TWiV 1357: Furin, Naturally with Paul Bieinasz

Welcome to This Week in Virology

0:00This Week in Virology, the podcast about viruses, the kind that make you sick.

0:10From Microbe TV, this is TWIV, This Week in Virology, episode 1357, recorded on September 11th, 2026. I'm Vincent Racaniello, and you're listening to the podcast all about viruses. Joining me today from Ann Arbor, Michigan, Kathy Spindler. Hi, everybody. Here, it's 76 degrees, and it's just beautiful. Blue sky, white puffy clouds, really, really nice. Again, the app that I used to have

0:45that gave me the temperature in Celsius is not here anymore, so just imagine it's perfect. It's close to room temperature, so it must be close to 25-ish. Here in New York, it is 27. It's sunny. Yeah, it looks nice out, but I have no windows here, so I can't tell. Also joining us from Western Massachusetts, Alan Dove. Hello, and it is gorgeous here, too, 77 Fahrenheit, 25 C, and it is a CAVU day, as they say in aviation, ceiling and visibility unlimited, clear blue

1:19skies, gorgeous light winds. CAVU, is that a thing, CAVU? Yeah. Yeah, ceiling and visibility unlimited. You'll love those conditions. Wow. Just means no clouds, and it's clear. Our guest today is right here in New York City on the other side of Manhattan from me. You can just take the cue and get there very reasonably. He's at Rockefeller University. He's been on TWIV before. Paul Beanash, welcome back. Hi, Vincent. Hi, everybody. Great to be with you. Lovely day here.

1:51Not greatly different weather here than on your side of the island. And taking the cue for those non-New Yorkers doesn't mean getting in a line in this case. It's a train. Yeah. So, yes, I am in my office. This is all natural light. Yeah, he's got a big window looking out on the East River, right? Nice. Yeah. It's very nice. I bet you watch the boats because they're so cool, right? I do. After a while, it gets a bit same old, same old. This time of year, you can have some really quite impressive, expensive yachts sailing by, and sometimes I

2:27Google them, and it's like a million dollars to spend a weekend on. Yes. You're near Hellgate, right? You can see the intersection of the three rivers? A little south of there. Right, but you could probably look to an angle and see. Yeah, we could. If you enjoy these programs, we'd love to have your support to produce them. You can go to microbe.tv slash contribute. And yes, today is 9-11. It's 25 years after that terrible incident here in New York City. And there are a lot of

3:01remembrances. This morning on my way in, I passed a high school in New Jersey. Many of the states had people who died in the towers on that day. And so this was a high school. They had put a flag in the ground for every person who had died. So there were thousands of flags. It was very moving. So RIP

Viral nuclease and genetic innovation

3:21everyone. Well, Paul is here because at ASV, he gave a talk, which was amazing. So first of all, just the two days before ASV, Theodora texted me. She said, make sure you go to Paul's talk. He's going to explain where the furin cleavage site came from. And so with that, I said, well, I would have gone anyway. So he gave a talk about that. And the reprint is out. And so I wanted to get him

3:55on to talk about it. The name of it, it's on BioArchive. So it's just under review now, I presume. The name is Genetic Innovation in Coronaviruses Driven by a Viral Nuclease. And before we get into the science, Paul, you got a bunch of authors on here. Can you tell us something about them? Yeah. So first author is Christopher Bianco, and he's responsible for the bulk of the work in the paper. He's a postdoc. He's been with me for a number of years. Before

4:28that, he was a graduate student with Ian Moore, but a terrific young scientist. Others on the paper, Alex Thable, he's our resident bioinformatics expert. Manevel Loder, Miranda Aldis, Michael Tartel, they all contributed in important but less large ways to the paper. And of course, my wife and scientific partner, Theodora Hatsyuanu, we sort of do everything together.

5:02So take us back to the very beginning and talk about how this got started. So, okay, it's a little convoluted. So you may have to bear with me for a minute. We like convoluted. So basically, towards the tail end of the pandemic, it became much easier to do genetics in coronaviruses. As you know, they have large RNA genomes and the systems for actually

5:36doing molecular biology in coronaviruses has been really quite difficult to handle. But a group came up with a method. It's called CEPERF. It's basically a circular polymerase chain reaction method where it's become infinitely easier to assemble recombinant coronavirus genomes.

6:00As retrovirologists, we are very used to carrying the entire genome of our virus on a single plasmid, transfect cells, get virus out. It's been frivily easy to do genetics. But when this method came out for studying coronaviruses, I thought, ah, this is something we can really do. We've been, historically, we're HIV retrovirologists. During the pandemic, we did a lot of work, as you know, on coronavirus neutralization. But I've been sort of looking for

6:31ways to get into really studying coronavirus molecular biology. And the advent of this tool enabling us to do recombinant work with coronavirus really spurred this. And so one of the things we thought we could do was basically to make recombinant viruses to measure the recombination rates between coronaviruses. It's something we know happens, but there aren't really good tight measurements of how frequently coronavirus genomes recombine.

7:03So at the time, SARS-CoV-2 was still a BSL-3 pathogen. So we built our first recombinant coronavirus system based on a common cold coronavirus, OC43. And the first virus we made, other than the wild-type virus and simple viruses carrying reporter genes, were basically viruses that had inactivated GFP reporter gene and inactivated in such a way that they had

7:34frameshift mutations introduced near the amino terminus and near the carboxy terminus. So that made viruses that made pieces of GFP, but not a functional GFP. But if you co-infected cells with those two viruses, if they recombined, they'd make a virus that made a functional green GFP. So that was sort of our first piece of coronavirus genetics. We haven't actually published that yet. And just for the general audience, GFP is green fluorescent protein, a very handy

8:08fluorescent assay. So when the GFP works, they glow. Right. Yes. It's been a while since I've been on TWIV. No, that's all right. I'm still calibrating where I have to pitch this. So you've got this frameshift. Right. So one of the things we found actually early on was that if we made these two viruses that signified recombination by acquiring green fluorescence, we found that if you change the

8:41nucleotide sequence of one of the GFPs, okay, we expected the recombination rate to go down. Okay. But actually the opposite happened. It went up.

8:55I probably won't get into the details of why we think that might be the case. It's only, we only have hypotheses. We don't have data. But we figured that maybe, maybe one of the reasons might be because what's happening is that double-stranded RNA is forming. And during replication, the sort of strand transfer event that accompanies recombination might be facilitated if there was breathing of the duplex RNA and the virus was trying to

9:27recombine with double-stranded RNA. And that would be facilitated if the duplex could breathe. Another idea was that perhaps when you have double-stranded RNA that's imperfectly matched, it might be more nuclease susceptible. So we did an experiment where we tried to measure the recombination rate between these two viruses that had the ability of the duplex to breathe,

10:01so we supposed. But we also made viruses that had mutant, wild-type mutant NSP15. The hypothesis being that perhaps the nuclease was nicking the duplex and facilitating recombination. That hypothesis proved to be completely erroneous. But it gave us some of the reagents that initiated the paper that we're going to talk about today. So one of the other things that we thought would be interesting was to

10:38actually try and measure the rates of other types of evolutionary events. So there's a long history of measuring nucleotide misincorporation into viruses as they replicate. But what's been much more difficult to study are rates of insertion and deletion? Because they are, at least were thought to be, much rarer events. We didn't know much about their frequency or much about the mechanisms accompanying them.

11:11Sequencing tools, until quite recently, weren't sufficiently developed to measure those rates. And so because we were building these tools with coronavirus OC43, what we then built was a tool that we thought could measure these insertion and deletion rates. And the way we did it was related to the tools that I just explained to you. But what in fact we did was to put a GFP gene into the viral

11:42genome, and then just knocked it out of frame. So it wouldn't make a green fluorescent protein. It would, it's the GFP gene is preceded by a peptide with a start codon that's out of frame with the bulk of the GFP gene. So it doesn't make green fluorescence. But if that virus acquires insertions or deletions in that particular spot, and of a particular length, it would put the GFP back into frame,

12:12and we get a green virus. And then in so doing, we could both measure the rate at which those events happen. And then by isolating those GFP positive viruses, we could see what actually happened and perhaps learn something about the mechanism. And that that's basically the genesis of figure one. And the elaboration of that experiment was to include a virus that did or didn't have NSP15, which we'd actually made for another experiment. And basically, because we had that

12:48virus, we incorporated it into this experiment. And that's what led to the finding, the central finding that's depicted in the first figure. That is basically that having a nuclease in the viral genome greatly increases the rate at which the virus acquires insertions that put that GFP reported gene back into frame. Were you surprised? We were actually, yes, quite surprised. The hypothesis was, in fact, and this had some basis in a previous

13:26publication, that it would perhaps affect the rate of deletion. So when you think nuclease, you think deletion, you don't really think insertion. But in fact, the opposite occurred. While we don't really have enough numbers to quantify the deletion rate in this experiment, it's basically a black and white result when it comes to insertions. So we were actually very surprised. Weren't expecting it at all. But that's what kicked off

13:58this entire line of investigation, that kind of serendipitous result. So why did you choose OC43? This is one of the common cold coronaviruses, right? Yes. So it's a virus that grows very well in cell culture. BSL-2 pathogen, so we could work with it without too many restrictions. We did have access to BSL-3, and we have more access to BSL-3 now. And now we can actually work on SARS-CoV-2 at BSL-2. But such was our impatience to become coronavirus molecular

14:34biologists and didn't have unlimited access to BSL-3. We started with OC43. It's an interesting virus in its own right, actually. And we found some other things that are quite fascinating about it, but that's for another day. So NSP-15, it was known to be a nuclease. And so what was known about it

The canonical role of NSP15

15:00before? So this is, in large part, the work of Susan Weiss, who's a long-time contributor to coronavirus biology, working with Volker Thiel, I think, initially, but then subsequently on her own. The canonical role of NSP-15 is basically to reduce the amount of double-stranded RNA in cells. So double-stranded RNA, as you know, is something that's sensed, causes cells to make interferon,

15:35bad for virus, sometimes bad for pathogenesis. And what was found is when you deleted or inactivate, you can't actually delete NSP-15, you have to mutationally inactivate it from the virus. The virus makes more double-stranded DNA, a more aggressive innate immune response. Not much effect on replication in cell culture, but very dramatic effects on replication in animals in a way that's really dependent on their innate immune response. I should say very clearly,

16:14we don't have any problems or any reason to doubt that as the canonical role of NSP-15. What we're describing here is an additional role, not anything that supplants what's previously been reported for NSP-15. There's a more recent paper from Vinny Menachery and Andrew Routh, who did some deep sequencing of SARS-CoV-2 wild type and NSP-15 mutant. They found something

16:48not very different to what we found. In fact, we used their publicly available sequence data sets in our paper. They found that NSP-15 affects the deletion rate and the recombination rate. The mechanisms underlying those effects aren't really worked out, but the effect is clear. I wouldn't feel very comfortable speculating on what's going on. I don't really have a clear idea

17:20how to explain their data, but it's clear that that happens too. So NSP-15 clearly chops up viral RNA, perhaps not equally all viral RNAs, but that has somewhat pervasive effects, right? Innate immune activation, change in the balance of viral RNA deletions. Also, subgenomic to full-length RNA ratios change a little bit when you complete NSP-15. So it's probably doing multiple things.

17:56I mean, this is a question I want to ask later, not now. You know, you have this canonical function and something new you discovered, but I want to wait a bit, okay? Because it's an obvious question, right? Anyway, Kathy, did you want to ask something, your purple question there? Oh, well, yeah. So you've described why you use the NSP-15 mutant. It was sort of something that you already had, but would there have been, and you were kind of surprised by the result, if it had not turned out to be that NSP-15 was having the effect that it does,

18:33did you have another candidate in mind? Like, next in line, we will try some other gene, or are there other possible candidates? Actually, no. For the phenotype we found,

18:49we weren't even thinking about that as something to look for, right? We, Chris, as I recall, said, well, let's put this NSP-15 mutant in because there's this paper saying it affects the amount of deletion and recombination. Let's see what effect it has on our assay. And you had it already. Yeah. And it, not only does it affect deletion, but it affects insertion, and that's what was surprising, right? We had no reason to think that it would affect insertions.

19:20So, these experiments were first done using GFP, visual GFP, and you find that NSP-15 knocks down the amount of insertion. Then you switch to deep sequencing. Can you describe that process and why you did that? Right. So, there are perhaps, I'm not quite sure in what order to say these things because the way they're presented in the paper isn't exactly the same as our thought

19:57process as we went through. Yeah, that's usually the case. I want to hear what you were thinking, yeah. So, you know, in this GFP virus experiment, it's actually incredibly laborious to go from one green cell to isolate a GFP-expressing virus from that because you have a well that has a hundred different viral species in, and you have to go through multiple rounds of limiting dilution to actually isolate the GFP-plus virus and sequence it and characterize what actually has gone on.

20:32So, that was the real motivation for switching to the deep sequencing method. But before doing the deep sequencing, we did have a piece of information that when we looked, when we sequenced these half dozen or so viruses that had inserts in them, the sequences of the inserts matched the viral genome. For five out of six of the inserts.

21:02For one out of six, 15 out of the 20 nucleotides matches the viral genome, and then there's another six nucleotides, sorry, another four nucleotides that match somewhere else in the viral genome. But the four could be a match by chance. We'll come on to all those calculations a bit later. But before doing the deep sequencing, we kind of knew that the inserts that this virus was picking up came from the viral genome. And we also knew that they happened when NSP-15 was present.

21:42But Chris said, you know, we've kind of had enough of isolating green viruses.

Deep sequencing the viral genome

21:49Couldn't we do this a bit more efficiently if we just sequenced the hell out of a virus stock?

21:57And I said, yeah, okay, well, give it a try. So, he grew up two virus stocks, wild-type virus, exactly the same viruses that we've been using with GFP reporters and wild-type and mutant virus stocks. Generated virus populations of rather normal size that you generate in cell culture. So, this virus grows really quite well, up to 10 to the 9 or so black-forming units per mil.

22:28And so, he took, I think, about 10 mils of virus, concentrated it to 1 mil, and took a fraction of that and sequenced it, but sequenced it at a depth that's kind of unprecedented. People look to get coverage of the entire viral genome. And, you know, if you get 10x to 100x coverage of your viral genome, you can be pretty confident that that's the sequence of the viral genome. What we want to do is characterize all of the variants,

23:02or as many of the variances that we could find in that population. So, we put it on a sequencing chip that would give hundreds of millions of reads.

23:14So, basically, we sequenced the virus at a depth such that each individual nucleotide in the genome was sequenced at least a million times. So, the total amount of sequencing data we get from each virus stock is, I think it's approximately equivalent to 30 human genomes, or 3 million viral genomes.

23:45So, when you're looking for low-frequency events, right, which we could do with the GFP virus, because you can put 100 million viruses on a monolayer of cells and pick out those individual cells or wells that had a green virus, that's, you know, you don't get to pick and choose what you sequence. You just have to sequence by brute force, and so that's what we did. So, essentially, what's happening with deep sequencing is what you want to do

24:17is sequence every single virus in your sample, and the way you accomplish that is to sequence the whole sample a million times, basically. Not exactly. You want to try, at least know, what the population size of the virus is in your sample, and have that somewhat coherent to the amount of sequencing you're doing, right? So, if you have a tube with 1,000 virus particles in it,

24:48absolutely pointless sequencing that hundreds of millions of times, because you just get the same sequence over and over again, right? We sort of calibrated it so that we would be sequencing a few percent-ish of the viruses in the sample, but millions of viruses in a sample that had tens of millions of viruses. So, we weren't going to be saturating. Right. You're limited by, I mean, millions of sequences.

25:20Sequence reads is a heck of a lot of sequencing, and you're limited by the technology to a certain extent, but the goal is to get as deeply into the individual viral particles as possible. So, effectively, we have 3 million-ish viral sequences. Right. They're not 3 million genomes completely sequenced. It's the equivalent of unlinked reads from 3 million viruses. And the other thing to bear in mind is people don't usually sequence this deep,

25:51because what people are mostly looking for are point mutations. And the error rate of sequencing when you're considering point mutations is about 10 to the minus 4-ish. So, it's kind of pointless to go much deeper than that, because you're just sequencing the error rate of your technique. But, of course, that error rate doesn't apply to insertion mutations, right? The error rates there are very different, and perhaps we'll get onto the sources of artifacts later. But bottom line is we could sequence these two virus populations,

26:24and we saw basically a result that confirmed what we found with the GFP viruses. You see many, many more insertions in the viral genome when you have NSP-15 present. And when you inspect those insertions, and now it's all sort of bioinformatics,

26:47you find that a large fraction of those inserts come themselves from the viral genome. So, at this point, I have to sort of mention, how are you sure that it comes from the viral genome? First thing you have to do is constrain yourself by length, because, say, inserts of 3, 4, 5, 6 nucleotides, random sequence, because of those lengths occur by chance in the viral genome.

27:21And we kind of, while we document them, we kind of ignore them for the process of analysis. We can't be sure about where they come from. The sort of the cutoff where you begin to get confident about the origin of your inserts is around 11 nucleotides. So, we did a fairly complicated analysis that shows that if you have a sequence of 11 nucleotides, and it perfectly matches the viral genome, the chances of that occurring by chance are less than 5%.

27:53So, we basically constructed a slightly more complicated algorithm than that to confine our analysis to inserts of 11 nucleotides or greater. And a huge fraction, probably 90% or more, have a perfect or near-perfect match to sequences in the viral genome.

28:16And they are, in the case of OC43, 87 times more abundant in the wild-type virus compared to the NSP15 mutant virus. And they're mostly from the plus strand also, right? Right. They are. So, the implications for that are a little complicated. So, we know that plus strands are more abundant in the viral genome,

28:49but it also suggests that they're incorporated during minor strand synthesis, right? Because, sorry, getting a little ahead of ourselves. Well, if we don't know exactly how they are inserted, right? Whether it's a ligation process, which is possible but unlikely, or a sort of a recombination-like process, where the virus is copying, making a copy of the viral genome,

29:19then jumping to a short oligonucleotide, which we suspect is the product of NSP15 cleavage of the viral genome, copying that oligonucleotide and then going back to copying the genome. That only makes sense for the inserts to be of plus strand origin if it happens during minor strand synthesis. Right. Because during minor strand synthesis, you're copying a plus strand, right?

29:53So, to get a plus strand insert, you have to copy another plus strand. If it was the other way around, it would look like you had an excess of minus strand inserts.

30:05I wonder if at this point it might be good, at least my understanding is that the NSP15 digestion of double-stranded RNA that you're talking about is predominantly digesting up viral double-stranded RNA that's being produced.

30:25So, I actually think that NSP15 is reducing double-stranded RNA indirectly, actually by digesting single-stranded RNA, just reducing the amount of RNA that's available to make double-stranded. Oh. But the net result is that then there's not as much double-stranded. Right. Or it could be double-stranded RNA that's breathing, right? Sure. Right. Typically, nucleases of this type are cutting, unpaired.

30:55Right. So, this is not a double-stranded RNA nuclease, right? We know this. Not really, no. Okay. Okay. Okay. But then, I guess I was trying to make the point that you're talking about digestion of viral sequences as opposed to host cellular sequences of RNA. Yes. Okay. Yes. So, we don't know for sure, but we think this is probably all going on inside the double-membrane vesicle in which coronavirus replication is occurring and from which host RNAs would be largely,

31:28but perhaps not entirely, excluded. So, next, you wanted to go to SARS-CoV-2, right?

Analyzing inserts in SARS-CoV-2

31:41Right. So, tell us how you did that. Okay. There's one other thing we noticed about the inserts before we jumped to SARS-CoV-2, and that is if you look at the sequences of the ends of the inserts, or actually look at the sequences of the inserts, there's two things of interest. First thing is that the inserts come from all over the viral genome, and they can end up at kind of any place in the viral genome.

32:12Okay. So, if you plot the distance, because when you confine yourself to 11 nucleotides, you can know what the destination of the insertion is, but you can also know what the origin of the insertion is. And if you calculate those distances, it's as if they're random positions on the viral genome. The distance, it's an unlinked process, the generation of the insert and the insertion of the insert. The other thing about the inserts is if you look at them in their origin context,

32:46so from where the inserts come, we know something about NSP15. It likes to cut RNA 3' to U nucleotides. Right? And if you look at the inserts in their origin configuration, they are highly enriched to having U at the ends. So they look like they're the products of NSP15 digestion, and they're inserted quasi-randomly around the genome.

33:15So then we knew all this before we even began to think about SARS-CoV-2. So we first looked at this publicly available sequence dataset from the Ralph and Minituri labs. They used a somewhat different method for acquiring their sequences, but they're the only group that had really published anything that was remotely deep enough for us to do this analysis of inserts. It still wasn't as deep as we had gone,

33:46but it was sufficiently deep that we could see a difference between their wild-type virus and NSP15 virus in terms of the number of inserts. It wasn't quite deep enough to do the types of analysis that I just explained about the nucleotides at the end of the inserts. So we generated another sequencing dataset on SARS-CoV-2, just wild-type virus, this time sequencing even deeper. So we did a billion reads from SARS-CoV-2,

34:19equivalent to about 40 million viral genomes. And when you look at those inserts, you find essentially the same characteristics. The inserts look like they are the products of NSP15 digestion, the distances between the insert origin and destination, are like two randomly chosen points on the viral genome. And then when we looked at both sequencing datasets,

34:52again, this goes back to the origin and destination of those inserts, we found that a subset of the inserts, right, not only had one origin, but actually looked like two oligonucleotides from two different parts of the genome had come together and been inserted into one spot in either the OC43 or the SARS-CoV-2 genome. So it's, you know,

35:25the potential for generating genetic diversity is quite remarkable in that respect. So there are some differences between SARS-CoV-2 and OC43. The length of the inserts tends to be a little shorter and the number of inserts is a little less. We don't really know why that's the case.

35:50Perhaps just differences in the inherent properties of the two enzymes, the amount of enzyme, its activity. But there is, there's this subtle difference, but in the important criteria, the fraction, the distance between inserts and their origin and destination, and the fact that they're NSP15 substrates is very, very clear in both viruses.

36:21And in both viruses, you see what seems to me, having been told for years that inserts are rare, that this is not rare. This is, this seems to be happening all the time, right? Right. So, so rare is a relative term. Of course. I've learned to, to use those words carefully because, you know, one in a, one in a million is rare, but if you have 10 million, it's common.

36:54It's right. So we, when we talk about these, and we try to use numbers, that's not in any way intended to be a reproach, but basically the insertion frequency is you get a few inserts per thousand genomes. So if you have a thousand virions, you're going to have a handful, probably around five or 10 genomes that have an insert in them. That's less rare than I expected. Right.

37:22And it's, it's, I was going to say, and, and compare that to the point mutations that you would see in that same number of genomes. Right. So the point, point mutations would be a bit more frequent, but not massively more frequent. So why do you think over time, we were misled to think that insertions or deletions are rare? Right. Right. So they are far more likely to be deleterious

37:56or even lethal to the virus. So the vast majority of these inserts will be purged when low multiplicity infection happens. Which means you won't isolate them then. Yeah. Yeah. Yeah. So we, sorry. You must know what fraction are deleterious just by looking at the sequence, right? Yeah.

38:19So we don't exactly. What I can say is that when we, which we should qualify this because we're doing this experiment under pretty defined conditions. So we're starting with a cloned viral genome. We're transfecting cells, have a very small founder population of virus, and we expand it over a small number of days to, with no immune selection, no other selection other than replication competence to a population of 10 to the 9-ish, right?

38:53Remember, though, that in an individual coronavirus-infected cell, right, there's not one genome, there's many genomes, okay? So these defect, if, say, 10 out of 1,000 genomes have a lethal insertion in them, then there's many, many complementing wild-type genomes that would maintain this in the population. So when we sequence, we're getting, you know, provided they're not present

39:23at extremely high frequency, lethal mutants can be carried in a viral population, and probably are newly generated all the time. So when we try to answer Vincent's question, kind of, just by counting what fraction of the inserts are multiples of three and don't have a stop codon, right, because they would clearly be protein truncating, only about a third of them are in frame. So most of them,

39:54most of them are actually, are probably lethal mutants. They're not particularly enriched in non-essential genes. So this really looks like if you did a random mutagenesis experiment and then sampled right away, this is probably what you get. There's not really been, no evidence actually of any selection pressure having been applied to these mutants under these experimental conditions. So it's probably a reasonably good estimate of the mutation frequency,

40:26not an estimate of what fraction actually would go on to survive.

40:34Of course, the inserts, even if they're in frame, they may be deleterious also. Right. A large fraction of those would certainly be deleterious. But this certainly represents a process that is generating a lot more insertion diversity, if you will, than naive folks like me might have expected. Right. So this is the food on which evolutionary pressure feeds, right? Right. So it probably is evolutionarily

41:04selected for. So then the NSP-15, it's got the immune suppression function, it's probably also have this function involved in evolution, right?

41:15Probably, yes. It's, it's, it's, you know, I've spent some time thinking about this, but I kind of tie myself up in knots in thinking about how much foresight can we endow viruses with in engineering their own evolutionary rates, right? Or how often do you have to have a selection pressure applied that selects four insertion mutants for a virus to maintain the ability

41:46to generate insertion mutants for that to be maintained? Yeah, well the problem here is that you, you make an active site amino acid change to knock out the nucleus activity and you, and you're going to affect both, you can't separate the two, right? Absolutely. So in that case it's, it becomes rather difficult to say that's the, that's the activity that evolution is selecting for. Right, because you select one you're going to select the other. So, I mean, you could do all the thought experiments as you say, as you want,

42:18and you're never going to get an answer. And this is kind of a long-standing problem in evolutionary biology. How much utility does a gene or protein have to provide in order to be selected for? And I think just intuitively, I mean, helping immune evasion is a huge benefit to the virus already. Yeah. And then this generation of extra viral diversity is maybe, you know, a bonus. Yeah. I think that that is a totally reasonable way to

42:49think about this thing. I mean, mutagenesis, anyway, you can imagine that the insertion is not as important as point mutations. But the other thing is, I was wondering if you would, if you're going to do experiments where you co-infect with two different coronaviruses and see if the insertions are swapped across genomes. Yes, we are thinking about how to do those experiments in a way that doesn't get us into trouble.

43:19Yeah, exactly. But it also is informed. I'm pretty sure we could do this with, you know, a virus carrying a GFP and look for the appearance of GFP inserts in the other virus that doesn't have GFP or something. Well, and you could do it with two other, like OC43 and one of the other common cold coronaviruses that people are not so wigged out about. Yeah, I think experiment one is to do it with OC43 and another OC43 carrying an innocuous gene.

43:50Yeah, yeah. It actually gets a little tricky when you start mixing coronaviruses. They are quite good at super infection exclusion. It might be that the first one in there just kicks off enough of an innate response to keep the other one away or sucks up all of an essential cofactor that stops the other replicas. Because we've been trying for unrelated reasons to generate double infected cells and it's more difficult than you might imagine.

44:21I wanted to ask you about this experiment where you do ultra deep sequencing on libraries from PCR

PCR controls and natural samples

44:30amplicons. why did you do that? So everything we've talked about so far is with purified virus. Virus grown in cell culture. We can isolate the virus particles. We can put a lot of stuff into the sequencing reaction. But we also wanted to know whether the same process happens when you for example have infected animals or infected humans. virus.

45:00And there when you harvest virus from a person or an animal most of the stuff that you get out is not viral RNA. And so the population size that you need to generate in order to get the sequencing depth to do this type of analysis becomes much more challenging and you kind of need to do a PCR step to amplify that

45:30nucleic acid up to a level that you can sequence. And that comes with a set of other problems.

45:40So interestingly you did a control on a sample with two RNA molecules. Can you explain what that does? Right. So the issue is when you start PCR amplifying things you've got polymerases copying nucleic acids and they have a mutation rate of their own. They in principle can also generate insertions. Obviously there's no NSP15 there so we're not worried about that but it is

46:11possible for all kinds of jiggery pokery polymerases jumping here at low frequencies. Right. But remember we're looking for low frequency rearrangements to occur. We reasoned that if there was variation in a starting population of let's say we took a sample from a mouse lung which is one of the things we did that had

46:42say a million RNA molecules in and we wanted to find the frequency with which there are inserts in those million molecules we still have to PCR amplify how do we distinguish what was there in the original sample with what we accidentally introduced when we PCR amplified it. So the way we addressed that was to go to our purified virus where we kind of know what the insertion mutant frequency

47:12is and reasoned that if we just dilute those 10 to the 9 molecules down to one or two molecules all that variation in there will be erased plus by PCR amplifying those one or two two is a calculated number we can't know the difference between one, two or three molecules in a PCR reaction we know there's at least one because we've got a PCR product but we don't know if there's one, two, three or maybe even four

47:43but because of our insertion mutant frequency is sort of in the one in the hundreds right if we go down to those numbers we shouldn't find insertion mutants when we go down to that level so we basically did side-by-side experiment PCR amplify our complex and complex 10 to the 9 molecules and PCR amplify about two molecules and what you find is when you amplify those two molecules you do in

48:13fact find some rearrangements right but most actually all of those rearrangements come from within the sequence you're amplifying right so if we're looking for real insertions right we look for insertions into the PCR amplicon that come from viral genome regions that are outside the bit you're amplifying right because your PCR is only amplifying a piece of the viral genome right exactly and so when we do that all the

48:44artifacts that you generate in your amplicon melt away you know the insertions don't come from elsewhere the artifactual insertions come from where your PCR are amplifying all right so then you use that to look at virus from mice and humans right yeah and what did you find there basically basically same thing lots lots of insertion mutations

49:15it doesn't really matter the context where you you're getting the viral genomes from it does become a little a little more difficult because when we when we're harvesting from a mouse we know the number of viral RNA molecules in the samples that we generate but then when we start looking sequence databases so SARS-CoV-2 sequences that have been deposited in

49:46sequence read archives you're basically pooling data from many individual people you don't have any real idea what the population size viral population size is in those individuals but by applying those criteria that I've just talked about and then looking at the inserts looking for the same biases that we found associated with NSP-15 driven mutagenesis in our cell culture experiments you can find the same things

50:17both in the mouse and in the human so it's not something that's just happening in cell culture and of course you should do this in bats as well right reviewer number three sitting over here yeah we would like to do that but the logistical challenges are even more more of a problem and we would not have really any idea of the population size in a bat

50:47guano sample there's no it could be measured there are no databases from bats existing not of the depth that we would need to do this type of analysis but obviously we're going to get to why that's important but that would be something interesting to do right right but to be frank though given that we found this process occurs equivalently in OC43 in SARS-CoV-2 in mice and in humans I'm not

51:17particularly worried that it doesn't happen everywhere neither am I but you know you get pushback unless you show it because we're going to make some claims about this and that you can imagine anyway yeah so let's talk about so then you find furin cleavage sites inserted and did they just jump out at you

Furin cleavage sites and evolution

51:45or did you look for them no we looked for them so you know we're not we haven't been living under a rock we're not unaware of the controversy that's been associated with this furin cleavage site and you know the first thing that I've been thinking about this for a long time actually because you know it's been a kind of an argument that's largely been dominated by people just

52:16insulting each other yes how can you be such an idiot to think that that arose naturally how can you be such an idiot to think that it didn't arise naturally and so in trying to sort of bring some quantitation some rational basis for arguing rather than just having a go at each other the first piece of math I did was what are the chances of a furin cleavage

52:47site just appearing by chance can I just jump in with a quick background or for people who might have been living under a rock as you say so that the significance of the furin cleavage site is this is a an enzyme cleavage site that is found in SARS-CoV-2 that is not found in right other closely related coronaviruses and a major argument of the folks claiming that this originated in a lab is you have to make this significant change to insert this furin cleavage site

53:18that had to have been done in a lab you couldn't have had that arise naturally with you know point mutations or what have you and so that could have arisen if you got an insertion but then there's this whole thing about well but insertions are so darn rare how could that happen so this is what we're getting to now yeah so I apologize I guess I assumed your listeners would most of our listeners are down with this but we always like to be sure yeah fair

53:48enough fair enough so the other point which is sort of amplifies what you said is we when people look at an alignment of sorbeco viruses that include SARS COVID-2 and this furin cleavage site sticks out like a sore thumb right it's a little bit of a rawsash test the phrase I used in my ASV talk right so if you're conspiratorially minded you think

54:19that you know all these other sorbeco viruses don't have it looks like it's engineered but on the other hand if you're if you're not inclined to think in that way you think well viruses they make insertion mutations that they're pretty rare but the population size is pretty large so maybe it's not that surprising and if there's a selective advantage right so totally reasonable to look at that rawsash test

54:49and think something else basically this what this paper does is bring some numbers to this debate and so the first thing to not the first thing but one thing to bear in mind is that the generation of a furin cleavage site is not difficult right so the minimally actually this isn't a very good guide

55:19to a furin cleavage site but canonically minimally all you need is two arginines RXXR that's thought to be a requirement for a furin cleavage site actually you can get furin cleavage with only one if the other amino acids cooperate and not all RXXRs are in fact furin cleavage sites where XX is any amino acid right but any amino acid isn't exactly right there's some scoring

55:50algorithms that help you predict but I don't you know without having tested them exhaustively the point is this is not some massive engineering challenge exactly so you can actually do math and figure out how often an RXXR motif would appear in random sequence right and you can know there's 64 codons and six of them encode arginine and just multiply those two fractions together and about 1% of random

56:2112 base sequences include RXXR motifs or predicted for in cleavage sites okay so you can use numbers like that coupled with the fact there are approximately 30,000 different places in the coronavirus genome where an assertion mutation can arise we can know how often insertions arise from the

56:52measurements that we've made and it is possible to do a calculation for how frequently based on purely theoretical grounds it would be for a furion cleavage site to occur at one particular position in the coronavirus genome and the number is not shockingly large when you consider the population size of coronaviruses and the number is actually somewhat consistent with what we find

57:23when we deeply sequence these coronavirus populations if you sequence you know several million viral genomes you will by chance find a furion cleavage site approximately at the spot where SARS-CoV-2 differs from other sarbeco virus ancestors or other sarbeco virus cousins let's put it that way and so just by

57:55looking at our deep sequencing data sets which don't sample the entire population right we found a couple of new furion cleavage sites precisely at the place where the existing furion cleavage site exists so if you think furion cleavage site is lightning striking in one particular spot you know we've got it struck there at least three times before looking any

58:25more closely but the bottom line is yes furion cleavage sites are not difficult to make insertions are not difficult to make there are only about 30,000 different places in the genome where insertions can occur and so just by chance it's sort of inescapable that in viral populations of a particular size or greater that they contain viruses with new furion cleavage sites

58:56at that spot when we go back to our SARS-CoV-2 viral stock and sequence even deeper and the way we do this is to not just put the viral RNA on the sequencer but do a set of PCR reactions where we're putting multiple aliquots of 10 to the 8 or 10 to the 9 RNA molecules PCR those and then just focusing on that region of the spike protein

59:26and then sequence those we find many more furion cleavage sites and actually the ones in the paper are very likely an underestimate because we imposed on those sites the requirement that we found a perfect match in the viral genome because of this the steps we're going to rigorously exclude PCR artifacts we absolutely think

59:57we're also probably excluding some naturally occurring furion cleavage sites we can't be sure that they're not PCR artifacts but we strongly suspect that there are way more than we report in the paper so basically you show that acquiring a furion cleavage site is not rate limiting so what does this do for the origins does it tell us how SARS-CoV-2 FCS

1:00:28arose or does it just generally remove it as evidence for engineering in your opinion so I think the latter you know any individual event right must by definition be extraordinarily rare right but there are many different ways to generate a furion cleavage site so cumulative and it happens with such frequency

1:00:58in unselected viral populations that we shouldn't be surprised to find a furion cleavage site there provided it endows the virus with a selective advantage clearly if you do the math right in a sample of you know a sample

1:01:29of the size that you would find in a swab from a single infected individual you would expect based on these findings to find multiple viruses with new furion cleavage sites there the only thing that's stopping them from rising to prominence is whether there's a selective pressure that enriches for them right which would explain why so many other coronaviruses don't have that because they don't need it to complete their

1:01:59life cycle right so there's another point we need to bear in mind here though that it's not always advantageous for SARS-CoV-2 to have a furion cleavage site I can show you many cell culture experiments where the virus was selected to lose it there's very likely sort of a balancing act between the stability of the spike trimer and its fusogenicity and whether it's more important to have

1:02:29spike stability or more important to have fusogenicity could absolutely be on a knife edge in terms of what what's advantageous so you know now if you look for a furion cleavage sites arising in say OC43 would you expect to see the same thing so so we haven't specifically looked at that particular spot in the

1:03:00in the OC43 spike protein but they occur randomly across the OC43 genome in our sequencing data set at actually somewhat higher frequency than in SARS-CoV-2 because OC43 tends to generate slightly longer inserts so so we haven't done the specific analysis you asked for but there's no reason to think it would be really

1:03:30any different right so Eddie Holmes has always argued that the absence of a furion cleavage site in the known sarbeco viruses other than SARS-CoV-2 is a sampling artifact so do your results bear on that

1:03:49depends what you mean by a sampling artifact he means we haven't sampled enough we only have a few hundred sarbecos yes I mean and they're all consensus sequences as well each one of those hundred is probably generated from a probably from an Illumina sequencing data set that has a depth of way less than the depth that we analyze so that their consensus sequences is one thing now

1:04:21if we if we searched hard enough could we find a sarbeco virus in a particular host for which having a furine cleavage site is advantageous and so that's the dominant species absolutely that's possible I don't know enough about what the environment of a bat gut is what the immune selective pressure what the the advantage of having

1:04:52a stable spike trimer versus one that's more fusogenic how that plays out in bats or in raccoon dogs or in whatever other species you choose to analyze yeah but yeah I am I am pretty sure that if you took those hundreds hundreds of sarbeco viruses in bats and sequenced all of them at the depth that we have

1:05:23sequenced you would find furine cleavage sites in all of them okay I don't think there's any doubt about that right just from the if you make if you make the minimal assumption that the mutation insertion frequency that we find in OC43 and SARS CoV2 is basically the same in bats arbeco viruses so the

1:05:54reason this is significant is because the you know the argument has been from some people the FCS couldn't have arisen naturally and now you have said it can and that's that's the bottom line and that's if you put another way as you said at the end of your ASV talk it's because those people are saying it hasn't been found when you look at hundreds of sarbeco virus genomes but come back when you've looked at a billion genomes and see what so it's

1:06:26important to recognize what those people don't say when they say it's never been found in a hundred 800 sarbeco viruses that statement is loaded with the implication that bats sarbeco viruses don't or can't generate furin cleavage sites I think without actually sequencing bats sarbeco viruses I think we've falsified that premise that

1:06:56doesn't negate the likelihood that having a furin cleavage site is probably negative in the majority of circumstances in bat coronaviruses but you know we don't have to speculate for very long to think that you know a transmission event from a bat to a raccoon dog minimal

1:07:27circulation in some intermediate species or even in humans that mutant that generated a furin cleavage site and it doesn't really matter what the furin cleavage site or the precise sequence of the furin cleavage site was whichever sequence it was it was going to be a vanishingly rare individual event right yeah but if it has a selective advantage it will rise to the top you know the other thing we should bear in mind is every every

1:07:58dominant variant of SARS-CoV-2 Delta Omicron that came to be virtually every every sequence in the world at some point subsequently began its existence as one molecule of RNA in one person's lung at one time point and that single event vanishingly improbable as a single event seeded an entire

1:08:28global spread of an individual virus so arguments based on the probability of something happening being extremely remote just don't really hold any water because everything that happens in biology is extremely rare so at the end of your ASV talk which I had a chance to listen to the recording of you

1:09:00said human intuition generally fails in accounting for phenomena impacted by large populations or long periods of time and so and that rare events like one in 10 million become common when large populations of greater than that are sampled so and that's the amount of virus that would be in one individual yeah absolutely this is so I never thought the urine cleavage site was the

1:09:30best evidence for lab but many people thought so and the reason is that no one had something to put it in that was very close to SARS COV-2 but I do think this is like a nail in the coffin now because I think this removes it completely but I'm really interested when this is published to see how people so Richard Ebright is incredibly negative on the FCS he says it's clearly a mark of lab engineering I want to see what his response is right

1:10:01so so obviously so I've experienced some of some of the pushback from the from the lab leak fraternity who are still pretty active on on social media and they're basically what what they say is that ah okay so you've you've shown that furian cleavage sites can arise naturally we never

1:10:31doubted that but what you haven't shown is that furian cleavage site arose naturally this is classic denialist strategy oh no we already agreed to that what we're now going to fall back to is you know you can't disprove what we're arguing which of course you can't because you can't prove a negative right but but as I as I've just argued any individual event in biology is impossibly rare right here's a this is a kind of

1:11:01cool example um there's a a paper by Harrison and Sachs in PNAS and it argues about the lab origin of SARS COVID-2 and they talk about the furin cleavage site and how it's not found in hundreds of sarbeco viruses and oh look at the sequences of these furin cleavage sites that you find in epithelial sodium transporters that are being worked on at UNC yes

1:11:32and could and could have been top candidate for choice for insertion into into SARS COVID-2 they kind of gloss over the fact that half of the sequence that they identify was already present in other sarbeco viruses by the by but one of their favorite candidates is the epithelial sodium transporter furin cleavage site from the mouse epithelial sodium transporter and if you look at the amino acid sequence of that

1:12:02furin cleavage site it spells out something very interesting spells out SARS ass what's the probability of that happening yeah vanished it's impossible that that happened but it happened years ago I saw a bioinformatics analysis of searching for Elvis in the genome and they determined that Elvis is actually quite commonly distributed

1:12:33so some of your insertions are from the host right maybe so it becomes so we have a category of insertions that are difficult to classify right so I talked at length about how you need 11 nucleotides to to be pretty confident that it came from the viral genome now when you go to the human

1:13:03genome that length becomes quite a bit longer right so we do have inserts of 11 nucleotides or more that match the human genome but don't match the sarbecovirus genome we're still actually working on those a little bit but it's possible to have chance matches to the human genome with sequences that are quite that are a bit longer sort of 15

1:13:3416 or so basically how long do you need to make a PCR primer in order to only amplify one bit of human DNA so I you know in the preprint I don't want to be necessarily held to the frequency that that's the number of inserts that come from the human genome what we suspect there are there's a large category of inserts where it's ambiguous where they come from and what we suspect is happening there is

1:14:05that they're composed of inserts of less than 11 nucleotides that are actually chimeras probably come from the viral genome and are inserted as chimeras but it's difficult it's nearly impossible to prove that statistically because each component of the chimera could occur by chance multiple places so there will always be some ambiguity nevertheless it is

1:14:35absolutely possible that a small fraction it's less than 1% come from the human genome because I think some people have published on this already saying that there are some bits that look like human sequences right right so the for example the 12 nucleotides that compose the furin cleavage site there are multiple matches to that sequence in the human genome but there would be matches

1:15:05by chance as well that's the issue there so other viruses encode nucleases right influenza bunya viruses do you think they might contribute to insertions as well absolutely possible we we're thinking about HIV actually which carries a nuclease right along with its polymerase and chops up the RNA into oligonucleotides right after it's reverse

1:15:36transcribed RNA-SH right yeah so that might be our next port of call in terms of trying to extend this to other viral systems but yes and perhaps so you know getting into the realms of speculation it might be that chopping up the genome into oligonucleotides creates a sort of a freely diffusable pool of

1:16:07sequences that can more easily invade and elongating polymerase as opposed to a longer viral genome that might be more difficult to insert itself into an active site so yes you're absolutely right it might not stop at coronaviruses hey why limit a good thing to one virus yeah is there anything that we missed we should talk about Paul I don't think so

1:16:37I think we've given this a pretty good thrashing well I wouldn't say we thrashed it we looked at it pretty closely yes I think the data are the conclusions are justified from the data how about that well yeah I think that's right there's probably one sentence in the discussion that the lab leak community took exception to and it's basically whether we're talking about a

1:17:08furion cleavage site versus the furion cleavage site and so the final published version will probably make it clearer that we're talking about a furion cleavage site rather than the furion cleavage site but if that's the worst thing about this paper then I'm happy with that okay Paul thank you so much thank you for joining us for Rockefeller University I hope you get published soon and as I said looking

1:17:39forward to more all right very nice talking to you thank you see you around bye take care bye bye well as you know the won't satisfy anyone because everyone's if you've made up your mind already you're not going to be convinced you can't reason somebody out of a position they didn't reason themselves into let's do

Listener emails and scientific integrity

1:18:03a couple of emails and then we can do our picks I'll take the first one because it's short Anthony writes is saying I'm making this up now forbidden on Twiv so just to give you context Rich likes to say I'm making this up now when he's not sure about something right and someone wrote and said he really shouldn't say that because scientists have enough with

1:18:33certain people scientists are already not trusted so I talked about it and Anthony writes fabricating receiving question mark an idea peg is the shared sacred spring of both science and art a scientist searches reality for a hole that fits the peg the artist uses her talent and their talent and skill to craft the setting for the inspiration I would not say it's forbidden I would just say if you want to use the expression

1:19:03you just say I'm just not sure the facts here I'm just not sure of the situation that's all or I'm hypothesizing I'm hypothesizing you can say Rich can say I'm making it up and then you can say I'm hypothesizing yeah I'm postulating Alan can you take the next one okay so this is a long email that I will partially summarize I'll give the intro and then I think I get the gist of it Dallas writes dear wise

1:19:34men presumably ladies and gentlemen you you happen to read a letter that pushed the idea of using legumes and more vegan diets to avoid the massive amounts of energy required to synthesize fertilizers it sounds good in theory as long as you don't note that humanity is already using most of the best land area and fresh water supplies to feed ourselves on this finite planet that makes the efficiency of converting sunlight into protein very important which puts it in my thermodynamic wheelhouse the probable

1:20:05activists who prepared the letter that ignored the land area probably wouldn't believe someone like me saying he is missing the 800 pound gorilla in the court of reality so I asked Google AI and so there's a long discussion of the amount of energy required for the Haber-Bosch process to fix nitrogen which is used to generate I think half of the nitrogen that is now consumed by people in crops and gives a table table one

1:20:35here calculating protein yield per year of sun and pointing out that if you use solar panels and electrolysis for the Haber-Bosch process you generate more protein yield for an amount of sun versus natural biological nitrogen fixation and then goes into some depth explaining why the solar industrial pathway wins now the

1:21:06bottom line because solar panels capture sunlight with far greater efficiency and because artificial photosynthesis systems coupled to electrochemical reactors do not lose energy maintaining living plant tissue the solar powered electrochemical route produces multifold higher yields of ammonia and subsequent protein per unit of solar radiation compared to biological systems if you'd like to explore this further I can break down the exact chemical formulas and energy calculations for both pathways or show you how synthetic biology is trying to

1:21:37bridge this gap concludes to handle the coming 2.5 plus billion more mouths to feed as people get older we can't let the activists disrupt progress like Greenpeace did with their anti-GMO anti-golden rice campaign that killed and blinded millions of children over 100 Nobel Prize winners suggested that Greenpeace created crimes against humanity and Dallas is from Huntington Beach California thank you for that deep dive into a topic that I'm sure Dixon would have

1:22:08found fascinating well Dixon when he when he wrote that you know most of the land is being used already that's exactly Dixon's wheelhouse right yeah and his argument was most of the land that we could farm is now being farmed and the problem is that it is being farmed badly in a lot of cases and so this you know the idea of increasing the efficiency with which we turn sunlight into protein certainly was something he was very interested in yeah I

1:22:39didn't know that you could use solar and electrolysis for Haberbosch you know I thought you had to use all that heat yeah etc but we also had a letter somewhere else about this as well uh what was it Haberbosch process came up on an earlier episode and I forget the context so I made it a pick but yeah I don't I made it a pick you know so this paper with

1:23:10the nitro the nitroplast right in nitrogen fixation and then I started talking about Haberbosch and we did it on TWIM but someone said that um you know the half the number I quoted half of your nitrogen molecules come from Haberbosch someone wrote a long email you know suggesting that that wasn't exactly correct and I don't know where that email was written to because it wasn't written here anyway so we've gotten two on that already

1:23:41uh Kathy can you take the next one James writes on today's TWIV 1355 a pick was the actions being taken against Dr. Bob Gary a past guest on the show he mentioned that Dr. Ralph Barrick another TWIV past guest had also been similarly mistreated there was an article in science in May on Dr. Barrick authored by a science writer who it appeared has no formal education or experience in doing actual science he has a BA in science writing after the end of his

1:24:11article the author shows an extensive list of leading scientists who object to the government's actions but the reader must notice these afterthoughts the science article is at URL and he gives a URL which you're going to have to fix before you post it anyway as a retired engineer I have relied on TWIV since 2019 for scientifically well-vetted information on virology immunology epidemiology and the like would you guys and gals please devote a good chunk of time on one of the shows to talk

1:24:42about the actions against doctors barrack and gary and other similar government actions of project 2025 inspired defunding actions please identify both the politicians and scientists who are colluding in these activities and talk about the impacts on basic and applied research as well as where or if government funding might be replaced with research and development funding from non-government organizations thank you very much for all the work you do both in the research and

1:25:13development of science and technologies and the communication of same to us life science challenge viewers the availability of your subject matter expertise has been critical during these past six years jim so to spending a lot of time talking about the mistreatment of Bob Gary or Ralph Beric or others I think

1:25:44it is adequately covered in articles in science and nature and so forth so and then trying to identify the politicians and scientists who are colluding in these activities I think that is going to be somewhat difficult at least with respect to both

1:26:15categories politicians and scientists and talk about their impacts on basic and applied research and where it's going to be replaced and to be totally pessimistic and Debbie Downer about it I think the impacts on basic and applied research are already huge and where or if NGOs are going to be able to replace

1:26:46that funding I don't think that's possible so one analogy I make for people is okay you can think about the Kennedy Center and the damage that's been done to the management and organization of the Kennedy Center and if you were going to say well how long would it take to fix that in an ideal world and you might say oh I don't know maybe five years or maybe that's being too generous maybe ten years well

1:27:17personally I think the damage to research in not just virology or biomedical science but everything ecology the climate energy etc is way beyond that and so I think we're looking at 50 or more years and that would be if you had

1:27:47government funding coming in to save the day so I think what you're asking is not possible for us to do but maybe Vincent or Alan are less pessimistic I am not less pessimistic the problem with funding pulling the rug out from underfunding in basic research which is what we're talking about here I mean this has just been slashed

1:28:18arbitrarily and capriciously and deeply and there was the whole doge nonsense which shed thousands of employees from the federal government many of them in research and public health this is a generational destruction of American science which formerly led the world for generations I mean going back to World War II Van Iver Bush designed this research enterprise and basically told us how to use it

1:28:49and it's been going great and has fed I mean that's why America owned the 20th century was our undisputed lead in science and that has been destroyed and it's not you know you can say well maybe NGOs maybe a couple of philanthropists could come in maybe private industry could none of that is going to replace the structure

1:29:19the depth of expertise the sheer quantity of funding I mean it is not really the issue yes to say nothing of the destroyed morale the destroyed morale and the reason this is generational is yeah I mean Elon Musk is a trillionaire he could write a check for the entire NIH budget for the next 10 years and not feel it that's not the problem the problem is that the people doing the research have been jerked around enough

1:29:50and they're not stupid that they are going on to other things or retiring early and the next generation of scientists which we need to continue this is already I'm the parent of a junior in college okay and she's in math and computer science and she's looking at her job prospects and trying to figure out well do I want to go to finance or do I want to go to you know biotechnology government

The future of science funding

1:30:18funded research is not really on her radar because that's not necessarily a good thing to go into now so you lose a whole generation of scientists and then you're going to have to rebuild the whole thing from scratch and I think Kathy's 50 year estimate is completely reasonable I think that's the best we can do we can tell you what's been cut it's hard to predict but as Alan said we we know areas are already and that assumes that

1:30:48in 2028 responsible management comes in and we don't do this stupid crap again for another 50 years which I don't know how reasonable that assumption is I feel that even if we do change we're going to get them back again at some point in the future because now they're embedded in maybe US I don't know if we

1:31:13those individuals I would say James you should take a look at the article on Bob Gary it's open access it's in Bloomberg it's a very long comprehensive article and it tells you exactly what's going on with him and really similar things are going on with Dr. Barrick there's no case against either it is simply vindictive as is the case with Tony Fauci they have nothing on any of these individuals but they

1:31:44will financially destroy them by having a government investigate them they're going to have to hire many lawyers and their life will be ruined basically already the Gary lab is shut down the barrack lab is shut down they don't have any more grants and so going litigation having a lawsuit anyway so they're in big trouble and it's not going to end at them unfortunately because we have vindictive individuals

1:32:14in power right now and you know prosecution shouldn't be just for vindictiveness but that's what's happening all right let's do our picks of the week so we can end on

Picks of the week

1:32:25a lighter note Kathy what do you have for us okay let me get to my pick I picked the Jurassic Museum of Asturias which I was lucky enough to go to in August Asturias is one of the geographic regions of northern Spain on the coast and this Jurassic Museum is made in the shape of a dinosaur footprint so it has three big lobes to it and I put in the link to it

1:32:56from Wikipedia and so that's in English and so I was looking at the exhibits I think it was mostly in one of the lobes of the footprint and then kind of turned the corner and I should tell you that many of the dinosaur skeletons and so forth in the museum are reconstructions they're not actual dinosaur bones and so I turned the corner and there's skeleton casts

1:33:27of a male and female T-Rex doing something together that I never seen before in a museum and I looked at my friend Isabel Novea who was a virologist at the University of Toledo and that's how I know her and she's moved back to her home in Oviedo which is in Asturias and she took me to this and I looked at her and she looked at me and kind of smiled and laughed like yeah Kathy you figured it out and then

1:33:58there's a big mural on the wall showing them and if you go to this Wikipedia site and you scroll down far enough you'll you'll see them the other thing that was even more exciting and part of the reason why this museum is where it is is that that whole region of Spain is rich in dinosaur abelia I mean memorabilia I don't know what you would call it but dinosaur stuff and so just a little over three kilometers away

1:34:30then we drove to this site on the beach where there are dinosaur footprints and I got to stand in dinosaur footprints I never I just got goosebumps again I never in my life would have dreamed that that was possible and as we walked there there were all these ferns and flowers and stuff that made it just seem kind of Jurassic and it is right on the beach and

1:35:00several times a year there's a super super high tide and the water washes over these footprints and so that means that there's not really anything that can be done to protect them and they've lasted for thousands of years and so you go down some steps and you're now at beach level and you can stand in the footprints so if you go to Asturias which has lots of things that Isabelle

1:35:30took me to I went to one of the places that has cave paintings so I got to see some of those famous ancient cave paintings but go to this dinosaur museum I'm happy to tell you more about it how to find the footprints you've got to go to the beach and see them that's very cool yeah Jurassic porn yes nice and by the way if you ever want to stand in dinosaur footprints again without getting on a plane well

1:36:01maybe you would have to get on a plane if you ever find yourself in western Massachusetts we have some just up the road from me along the Connecticut River there's preserved dinosaur footprints well not preserved they just happen to be there next to Connecticut right right right Alan what do you have for us I have a book I read recently this is nonfiction it's called Grand Central Winter by Lee Stringer with a forward by Kurt Vonnegut Lee Stringer in the

1:36:31mid 1980s he was homeless and addicted to crack cocaine and huddling under a platform in Grand Central Station to have a place to stay he had a little hovel he hollowed out there and he was looking for something to tamp down his crack pipe with and he found an old pencil and then he started carrying that around behind his ear and he found a composition book a little while later and he happened to start writing and then he kept writing and he really liked it

1:37:02and he essentially over a period of years and with a couple of setbacks along the way he wrote himself out of his addiction and into a job and you can see why when you read this book he is a really excellent observer and essayist and there's just you know these poignant insights on life on the street and some of it is stuff you expect

1:37:32and some of it is stuff you really didn't expect and it's all fascinating and it's taking place so this came out several years ago and actually probably about 20 years ago now but the events he's recounting in these essays took place in New York City in the late 80s to early 1990s so if you were in New York City in the late 80s to early 90s you may see a lot of familiar stuff he was

1:38:03the editor of Street News for a while if you remember that the paper sold on the subway by homeless people so a lot of really cool stories and very very well written wow that's cool and they're essays so you can read one in 15 minutes and set it aside and pick it up again I read this at the gym I passed the guy on the street this morning he was sitting in front of Penn

1:38:35Station he was just sitting on the ground it's still warm out so you can do that he was all hunched over and he had a sign next to him said I'm losing hope oh

1:38:50and I just I walked by and I just wanted to go back and do something right because I've never seen just that kind of despair I don't know if it was real or not I assume it was right but this is this guy this guy lived that yeah yeah but it's it's really it's an uplifting book yeah sounds like yeah yeah I have two

1:39:20videos I've been watching these videos on olive oil because apparently a lot of olive oil is fake okay so I give you two videos which are not made by AI okay because a lot of them are made by AI and it drives me crazy and so one how to choose a real extra virgin olive oil this is by a guy who's an MD PhD and he says I present you with real data he reads papers about this and so he has a scientific

1:39:50approach to it he says the end so a lot of these oils are like canola oil with a little bit of olive oil in it right they're mixed and so he says you can tell a real olive oil the key is when you swallow it in the back of your throat you get this spicy taste and that's telling you that it's real but even he says extra virgin which is supposed to be the first pressing you know and all he said that gets faked all the time anyway

1:40:21so that's a good one then there's a short one by a cook how to tell if your olive oil is fake you basically put a little bit in a bowl and you stick it in the fridge and if it's real olive oil the next day it's going to be hard and if it's not it's going to be still liquid so that seemed pretty good and the video description she does say this isn't a perfectly reliable test does it depend on the temperature of your refrigerator no well I'm going to try it with olive oil refrigerator or

1:40:52freezer I forgot what you said I don't want to play it now but I will try it with olive oil and canola oil I don't have any canola oil maybe I have to try something else you can report back to us tell us the data I tell you the data yes anyway I thought it was funny that an MB PhD is doing the other one too he seems I mean he's got very corny jokes but I think it's interesting and I just think it's too bad if olive oil is faked I mean

1:41:22oh my gosh just the consumerist thing about this world is getting out of control right I mean I was I've been watching a lot of videos about this the crash of the Amazon plane in Miami right and they had a they had a cargo of 32,000 pounds of contact lenses and I'm like so your contact lens is just another commodity you know like everything else it's kind of sad I think we have a listener pick from Kathy

1:41:53I heard your intro on episode 1355 about the derision of the scientist Adionath's work during her lifetime he's a woman who just died and she had solved the ribosome structure got a Nobel prize I recently read a great book called I Told You So by Matt Kaplan it looks at modern scientists like Catalan Currico and her struggles in historical doctors like Inez Semmelweis revealing the professional and personal attacks they endured it was eye opening and well

1:42:24written also Kaplan makes

1:42:29vitriol in modern research I hope you enjoy the read yeah that's what Paul was talking about the vitriol in if you're in cleavage sight people can't just calmly talk about it they have to say you're stupid to think that come on that's not what it's about just have calm measured discussions people I guess think if you're screaming that you have more of an impact but it doesn't work that way all right that's TWIV

1:42:591357 you can find the show notes at microbe.tv slash TWIV you can send your questions and comments and pics of the week to TWIV at microbe.tv and if you enjoy these programs we'd love your support microbe.tv slash contribute Kathy Spindler is Professor Emerita at the University of Michigan in Ann Arbor thank you Kathy thanks this is a lot of fun and you told me to point out that the episode number is

1:43:29also 1357 which is the first four odd numbers nice pretty cool Alan Dove is at alandove.com turbidblack.com thank you Alan thank you it's always a pleasure I'm Vincent Racaniello you can find me at microbe.tv I'd like to thank the American Society for Virology and the American Society for Microbiology for their support of TWIV Ronald Jenkins for the music and Jolene Ramsey for the timestamps

1:44:00you've been listening to This Week in Virology thanks for joining us we'll be back next week another TWIV is viral you you you you you you you