethink ^{(♔ ♕)} 10:04, 2 August 2021 (UTC)

re: so they don't actually have the virus? Maybe a bit of a stupid/elementary question, but if they took droppings from bats within which they found this virus, that thing would be a real virus right?...why can't you just multiply it in a petri dish with some suitable host cells or something? Then you'd have a lot of normal virus, right? Unfortunately, it is not that easy. Many long and detailed papers have been written about how best to do this. Every step in the process of sampling, growing, identifying, creates "filters" that affect how we view the natural world. Many viruses we sample this way are not compatible with the conditions in vitro. We also have to use lots of antifungals and antibiotics to make sure that such cultures are not overgrown with contaminants. We are creating the perfect environment for the growth of these other non-virus pathogens (warm, wet, dark, exactly the temperature of a human body), and so we are truly "inviting" the bacteria and fungus in these samples to grow as well. They don't need the cells, they just love the conditions we grow the cells in. But the antifungals and antibiotics can actually change the way the cells act, putting them on high alert to invading pathogens, which makes them mount defenses against the viruses we hope to grow there. Likewise, the way we identify cells is itself a filter, because not all viruses make pretty "plaques" on cell surfaces (the so-called "plaque assay" which is often used to identify and isolate viruses). These are just individual examples of an overall property of this type of experiment. And I don't think these examples necessarily apply to RaTG-13, just saying these are the reasons you wouldn't do these experiments in the first place.

Instead, many researchers have, just in the last decade or so, come up with novel ways of hunting viruses that avoid all these problems! Ways that are unaffected by some of these sampling biases and filters. What they do is, they get the sample from the animal, they mix it up really well so that it releases some of its viruses, then they combine it with Trizol, a reagent that is useful in separating out RNA/DNA from everything else. Unfortunately, this has the effect of killing all the cells/viruses in the sample. Then, you add DNAses (enzymes which destroy DNA but not RNA) and add them to the sample. Then, you use any of several processes to randomly "fragment" the RNA (sonic vibrations, radiation, enzymes, all of these work) You want to try and get the RNA to be small enough pieces but not so small that it's unusable. Then, you use a process called "shotgun sequencing" or "parallel sequencing" to find out the sequences of all these little pieces. Since the fragmenting process was inherently random, and you expect to have many multiple copies of the virus in your sample, you should be able to then computationally reassemble all these pieces into a complete genome. It doesn't always work, and for wild samples like this it's even more difficult, because you have less virus than a sick human patient (which is what these techniques were developed on). What they also do, is use known coronaviruses as a "guide" to help assemble. This allows them to more specifically examine the parts of the genome which diverge from known coronaviruses, as the reads will "align" onto this template and show clearly where the nucleotides are different. This is also probably why the researchers were able to first release the polymerase (BtCoV/4991) before later releasing the whole thing (RaTG-13). They had the most confidence in the polymerase assembly (which was most similar to the template).

BTW, the other reason for this is that RNA is really really fragile. In a sample of bat guano, there are probably millions of RNAses, waiting to chew that genome up. RNA is more vulnerable than DNA, because it isn't double-stranded. That's why Trizol is so great. It isolates the RNA and denatures all those nasty RNA-killing enzymes. If you just froze the guano at -80dC, it might still have live virus in it for like a month, a few months at most. But after that, all the virus in there is dead. Just freezing it once and thawing it once immediately kills 90% of the virus in a sample, because the ice crystals are so brutal to molecular structures. And that's WITH additives (like DMSO) to try and minimize this destruction. That's why we grow large amounts of virus (1 or 2 million particles per mL) when we freeze it down as a pure stock. We expect to lose 90% (or 1 log). RNA in trizol, however, can last years.

re: reverse genetics--I presume this process is used to learn more about infectious diseases? Since the process seems time consuming and difficult, how do researchers identify which samples seem promising to work on? Money. It's all about the benjamins. What do people give you money to work on? Where is the funding? But it's a direct product of the state of the field. Which viruses are the most likely to result in a good publication in a high impact journal, that will elevate your status and help you advance in the world and become more respected? Before this pandemic, nobody really cared about coronaviruses. Sure, SARS-1 happened, but that was 17 years ago! All the funding had dried up, vaccine candidates were dying on the vine. Plainly speaking, all the politicians and funding people didn't really see the benefit of sinking money into this field, and so grants weren't around. The EHA/WIV partnership was basically nothing in terms of money allotments when it comes to biomedical research. I think it was like $500k total? That's like half of one R01 grant [2], the basic minimum to run a small lab in the US, with maybe a grad student, a tech, and a post-doc. And these sampling expeditions are expensive. So you have to be careful how you spend that money. There is also a sort of "trial and error" to this, where you do it on one related virus, and that helps you figure it out for others. But overall, it just makes sense to use these pseudovirus and chimeric approaches, where you don't end up beating your head against the wall, and you end up saving money as well. You don't need the full virus to do experiments.

Re: Ralph Baric sentence, do you mean reading materials about why it makes sense to do these chimeras and pseudoviruses? Well the original 2015 paper is helpful: [3]

recent metagenomics studies have identified sequences of closely related SARS-like viruses circulating in Chinese bat populations that may pose a future threat1,6. However, sequence data alone provides minimal insights to identify and prepare for future prepandemic viruses. Therefore, to examine the emergence potential (that is, the potential to infect humans) of circulating bat CoVs, we built a chimeric virus encoding a novel, zoonotic CoV spike protein—from the RsSHC014-CoV sequence that was isolated from Chinese horseshoe bats1—in the context of the SARS-CoV mouse-adapted backbone. The hybrid virus allowed us to evaluate the ability of the novel spike protein to cause disease independently of other necessary adaptive mutations in its natural backbone. Using this approach, we characterized CoV infection mediated by the SHC014 spike protein in primary human airway cells and in vivo, and tested the efficacy of available immune therapeutics against SHC014-CoV. Together, the strategy translates metagenomics data to help predict and prepare for future emergent viruses.

But also there's this paper from another lab in the US: [4]

Every year, additional CoV sequences are discovered. However, there is a massive knowledge gap in the field, as very little work is performed after the viral sequences are published. Therefore, it is unknown whether these viruses have the potential to emerge in human populations. Current methods for studying betacoronaviruses are technically demanding. Viral isolation from field samples is rarely successful and reverse genetics recovery of recombinant virus is labour intensive and expensive, as the synthesis of a single genome can cost upwards of US$15,000. These limitations are prohibitive to studying CoVs at the scale at which they are discovered....Additionally, a previous study showed that replacing the RBD of the lineage B bat virus Rp3 allowed the virus to enter cells expressing human ACE216. We therefore developed a method to functionally test the RBDs from lineage B betacoronaviruses in place of the SARS-CoV spike RBD.

There is also this book: [5]

[Baric] explained that the original vaccine target for the SARS-CoV outbreak 2002-2004 strain was 99 percent identical between human and civet (Ge et al., 2013). However, metagenomic sequencing showed that bat SARS-like CoV (SL-CoV) with 65 percent to 95 percent sequence homology, can constitute a large pool of strains with pandemic potential against which countermeasures need to be developed. To evaluate whether the existing vaccine and drugs work on these strains, Baric's team and others used two types of approaches. The first was based on the production of CoV pseudotypes coated with virus spike-like proteins that can potentially engage the human angiotensin converting enzyme II (ACE2), which is the SARS-CoV cellular receptor molecule. This method constitutes a safe and ethical research alternative approach. Similarly, chimeric recombinant viruses that encode spike-like proteins as part of the virus particle can also be used. While studies using pseudotypes and structure-based prediction confirmed the existence of a bat SL-CoV that can infect human cells, only studies using GoF chimeric virus identified an additional bat SL-CoV as a potential threat. Baric noted that both bat SL-CoV were less virulent in a mouse model. Importantly for public health implications, data further showed that existing vaccine and human monoclonal antibody therapy failed to protect against these two newly identified bat SL-CoVs, leading Baric to point out that “we are vulnerable” to SL-CoV bat strains that currently exist in nature.

Re: Does high similarity with RaTG13 indicate anything about where SARS-CoV-2 came from? I mean, would it be a reasonable conclusion that it originates in an animal in or near the cave they found RaTG13? (the area they found RpYN06 seems to be near, too)? Yes! it does tell us something about the origin of the virus. That RATG-13 and SARS-2 likely share a common ancestor. And RpYN06 also tells us something! That an ancestor of SARS-2 likely, at some point in the past, took part of the genome of an ancestor of RpYN06 while they were infecting the same cell, likely through genetic recombination (which we know occurs in coronaviruses in the wild). Because one big chunk of RpYN06's genome (ORF1, ~20 thousand bases out of 29.9 thousand) is 97% identical to SARS-2, more identical in that region than any other known virus. This is so-called "identity by descent."

The thing about this, though, is that we shouldn't restrict ourselves just to the bat caves in Yunnan. Many of the closely related viruses have also been found in Thailand, Malaysia, Cambodia, etc. and others have been found in Zhejiang, an eastern province of China along the coast. The family of bats these viruses have been found in various places all over Southeast Asia, including Hubei province (where Wuhan is) [6]. In fact, other studies in the past have identified these exact bats carrying sars-like coronaviruses in Hubei province [7] (see Figure 1) and [8] (see Figures 1 and 3). I would say that the fact that these Yunnan bat caves is where these most closely-related viruses (like 96%) have been found is probably more because that's where people have most recently been looking. There's no reason to assume that's the only place this virus could have come from in Southern China. Bat migration patterns don't really care about these geographic restrictions, either. We know that at least some Rhinolophus bat species (the genus of bats where many of these viruses are found) migrate hundreds of kilometers between roosts [9]. We also know that the viruses don't really care to remain inside one host species, they move around among different bat hosts ([10] see figure 5).

All of which to say, a Hubei province origin is absolutely still a large possibility, and I would even say somewhat likely.

I hope that helped some! And sorry for the delay.--Shibbolethink ^{(♔ ♕)} 11:12, 2 August 2021 (UTC)

Thanks Shibb!! I'm slowly making my way through it all and the given links (through much of it but not the Ralph Baric stuff yet). And no problem, it's very hard to complain when you're getting free teaching from a virologist ;) ProcrastinatingReader (talk) 16:36, 7 August 2021 (UTC)

Ive heard Covid19 was only sequenced using Illumina COVIDSeq Test. Where there's conflict of interest between various board directors sitting on the FDA and other company boards. It begs the question is "trusting the science" dependent on trusting Illuminas computer program? Is there outside peer review and scrutiny? Ive heard a computer programmer say once he has to program for quantum computing hes moving into management from trying to program a binary choice thats not there. It begs the question is quantum computing so compartmentalized its a intended or unintended hoax? Ive heard this Illumina argument presented as the possibility that not only are we putting all of the trust into Illumina to tell us the virus is real or unreal. But what exactly a vaccine is programming our cells to do. However, I think the current vaccines are similar enough in effect to Kawasaki disease cytokine storm to add credibility to a wild strain argument. Where, I believe, the main target for gain of function to be the spike protein?— Preceding unsigned comment added by 2605:a601:a0c6:1200:709e:3489:340c:8d9 (talk) 18:19, 9 August 2021 (UTC)

Reference error at COVID-19 misinformation

In this edit you added a reference titled "nih-iver-2021-01" but this reference is not defined, so it is causing a reference error. Can you fix this? Velayinosu (talk) 01:06, 20 August 2021 (UTC)

Velayinosu, fixed  — Shibbolethink ^{(♔ ♕)} 01:41, 20 August 2021 (UTC)

Appropriate page

Excuse me I would like to know which is the appropriate page where should I write on. Datafiller (talk) 22:25, 28 August 2021 (UTC)

I answered this question on your talk page, please reply there. Thanks.— Shibbolethink ^{(♔ ♕)} 22:26, 28 August 2021 (UTC)

Your DS notice

Can I humbly suggest, in place of your green DS awareness declaration: {{Ds/aware|covid|cam|cc|ps}} and whatever other topic codes suit your fancy? Anyone trying to post an alert on your talk page will eventually get the big bad red edit notice, and it'll warn them not to alert you to whatever topics you include in this template. Firefangledfeathers (talk) 18:40, 9 August 2021 (UTC)

Firefangledfeathers, I mean I appreciate the suggestion as always, of course. But I haven't had anybody actually try to put a DS notice on my talk page, like...ever. So I might just give them to myself and call it a day :P or like you said, use Ds/awares and just make them hidden. I don't want to make life harder for other people :)--Shibbolethink ^{(♔ ♕)} 15:45, 12 August 2021 (UTC)

True wisdom Firefangledfeathers (talk) 17:19, 12 August 2021 (UTC)

Special:Diff/1001780023 (not to avoid receiving it, that I don't find to be a problem, more because it applies to everyone editing on the topic and I regularly issue it myself) —PaleoNeonate – 02:27, 13 August 2021 (UTC)

PaleoNeonate, Yes I know what you mean. If I'm going to do it to others, might as well know what it feels like doing it to myself. This is a general principle I have applied to most of my life! The exception being surgery. I may be crazy, but I am certainly not crazy enough to match the russians.--Shibbolethink ^{(♔ ♕)} 12:11, 13 August 2021 (UTC)

Oh yes, I remember of this, amazing... —PaleoNeonate – 21:12, 13 August 2021 (UTC)

Administrators' newsletter – September 2021

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WikiProject Medicine Newsletter - September 2021

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Thanks, Ajpolino (talk) 05:50, 9 September 2021 (UTC)