TWiV 1347: No Short Answers for Long COVID

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 1347, recorded on August 7th, 2026. I'm Vincent Racaniello and you're listening to the podcast all about viruses. Joining me today from Madison, New Jersey. Brianne Barker. Hi. It's great to be here. It is very sunny, 90 degrees Fahrenheit, and extremely humid here. Let's see what it is here. 33, mostly cloudy here. We have a severe thunderstorm watch for the remainder of the afternoon. Also joining us from Western Massachusetts, Alan Dove. Good to be here. And here in New England, it's, well, Western Mass anyway, it's 90 Fahrenheit, 32 C.

1:03Heat index with the humidity feels like 95. It's really not very pleasant weather. And I think tonight, chance of thunderstorms. But it was even hotter and more humid in Minneapolis, right, Brianna? Oh, it was. Last week. Very hot and very humid. I kind of like it. I kind of like it. I like it if my hotel is not quite as long of a walk to the conference. Yeah, well, that's a problem. But I like this August doldrums. It's kind of cool. You know, this introduction reminds me, we were doing a twip last Friday, and I introduced Daniel, and then we started getting into his bow tie, and I forgot about Christina.

1:46All of a sudden, I see her waving, and I'm like, oh, my God. We took like seven minutes to talk about that. Anyway, speaking of TWIP, if you're interested in cyclospora, understanding it, not getting it, we just released a TWIP on it to explain things. It's very hard to wash off lettuce. It sticks. Yeah. Yeah. So what do you do? Yeah, that's true of leafy vegetables generally. really. And if you ask people who study food safety, you know, are there things that you won't eat? The very first thing on their list is always sprouts. Sprouts. Sprouts. Because you cannot wash them and there's no kill step in their preparation.

2:27And their preparation is leaving them sit in room temperature water for days. It's just, I'm not citing a culprit here. I'm not saying I know where the cyclospora is coming from. And they also all grow close to the ground. And so if If there's any kind of water contamination, it's going to get to those vegetables very easily. Lettuce is hard, too, to wash. But a lot of people... I had a salad on the plane back from Minneapolis. I didn't need it because I didn't trust it. Who knows where it's coming from? Yeah, I largely avoid salads. I prefer cooked vegetables. Yeah. If you enjoy these science programs, we would love your support.

3:12Go to microbe.tv slash contribute. And don't forget the Rosenfeld Lab in Philadelphia is recruiting a postdoc and technician to work on enterovirus pathogenesis and immunobiology. You can find more about that, those positions by emailing amy at rosenfelda at mlhs.org. and under news, I just have it all sucks. I'm not going to go into anything specific. That's all you need to know. It's all you need to know. We'll go right into our science. I got an email from somebody suggesting, you know, you should cover this on TWIV. I was like, oh, God, yeah, no. It's just too much. Everybody knows what's going on, and we have other programs that address it.

3:58Rasmussen Reports addresses a lot of the other stuff going on, so go check that out. Let's do some science. Today is apparently all about long COVID. I found, well, so last week, Brienne and I were having lunch in Minneapolis, and she mentioned this paper. So I said, let's do it. And then I was looking through the literature, and I came across the second paper. I said, why not? Let's make a double long COVID. So the first one, they don't have to be in any particular order, but this is the order. This is a nature paper. It is entitled Virus Reactivation in Acute and Long COVID. Oh, a lot of authors.

4:47Oh, my gosh. So first author, surprisingly not co-first authors or anything. It's Cole McGuire. And the senior author is Esther Melamed. and the list of affiliations appears at the end of the paper, of course, but not the end of the end, so I can't just skip to it. We're going to scroll down to this. Right. The team is from the University of Texas at Austin, Clinical and Data Coordinating Center, Precision Vaccine Programs at Boston Children's, Emory School of Medicine in Atlanta, Baylor College of Medicine in Houston, University of California, Los Angeles, UC San Francisco, Chan Zuckerberg Biohub in San Francisco, Harvard Medical School, Case Western Reserve University, Drexel University in Philadelphia, Stanford University in Palo Alto, Mount Sinai School of Medicine in New York, Oklahoma University Health Sciences Center in Oklahoma City, Oregon Health Sciences University in Portland, University of Arizona Tucson, University of Florida and Gainesville, Yale School of Medicine in New Haven, National Institute of Health in Bethesda, La Jolla Institute for Immunology, University of Washington, Seattle, the Broad Institute of MIT and Harvard.

6:06And I want to mention that one of the authors on this is the IMPAC, I-M-P-A-C-C Network. And I'm absolutely not going to read the authors list on that because that would be the entire episode. A lot of people. Yes. And if people think this sounds at all familiar, I was quite surprised by the number of online posts and news articles about this paper that I saw yesterday. So people may have heard some coverage of this paper. Yeah. Right. So we all harbor lots of chronic viral infections, like 8 to 12 is what—8 to 12 herpes viruses alone. And then there are others like aneloviridae. And they infect you your whole life.

6:57You're not going to get rid of them. And sometimes they exacerbate when you're immunosuppressed or you have cancers. So typically they're dormant or low level. and then things can make them reactivate. Stress, sleep deprivation, you got to get your sleep. Surgery, hormonal imbalances, critical illness, et cetera. And so SARS-CoV-2 is a critical illness, right, or can be. And so it stresses your body and maybe reactivates many of the latent viruses that you have in you. And in fact, there has been evidence published already that herpes viruses like Epstein-Barr virus, cytomegalovirus, human herpes viruses, 6 and 8, are all turned up by SARS-CoV-2, and they may be associated with worse clinical outcomes.

7:49But, you know, these studies have not been extensive. And here, this paper is leveraging IMPACT, immunophenotyping assessment in a COVID-19 cohort with 1 ,100 people. And so these are bigger numbers than we've had before. So it's an interesting study. So this cohort has 1 ,154 patients hospitalized for COVID in 20 different hospitals between May 2020 and March 2021, so very early in the pandemic. And we're going to – it's going to become important in this context that these are hospitalized patients and this is early in the pandemic. Yes. So I think with both papers that we're going to talk about today, who are these patients is really key.

8:39And so the fact that these were hospitalized patients and thus perhaps had more severe infection than others is key to remember. And so in both cases, we are probably going to ask how representative the patient populations are. But at the same time, in both cases, if you think about it, these are interesting and important questions. And you can sort of think to yourself, well, how would you answer that? And it's hard to imagine ways to answer it that don't have questions about what's the right population. Yeah. And these are all unvaccinated patients because the vaccine didn't exist when they were hospitalized.

9:22So they classify these patients, the groups, based on symptoms, mild, moderate, severe, critical, fatal. Yeah. But I mean, mild is still you were hospitalized three to five days. Yeah. A mild hospitalization.

9:41So then they got PBMCs, nasal swabs. And some of the patients were mechanically ventilated. They got endotracheal aspirates. and they did bulk RNA sequencing on these samples at up to 10 visits during one year post-hospital admission. So they got follow-up as well. They also look at whole blood immune cell populations by cytometry. They look at antibodies to EBV and CMV in the serum. They look at serum cytokine levels and the plasma proteome and metabolome by mass spec. Big data, that's what this is called. Yes. Let's see if we can make something of it. A lot of statistics are necessary for this.

10:27Okay, so first they take the RNA-seq data, and they look for any human infecting virus RNA, and they can find viral RNA, nasal endotracheal aspirates, and PBMC samples for a bunch of human viruses. Of course, beyond SARS-CoV-2, they find plenty of SARS-CoV-2. They have EBV, Epstein-Barr virus, cytomegalovirus, human herpes virus 6, human alpha herpes 1 and 2, which is HSV 1 and 2, and some aneloviridae and enteroviruses. Aneloviridae are interesting viruses that infect most of us, if not all of us. They're small, single-stranded, circular DNA viruses and just everywhere. So they find all of these sequences in their database.

11:18I was going to say, and the aneloviruses are not known to have disease associations. That's right. Yeah, I mean, if you get a pint of blood, it's got aneloviruses in it, racinelloviruses. Likely including one of Vincent's favorite viruses to pronounce. Torquitaino. Thank you. Torquitainovirus. It's an anelovirus, a racinelloviruses. They've left the I out because it would be racinelloviruses, so it's not quite right. But it's got two L's. It has two L's. Look, A-N-E-L-L-O. And yellow. If you put an I in, it would be the last letters of my name. Yes. Okay, I won't dwell on that anymore. So these, aside from SARS-CoV-2, these are chronically infecting virus.

12:02They infect a lot of people for their whole lives. Okay. Now, the herpes viruses, they found these in multiple compartments during acute COVID. That's the first 40 days after admission. And less frequently during the convalescent period, which would be two months or more post-admission. So they've gone up during acute COVID and then back down again. And as I said, they found aneloviridae and enteroviruses. They don't speciate the enteroviruses. They just don't have enough data for that.

12:40Each virus had unique temporal dynamics relative to the hospital admission. For example, EBV reactivated early in the disease course, and then it slowly went down over time. But an aloe vera day, the frequency remained constant until day 20 and then slowly went down. Yeah. And that's sort of interesting because I don't know. I mean, I guess because I'd read this and thought about it, I was like, of course, I would expect the viruses to reactivate. reactivate. But I don't know that you actually would have said that for certain because, you know, it makes sense that there may be some reactivation because there's physiological stress.

13:25And so your immune system may not be combating those viruses. And so they may, you know, get to the mouse, the mice may get to play while the cat's away, you know, kind of thing. But you could also imagine that perhaps because of the SARS-CoV-2 infection, you have a lot of innate immune responses, you have a lot of interferon, and so perhaps that would have dampened other viruses. So here, seeing that, in fact, these other viruses are not being dampened by that innate immune response is interesting. The stress seems to be, and I'm saying stress very broadly, seems to be allowing for expansion or reactivation of these viruses.

14:11And the differences in the temporal dynamics start to make me think about things like susceptibility of these viruses to different types of immune responses, which isn't really what they're getting at here, but just makes my brain want to start hypothesizing. This was also the point in the paper where I started thinking, what would it look like if you did this profiling for people hospitalized for flu or for some other viral infection? And is this a pattern? Because I wasn't aware of this being observed anywhere else, but I'm not aware that anybody's looked anywhere else. It might be interesting if this is just like you get a severe viral infection and these resident viruses reactivate?

14:57I don't know. Or any kind of severe illness, right? Right. Yeah. I mean, what if it was a hospitalization for a non-infectious type of thing? Yes. I bet it would go up. Yeah. People don't look for it, though. You're absolutely right. I mean, it's expensive to do this work. Yes. And so you need to be funded to do it. You can't just be doing it. But I think any kind of stress would result in reactivation. And so SARS-CoV-2 is a certain kind of stress, but I don't think it's unique at all. Okay. I think a lot of things can do this. So they also look across these compartments, right? They have limited compartments that they've sampled.

15:32So the EBV were more common in the PBMCs and the HSV-1 in the nasal and endotracheal aspirate samples. Among participants with viral reactivation, about half of them had reactivation, 550. Most had only one virus detected in the acute period. It's interesting, 67%, just one virus. So, you know, we're talking about a lot of viruses here, but most people only had one. It's not like you had an Occam's razor worth of infection. Yeah, that starts to get at sort of the thing that I was thinking about a lot with both of these papers, which is that when you look at humans like this, there's an awful lot of heterogeneity in many different cases.

16:19And so, again, what made particular viruses reactivate in particular people is an interesting question. It's a very interesting question, yeah. I mean, I think it's probably stochastic, but who knows. They also had another cohort of whole-blood RNA-seq data from COVID-19 patients, and they saw similar things. So some of our EBV, CMV, and LVIRID going up, as they saw here. Okay. Then they want to know how, remember, we're looking at the number of viral transcripts in a sample, and that's kind of their indicator of the level of virus replication. They want to know how that correlates with COVID-19 severity.

17:06and so they do see so remember this is all associative now nothing proves anything it could be that the viruses are doing something or it could be they're just a consequence of the the SARS-CoV-2 infection again it's all in patients who are hospitalized so variation in what severity means isn't really capturing the whole variation but I do think this is a worthy study because it makes people think You see it got a lot of attention, so people are going to be thinking about it. And there will be more studies done like this, and I think that's good. So they see associations between COVID severity and herpesvirus and anellovirus transcripts.

17:45All right. And they can break that down in the different compartments. And interestingly, those with CMV transcripts in any respiratory compartment were more likely to die within a year. I don't know what that means. Right? Right. May not mean anything. Maybe it's an association at this point. It's also the same with people with detectable nasal Epstein-Barr virus. Right. Then they look to see if the transcripts vary with age. They saw a positive association between anelovirity and PBMCs and increasing age. I guess that's not surprising, right? You're increasingly immunosenescent. Right. Right.

18:34And Hispanic ethnicity was, I'm glad they use ethnicity and not race, for God's sake. Thank you very much. Hispanic ethnicity was significantly associated with both CMV and EBV. And I've known that before for CMV. I showed a picture of that. I didn't know for EMV. But there's no association between the number of viral transcripts of biological sex or treatment with remdesivir or steroids. So the steroids is very interesting because, as you know, very early on we started to use dexamethasone to treat people who are in the inflammatory phase, typically hospitalized patients in the inflammatory phase, which would be immunosuppressive.

19:16But they didn't see an association. But that's with the levels. But I don't know. So they make a big deal that this shows that immunosuppression is not the answer to this. But I wonder how much the steroids played in triggering the reactivation in the first place. Yeah, I think that it's hard to say in these patients how much immunosuppression was happening before all of this. Were these patients more immunosuppressed for other reasons that led to severe COVID? I think that there are lots of additional variations, but I'm really excited to have this as a starting point for people to look at more questions.

19:58Then they looked at the association of viral transcripts with comorbidities, medications, and clinical complications. All right. So the anelovirid and the PBMCs were associated with the history of solid organ transplantation, immunosuppressive medications, shock, intense ICU admission, and myocardial infarction. Well, stresses, right? Yep.

20:28So medication-mediated immunosuppression, as I said, was associated with aneloviridae, but it's only 17 % of the aneloviridae-positive patients. So they say, you know, presence of this virus is not exclusive to long-term medication-associated immunosuppression. They make a big point about that. So for the herpes virus, the CMV in the nasal compartment was linked to pneumothorax. But in the PBMC, CMV was associated with bacteremia, pulmonary vascular disease, renal complications, shock, and stroke. EBV in the nasal compartment were associated with ICU care and shock. EBV and PBMCs with liver failure, concovers.

21:12So this goes on and on. To me, this sounds all random. It doesn't sound just okay. hey, it's just what happened, and these viruses are turned on in all these patients, so you're going to get these random associations, but we'll have to see. We'll have to do more. Okay, now some immune responses. So they have a lot of sequence information, and they do some flow and stuff, so they can look at host immune responses. So the people with elevated EBV transcripts in the PBMCs, they have persistently elevated antiviral IgG and IgA. People with CMV had higher CMV at baseline, actually. It's interesting.

21:56And then they do mass spec proteomics. They find that plasma, herpes simplex 1 proteins are more common in participants with simplex transcripts and nasal swabs. And then they also find associations between EBV transcripts and PBMCs and increased proportions of B-cell plasma blasts, which are the host of the virus. And they find that CMV transcripts are associated with an increase in CD4 and CD8 central memory T-cell frequency and a reduction in CD27 low effector memory CD4 T-cells. Why would that be, Brian? again? So either those cells are getting pushed really hard, and so they're not so much effector memory, and they're really becoming effector cells, or they're not getting pushed hard enough, and they're staying as central memory cells.

22:55So sort of Goldilocks, I guess. Okay. Next, they look at whether herpesvirus and anellovirus transcripts are associated with inflammatory protein changes. So this requires modeling. They compare longitudinal cytokine dynamics in patients with or without viral reactivation, and they could therefore identify severity-independent cytokine changes association with different virus reactivations, right? So different viruses have different cytokine profiles, although they do overlap. I'm not going to read them all to you, but for example, EBV in PBMCs was associated with a bunch of cytokines, all of which are linked to COVID-19 severity.

23:41So that could be a connection there, right? But only one of these, CXCL10, was associated with EBV in the nasal RNA sequences. And then there in that nasal compartment, EBV was associated with other cytokines, including interferons. So the idea would be that the host response varies in different compartments, which is not surprising, right? Simplex and CMV were also associated with different sets of serum cytokines. But interestingly, in Nelaviridae, we're only associated with increases in CXCL11 and IL18 and decreases in TNFSF11. So the other viruses, Lots of different cytokines going up, but anello kind of mild, which I don't know what that means.

24:32Maybe the hosts are used to having anello viruses all the time. And again, another place where it's interesting. And you look at plasma metabolites, great small molecules that you can pick up in the plasma. They see viral reactivation is associated with changes in these, particularly in amino acid and lipid metabolism. CMV is associated with the greatest number of metabolome changes, increased urea, and this compound called T-MAP. CMV and anelovir, they are associated with increased long-chain fatty acids. And there's a shared signature, metabolite signature, across multiple viruses with specific changes that we don't need to talk about.

25:18So then they want to see if they can identify a signature for each virus that's independent of COVID severity and participant demographics. And so in the PBMC transcriptomics, viral detection in either the PBMC or the nasal compartments are associated with changes in PBMC gene expression. And they see downregulation of diverse RNA processing and protein translation pathways. EBV, CMV, and simplex were associated with upregulation of cellular replication pathways, which is not surprising. That's what viruses do. So that's in PBMCs. Now, in the nasal compartment, EBV, CMV, and herpes simplex had the strongest associations with gene expression changes in the upper airway.

26:13Well, that would make sense also, right? These were all associated with upregulation of interleukin signaling, including IL-10, lymphocyte immunoregulatory interactions, and neutrophil degranulation. Upper airway, CMV, and EBV transcripts were also associated with increased antiparasite and phagocytosis pathways. To give you an idea. And then finally, we get to long COVID. Yes. This part particularly surprised me because when I saw all of those news articles yesterday, most of them were talking about this paper in relation to long COVID. Yeah, right. And I was sort of like, wait, that was it? It was a shorter portion of the paper than I had expected from the way I was sold this paper.

27:05Yeah. Yeah, I don't know why that is. Maybe the press release is emphasized. Yeah. So the press, I didn't see the press release on this, but I can imagine that it would probably emphasize the thing that is going to get the most clicks, which is the long COVID link here. Because otherwise, I mean, okay, viral reactivation in acute COVID-19 patients, it's fascinating as a subject to us, but to the general public, it's like, yeah, that was five years ago. Who cares? So long COVID is the selling point here, but it's also a very little part of the paper. Yeah. All right. So we already have previous papers linking long COVID with reactivation.

27:53So they use their data. They have patient reported outcome groups, right, where the patients self-report on their long COVID signal. And this ranges from minimal to no defects, to physical disability or fatigue, to cognitive impairment and global deficits, It's both physical and cognitive, right? One thing I do really like about this is with this study, they're looking at a bunch of COVID patients. And so they have some patients who basically have sort of minimal to no deficit. And so whereas the other paper we're going to talk about are people who are self-reported long COVID patients. So it is sort of nice to have this different way of looking at these patients, but first looking at COVID patients and then being able to see those who eventually progress to long COVID or not versus those who are self-reported long COVID patients.

28:50Okay. So first of all, when they look at viral reactivation during acute COVID, they see no relationship with these different pro groups, self-reporting, patient-reported outcome groups, pro, right? No. But they say, our sample size is small and some people died and left the study, so really can't make much of that. So then they look at the correlation between virus during the convalescent period, right, after the acute COVID and these different pro groups. So the anelovirdi and enteroviruses were the most frequently detected virus in convalescent samples. And the anelos were more prevalent in people in the physical pro group, right, high scores on these different physical surveys, which suggests that maybe, they say, maybe Anello's could be a potential signature for persistent physical disability in patients with long COVID.

29:51Maybe. You'd have to do more studies because the numbers aren't. So another very diagnostic signature of persistent physical disability is physical disability that persists. Yes. And it's not like we really need a blood test to tell that. Right. Well, it would be nice because it depends how you measure the – if you say someone fill out a form and they said, yeah, I can't do that, it's better to have a test. Yeah. Yeah. Right. It definitely is also helpful with having physicians believe you. Sure. I think that that's helpful. And it gives us some interesting thoughts about, well, maybe we should know some more things about these annulloviruses.

30:32Yes. All right. So that's the data, basically. So a number of limitations here, right? First, we mentioned that they're all hospitalized. They're unvaccinated. They were infected with the original SARS-CoV-2. And now it's different strain. Who knows how that impacts it? And this was actually, the cohort was enrolled before long COVID was really well-defined. And so it's different now. And so it should probably be reassessed. They only looked at three compartments, right? It would be nice to look at others. Not as easy to look at others. And then these endotracheal samples, they come from the mechanically ventilated patients, which is a really small subset, right?

31:22And that could be a bias. It's also a little bit stressful. Yeah. It's a small subset with very, very severe problems. And as for the long COVID, right, the association, as we said, between Anello and the physical pro-group, this is the headline. But it's an association. And the paper explicitly says we cannot establish causation. I hope the newspapers didn't. But these aneloviridae are troublesome because 80 % to 90 % of the people have him, and there's no pathogenic mechanism there. So we don't know if they're drivers or just passengers. We have no idea. Yeah, and it's, I mean, if you reverse the causality on this, that the physical disability is causing the aneloviruses to stay reactivated, that's totally believable.

32:12True. We can't tell. So only 17 % of these anelopositive patients were on immunosuppressive medications. But as I said, these hospitalized patients often got dexamethasone. So maybe that's driving it too. And I don't think they looked at that. And then so this is, the whole paper is really associative. You know, the reactivation correlates with disease severity, cytokine levels, metabolomic changes, and long COVID. but are the viruses doing this or just growing in a disrupted immune environment, right? We don't know. So what are the clinical implications? The authors actually say in the discussion that maybe antiviral therapies for herpesvirus could be used, but there's no causality evidence.

33:03You can't give antiviral unless you have causality evidence based on just this. But it's something you might work towards. That's totally fine. And then, of course, the aneloviridae, we have no antivirals because they don't do anything to anyone. And I doubt anyone would develop them, right? So it's an interesting paper, though. And I said, as we have said, it needs to be done and it will stimulate more similar work. Yeah. Most of all, with both of these papers, I'm excited that they were done. and I'm excited that they start to provide information about long COVID to potentially tell us what's going on and potentially in the future help some patients.

33:45And I think next time... Go ahead. Neither of these papers provides answers, but they both raise interesting questions. Yeah, that's good. That's what science does, right? But you can bet next time you get sick, you're probably reactivating some viruses. Sure. You all have them. You had 8 to 12 at least chronic viruses. So now we have another long COVID paper. This is in Cell. And this one, this is the one that Brianne said over lunch last week that she really enjoyed this. So I said, well, then we have to do it if Brianne enjoyed it. I saw a talk from the senior author of this paper presenting these data.

34:27And as I was saying in the pre-show, my number one take-home message from seeing this talk was, wow, I really don't want long COVID. Right. So this is an article called A Causal Link. Now, I object to causal, but we'll get to that. A causal link between autoantibodies and neurological symptoms in long COVID. There are three co-first authors, Kayla Santos-Gueres de Sa, Julio Silva, and Rafael Beari-Olmos. And the senior author is Akiko Iwasaki. The team is from Yale University School of Medicine in New Haven, Seltrend GmbH in Luchenwald, Germany, Ceromics Systems in Woburn, Massachusetts, Mount Sinai School of Medicine in New York, and Universitatsmedizin, Berlin.

35:14Interesting. Ceromics. S-E-R-O-M, capital Y. Y-X. Little X. That's very interesting. The Y is like an antibody, I guess. Yeah, exactly. I don't know if that was the intent, but that's how I read it. That's what I would do. So two of us, right? All right, so long COVID again. 10 % of individuals after SARS-CoV-2 develop long COVID, and you get lots of neurological symptoms, which are coming from different parts of the brain. Women are more likely than men to get long COVID. and other post-acute infection syndromes like ME, CFS. So that suggests some sex differences. And the authors say because many autoimmune diseases like MS, rheumatoid arthritis, systemic lupus, Sjogren's syndrome, are more prevalent in women, they say, well, maybe autoimmunity plays a role in these long COVIDs and other post-acute infection syndromes.

36:16And there is some data supporting an autoimmune etiology. So autoantibodies, AABs, have been found in patients with ME-CFS along COVID. And some of these antibodies target G-protein-coupled receptors and GABA receptors that are involved in neurological events. And you could imagine that they could be associated with symptoms. And they also point out that infections naturally trigger the production of antibodies, which we know, right? Everybody knows that or should know that. That's how vaccines work. But also, you may get autoantibodies by bystander activation or molecular mimicry. And you see these autoantibodies during the acute phase of SARS-CoV-2 infection.

37:12and other retrospective studies of COVID patients have reported that 20 % to 40 % increased risk of multiple new-onset autoimmune diseases after SARS-CoV-2 infection. That's a lot. It is. I don't know the data, but I would suspect that if you looked at, again, flu or a number of different infections, you might see some increase in autoantibodies during the acute phase of the infection. Really, part of this is that when you make B cells, you're always going to make some B cells that are autoreactive, that are making autoreactive antibodies. And we have multiple mechanisms to sort of shut those B cells down and make sure they're not actually going to produce those autoreactive antibodies.

38:04And during some of the parts of acute infection, you may start to, you know, those B cells might get turned on a little bit. Those B cells might start producing their autoantibodies. And then, as it says here, when the immune system returns to homeostasis, you'll start to have those autoantibodies go down. The B cells will sort of chill out. If everything works perfectly. If everything works perfectly. But especially if you're infected with a virus that has an antigen that's similar to some autoantigen, then you may end up with a persistent autoimmune response, and that's where these conditions come from.

38:45So, Brian, does everyone have B cells that recognize autoantigen? Everyone has B cells that recognize self, yes. And in the best condition, they are never activated, right? In the best condition, they are never activated or they're somehow, they're cholerized to stop them from really making many antibodies. So what is bystander activation when these are turned on by being next to a B cell that's being activated? Yeah, maybe you are making so many cytokines that are trying to turn on the SARS-CoV-2 B cells that some of the autoreactive B cells get turned on by accident. Perfect name, bystander.

39:23Yes. You're just standing there on the sidewalk and something happens to you, right? Yes. Okay. So in this paper, this is what they want to look at, long COVID and autoantibodies. And they have two cohorts. These are patients with neurological symptoms with long COVID. They have a Mount Sinai-Yale long COVID MyLC cohort. And they have another one that they use for validation called MyLC2. Yeah. So the two things I would say, I like that they point out right from the get-go, you know, the heterogeneity of long COVID poses significant challenges. And I know that's something that I've heard a lot in presentations about long COVID as well.

40:05So here they are kind of lumping together all types of long COVID, and there likely are maybe different things going on in different groups of patients. But these patients are sort of patients who self-reported to become part of a long COVID, these long COVID cohorts. So that's, you know, one big difference with this in the previous paper. And another difference is the sizes of the samples. We're down about an order of magnitude here. We're talking about, you know, 82 patients and 35 controls in one set of data and so forth. Right. So they have 82 long COVID patients, 35 healthy controls, and then they have 30 convalescent controls.

40:49These are people who got COVID but didn't develop long COVID. These are mostly women, 70 % women, 45 years of age on average. and so most acute infections in the long COVID group occurred between week 7 through 18 of 2020 and that's the WA1 strain circulating and again that's another limitation that this is all done with infections with that and it could be different now. I mean it is also kind of nice that because I was thinking about who these healthy controls were versus convalescent patients And if you're looking at controls that are from that timing, you can perhaps find people who have not ever had SARS-CoV-2.

41:32That's right. Right. The samples are taken on an average 416 days after the acute symptoms. Only 13 % of the patients had acute, severe acute disease. So these patients had neurological symptoms. Half of them had five or more symptoms. And for this study, they look at the following kinds of neurological symptoms, brain fog, confusion, dizziness, loss of memory, sleep disturbance, alterations in mood, pins and needles, disorientation, hearing, fainting, and numbness of the face. And the most common symptoms were fatigue. 87 % had that brain fog and headache, loss of memory, dizziness, sleep disturbance, and confusion.

42:22And none of these individuals had an autoimmune diagnosis at the time of enrollment. So first question is, do these patients have autoantibodies? So they purify total IgG from plasma, and they do immunofluorescence using sections of healthy human tissues. And there are very nice pictures here that show very clear immunofluorescence. So the purified total IgG from a subset of long COVID patients show increased reactivity against different parts of the brain, including the human locus coeruleus, the human thalamus, the mouse sciatic nerve. So obviously they got some mouse tissues in there and the mouse meninges.

43:13And so the coeruleus is the primary source of norepinephrine, noradrenaline, right? And it's involved in many, many things, including the sleep cycle. So it could be why these patients have sleep disturbances. So that's a really interesting finding. And what the finding is that these antibodies are reacting with these parts of the brain. And also, in addition to the surrelius, they're reacting with, I said that, the thalamus, the sciatic nerve of the mouse. So you see, it's a diffuse staining. There are a few cells showing distinct labeling with the antibodies. And on average, 94 % of healthy and 85 % of convalescent controls tested positive for at least one neurological tissue.

44:01So that's not such a big difference, right? A lot of convalescent people are making autoantibodies apparently, right? Yeah. I mean, I think it's not surprising that they make the autoantibodies. I think that the amounts are sort of interesting, but they don't totally quantitate that. And throughout this paper, they display data as ROC curves a lot, which are kind of important when thinking about diagnostics and whether or not this particular test actually distinguishes between two groups of patients. And so basically here you're plotting the sensitivity versus the one minus specificity about how many true positives you have versus how many false positives you have using that criteria as a distinguisher.

45:03And they basically have a line, a diagonal line, which is what you would see if this was random. And so that would be the relationship that you would see. And basically, if the curve actually goes into the upper part of the line, so it's above the diagonal line, more to the left and higher, that is better. And so while it's really hard to sort of list that as one particular value, sometimes you talk about the area under that curve. In some of these studies, when you actually look at that kind of multi-component analysis, there is something that's pretty striking, particularly like the mouse meninges.

45:53Of course, it's mouse, right? Yeah, exactly. So then they also do another quantification. They calculate how many tissues per individuals that reacted with the serum. So the long-coverage patients had reactivity to an average of 3.6 human neurological tissues per individual compared with healthy controls, 2.4 tissues, Convalescent control is 2.5. So again, as Brian said, the graphs are better at showing this because those numbers don't sound all that different. Half of the LC individuals were positive for at least one human peripheral tissue. So antibodies to at least one tissue with a mean of 3.41 tissues per individual.

46:44And they also found some antibodies to peripheral human tissues like adrenal gland, heart muscle, parathyroid and thyroid tissues, and they have antibodies against human peripheral nervous system tissues and central nervous system tissues, as we have talked about. Do these antibodies against nervous system tissues correlate with long COVID symptoms? there is an overall increase in odor activity in the LC group. You can see it in the effect size of the mean of the fluorescence or the frequency of individuals with positive symptoms who had antibodies. And particularly the antibodies against the human locus coeruleus were significantly associated with loss of taste and smell, nausea, skin lesions, joint pain, and joint swelling.

47:42All right. Then they want to know what these antibodies are recognizing. So they do ELISAs with plasma samples from 29 healthy controls, 24 convalescent controls, and 72 individuals with LC, and they can identify specific proteins. proteins, one of them is NMD-R2C, which is, I don't know, I forgot what it is. Does anyone remember what that is? NMD, it's a neural protein. Right. I think it's probably a receptor. Probably, yeah. And then they see if they can stratify the patients based on the antibody profile recognizing certain kinds of proteins. And so they looked at G-coupled protein receptors and ionotropic receptors.

48:41If I find two clusters of patients with distinct antibody signatures, one has a homogeneous pattern of reactivity with positivity against beta-1 and beta-2 adrenergic receptors and a couple of other. And cluster two has a more heterogeneous profile with lots of other targets as well. All right. But none of these clusters had any significant association with any symptoms at all. So that didn't mean anything apparently. They also, and this gets difficult, they have this human protein microarray and they screen the plasma against that. It's got 21 ,000 full-length human proteins on it. So you can see And you know where each one is.

49:27So if your antibody is lighting one up, you can tell what protein that is.

49:36And it's kind of inconclusive. So people with LC and convalescent controls both exhibit a higher frequency of autoreactive antibodies, just like we saw before in the staining. But there was also poor discrimination between the convalescent and long COVID groups. There were many thousands of hits unique to the long COVID group. The brain tissue was the top target of these, but also testes and lymphoid tissues. And so they write, given the predominance of females in our cohort, the relevance of the presence of antibodies targeting male-specific tissues remains unclear. So this is one of the, it's what you see.

50:17That's what the data say, but we don't know what it means, right? All right, so what do these proteins do? What are the pathways? So they do pathway analysis, and they find these proteins are involved in hormonal signaling, cell adhesion, synaptic function, neurogenesis, and regulation of neuronal apoptosis. So those would make sense in terms of some of the symptoms, obviously. They also found clusters of—they could cluster the patients in terms of reactivity to neural proteins, Like one cluster has reactivity to proteins enriched in glandular and epithelial cell types, and the other cluster preferentially targets proteins in neurological cell populations.

51:01And in fact, that cluster has a higher proportion of patients reporting new onset pain. So there's a little bit of a correlation there. I also find that headache is correlated with multiple autoantibody targets as well. They also see that they want to know if any of these autoantibodies overlap with autoantibodies found in other autoimmune diseases. And they use systemic lupus erythematosus as an example, SLE. And they find that a subset of individuals with long COVID have a similar range of hits as patients with SLE. And they have some common antigens between the two diseases. But most of them were not in common at all.

51:44So maybe there's some commonality, but not much. Yeah. I think that one of the things I get from all of these data, and they mentioned this themselves, is the heterogeneity. So they've done some work on clustering their long COVID patients, and they keep finding different clusters, which is cool. But when you start with a relatively small cohort and then start clustering it into smaller groups, it becomes very hard to get statistical significance to know what's going on. And so when they put the entire cohort together, we know that it's a mixture of heterogeneous individuals. And so that might sort of dilute out a signal.

52:29And so in sort of a perfect world where you had everything possible and all the money and things like that, it would be great to look at some huge cohort and be able to actually divide them into unique forms of long COVID and see, are some of them related to the viruses we saw in the previous paper? Are some of them related to these autoantibodies? I mean, the fact that there is this much heterogeneity and they still, in certain places, can start to see a signal is kind of interesting, but there's obviously a lot more that needs to happen. Yeah. If you could do this with a whole cohort of millions of long COVID sufferers, you might get some interesting information.

53:12So I'm going to skip through a lot of this. They basically say we don't observe a striking quantitative difference in the number of autoantibodies between long COVID and convalescent samples across all of these different screenings that we did. There's a lot of heterogeneity and reactivity, as Brianne has just told you, and they said that's a major challenge for identifying a single shared target with robust diagnostic utility or association with clinical symptoms. But our data suggests that although autoreactivity may manifest, the composition of these antibodies is different in long COVID. And it may be, I mean, obviously everybody would like to have a single test for a single thing that gives you a clear answer, oh, this is long COVID, and then we can target that pathway.

54:00But as Brianne has discussed about the heterogeneity, that actually suggests that maybe there are many roads to long COVID, that autoantibodies against a whole bunch of different things could lead to this outcome. Because when you look at the symptoms of it, I mean, it's not like you get one very clear symptom of long COVID. You get all these things going wrong in different places. And so it's quite possible that you're not going to get any kind of a clean diagnostic test, you're going to see a general dysfunction going on, and that may be as good as it gets. Well, I mean, if you had 20 targets, let's say, you could make a test for all 20.

54:42You could make a test for 20 targets, yeah. But it's, yeah, we're not there yet, obviously. One of the top protein hits, right, the target of one of these autoantibodies is proteins from what are called the mediator complex. And the mediator complex is an interesting name. It regulates transcription, right, of DNA to mRNA. And they find that antibodies that target these mediator, in particular one of the mediator proteins called MED20, these antibodies bind multiple FC gamma receptors. And so they say maybe there's effector functions going on here with these antibodies. They could be pathogenic, right?

55:30So they do some assays. They do an antibody-dependent complement deposition assay and an antibody-dependent cellular phagocytosis assay, which would involve the antibodies binding to the FC receptor, right? They see observed phagocytosis, right, with these MED20 antibodies. And this character of these antibodies is positively associated with fever and urinary difficulty. difficulty. But that's all they say. They don't go into it anymore. So there is this increase in this particular kind of FC function, but we don't know it. Yeah. And so I think that this goes back to their previous point of maybe the antibodies are different in these patients.

56:12Yeah. They did this other cohort that we mentioned, this independent cohort. They did a lot of these assays with them and they basically find the same things. Okay. So that's very good. But now we get to the experiment, the actual experiment, which is, and this I know Breanne liked very much, although it's also got a lot of problems with it. Oh, absolutely. But it's an interesting experiment. So they make a mouse model, a passive transfer model. They give mice total IgG from these patients intraperitoneally. And each mouse gets IgG from a different donor. And then they They see that the IgG gets into the brain.

56:555 % of the human IgG actually crosses the barrier. And then they run assays on these mice to see if they have... I mean, these assays are all surrogates for long COVID, obviously, but they have a pain sensitivity. You put your mouse on a hot plate and you see how long it is before they jump or something or run away. So three days post-injection, mice display increased thermal pain sensitivity. 85 % of the mice with the pain phenotype got IgG from participants with long COVID with self-reported new onset pain. But also, others here also do this to a lower lesser extent. So if you had asked me about this experiment before I saw any of the data, before I read this paper, I would say there is no way this would work because we're taking total IgG.

57:50So it's not purified against any particular target. So there's the heterogeneity there. And you've got this heterogeneous group of patients. And you're putting human antibodies into mice. And you're trying really to measure things that are sort of symptoms as signs. you know you're trying to quantitate things that maybe aren't so quantitative there is a 0 % chance you will see anything at all in this and so the data aren't you know dramatic but the fact that with all that heterogeneity they see something to me is sort of striking we don't know if it's the antibodies that's doing that true exactly because they didn't really fractionate it Right.

58:38Yeah, this reminded me, I think there's a Mark Twain quote about teaching a dog to sing, and it's not done well, but you're amazed that it's done at all? Yes. So reading this, I thought, okay, this is really not much of a result, but they got a result? Yeah, exactly. Wow. And if, I mean, there are some antibodies that, or something in the serum that could be the antibodies that is somehow changing, you know, a mouse's ability to stand a hot plate and to grip things. Like, I don't want that serum. No. Yeah, there is something. Yeah, so that's the other thing. What they successfully prove here, I think, is that there's something up with this serum.

59:25Yeah. They also show that half of the mice scoring positive for pain, receiving IgG from patients who reported weakness and 90 % from patients with dysautonomia. But the thing is, it's interesting is all of these symptoms of this hot plate stuff goes down to baseline after six days. Yes. Right? So that's not long COVID. Something else is going on. it. The other thing they do is this grip strength. They do this after injection, shows a trend of muscle weakness in mice injected from people with long COVID. And that is associated with tinnitus in people, headaches, and those two. So tinnitus and headaches are associated with the grip strength.

1:00:12And if I'm remembering correctly, the way they test the grip strength is they take the sort of lid of the mouse cage with all of the bars on it. Yes. And they put the mouse on it and basically see how long it takes them to fall off. Yes. Yeah, we used to do that with polio. Remember, if they couldn't grip the cage, they were paralyzed. Yeah. But look, grip strength, tinnitus, headaches. I mean, what's the correlation? I don't get it. This doesn't get us to mechanism or even really into the ballpark. It's just, but as Brianne pointed out, I mean, the fact that anything happened here is just amazing.

1:00:50Amazing. Then they do a treadmill test, right? So they have mice on a treadmill and, right? I'm sorry. Yeah. Increased fatigue-like behavior in the treadmill test.

1:01:08But that didn't correlate with anything from the patients. They also can evaluate anxiety-like behavior and locomotion by open field and elevated maze tests and also blood pressure and heart rate. That's easy to measure in the mouse, and there's no differences in the groups in those. They want to know how these antibodies are inducing pain, so they look to see what they're staining in the mice, and they find some intraepidermal nerve fiber staining, which may be where these antibodies are doing that. But that's basically, so basically the passive transfer of IgG from patients with long COVID, it gives heightened pain sensitivity, fatigue-like behavior, loss of balance and coordination, and muscle weakness, which is associated with symptoms reported by the patients.

1:02:09And then they see widespread neuron activation, which is consistent with chronic pain and fatigue by whole brain mapping. So that's the paper. So again, this is very interesting. There are a lot of issues, right? There are issues with the cohort, which is pretty small. It's mostly women, even though they mostly get the disease. I don't know if that's generalizable. It's the original virus. And there's no longitudinal sampling, right? You don't know when these antibodies. You don't know the time course of this antibody production. and the mice, it's not purified IgG. And actually, we didn't go into it, but the phenotypes in the mice are pretty modest and short-lived.

1:02:57Yeah. So I don't know the clinical relevance of that. They only use female mice, but... Well, that's appropriate for the, I mean, to reflect the sex distribution of your patient's appetite. Men do get long COVID also. Of course they do, yes. We're not going to learn much about it. Though I was actually kind of pleased to see people using female mice because the default has been you use male mice for everything. Yes. This is a good trend. Yes. And these mouse behaviors are proxies, right? Yes. It's thermal hyperalgesia and treadmill fatigue are not the same as brain fog, POTS, or chronic pain. But I do also appreciate the fact that at least, you know, especially at the beginning of the COVID pandemic, there were patients who were reporting these symptoms who were not getting believed.

1:03:44Yeah. And I think that that's also true with a number of the other post-acute infection syndromes. And so the fact that there is some attempt and ability to show, yeah, there's something up and these things that may be hard to study and hard to quantitate. Yes, we can start to see there's something there. As Alan said, there's something in this serum. Yeah. And there's plenty that we still need to know about it. But I look at both of these papers and I think about those patients who were not supported and were not believed that something was going on. And I say, OK, look, there's legit evidence here.

1:04:29We should be believing these patients. There's something going on even if we don't understand all of it. Even if we shouldn't use the word causal in our title. Sure. Go ahead. I was going to say, and yes, this is definitely worthy of continued study. Of course, of course. No, it raises a lot of questions, which is always good for science. The other thing is these antibodies may arise from tissue damage. They may not cause it, right? So that's something you'd have to evaluate. I don't know. This transcription factor is a nuclear protein. I don't know how that would be involved. Yeah, I don't know that transcription factor particularly well.

1:05:09But it's not on the surface, so that's an issue. And then, of course, long COVID, they just call it long COVID, but of course it's multiple distinct types. And it doesn't mean that this drives all of long COVID, right? It may be relevant for a minority. So anyway, so the authors acknowledge all these issues, but that's fine. Most papers do have issues, and that's why we keep doing science, because we address them all. But it's nevertheless two interesting papers on long COVID, right? Yep. Okay, let's do some email. Oh, Alan, this first one's for you. Yes, I will take this first one, and it will reveal that I am not caught up on TWIV episodes.

1:05:54So I wasn't on this episode. Oh, you weren't on this episode. I hadn't listened to this one yet. So Bill writes, Dear TWIVers, I was listening to TWIV 1339 where you were discussing aviation safety, and the term controlled flight into terrain came up. You commented that this meant intentionally crashing a plane. As a pilot, I'd like to correct that. Controlled means that the controls of the plane were working correctly, so things like the hydraulic and engine systems were in good order. It ordinarily does not mean an intentional crash, however. These types of accidents usually occur in clouds, where the pilots can't see the terrain and inadvertently fly into it.

1:06:30Modern airplanes have systems to warn pilots before this occurs, and as a result, such accidents are rare. those systems came to be from studying these accidents. Thanks for all you do, Bill, who's in Issaquah, Washington. Well, Bill, I'm going to provide a correction to your correction. These accidents are actually not rare if you are looking outside of the airline industry into general aviation, where things like, you know, built-in TCAS are not common at all. So in the planes that I used to fly at little Pipers and Cessnas, you don't get Betty coming on saying, you know, climb, roll right. You just, you know, hum along till all of a sudden, what's a mountain goat doing up in a cloud bank?

1:07:16And they also, CFIT encompasses accidents where the pilot is in control in a situation where they accidentally navigate the plane into the ground, even if they might have been able to see it, even if they weren't in the clouds. So a classic situation is in the landing pattern. When you come around and you're making your turns low to the ground, if you stall the wing there, you can undergo controlled flight into terrain. You have control of the aircraft, but not really because you just stalled. And this actually happens. It's a common cause of lethal General Aviation Act crashes. Wow. Well, I'm sorry you weren't on that episode because we all figured it meant you did it on purpose.

1:08:04Nope. It's exceptionally rare. There are cases, obviously, of pilots committing suicide, but that's exceptionally rare. This term is used broadly for everything was working fine and the pilot crashed the plane for various reasons. Well, thank you, Bill, and thank you, Alan. You're welcome. Breanne, can you take the next one? Sure. Carolyn writes, if I were to make a post stating this, would it be correct? And here's her post in quotes. Since I keep reading about common sense and that being a big deal when it comes to pandemics, I thought I would mention that if scientists learn how to mutate a virus from nature, the nature is the teacher, right?

1:08:47So if a student can do it, shouldn't the teacher be able to do it too? Just because man made a cleavage site in a mouse MERS virus doesn't mean man made the same or a similar cleavage site in SARS-CoV-2. A study out of Germany was released right before the Dr. Fauci diary was released, stating that they show this cleavage site was not man-made. When they tried it with that virus, it was too weak to keep circulating. Or that was my understanding. So if Nietzsche did it with SARS-CoV-2, the teacher did it better. There have been others who presented strong evidence that the cleavage site was not man-made.

1:09:27But Rand Paul never mentions that. He never mentions his wife investing in remdesivir that somehow is poison. But that is another common sense story for another day. As for hashtag Rand Paul, I hope you end up with the life you deserve. And that's the end of her quote. I have to make sure I'm understanding this right. I would ask a virologist friend, but he works for the NIH, and I don't want to jeopardize his job in any way, shape, or form. I'm a novice, but fascinated with virology. I just don't have a lot of time for it, and I'm not that great at learning so many terms. It's hard enough to keep up with all the IT terms I'm dealing with.

1:10:07Thanks, Carolyn. I think it's fine. Yeah, sure. I think it's right. Yep. There will be, we're going to have in a couple of weeks an even better paper showing that the furin cleavage site was not inserted. We already know it wasn't inserted, but this is, remember the talk from ASV brand? He's going to come on to TWIV and talk about it because that paper is in bioarchive, I believe. And there have been, I think, going to some of the other meetings I've gone to, looking broadly at the diversity of peering cleavage sites among various coronaviruses, it's sort of clear that it's coming in and out of a lot of different coronaviruses.

1:10:56VC writes, hi, Vincent and the TWIV team. Thank you for the great work, shining as a beacon of truth in a dark era of misinformation. I want to bring your attention to the work of Robert Schaefer at Stanford University, Bob created the Stanford HIV Drug Resistance Database. There's a link for that. It's a curated database that is free, has saved countless lives across the globe, and Bob's life is dedicated to HIV drug resistance. The impact of the database is more on resource-limited parts of the world where the therapy choices are limited to first-generation drugs. A clinician anywhere in the world could use the database to input the genotype and the predicted phenotype will help make treatment decisions.

1:11:40His work also led to the creation of a panel of HIV drug-resistant clones representing all clinically relevant resistant patterns for the different classes of HIV drugs. These clones are a great resource for researchers and companies developing new drugs as they act as a benchmark to test against existing drug resistance. I was fortunate to have Bob as my a postdoc mentor, and work on creating these clones. During the pandemic, Bob used his expertise to create a SARS-CoV-2 database. Bob is also working with Long COVID. I think it's worth having Bob on one of the TWIV episodes. Don't take my word for it.

1:12:15But if the TWIV team is convinced after reviewing his contributions to the field. Despite all of this great work, what impressed me most about Bob is the way he stood for truth with courage in a legal fight on an obvious patent. ABL, a company based in Luxembourg, threatened to sue Bob and Stanford for violating their patent on using computers to analyze medical data. Stanford lawyers settled with the company, agreeing to write on the database page that they are acknowledging the patent of this company without including Bob in the discussion. But he stood his ground, did not agree to make any changes to the database.

1:12:51He created a website about obvious patents and fought the lawsuit with donations from those who supported his cause and invalidated the obvious patent. John Cohen wrote about this case in Science. He gives a link for that. I've moved on to become a public health lab professional after my postdoc, but I still keep my virology interests alive. The intent of this email is not to be read in an episode, but to acknowledge the appreciation of the great work all of you have done. Well, you may not have that intent, but I like to read it. It seems fine. And then, P.S., if I have to do a listener pick, if I have to, are we forcing you to do it, Vici?

1:13:27I will go for PBS Eon's educational YouTube channel and digital media series about paleontology and natural history. And Alan will get a link for that. Thank you, Vici. I don't know how to say it. Vici? Do you think it's Vici or Vici? I was going to say Vici as in Veni, Vidi. Vici. Vici. Vici. i'm looking at this article now because the idea of as it what it says in this article of or in this letter of having a patent on using computers to analyze medical data ridiculous sounds so ridiculous it's way too broad yeah yeah but what do i know i'm not a patent lawyer. I don't want to go out of my...

1:14:11No, but I mean, it's great to... It is very annoying that the university lawyers would just bow to this and say, oh, yeah, okay, we'll settle, when the right thing to do was definitely to challenge this bogus patent that's just being used by trolls. Yep. In my opinion. Let's go back to you, Brian. This one's a little thorny, but we can do this. Me or Alan? I think I'm actually. Oh, sorry, Alan. Sorry. I mean, Brianne, if you want to do it, I'm all for it. I'm happy to have you do it. Okay. All right. James writes, I'm writing to you as a dedicated listener who turned to this week in virology as my primary source of truth during the height of the COVID-19 pandemic.

1:14:55I valued your team's expertise, committed to the scientific method and willingness to break down complex virology for the public when clear information was hard to find. However, reflecting on the show's coverage over the years, there are a few points where I feel the audience was not fully served. First, during the critical period when public trust was being established, the show omitted key context regarding gain-of-function research on coronaviruses in Wuhan, work that the team was aware of. Failing to proactively inform listeners about these research activities left a significant gap in the public's understanding of the full picture.

1:15:26It was frustrating to not know about what had been done when the other side discovered these activities. Second to this day, I'm actually going to answer this in sequence. We actually have discussed gain-of-function research for quite a number of years before the pandemic, going back at least to the Kawaoka and Ben Fouchier flu papers. We talked about the work going on in Wuhan. So this is not something that we hid in any way. Yeah. This is not a correct statement, James. I'm sorry. Second, continuing with James' letter, second to this day, TWIV consistently presents the natural origin hypothesis as the only credible explanation while dismissing the research-related hypothesis.

1:16:14While advocating for natural origin based on available biological evidence is a fair scientific stance, failing to thoroughly report on the alternative arguments, anomalies, and biosecurity concerns deprives listeners of a balanced view of an issue that remains unresolved across the wider scientific and intelligence communities. This is also not correct. This is an issue that is resolved across the scientific community and is largely resolved to the extent that I have access to the information in the intelligence community. My understanding is that the intelligence community says it is possible this came from a lab.

1:16:51And that's about where they leave it. Right. James Point has a sentence here, while advocating for natural origin based on available biological evidence. And I would say, yes, that's exactly. what's going on. That's the evidence that we as scientists should make our stances based upon. But what James is saying is that since non-scientists listen to TWIV, we should address the other part in more detail. Right. So this is the old, this I'm very, very familiar with as a reporter. You should have a balanced presentation. Well, okay. A balanced presentation does not mean both sides get equal time because it is entirely possible that one side is wrong.

1:17:36The truth is not always in between. And in this case, a mountain of accumulated evidence says that one side is almost certainly wrong. And no, we're not going to go amplifying wrong arguments that are not based on any credible hypothesis. Um, okay. Next, finally, fundamental science requires attempting to falsify one's own assumptions. The only way to rigorously test or disprove alternative theories is to engage with them head-on and investigate them with the same rigor, even when the potential outcome is uncomfortable or undesirable. Dismissing a hypothesis outright without actively trying to stress test it runs counter to the spirit of scientific inquiry.

1:18:20Here, I would point out that the folks who are arguing for a lab leak do not have a falsifiable hypothesis. Look closely at what they're saying and how they structure their arguments. It's always, yeah, but you can't prove. They're absolutely right. You can't prove that it didn't come from a lab because you can never prove a negative. Yeah, I will say that the data that exists looking at some of the sequence evolution, looking at some of the I know that there is a paper. I'm pretty sure that Michael Roby is part of it. And I can't remember all of the authors that has sort of the GPS information about early cases and where that was compared to the market and compared to the lab.

1:19:10some of the swab data, I actually would say to me, that feels like a pretty rigorous test. And to me, it in fact falsifies that theory. And so I find certain data to specifically falsify some of the things as best as I can understand them. Exactly. It's to the best we can determine all of the data. And every time somebody goes looking for a lab leak on this thing, they find more data that it wasn't. And the GPS data Brianne is referring to, which we've covered on TWIV, essentially shows if you plot the early cases on a map, they are a bullseye on the wet market, which is not next door to the lab.

1:19:58And you find, And the cases are right there. A lot of them were people who worked at the wet market. There were also people who didn't work at the wet market, but they lived in the neighborhood. And it's just one of many of these types of studies where you go looking for it coming from the lab. It's just not. Continuing the letter. I share this not out of hostility, but out of a genuine belief that scientific broadcasting is at its best when it presents all sides of an ongoing debate transparently. I hope you will consider addressing these perspectives in future episodes. And again, I don't think there are at this point really all sides or an ongoing debate.

1:20:40Sorry. Very early on. And in fact, I mean, we have discussed the hypotheses. Okay, it could be this, it could be that. It's not impossible, but it has become increasingly untenable to try to argue that this came from anywhere but nature. So on TWIFT 760, which was recorded in December 2019, I spoke with Peter Daszak, and he explained the gain of function that was happening in Wuhan because he was collaborating with them. And as a consequence, now his company has lost all of its NIH funding. But it was clearly there what exactly was going on. And then subsequently, we talked about it a lot. So it's not correct to say we haven't talked about the gain of function that was going on there.

1:21:23About a year ago, Alina Chan wrote an op-ed in the New York Times emphasizing that, in her opinion, this virus came from a lab. And she said 10 points on why this came from a lab. And we refuted them one by one on TWIV. So it is not fair of you to say, James, that we don't do this. We did almost a whole episode on that back then. And there since has not been anything new that we need to address. Right. It covered everything. It covered the lab in Wuhan, the fur and cleavage site, all these supposed cases of COVID that were not really COVID that happened in the lab, in the mines, in the woods and all this stuff.

1:22:02No, it's all not true. So I appreciate your writing, but we've done it. And maybe you just missed them. I don't know. Okay. John writes, Hello, esteemed hosts. As my first conference wraps up, I wanted to send thanks to the entire group. I was able to gain something out of every symposium and poster I encountered simply because of all the hours you have put into science communication on TWIV. So John came to ASV. He lives near Minneapolis. Minneapolis. He's been a listener for a long time. He actually registered and went to the conference. And is not a scientist. No, he fixes computers for a living.

1:22:42Every time you've paused to slow down and explain jargon, it helps people like me gain understanding that others might skip in similar science programming. And all your different specialties and guests you've invited on the show give me a depth of background to enjoy the varied material I came across at ASV 2026. So nice to meet Vincent and Brianne in person. Just being in their proximity was like getting a fundamental understanding of super spreaders. I talked to so many people just by being in their orbits for brief periods, and each conversation was pleasant and worthwhile. Here's to another good ASV in Minneapolis.

1:23:16Cheers. And to clarify, I don't think he meant that Vincent and Brianne are super spreaders. I don't think so, but I will say the line of folks who were taking selfies before and after was pretty long. It was very funny. And, you know, it's more like hypernodes. Right. You know that concept. Yes. So you could be super spreaders if you were carrying something. True. Yes, it could be. I mean, for Twiv, people would come up to us and then John is there and he would start talking to them, you know. It's a funny phenomenon. And I was once at a meeting and I was standing at a booth. Actually, it was Michelle Banks.

1:23:55She was selling her artwork. And people would come up to me and talk to me and then go look at her stuff. And she said, Vincent, you need to stay here all day. Okay, let's do one more. Vincent's burgeoning career as a booth, babe. Great. One more round. I think it's Brianne's. I think you skipped Brianne. Did I skip you? I think so. I'm sorry. It's okay. It's because you wanted me to read a really long one. Okay, go ahead. Sure. Howard writes, hello, Drs. Twiv. Kudos to all of you for your commitment to evidence and the nuance you almost always bring to your discussions. Even your occasional lapses from nuance are understandable in the current conditions of widespread misinformation.

1:24:38I just read the clear and thoughtful February Nature paper by 22 of the SAGO committee members. It triggered a couple of thoughts and a question. One, their closing paragraph could, with only a little editing, serve as TWIV's mission statement. Overall, perhaps the most important message to emerge from SAGO's first term is that the only rigorous scientific research, not conjecture or political opinion, will help to solve the mystery of where a virus estimated to have killed more than 20 million people by the end of 2022 and which cost the global economy up to$16 trillion came from. If the world truly wants answers, it must deliver the information and data that make rigorous scientific research possible.

1:25:24I like that. Two, this paragraph floored me. For many TWIV episodes, I was already aware of the lack of cooperation of the Chinese government in providing data on the origins and spread of SARS-CoV-2. But even then, the following statement astounded me. This type of blatant secrecy feeds fanciful speculation into potential conspiracies. And the paragraph is, no animal testing positive for SARS-CoV-2 has ever been reported in China. Yet it is unlikely that none was infected given the number of susceptible species that were in markets in the Wuhan region during the pandemic. And given the hundreds of infections were reported in species such as ferrets, tigers, domestic cats and white-tailed deer in the rest of the world from 2020 to 2023.

1:26:11three. I think that that is actually a really great point from our letter writer. And I will remind the letter writer and whoever else that with SARS-1, there was a multi-year period between when POMSivits were shown as testing positive and when that outbreak happened. And that was because the palm civet samples were taken at the time, there were no samples taken from the Wuhan markets. And so that same type of analysis isn't possible. And then the letter writer continues, number three, my question arises from the ambiguity of this sentence. Three others dissented from our decision to evaluate the possibility that SARS-CoV-2 had leaked from a laboratory on the basis of there being no evidence for this.

1:27:02This, to my reading, could either mean that the three dissenters were A, unhappy about the author's decision to evaluate the lab origin theory because there was no evidence to base such evaluation on, turning the exercise into one of pure speculation, or B, believed that there might have been evidence which the authors chose to ignore in their evaluation. Do any of you know which was the intended meaning? The first implies succumbing to speculation. The second implies participation in a cover-up. The difference is, in my opinion, material. And this is from Howard in Toronto, where it was 22C with 54 % humidity, clear skies, and clean air.

1:27:44A truly glorious day. My understanding is that they said there's no data. We're not going to evaluate the possibility. Yeah, and that's sort of his A. I'm pretty sure it was meaning A, yes. Yes. Yes. Yeah, I mean, you have to remember that. Well, I don't know if you have to remember anything. No, never mind. Sago, by the way, is the scientific advisory group for the origins of novel pathogens. It's a WHO group established to do just that. And remember, a few years ago, a group visited China to try and collect some data. It included Peter Daszak and Marion Koopmans and others. And they issued a report which was approved by the Chinese government before it was released and had to address the possibility of a lab leak also.

1:28:42But they also included other possibilities like it arrived in frozen food from somewhere. Which is also not supported by data, but that was insisted upon by the Chinese authorities who have been trying to float any hypothesis that would put the origin outside of China. And that is one thing that I would say, you know, that's kind of the stretch in the opposite direction from lab leak. It is equally unlikely that it came from anywhere besides China. But it came from a natural reservoir somewhere in China. All right. This one is from Brian. Hi, you wanted emails. This is about corvids and mirror tests.

1:29:23Pigeons are fine animals, but Angela said they are capable of mirror self-recognition while crows aren't. A 2016 Science Reports article found nutcrackers, a corvid, recognize their blurry reflection better than a clear one. Odd? Well, consider that they only see themselves in the environment in water reflections, shadows, that sort of thing. Humans assume a mirror shows us our real image, but it doesn't. It's inverted and it introduces distortions. Nutcrackers are familiar with their kind of mirrors, too. Farm pigs were able to find food they could only see in a mirror. A study tried to reproduce this with piglets in a research facility housed at optimal warmth, soft surfaces, music playing, and food available ad libitum.

1:30:09If you're familiar with pigs, you know how attractive this environment is. Few piglets responded to the potential food in the mirror. The researchers put an empty red bowl under the mirror to encourage pigs to move. They didn't really work. They attributed this to a lack of cognitive capacity. I suspect the piglets were satisfied staying put. I'm reminded of what Michael Levin, the cell biologist, said. When we try to determine animal intelligence, we are really applying an intelligence test to ourselves. That's great. And Brian provides some references for all of this. Oh, that's great. It's true.

1:30:49Cool. We're back to you, Alan. Yeah, and I think this is our final round we're wrapping up. Yeah, it's the last one. There's no more. Bob writes, in the middle of a trip to London and after a side trip to the Animals in War Memorial, very moving, I suggested we walk into nearby Hyde Park and see the fountains of the Italian garden. It was hot and we were already tired. The fountains were okay, but not worth the walk. But I checked out the statue that stood there and suddenly it was all worth it. It was Edward Jenner. Good to see this vaccine pioneer honored. Nice. Brianne. Greg writes, hello from Michigan's Upper Peninsula.

1:31:31Thank you for all the updates and truthful information. I, for one, appreciate the political banter on TWIV as factual and deserved. Unfortunately, it's become part of the medical landscape we are forced to endure. On a lighter note, I find some irony and many loose associations surrounding the Reekland cyclospora outbreaks in the United States. The implications of an alleged dirty political donation from Taylor Farms, the changing of a positive lab value to a false positive, and the historic circumstances of President Zachary Taylor, who died shortly after consuming a large bowl of cherries and milk on July 4, 1850, all reveal some very loose ties.

1:32:12History books tell us that President Taylor died five days later from cholera. Given the rapid onset of GI distress, his illness was not likely from cyclospora, but nonetheless probably resulted from poorly washed produce. Besides stool consistency, there are many loose facts implicating the Taylor name, the executive mansion, the location of Taylor's death, and poorly washed produce. In 1991, Taylor was exhumed to rule out foul play. I understand that poisoning was ruled out, but I'm not sure if cholera was ever confirmed as an official cause or contributing cause of death. Do either of you have any insights on cholera outbreaks in the U.S.

1:32:52during the pre-Civil War era? I recall reading that malaria was an issue in the U.S. around that time. Thanks to all you do to keep us educated, Greg. And Greg also asked, do your episodes count for CME credits? I do not, but Daniel has been thinking about it, and maybe we will do that for at least clinical update at some point. That makes sense. Yes. And Greg, when I was an undergraduate, I took some history of public health classes. And my professor was a woman named Margaret Humphreys. And I recall doing some readings about cholera earlier in U.S. history. And so I might start by doing some searches for her writings.

1:33:39There definitely are writings because I had to read them, but it was a couple years ago that I was an undergrad. There definitely was cholera in the United States at that time, and it was pretty widespread. Was there malaria also? Oh, yes. Cool. Well, not cool. Not cool. No. And yellow fever. Mm-hmm. All right. Thank you for the emails, folks. We appreciate them. Send more. Sure. They're fun. And even if you don't agree with us, it's fine. It's all about science. All right, let's do some pics of the week. Brianne, what do you have for us? I have something that I saw on social media recently and I found very interesting.

1:34:22It's the Merriam-Webster time traveler feature, which tells you when words were first used in print. And so you can put in whatever year you are interested in and find a list of the words that were first used in print that year. And when this made the rounds on social media, it was sort of put in your birth year and figure out which words were first used in your birth year. And I was very amused when I did that because some of the words that were first used in print during my birth year were antiviral or antiretroviral, lentivirus and interleukin, which all seemed a little useful. In fact, many of the years around when I was born seemed to be big years for virology and immunology.

1:35:12But I also found email, compact disc, food court, chill out and first world problem. All were from my birth year. I have used the word chill out a lot more frequently since learning this. And so I was very amused. And I know some of my friends have been looking up information here and have been amused as well. So I think you two know what word was first used in 1953, my birth year. I think I have an idea from Watson and Crick. Oh, DNA? No, no. HeLaCell. HeLaCell. Oh. HeLaCell. That's when they were first cultured and announced on national television. Yeah. That's cool. I've got assault weapon background radiation.

1:36:03Yeah. I think HeLaCell is perfect. I worked with HeLaCell for so many years. Yes. Ballpoint pen. Wow. 1953. Agarose, 1953. Cool. Who gets plaque assay? That's really, that's not me, but someone must get plaque assay. Maybe. Let's see. I think it was earlier, maybe, or later. Somewhere in the 50s, I believe. Maybe I'm wrong. Oh, immunosuppressant in mine. This is very cool, Brianna. Yes. I was just so amused that I have spent a lot of time thinking about lentiviruses and interleukin, and those were both from my birth year. Cool. Alan, what do you have for us? I have a book pick. We were talking before the show about how people aren't reading anymore, but hey, read.

1:36:53So this is a novel, The Incandescent, by Emily Tesh, and it is a lot of fun. It's a fantasy, dark academia story. it takes place in a magical boarding school no no not that magical boarding school this one it's so Dr. Walden is the director of magic at the Chetwood School which is a 600 year old boarding school in the UK and magic is one part of their curriculum because of course they exist in the modern world and you can't run a boarding school that only teaches magic the authorities would come down on you like a ton of bricks. You've got to match the national curriculum and so on. And most students there actually aren't there to take magic.

1:37:41They're there for this prestigious education. So when we first meet Dr. Walden, she's sitting at her desk late at night, filling out the risk assessment form for the invocation lab she's teaching the next morning. Because of course, if you're going to teach a group of teenagers how to summon a demon, there will be a risk assessment form. And the whole thing is just brilliantly done. This complete twisting of the magical boarding school tropes and really fun academic adventure slash romance story. Cool. That sounds wonderful. Definitely putting it on my list. Does this qualify as reading, Alan?

1:38:21Oh my gosh, this totally qualifies as reading, yes. So it will make your brain work, right? Yes. Okay. That's cool. And I like to read actual paper books. But you can get this in an e-book if you prefer. Yeah. When I travel, it's too heavy to carry books. Sure. Yeah. But if you get the e-book edition, you can read it on it. But Alan, I have so many books in my home. I don't know what to do with them. Well, donate them to your library. No, they don't want them. Really? Too many people want to donate books. Oh. Our local library does a book sale every year, used book sale. So they take donations of books.

1:39:02All right, I'll look into it. In this area, the American Association of University Women does a book sale to support scholarships for female scientists or for women in education. And if you can't find any place else, you can give them someplace like Goodwill. Yeah, they will take books? Oh, yeah. Huh, I didn't know that. All right, well, I can get rid of a lot of books. I have a lot of books. Which is a shame. When I left my office at Columbia, I threw all my books out. Because it was a Saturday. They told me I had to get out by Monday. And so I just threw them out. And then they didn't actually need my office for another six months.

1:39:45Because they lied to me. Which is becoming very commonplace in this world. My pick is in a Nature article, which is unfortunately paywalled, but it's pretty interesting. And I'll present to you what they have. This is called How Mosquitoes Are Conquering the World in Four Charts. Mosquito-borne diseases are breaking records as climate change and other human influences allow the insects to expand their range. By Mariana Lenjaro. And yes, climate change is happening. I don't care what you think. I don't care what alternative evidence you want me to present. It's all lies. The data are clear. Anyway, this is an article on just that.

1:40:28And the UK Health Security Agency warned people traveling to protect themselves against mosquito-borne diseases, not just for tropical countries anymore. Mosquitoes that carry various pathogens have spread to previously unaffected regions in Europe, Asia, the Americas, and Australia. And so they have a number of really cool graphs. So they have the first one called Malaria Remains a Deadly Disease After a Period of Slight Decline. Global malaria began to rise again in 2015, partly owing to extreme climate events and increased conflicts in endemic countries. They talk about specific diseases, chikungunya and dengue.

1:41:13Here's a graph. Dengue is a growing threat in the Americas. And you can see very little dengue in the 80s and now, well, 12 million cases in 2024, it looks like. So that's going up. And then mosquito invasions, new mosquitoes going to different places in the world. There's a graph of that. mosquitoes are spreading, and quickly, almost 50 mosquito species, around 25 % of those that transmit human pathogens, have been introduced to new regions of the world, many of which have become established. And you can see this graph reports on the y-axis the number of regions, the year on the x, and this is the number of regions mosquitoes are spreading to, either introduced and established.

1:42:02You can see it's going up. There's also another graph on the spreading rate, which is increasing as well. And the fourth graph, climate change is expanding with mosquito territories. And it's a map of the world showing months per year when temperature is conducive to disease transmission in 2019 and predicted in 2080. And you can see it is increasing because the red colors are 12 months a year where the temperature is conducive to disease transmission, and that's increasing. So it's a cool article. And by the way, even on the paywalled version, nature is actually pretty generous with the preview that they give you.

1:42:43You can get through this all the way down to the climate change map. Yeah, it's true. Just on the free site. Anyway, so this is a big problem, and it's going to get worse as the climate continues to change, and we will have more of this and diseases in places where they weren't before. They weren't before. And this is, you know, people don't believe in climate change. It's not. It is. It's happening. Come on. But you can deny anything. Just like RFK Jr. denied 40 things in his interview on CNN last Sunday. We have some listener picks. Vic Vici or Vici, Veni Vidi Vici, right? I will go for PBS Eons and Alan provided a link.

1:43:33Educational YouTube channel and digital media series about paleontology and natural history. Charles writes, 75F24C at almost 2 a.m. in very dark Chapel Hill, North Carolina. Rain is forecast later today. We still need the rain. What are you doing at 2 a.m.? You can't sleep? walking around the house. NPR's Weekend Edition Saturday ran an interview with Stephen Dobson on releasing Wolbachia carrying male mosquitoes to suppress Aedes albopictus. Half a million are going out in the D.C. area this summer. Releases in Singapore have knocked populations down by 90%. The males are fertile. It's cytoplasmic incompatibility, so the wild females they mate with lay eggs that never hatch.

1:44:13Species-specific, no known resistance, and it only touches an invasive that wasn't here before, 1985. In fact, I visited the place in Singapore where they make the mosquitoes. It was very cool. I spent many enjoyable years in biomedical engineering at University of Kentucky, so my ears perked up when he mentioned that this came out of his UK lab around 2005, injecting Wolbachia from one mosquito attic into another under 40x magnification and provides a link for that. In keeping with a recent trend, here's my next music pick from our generation. Credence, Clearwater Revival's Cosmos Factory. Great album, but it doesn't include my favorite CCR song, Fortunate Son.

1:44:55That one's on Willie and the Poor Boys. I always enjoy hearing it played at political rallies by people who seem not to have listened past the chorus. Thank you, Charles. And yeah, I like CCR very much. I bought their album in college, and those songs are just burnt into my head. I can play them and remember all the lyrics. Who, what is the name of that song? Something Rain. Oh, it's like. Well, Purple Rain would be my generation's answer to that. No, it's like, who can stop the rain? Who'll stop the rain. Who'll stop the rain. Who'll stop the rain. And also. I like Charles's signature line too.

1:45:41freedom from responsibility is not liberty it is childhood that's good alright that is TWIV 1347 you can find the show notes at microbe.tv slash TWIV you can send us questions and comments to the picks of the week to TWIV at microbe.tv and if you enjoy these programs we'd love your support microbe.tv slash contribute Alan Alandove is at alandove.com, also turbidplack.com. Thank you, Alan. Thank you. It's always a pleasure. Breanne Barker is at Drew University Bioprof Barker on Blue Sky. Thank you, Breanne. Thanks. I learned a lot. I was at Drew two weeks ago, right? Yes. And we did a TWIV in front of an interesting audience of the Governor's High School of Science.

1:46:33What is it called? The Governor's School of Sciences Scholars. So top 60 New Jersey rising high school seniors. So top 60 in the sciences. So Vida and I did a paper in front of them, and then they asked questions at the end. And I was just amazed by how good the questions were. So that'll be released over the coming weeks. I'm not sure when yet. 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 Jenkes for the music, and Jolene Ramsey for the timestamps. You've been listening to This Week in Virology.

1:47:13Thanks for joining us. We'll be back next week. Another TWIV is viral.