TWiV 1373: Challenging Dengue and RIGging Flu

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

0:10From Microbe TV, this is TWiB. This Week in Virology, episode 1373, recorded on November 6th, 2026. I'm Vincent Racaniello, and you're listening to the podcast all about viruses. Our numbers are starting to sound like actual years. Way back in 1373, your mom and I joining me today from Madison, New Jersey, Breanne Barker. Hi, great to be here. And great to be here for 1373, even if I wasn't here for the other 1373 year. It's pretty gloomy and rainy out in Madison, and it's 66. But that's all right. Yeah, rain is okay as long as you're inside. It's 19 here in Chelsea. It's just drizzling. It's going to drizzle until about 9 p.m.

1:09I woke up in the middle of the night and I heard it on the roof. And that's great because it soothes you and puts you back to sleep. Also joining us today from Ann Arbor, Michigan, Kathy Spindler. Hi, everybody. Here it's 45 degrees, but the feels-like temperature is 32. And I can vouch for that. It's been sunny and cloudy and sunny and cloudy, and I should have dressed a slight bit warmer when I walked in just now. And I'm trying to find out what that is in Celsius, but it's not coming up quickly. So, yeah. We had an email on clinical update yesterday from a guy who said, you know, I'm a long-time, who have a listener, my favorite temperature is 28C.

1:51Also joining us from Western Massachusetts, Alan Dove. Good to be here. And here in Western Mass, it's 63 Fahrenheit 17 C, also raining, supposed to rain through the night into tomorrow and often on Sunday. And I think that's great because it's been super, super dry lately. Also, when it rains, you don't have to go out and work outside and go out for any reason. You know, it's good. It's a good excuse to stay inside. Yeah, I actually had to mow the leaves on Thursday this week and blow them out of the gutters. They'll be back, right? Oh, they'll be. Yeah. We're actually, we'll be, I think we're about three quarters done with leaves now.

2:30Down here, it's not even a quarter. There's a lot on the ground, but there's more up there. The maples are done. The white oaks are mostly done. And now it's the red oaks. I have to agree that I think 82 is my favorite temperature. Not just because it's a palindrome with 28C, 82 Fahrenheit, but just because I like 82. It's not too warm, not too cold. That's a good temperature. It's a good temperature, yeah. And the temperature here in C is 7 Celsius. So feeling like that. Pretty chilly. Yeah. If you enjoy these programs, what are these programs about? Well, you should know because you're listening.

3:09You know this. But if you're new, we do science podcasts. This one's about viruses. And there's a whole bunch of other ones. And if you like science and like people explaining science in a way that you can understand, we'd love your support to make these podcasts. You can go to microbe.tv slash contribute. We have an ASV PSA, Kathy. Yeah, it's been a while, right? So this is for the American Society for Virology meeting in Madison, Wisconsin, July 19th to 22nd, 2027. And applications are open for this ASV meeting. if you want to be a global scholar. And these are available to applicants doing virology research in low - to middle-income countries.

3:54So to get more information, which you should do right away because the deadline is November 30th, go to the ASV website, asv.org, click on ASV 2027 in the upper right, and then there's a dropdown to the Global Scholar Awards. So it's a great opportunity to present your work, and interact with others doing cutting-edge virology research. A couple of news items. Of course, we don't have to remind you that we just had an election and the effect on science and public health should be more positive. But we have an article from CIDRAP, Democratic Doctors Win Big in 2026 Midterm Elections by Stephanie Suchere.

4:42Among the winners who helped flip the House and Senate, during the midterms were six doctors and public health professionals. These were supported by a 3.14, the Pi Action Fund, which is dedicated to bolstering Democratic candidates with STEM backgrounds. Cool. So we have Amy Acton, MPH, the new governor of Ohio, Abdul El-Sayed, of course, M.D.D. Phil, the newly elected senator from Michigan, and they also have public health backgrounds. pediatrician Ann Andrews, MD, also defeated Darlene Graham in South Carolina to flip a Senate seat. And so now with some medicine public health strength, there are going to be calls for swift action against RFK Jr.

5:36So we'll see what happens there. But it's really nice that we now, because the last time, what was this quote? The last time an MD was I don't remember how but the last time an MD was something in the Senate, that's when Medicare and Medicaid was formed. So it has something to do with an MD being elected. Well, on the other hand, there have been MDs in Congress and they're kind of a mixed bag. Yeah, I understand. You know, because it's... They're that one from Kentucky. Yeah, yeah. They're... Okay, yeah, I'm married to an MD and big fan of doctors in general, but there are some MDs who are not necessarily on the side of public health.

6:23But these folks, great. The guy in Louisiana wasn't so great. I agree. He's a doc also. Also, we have Richard Pan in California. He's a pediatrician. Mitchell Berman is a registered nurse in Wisconsin. Christina Knickerbocker is an oncology nurse practitioner in Ohio. and Dr. Adam Hanway is a surgeon, New Jersey 12th District. So there's also a woman, Jasmine Clark, who won a House of Representatives seat in Georgia, who has a PhD in microbiology. Nice. That's cool. I've got to say, though, it's good they're putting a lot of MDs and, I guess, a nurse into Congress, given how old a lot of the others are.

7:04Yes. Well, someone wrote in a letter here and said, When are you going to elect a president who's not 80? 2028, I hope. One hopes. Yeah. Right. Yeah. And another paper, another article, a news article from CIDRAP also. This is relevant because we're doing a dengue paper today. We now have 485 cases of dengue in Florida. These are locally acquired dengue. So the virus is there in Florida. It's, as we say, autochthonously acquired. People are infected. Mosquitoes are spreading it among people. That's high. Last year, there were 62 cases. And to be clear, this is not, as we'll talk about today in the paper, this is not a problem of people not getting the vaccine because there isn't really a good one for dengue.

7:57Right. Well, and I think when I started teaching at Drew, there was zero autochthonous dengue in Florida. That could be, yes. This is more likely a climate issue. You said 485 is the total in Florida now, and what was it this time last year? 62, I think. Ah, okay. Wow. Yeah, I mean, there have been increasing numbers. As Brianne said, at one point there weren't any. There were 80s Egypti for sure, but then you need to have a certain burden of infected people to maintain it, and then so that's been achieved. And I suspect it will move farther and further north as the mosquito range moves north as well.

8:41So, you know, what are you going to do? Okay, let's move on to the literature. And our first paper is a dengue paper. In Science Translational Medicine, Dengue Virus Controlled Human Challenge Reveals That Pre-existing Immunity Shapes Viral Replication and Broadly Protective Responses. And I think this may be a record. There are 10 co-first authors. Oh, wow. Because apparently all the children are above average. No, two are first authors and the next eight are co-second authors. Are they? That's the way I interpreted it. Yeah. Oh, yes. It's a one dagger and it's a two dagger. Oh, I see. Still eight co-second authors.

9:26Eight co-second authors. Okay, then I'm not going to read the eight co-second authors. The co-first authors are Camila Odio and Rosemary Ogo, and the senior author is Lea Katselnik. And everybody else is a second author. Actually, no, there are people who didn't even make the 10. So among the authors are Daniela Weiskopf from Scripps, who you may remember was on TWIV. She works on T-cells, and you'll see why she's on this paper. So I remember that, talking about COVID T-cell responses. All right, Kathy, do you have a summary for us? Yes. So dengue causes at least 50 million cases worldwide every year.

10:06And there's really no good therapeutic and no widely available efficacious vaccine. I added the word efficacious because I don't think all they said was not widely available. And I don't think that's enough of an explanation. And this is partly due to the immune complications that are caused by a repeat infection, which I'll talk about in a minute, and also partly because there are not good animal models. So we've talked about this a lot in the past. There are four serotypes of dengue, numbered 1, 2, 3, and 4. If you've had an infection by one of them, and then you get infected by a virus of another serotype, you can get a much more serious disease than if you had not had dengue previously.

10:50This is kind of counterintuitive because you might think there'd be cross-reactivity and antibodies from the first infection would protect you. But in fact, in sort of a diabolical twist, the antibodies produced in the first infection are kind of weak, and so they don't neutralize the virus, but they bind just enough to cause problems and get into immune cells, kind of like a Trojan horse, where they replicate high levels and make for a much worse disease. And so these authors have performed a phase one clinical trial. And in this, they're using sort of a challenge model in that they're going to be giving a highly attenuated type 3 dengue virus, which is a vaccine candidate because it's highly attenuated, but it's live.

11:46And so they do that, and they give it to 45 individuals that they group into three groups. But all of the individuals are going to get this highly attenuated dengue 3 virus. So the three groups are based on serology. They measured their serum before they start the infection and determine whether or not they have antibodies to dengue 1, 2, 3, or 4, I guess. They test for all four of them. So the groups that they set up then are they never had a dengue infection, so they have no antibodies. They had a single infection, so they have antibodies to 1, 2, or 4. Or they had multiple previous infections, so more than one of those, 1, 2, and I think maybe even 3 is included in that poly group.

12:41And so as they say in the materials and methods, they were going for two outcomes. One is basically the safety of this vaccine, which is effectively a challenge when they're giving it to someone who has already had a prior dengue virus infection, because you want to avoid this immune response. Oh, which I forgot to say is called antibody-dependent enhancement. The antibodies enhance the infection. And so that's also sometimes called ADE for short. And ADE is sort of a unique thing in dengue. Right. It's not like people should decide that this means all of immune responses are bad. Right. No, this is a weird one.

13:25Right. So, yeah, so they're going to test the safety because they don't want to be inducing ADE in somebody just by giving them this attenuated dengue vaccine three strain. And then the other major outcome they're looking for is how does the prior dengue virus immunity influence protection against the vaccine strain? And they measure virus titers and peak viremia, and they measure a number of other things. And so in the results, which I'll just state in brief, they confirmed multiple hypotheses that have been based on the natural history of dengue virus infections. but here with some real data with controlled situation of infecting patients and so forth with this vaccine strain.

14:19And then they revealed the optimal conditions for inducing potent and lasting immunity against dengue 1 to 4. They found that rapid activation of functions that are required to control an antibody-dependent enhanced viral infection, whatever you do to deal with that can come at the expense of generating a high-quality, lasting adaptive immunity. And they also found that if they gave early, through this, that giving early therapeutic immunomodulation may limit viral replication and thus also improve clinical outcomes and long-term immune responses. So there's a lot more to it. And I'm sure we'll get into more of those details.

15:09Yeah. And I just realized I didn't give the author affiliations. So just for credit, the team is from the NIH in Bethesda, Maryland, University of Cambridge, UK, La Jolla Institute for Immunology in La Jolla, Frederick National Laboratory for Cancer Research in Frederick, Maryland, University of North Carolina, Chapel Hill, the NIH campus in Phnom Penh, Cambodia, and let's see, Johns Hopkins Bloomberg School of Public Health in Baltimore. So as you have gathered from this discussion, the risk of severe dengue, so they're broadly speaking, there's a regular dengue where you get sick in various ways, but there's not the hemorrhagic life-threatening disease, which is associated with the severe dengue.

16:00And that's greatest risk during a second infection with a different dengue serotype because the different serotype induces a memory antibody response to the first serotype. Those antibodies don't neutralize the other serotype well, but they bind to it and they They allow for FC receptor-mediated phagocytosis and more viremia and fluid leak, shock, hemorrhage, organ failure, and death. Yeah. So my understanding is that this is basically because the virus normally isn't able to infect those myeloid cells. The virus doesn't have terpism for those cells. But because the antibodies sort of let the virus in, you get a different set of cells infected in secondary dengue or in enhanced antibody enhancement.

16:48And so it's sort of the infection of another new cell type that's really causing the problem. And this has, in fact, destroyed the prospects of multiple efforts to develop a dengue vaccine, including some rather high-profile failures where people discovered a little late that, oh, the vaccine is making it worse than people who've had the disease. So this severe dengue has a number of characteristics that will become relevant. dysregulated cytokine responses, reduced antigen presentation, CD8 T cells with weak killing activity, et cetera. But also, if you survive this secondary dengue, you get very broadly protective antibodies that target epitopes that are special, that they span the virion.

17:44And we're going to see they basically span glycoproteins. And very good effector T cells that are very cytotoxic. So in terms of challenge, there have been, well, first of all, there's a lot of data on dengue from natural infection, animal and vaccine studies, and they have identified correlates of protection, but they have limitations, and no single study has looked at the features of immunity that control versus exacerbate infection. So, you know, there have been some correlates made, but they're not, as they say here, they're not perfect. The longevity is unknown, and maybe you need multiple exposures.

18:28If you get the first infection, you get strongly neutralizing antibodies, but they're serotype-specific, right? And that's why when you get a second infection, those don't help you. But multiple exposures can induce these special, broadly cross-reactive antibodies that I just briefly mentioned, And they target the envelope dimer epitope of multiple serotypes. So the envelope protein is present on the surface as a dimer, and there's an epitope that spans dimer. It's called the envelope dimer epitope of ETE. And antibodies against that are broadly protective against multiple serotypes. But unfortunately, you have to survive a second infection to get those.

19:09It would be nice to know how to get them, though. So in this paper, the work was done between January—I'm sorry, cancel that and let's start again. Between January 2000 and 2025, 11 dengue challenge studies have been done. It's a lot. And they've identified features of protective and enhancing immunity. But the authors say they're pretty small. The sample size was small. They were not really designed to compare people who have no exposures or one or more than one exposure. Okay. So how do we feel about them calling all of these things challenge studies? When I first saw this paper, I saw dengue challenge study, and I was shocked, and I clicked right away.

19:56And here they're talking about 11 other dengue challenge studies. And all of these are not using wild-type dengue. They're all using some kind of attenuated form. And so that is definitely not what I expected when seeing the words challenge study. It makes me a lot less worried, makes it a lot safer. But I guess I hadn't really thought about calling attenuated virus exposure a challenge study. Yeah, I felt the same way. I mean, reading the title of this paper, I was like, challenge study with dengue what the heck um and then oh with this strain that's a vaccine you challenged with a vaccine no that's a vaccine trial i'm sorry i really is but i i see i see where they're going with it because they're looking it's the challenge it's a challenge and especially in the people who've had an exposure to dengue before.

20:58And given what I mentioned about the history of dengue vaccines, I mean, you could think of that as a challenge because maybe things are going to go pear-shaped. So yeah, I see the wiggle room there. I don't... It felt clickbaity. But it's also, it's all you can do with dengue. You can't give them wild-tech virus, right? I don't know about these other 11 challenge studies. I didn't look into them, but yeah. At least in this one, they mitigate it in the title or try to by using the word controlled. Yes. Controlled human chance. All right. So in this study, they have more people, as you'll see, they're going to enroll people with specific and different dengue immunity.

21:41And then they say, we're going to expose them to a full-length live attenuated vaccine and compare acute and convalescent responses. Okay. Now, I don't know why they say full-length vaccine because it's an infectious attenuated vaccine. It's not even full-length. It's missing 30 bases in the non-coding region. So I don't know why they keep saying full-length. Like I say, oh, we made a full-length cDNA clone of this. Yeah, okay. But why? Does anyone care about that? Were any of the other CHALN11 studies that they mentioned with much more truncated portions of virus? I don't know. I mean, I don't think you can take much more out and get an infectious virus.

22:27I mean, this one, the one they use here has a 30-base deletion in the 3-prime end. That attenuates it. It's very cool, but I don't know that you could take everything out. Anyway, just a little pet peeve of mine. Don't worry about it. Okay, the results. They have a challenge study with this monovailant dengue 3 vaccine candidate in 45 healthy adults. And these adults have had no previous infection. They have had one heterotypic, which means a different serotype, right? Or they've had multiple prior dengue infections. And if you've ever had a dengue vaccine or a challenge strain, as they call it, you couldn't be part of this study.

23:07You had to get naturally infected. And if you were exposed to one strain previously, it couldn't have been dengue 3. That's right. So this is a R-DEN3-Delta3031-7164, all right, that's got a 30-base deletion in the 3-prime non-coding region, has all the viral proteins, and it's got attenuating mutations in non-coding regions, okay? So they measure everybody's immunity on day zero with antibodies and get their medical history. And then they all get a subcutaneous injection with this dengue 3 and the deltoid region of 10 to the 3 plus or minus 10 to the 0.5 PFU, plaque forming units. Okay, that's up here, deltoid region.

23:51But subcutaneous, not intramuscular. Interesting. Is that to sort of mimic what a mosquito does? I guess, yes. I guess. They also, they mentioned some stuff about the release rate of a vaccine affecting response later in the paper, I think. And I was wondering if it was something about they want the virus to get in a little more slowly than it would if you injected IM like we normally do with a flu shot. I don't know. I didn't pay attention to it. I was just, I don't know. Yeah.

24:28So three naive individuals that didn't have any previous, they got a slightly lower dose. Okay. And then they collect samples at many days, 0, 1, 3, 6, 9, 12, 15, 28, 57, 90, 180, and 365 days a year after immunization. And this is all done at NIH in Maryland. Okay. Okay. So everybody was okay, pretty much. There was one person who got a staph infection in the foot, which had nothing to do with this virus, which shows you that stuff happens, right? And shows you that they're also reporting pretty widely things that happened. Yes. But, you know, the naysayers would say, ah, that vaccine caused that staph aureus infection.

25:16No, it didn't. And it wasn't that that infection was a long time after the challenge. Yeah, it's 328 days. 328 days, yeah. In the foot. In the foot, yes. And it was staph aureus. Okay. So as they say, as we had hypothesized, the mean peak viral RNAemia, so they measure RNA in the blood by PCR. So they're going to call it viremia, but they say, okay, it's RNAemia, but we're going to call it viremia. I don't know why. Why don't you call it RNAemia? I thought that line was maybe for TWIV. Yeah, maybe. So we wouldn't get all worked up about, they're not measuring virus. It's not viremia. They want to be able to use the term viremia.

25:59The RNAemia of the dengue 3 strain, that's the one they gave people, was higher in heterotypic than in naive individuals. Okay? Think about that. It's higher in heterotypic. These are people that have either dengue 1 or 2 or 4 antibodies, right? rather than people who have never seen dengue. You would think it's higher in the people who never saw dengue, but no, that's because of this antibody-dependent enhancement. Yeah, so it looks like they're seeing this dengue-3 virus is replicating better in people, presumably because of the antibody-dependent enhancement. Right, so that's right away. Put another way, it tells us their model is mimicking the real situation, as far as we can tell.

26:42Both of the naive and heterotypic groups have increases in the titers of neutralizing antibodies. to dengue 1 and 4 at days 28 and 57, right? That's so you're getting, remember, they put in dengue 3, so you're getting antibodies to 1 and 4. The heterotypic group had the largest increases of antibodies. All right, so basically this shows you that heterotypic immunity is associated with increased immunity and immunogenicity, right? Higher titers of antibodies in the heterotypic group. And maybe it's because they have more virus, but maybe it's more than that, as we will see. And what's also notable is that the polytypic group, so the group that has had multiple infections, does not have as much of an antibody response.

27:34So their antibody response is a bit lower. And so, again, this might be sort of this question of, well, maybe they don't have as much enhancement. Maybe they have some of those good antibodies that were mentioned earlier, the EDE envelope dimer epitope antibody. So maybe, again, this is starting to be a sign of some of that stuff happening. All right. So next they look at clinical outcomes and see if they're varying across these different groups. So the naive individuals had more dengue-like rash. That's part of the pathogenesis. which is known before. So if you never had dengue, you get a rash more often.

28:19But when they remove the low-dose individuals, that effect goes away. Remember, three people got low-dose because they were naive.

28:30The low polytypic group also. So they divided these people into low. I didn't tell you this because too much information. But they divided into naive and heterotypic and polytypic and different levels. Okay. So the low polytypic group had more reports of feeling feverish. Okay. And both the naive and heterotypic individuals had mild but significant decreases in neutrophil and lymphocyte counts. It's worth noting that the low polytypic group is three people. Yeah, there's small numbers. And it's low antibody titer. Yes. Is what the low means. That's right. That's right. So they want to know if these clinical changes are associated with viremia, right?

29:23So they compare that with viremia. And they find that increasing viremia is associated with established markers of infection, including symptoms, symptoms, reduction in neutrophil and platelet counts, increasing plasma blast responses. So they conclude the virus strain, as Alan said before, mimics natural dengue infection and prior immunity influences clinical presentation. Okay. Then there's a section called distinct pre-existing immune signatures shaped viral load kinetics and host responses. All right, so three existing, so what you have before you come into this trial. So they want to know the effect of baseline antibody and cellular profiles on infection dynamics and immunogenicity, so the immune response you get after getting the vaccine.

30:18So they have functional assays, right? They have plaque neutralization assays and T-cell assays. They're doing intracellular cytokine staining, okay? then they use statistics to say, do these go with each other? The strongest predictor, linear predictor of viremia, was fold enhancement of dengue 3 infection by sera at a 1 to 10 dilution. So fold enhancement means, the way they assay that is they use cells that express FC receptors, and all three FC receptors. And so they're getting it. The virus doesn't get into these cells unless antibody-dependent enhancement is happening. Right. So then they can make serum dilutions, and they find that a 1 to 10 dilution, the enhancement of infection is a predictor of viremia.

31:14Yeah, so you're most likely to have a higher level of viremia if you have more antibodies that can do this enhancement at a 1 to 10 dilution. Right. Right, right. And that's... I was just going to say, reading through it, for me, I had to, a couple of times when I read enhancement, I thought, oh, that's making it better. But no, it's making the virus, from the virus's standpoint, it's enhancing the infection. Yeah. And so an important aspect of that is that means that this system that they've got in it, a challenge, if you will, in humans, is giving you results that are consistent with a cell culture system that's very much easier to work with than corralling a bunch of human volunteers.

31:54So you can now potentially assay for immune responses in other vaccines you're testing and see if you get this enhancement with this simple cell culture system. Okay, continuing with this line, neutralizing antibody titers to dengue 3 were associated with protection against viremia. Okay. And participants with heterotypic or low polytypic immunity had higher enhancing antibody levels and low mature neutralizing antibody levels and showed increased viral load. Yeah. And so here we've got antibodies doing two different things. We've got some enhancing antibodies that are bad for the patient, and we've got some neutralizing antibodies that are good for the patient.

32:44And they both have a statistical association with that outcome of being bad or good.

32:56All right. Some other things that they try and associate with viremia. One of them is interferon gamma-secreting CDA-positive T cells. They were found in only four participants, including one naive participant, and they're associated with reduced viremia, right? That's something that's been known before, right? These are going to kill virus-infected cells and so reduce viral load. The participant with the highest viremia was in the low polytypic group and had no dengue T cells or mature neutralizing antibodies. Well, they had evidence of exposure to other flaviviruses, they say. Okay. Now, neutralizing antibody breath and non-structural protein 1 antibody breath, having neutralizing and non-structural protein 1 antibody breath to more dengue serotypes and having the highest neutralizing antibody to mature dengue 3 were protective.

34:05So also, NS1 protein, it's the secreted protein. Having antibodies to that is also good for neutralization. Okay, then they looked at the expansion of neutralizing antibodies that target partially mature and mature dengue virus 1 and 4 variants and dengue-specific T cells that are induced by challenge. So now we want to know not what's at the baseline, but what is induced by challenge. So when dengue viruses are produced, they're a mixture of immature and mature particles. And you can measure both of them in their epitopes on both of them. And here they're specifically looking at antibodies against other serotypes, not the one that was used for challenge.

34:49Dengue 1, 2, and 4. And I want to skip most of this and just get to the summary. You know, it's hard to read this paper because they just tell you the results sentence after sentence. And it's hard, I found. But I'm happy they have sometimes summaries. What they find is that baseline immunity influences both viral replication and the resulting adaptive response. Okay? So whatever you start with influences what the virus is doing in terms of viremia and also the new antibodies that are going to be produced, and T cells. Which makes sense because viremia should be driving some of that adaptive response.

35:32Yeah. Right. Okay, so the next question is they want to know whether viremia contributes to better post-vaccination immune responses, which Brianne just said, right? So they stratify the participants based on the viremia, right? And they identify groups with almost no viremia, that's 8, with viremia of similar magnitude to the naive group, that's 12, and with greater viremia than other groups, that's 11. So three groups based on no or medium or high, kind of like Goldilocks, right? All right, most of the participants with high and intermediate viremia had heterotypic immunity. Multiple strains could be neutralized, whereas most people with low viremia had polytypic immunity.

36:34So polytypic immunity seems to be reducing viral load, perhaps? Maybe, yeah. Heterotypic is just one, right? Yeah. One other. Okay. So polytypic is, I don't know how many is polytypic. I mean, I think it's more than one. More than one, yeah. But again, that's a situation where you could see those EDE epitopes. And the way I read it, at least initially, I thought that the more than one could include a dengue three response, but I might be wrong about that. I think so too. Okay. Okay. Now, also here, there's a lot of data that I want to just summarize. I'll tell you why. Here, for example, let me read this to you, and you'll see what I want to summarize.

37:21By days 28 and 90, those with intermediate viremia had the greatest expansion of dengue-specific CD8 cells, especially T-effector memory re-expressing, Tamra CD45, which has previously been associated with protective post-secondary immunity. I mean, it would take me an hour to decode that. And so I'm going to give you their summary. Among individuals with pre-existing dengue immunity, those with the highest viremia develop weaker long-term antibody and T-cell responses, whereas intermediate viremia induce the most potent and enduring antibody and T-cell responses. So once again, like Goldilocks.

37:58Mm-hmm. Yeah. You would think that the highest viremia would do better though, right? I mean, maybe, but the highest viremia patients are probably the ones who are seeing enhancement. And so they're getting that virus coming from monocytes and dendritic cells. And those monocytes and dendritic cells may be doing a poor job of turning on other adaptive responses. Well, also enhancement is associated with dysregulated immune responses. Yeah, that's exactly what I was thinking. Yeah. Okay. You cut out. I'm sorry if I said what you just said. Oh, no worries. Of every episode that I record now, at least one person cuts out for 20 seconds.

38:43I don't know why. It's ever since we switched to Fios, which is faster than what we used to have. We went from 200 megabits to a gig. And so I have to imagine what they're saying. It's all recorded and it all comes out fine. I just don't hear it. And in this case, you said exactly what I said. We just said it in different words. It's all good. But it's fine because people do not – Peter Palazzo always said, Vincent, it's okay to repeat yourself, especially to medical students. They don't listen. Okay. Next section, preexisting humoral immunity memory predicts predicted viral load. So they say the people with intermediate viremia, Maybe they had better existing immunity, both humoral and cellular, than the people who had high viremia.

39:35And they're able to refine it during the secondary response. Okay, so they used multinomial elastic net regression. I wrote that down because I thought it was pretty funny. To identify, it's a statistical procedure. That's good enough to identify which of the 80 distinct immune measures that they measured best discriminated between viremia responses. They found that low viremia is predicted by measures of potent neutralizing antibodies and targeting conserved quaternary structure epitopes, including this EDE epitope that we talked about before. So low viremia makes those antibodies. The people with intermediate and high viremia had some enhancing antibodies, which Brianne predicted, right, and lower levels of mature neutralizing antibodies.

40:33Intermediate viremia is associated with binding antibodies to NS1 of multiple dengue serotypes and living in a dengue endemic area more recently. So multiple dengue infections. And high viremia was best predicted by enhancing antibodies measured, the 1 to 10 dilution, as I told you before. All right. I think as a summary, throughout the paragraph, they kind of reverse the order of how they say it. But the bold heading is pre-existing humoral immunity memory predicted viral load. And so I think that was the clearest way to repeat it. It's just that the thing that was the predictor was being in intermediate immunity.

41:15Right. Yes. Right. So the next heading is, there were marked differences in acute immune responses across the viremia group. Right? So as I said before, other studies have shown that antibody-dependent enhancement inhibits intracellular antiviral responses and produces a barren immune signaling, enabling the virus to replicate more without triggering a normal, innate, and adaptive response. That's what I tried to say before, but in much nicer words. Got it. That's what I repeated. And so the high viremia group had elevated viremia at days 6 and 9 compared with the naive and low, and that's consistent with antibody-dependent enhancement.

42:00And so they want to evaluate the systemic response in those people. So they measure clinical outcomes, biomarkers, serum cytokines, chemokines, growth factors, et cetera, all right? And they go through all of them here. I'm not going to read them to you, but I will read to you the summary. Enhancing antibodies are the more important predictor of the inflammatory cascade that drives these biomarker responses that they measured here. So the enhancing antibodies are driving this aberrant immune response. And once again, this is showing that their models are lining up with the measures that they should line up with, I think.

42:46Okay, now the next section, distinct acute responses were observed across viremia groups, which modulated long-term cellular and antibody profiles. Okay, so now we're saying that what's happening in the acute phase here is going to influence the antibodies and cells that are produced. All right. So this is to understand the differences in plasma blast expansion, right? That's the expansion of the plasma cells to make antibodies. So they look at interferon gamma and IL-12 signaling, right, which have a role in this plasma mat, plasma blast differentiation. Okay. Interferon gamma and IL-12. So they measure IL-12 slash IL-23.

43:30I guess they go together in this process? They share a protein chain. So sometimes when you do the measurement, you actually can't tell if you're measuring both, like one or the other or both. Okay. So the concentration of that on day nine was associated with the plasma blast expansion on day 15. It's partially mediated by viremia, suggesting that this IL-12-driven B-cell activation and differentiation is amplified by viremia, but it doesn't depend on it. Now, this IL-12 interferon gamma axis has been shown in salmonella infections to promote short-lived plasmoblast differentiation. and it shifts the immune response away from high affinity long-lived plasma cells.

44:23Very interesting, right? This they observed in the high viremia groups. They have a greater expansion of plasmoblasts and a more rapid decay of neutralizing antibodies because they're not making long-lived antibodies anymore because of this IL-12-driven differentiation. And I can imagine that that IL-12-driven differentiation is related to the other kinds of cytokine changes that they saw in the previous part.

44:55All right. So there's an earlier increase in serum interferon gamma in this intermediate day six compared with the high viremia group. the intravenient viremia group with the high viremia group. And they said this suggests a more rapid activation of the T cell response, right? So they tested whether there was a difference in interferon-producing T cells between the groups. They find that levels of interferon gamma positive CD4 and CD8 cells are similar across all these groups at day 15. but only the high viremia group had a rapid decline in interferon gamma-activated T cells by days 28 and 90.

45:40And so they say maybe there was a delayed type 1 interferon response, and that impaired the survival of these activated T cells. The intermediate viremia group had their peak interferon gamma cells at day 28. So they said more gradual but enduring response, which may have contributed to them developing robust, long-lived immunity. So they say that both interferon gamma and IL-12 are having distinct roles in shaping the immune response to dengue infection, depending on viremia levels. Did I get that right, Brianne? Yeah, I think so. So those are the data. Let's summarize them. So secondary dengue can be modeled in a challenge study, right?

46:27Okay.

46:31Only sequential exposure gives you potently, broadly active antibodies like the EDE antibodies that have been associated with protection. So heterotypic exposure is important. So if you make a vaccine, you have to have all the serotypes, right? You have to make a response to all of them. People with limited prior exposure, the heterotypic or low polytypic, had the highest neutralizing antibody titers and percentages of dengue-specific CD8 cells after challenge. But among that group, those who better controlled replication had the most persistent neutralizing antibodies because of the IL-12 effect that we talked about before.

47:11So one of the ideas here is that controlling viral replication is associated with early innate and adaptive activation, a balanced pro and anti-inflammatory cytokine response, good Th1 cell profile, et cetera, and moderate IL-12 production. So you don't make these plasma blasts that make crappy antibodies, right? Enhanced replication gives you more IL-12 production and delayed interferon gamma. So you get these low affinity plasma blasts. So this is all important for understanding how a vaccine should work, right? You should make sure a vaccine does the right thing. Right. Well, going back to Alan's point about Goldilocks, it sort of means that, you know, as you're developing that vaccine, you shouldn't necessarily optimize replication level or antibody level or something like that, because in fact, you need to have this balance between a few different types of responses to get the best long-term memory response.

48:12And this paper also validates a set of tools for assaying your vaccine candidates in easier ways than doing the challenge studies, which you would presumably then do to test safety of the vaccine. But this is outlining the correlates of protection that you're looking for. They also point out that any amount of antibody-dependent enhancement, even mild, hampers the antiviral responses. And that's associated with delayed and sustained inflammation that can get worse and worse. And so they're saying maybe we could use immunomodulators in these cases of disease, maybe together with antivirals, right?

49:01soon after you see that someone is getting this enhanced disease. And they say such drugs will soon be evaluated for preventing progression to severe dengue in phase 3 trials. But it's important that they also have the data from this paper when they do that because immunomodulatory drugs can mean a lot of different things. And this describes some of the immune responses that perhaps should not be modulated or that are very important that you keep to get a good long-term memory response, even if you're modulating other responses. Right. And finally, they see minimal replication in people with these potent EDE antibodies, right?

49:42They say vaccines need to induce those. Those are really important. If you have enhancing antibodies and you don't have neutralizing antibodies, you could have antibodies to NS1 to multiple serratides if you've lived in a dengue endemic area, and that gives you partial control. So that's good in a way. So they say dengue and other vaccines that induce humoral and possibly cellular immunity to non-structural proteins like NS1 may not reduce viremia, but may prevent severe disease and make you do a better adaptive response, right, which is inhibited during severe disease. So they say this work confirms multiple hypotheses, reveals optimal conditions for inducing immunity, counter to existing hypotheses, we found that the rapid activation of the adaptive effective functions come at the expense of developing high-quality, enduring adaptive immunity.

50:42Very interesting. Yeah, I feel like they're playing around with the word adaptive in interesting ways here because sometimes the antibodies are adaptive and sometimes they're not. I think my students would be very confused by that.

50:59That's it. That's interesting. Yeah. Full length. A full length challenge virus. Full length, yes. Do you think it's a full length challenge virus because one of the other vaccine candidates was partially the yellow fever? Yeah, probably. Yes, that's exactly right. So the Dengvaxia, the first one was just the structural proteins dropped into the yellow fever backbone. Yes. So it wasn't full length Dengvaxia. Perfect. I removed my complaints, but they should have explained that, right? Well, who's going to know that except the 10 people that work on Dengue? Right? All of whom are second authors on this paper.

51:36It depends who you're writing for. I think when you write a paper, you need to do more broadly than your audience, right? Because you never know who's going to read it. Maybe Billy Holmes is going to read it. I don't know. Is that his name, Billy Holmes? No. Oh, the guy in the movie? Theranos. Oh. No, Elizabeth Holmes was her name. Elizabeth Holmes. He's Billy something. Because he listens to everything at 2X. Right. Let's see. It's in that red. Performatively. Billy Evans. I still don't know how you listen to things at 2X and don't have them sound like chipmunks. Oh, you've never – I listen to everything at 1.5X.

52:162X is another ballgame. Okay. I can send you this link that Vincent sent that plays Vincent at 2X and these other two people at 2X. Okay. supposedly Billy Evans listened to it. There's a Times article about this. The Times writer found all the people who made the lectures he's listening to, and they wrote me and asked me questions. And one of them was, do you think, first of all, is this hard material? I said, no, it's not hard. You just have to listen and think about it, which people don't want to do anymore. They actually published that answer. And then he said, can you understand this material at 2X?

52:56I said, no, absolutely not. I said, give me Billy after he listens to it at 2X, and I'll demonstrate how little comprehension. Give him a test. Yeah. I'll give him a test. A very simple test. You're not going to get it because it's not all about fast, okay? You have to listen to something. As you guys know, I'm not telling you anything you don't know. You listen to it, and you stop, and you think about it. And if you don't get it, you go back and do it again. Every time you don't get something, you stop. You don't just plow through it at 2X. You're not going to understand. Anyway, we have another paper.

53:25This is now jumping to a negative strand virus. I think we may have just lost a listener there. Sorry, Billy. Who's that? Billy. I don't care. He may not continue listening. He listens to Vincent's lectures. And this was back in the past. Oh, Vincent's lectures, yes. A 2011 lecture. Okay. Yeah, he listened to an old lecture in 2023. Well, I think, not having seen the movie, but knowing the timeline of all this, I think they were trying to simulate or when he would actually have been listening to this. No, it was just, they went into their house and they lived their normal lives and they just filmed them.

54:01There was no script or anything. But I thought... It's a documentary. Well, but I thought, I mean, from the 60 Minutes thing, it says that, you know, she started having her ideas in 2012 or 2013. So depending on when he got involved with learning more of this stuff. Look, all I know is that the thing was recorded in 2023. And I thought it was the month before she went to jail and they were just living their life and they let some people in to record their life. But I don't know. You might be right. Yeah, maybe there's some flashback or something. I'm pretty sure all of it is not documentary. Yeah.

54:40Yes. Oh, yeah. Yeah. Well, I saw a quote by the director who said, I have no idea what's going on in this house. Yeah. Because they're playing games, I think. Yeah. Anyway, it doesn't really matter. I'm glad he's listening to virology lectures, but he really should listen to a current one, right? Not a 2011. If he's listening in 2023. If not, I apologize, Billy. Keep it up. Okay, cell reports. Influenza virus antagonizes. No. No, but I understand why you use the word. Self-sensing by rig eye to enhance viral replication. The other night on my live stream, you'll like this. I was talking about photons.

55:22And my pick today is photons. I'm so excited about your pick. This is so cool. So to a photon, a photon thinks it's at the Big Bang and here today, right? And someone said, Vincent, the photon would be upset if he heard you saying that a photon is thinking. I said, you're right. Thank you for listening. So the other part of the title is the last part where it says self-sensing by rig eye to enhance viral replication. That, again, gives agency to something that doesn't have agency. And so it should be influenza virus. I don't know how to fix that part. Antagonizes self-sensing by rig eye, enhancing viral replication.

56:04I like that.

56:34program that can predict statistically what the next word is in a sentence. And maybe there are other ways of cognition besides the one that we as self-centered humans think is the only way to think. Well, I don't think it's a matter of cognition, though, because you just said the virus figured it out. But it was evolution that selected for this, right? But the virus was the participant in evolution that collectively— But it's not the agent. It's, you know, I will be as pedantic and semantic as Vincent on this because every time they use to as in the infinitive phrase, when it's really, they should change it to where they signal via rig eye, suppressing viral replication, not where they signal via rig eye to suppress viral replication.

57:28Okay. There it's trying to say that that's exactly why the virus is doing it. And no, it's the result of the virus doing it. The virus collectively sensed a problem and solved it. No. No, the viral titers were at a certain level. Something happened. A mutation arose. Some viruses grew better. And those were selected for it. You know how that's how it works. Yes, I know that's how it works. But I'm just saying that could be interpreted as a form of intelligence. Well, and I have to say that I was just listening to an episode where someone is complaining about the use of the word as instead of because.

58:11and the host of the show said using that in that way is really declining and maybe sort of a kind of holdover from British or maybe there's still some British English that does that. And I'm going to write to them and say, no, read half a dozen science papers and you'll see that everybody's using as when they mean because or since.

59:09Yeah. and thus became the dominant population. And that's just a lot of words when you're lecturing. Yes. You could find other ways to do it. But you understand it, but your students do not. So if you say antagonize to them, they're going to get used to you anthropomorphize. I do agree that a disclaimer is in order, but I also feel that we don't have to be quite so adamant about not anthropomorphizing. Look, I'm just going to complain about it whenever I see it. That's fine. By the way, I was discussing something with Claude at one point, and it anthropomorphized, and I said, you can't do that.

59:39And he said, you're right. You're right. You're absolutely right. Of course, because it always concedes. Yeah. It always concedes. All right. Anyway, Alan, the author. Yeah. So the first two authors are Mitchell Ledwith and Thomas Nipper. The senior author is Andrew Mealy. They're from the University of Wisconsin-Madison, the Institute Pasteur in Paris, and Dalhousie University in Halifax. Andrew Mealy was on TWIV. Mm-hmm. Yeah. He tells you that he pronounces it to rhyme with Bailey. Bailey. That's it. Mm-hmm. Yeah, I can't remember. But he's the one who said science will break your heart. He is.

1:00:15Wonderful. Okay, Kat, do you have a summary, Kat? It's very short. Okay. So the authors developed quantitative approaches to examine influenza virus infection, RIG-I, which is an immune sensor of RNAs. And they show that host cellular non-coding RNAs interact with RIG-I routinely. And during an infection, this interaction of RIGI and the cellular non-coding RNAs makes them become immunogenic, suppressing viral replication. But the influenza virus, now anthropomorphizing, has a way to counteract this, antagonizing these so-called self-RNAs. And that thwarts the cellular immune defense. and they go on to say that there may be other viruses that do this as well.

1:01:06So basically, the bottom line is there's a new viral countermeasure that disrupts this rig-eye activation and signaling by the host. Well, and to me, I was also like, what do you mean? Or thinking about the fact that the host sensing was antiviral was also an interesting piece to be thinking about with this paper. Very interesting. Yeah, it's an interesting twist for sure. And I just want to ask a question about antagonizing. So we can't refer to things as receptor agonists and antagonists? No, that's a noun, right? That's okay. Right, but an antagonist antagonizes. Yeah, but we don't usually say that, right?

1:01:48We say receptor antagonist blocks, whatever. I mean, look, there's always going to be some... Sorry, I'm just antagonizing you. No, no, it's fine. I just try and be consistent because I believe it. All right? And if I say it enough and eventually one person is going to listen. Okay? And agree. It might just be me. And also you wrote a textbook, which, yeah. So in the book we never do that. Viruses do this. We make a very big effort to take those words out and phrase it some other way. And that's why I give a disclaimer. Yeah. Yes, and Jane is originally British, and her English is impeccable. Her grammar is impeccable, and she picks it up immediately.

1:02:36She says, you know, you can't do this. Also, the because and as thing, she's very big on. All right, so if you've listened to Twiv, you know that we talk about how cells have pathways to sense foreign nucleic acids, right? And in this one, we're focusing on the RIG-I-like receptors. So these are RNA sensors that surveil. Surveil. I like that word, surveil. Is that a real word? It is a real word. But it's not survey. It's different from survey. No, it's surveil. Surveil the cytoplasm for foreign RNAs. There's another one in the cytoplasm, MDA5, that recognizes double-stranded RNAs. And then there's yet another one.

1:03:20But RIG-I recognizes shorter double-stranded RNAs, and they have to have a 5' phosphate, a diphosphate, or a triphosphate. And that's how they distinguish them from cell RNAs, which typically don't have that at their 5' N. They're typically capped. Yeah, because most of them are capped or just don't have it. So then... So when RIG-I binds to one of these RNAs, it undergoes a conformational change and then initiates a signaling pathway, which eventually in the nucleus leads to the transcription of interferons and cytokines and so forth, right? The antiviral response. So RIG-I is always on, right?

1:04:04So when a virus comes in, it's going to pick it up right away, right? And for influenza, it's the big sensor controlling influenza virus infection. But what's interesting is that RIG-I does not only bind viral RNAs. It samples any double-stranded RNA, no matter where it came from. And in fact, the number of RNAs in a cell are way more than the number of RIG-I molecules. Okay, so RIG-I has to have some method to get around that. So what it does is it rapidly cycles on and off any RNA that's non-immunogenic, which means it doesn't have a 5' dye or triphosphate, basically. So it binds the RNA. If there's no phosphate, boom, goodbye, you're done.

1:04:53I'm going to get off. But when it finds a dye or triphosphate, it then goes through this conformational change to start interference. It's the bouncer saying, go, go, go, go. Hey, wait a second. Right. So that's what's happening here. So let me turn on scrolling. Sorry. I have to always enable scrolling. And so this is how you distinguish virus from host RNA. And there are people who have errors in self-sensing, and these cause diseases, right? They're called interferonopathies. Singleton-Merton syndrome, Eckart-Guthier syndrome. You cannot differentiate yourself from non-self. So you get chronic interference signaling and always inflammation, really bad, right?

1:05:50But we've always thought that it's bad to sense yourself. But as you're going to see in this paper, and other people have found this, now we're beginning to think that self-sensing is good. It can amplify antiviral responses. So that's what they look at in this paper. They're going to look at RNAs that are surveilled by RIG-I, and they're going to see what happens in infected cells. So they develop a very cool method for immunoprecipitating RIG-I or any protein bound to an RNA and then sequencing that RNA. I'm not going to go through it, but it's really cool. It's called CLIP-SEQ, Cross-Linking Immunoprecipitation Sequence.

1:06:35Well, they didn't develop that, but they developed a method to make it quantifiable, which I don't want to go into, but it's pretty cool. So then they compare RNAs bound by RIG-I in an infected cell or an uninfected cell or with VSV, which doesn't have a dye or a triphosphate. Okay, so then they see, okay, RIG-I binds influenza virus RNA. The binding is at the end of the genome, right? And these are RNAs that have 5' triphosphate. But the vast majority of all the RNAs that they sequence are derived from the host. So even if you pull out RIGI from an infected cell, most of the RNAs are from the host.

1:07:16And what are these RNAs? They are mostly non-coding RNAs that are made by POL3, RNA polymerase 3. And some of them have names I've never heard of before, like YRNAs, VTRNAs. I've heard of U6 and ALU and TRNAs. So you've heard of TRNAs probably. These are all bound by rig eye. I had to look up what the YRNA was. And it gets its name from cytoplasmic, from the Y of cytoplasmic. So it's found in SLE, but it's opposed to nuclear RNAs found in SLE. Got it. Lupus. Yeah. And I think the key thing here is that these RNAs are double-stranded. Yeah. And so that's part of why this is some population of non-coding RNA, because at least some of this needs to be not – we use the coding strand so that you can get double-stranded RNA.

1:08:18Right. So RIG-I at background levels is sticking a bit more to these PAL3-encoded small double-stranded RNAs, even though they're not virus. And then during an infection, it's sticking to viral and these host RNAs. Right. So it changes during infection, as we will see. But yes. But, yeah, in an infected cell, it is also detecting viral RNAs. And the idea is that detecting the cell RNAs help to amplify the interferon response. So basically what they find so far is RIG-I is always binding and surveilling cellular RNAs, whether or not the cell is infected, particularly PUL3 RNAs. And even in an infected cell, most of the RNAs that RIG-I is sampling are host RNAs, which I didn't know.

1:09:13It's just very interesting, right? I had no idea. I had somehow, but it was stupid of me to think that the virus infects the cell and immediately RIG-I just goes to that and grabs it. Yeah, of course not. Yeah, their point about the RNA concentration in the cell is like, well, oh yeah, of course. Of course it would be like fully bound to lots of other RNAs. But I was the same way of insight. Yeah. So the influenza virus. Yeah, what they're going to get to here is the effect of this background binding on cellular RNAs, the effect that has on the immune response against the virus. So they turn to a viral protein, the nuclear protein, NP, that's known to bind viral RNA.

1:09:57I don't know how they got here, right? But they don't tell us how they got here. They just said, we know that NP binds viral RNA. We know it binds kind of agnostically. There's no sequence-specific binding. And so we wondered whether NP would play some role. So they first ask whether NP interacts with rig-I, right? And it does. They do co-precipitations, and they do interact, and it needs RNA for the two of them to interact. If you destroy the RNA in this co-immunoprecipitation, the RIGI and NP do not interact. Okay. So then they say, well, maybe NP is also interacting with cellular RNAs in addition to the viral genome, right?

1:10:49So in an influenza virus particle, all the RNAs, the viral RNAs are coated with NP. So they immunoprecipitate NP from infected cells, and they sequence the RNAs that are bound to it. And they find, yeah, NP is bound to lots of small RNAs, and there's the same ones that rig-I is binding to in an uninfected cell. They also find viral sequences, of course, but 25 % of the bound sequences are host-derived. and many of them belong to this non-coding family that we've just talked about. No sequence motifs or anything like that. So it's just the way NP binds to RNAs. Okay, so a quarter of these NP RNAs have host RNA.

1:11:42Is this intrinsic or is it infection specific? So they look at NP-bound RNAs under multiple conditions. They do infection. They do just producing the protein on its own.

1:12:00And on infection, about 30 % of the NP-bound reads are influenza virus-derived. 20 % are influenza-derived when NP is pre-expressed before infection. And it looks like NP binds a collection of RNAs compared to the input. And this differs when NP is expressed alone or compared to infection. So infection makes a difference. Yeah. And I was a little confused by this experiment because there's not actually a physiological situation where NP is ever alone.

1:12:38No, but they wanted to know whether it would bind on its own without a viral influence, right? That's all. Right. So they're trying to find out if this is a characteristic intrinsic to just NP, or if it needs to occur in the context of a full viral infection, and it appears to need a full viral infection, which, as Brianne pointed out, is the only time you'd ever get NP in the cell anyway. But importantly, when you compare NP alone versus infected cell, you have different RNAs. They have 1 ,400 different RNAs that are differentially bound between induced and infected samples so that NP is induced on its own versus infected.

1:13:17So they say influenza infection reshapes the population of RNAs that are bound by NP. So that's why that's an important experiment. Yeah, so I would say that is some other part of the virus along with NP is influencing which RNAs are bound. Yeah, most likely, yes. It's not NP alone. Yeah, exactly. NP plus mystery part of the virus. We're not going to find that out in this paper. Yeah, I'm sure we'll find out. And also they do a nice experiment to show whether subcellular localization matters, right? NP is made in the cytosol, but it goes into the nucleus. And wherever NP is, it will bind RNA, basically.

1:14:00So that's pretty cool. All right. So it turns out that rig-I and NP bind many of the same RNAs, right? They're produced by POL3. And by the way, these POL3 transcripts, they're made with 3 prime, 5 prime triphosphate, but then it's cut off. but there is a time when they do have 5' triphosphate. So they want to know, does NP bind? So they call these immunogenic host RNAs, meaning that they have a 5' N that will stimulate, that will bind and activate rig I, right? So they have a reporter assay that looks for activation of interferon promoter by the right RNA. So they take NP-bound RNAs isolated from infected cells versus uninfected cells.

1:14:54And when you take those RNAs and throw them into this reporter cell line, the NP-bound RNAs from infected cells upregulate this interferon promoter compared to RNA extracted from uninfected cells. And this is where I was sort of getting to that question of sort of what's going on with the actual NP and infection and what's up with the NP without infection. Because what's interesting here is there's these unique RNAs that are being bound by NP in the situation of infection that are giving you this unique immune response. And so in my mind, this is the NP plus mystery protein gives you the unique immune response.

1:15:44And I'm less certain that I care about what just NP leads to. Yeah. But there's a difference between infected and unaffected cells. That's the key. Because if you just put NP into cells that are not infected and pull it out, it does not stimulate this interferon promoter. Yeah. And so I'm mostly like, what are these different things that make it simulate interferon promoter? Well, that's a very interesting question. Well, I mean, I think the reason they did the NP alone versus infected is to find out that, in fact, there is a protein X there that is, or whatever. I mean, they do another experiment where they take these host RNAs pulled out with NP, and they treat them with RNAs.

1:16:28And they don't activate anymore, which makes sense. and they do another one where they remove the 5' triphosphate and it doesn't activate anymore. So they say MP binds immunogenic host RNAs that signal via RIGI to activate type 1 interferon response. And that only happens in infected cells. That's the key. If you put MP into an unaffected cell, this does not happen at all. So they purify these RNAs via a different approach, antisense oligonucleotide capture. And I guess it's easier for them to get the RNA and do experiments with it. And they demonstrate that. So they know the sequence of some of these RNAs.

1:17:13So they can make antisense oligonucleotides and easily pull them out. And they show that these RNAs stimulate the interferon reporter cell lines. But only if they're from infected cells. If you take these RNAs from unaffected cells, they will not stimulate the interferon promoter through RIG-I, of course. They actually do an experiment in which they measure the 5' end, what's present at the 5' end in terms of phosphate. and they see that viral infection causes an increase in the percentage of these short double-stranded RNAs that retain a 5' triphosphate. So, Brianne, whatever your viral protein is, it's making more of these little RNAs have a 5' triphosphate.

1:18:06Yeah. I also wonder, so I'm saying it's a viral protein, I guess, but we don't also know is— It's a process, right? Yeah. Or is it viral genome being present? And there are, as you start to think through all of that, you could imagine ways that you could start to experimentally test some of those things. But, yeah, so it could be viral protein, some other viral protein, or it could be the fact that there is some rig eye that's being occupied by viral genome. Right.

1:18:45So the idea now is that NP is binding to these immunogenic RNAs that would normally be sensed by rig-eye. So is NP interfering with sensing, right? That would be an antagonist of sensing. I can use that as an answer. Yes. That's okay. So they want to test whether NP interferes with rig-eye sensing, which they cannot do in infected cells because you need NP for viral replication. So they have this reporter cell line that is a promoter that will respond to triphosphate double-stranded RNA, and they put NP in that cell. And they see that if they have a cytoplasmic version of NP, so NP normally will go in the nucleus.

1:19:33So they make a version that stays in the cytoplasm. When they do that, this completely abolishes sensing of RNAs. So yes, NP is antagonizing sensing by binding to these little triphosphate. So somehow the virus infection is causing a higher level of triphosphates on these RNAs, and NP is binding them to prevent sensing. Right. The question is, what's the function of the virus making these, having these, I'm anthropomorphizing. Yeah, well, and again, it gets hard because they don't actually show this happening in an infected cell here? Because we know in an infected cell, you make a lot of, you get a lot of stuff getting sensed.

1:20:19Here they show you have NP, but no infection, you block sensing. Well, but in infection, you just told us a lot of stuff got sensed. Well, maybe they can make an NP that will bind the viral genome, but not these RNAs. And it may be that some side effect of infection is causing an overall increase in 5' phosphorylation of small RNAs because the flu virus has small RNAs, and that would have to be part of its life cycle. Yeah. I mean, it's probably relevant to virus replication. Yes. By the way, they do this in other cells. They do it in lung cells. They do it in primary human bronchial epithelial cells.

1:21:04They go as far as they can. They even differentiate to be on an air-liquid interface, and they find the same effect. So they don't go in an animal, but they go as far as they can to show that this works. So basically, you have these RNAs that become immunogenic during virus infections because they have more phosphates at their five prime ends. They would normally be sensed by rig I, but now NP is binding to them and blocking sensing. But how do we know those RNAs happened as a result of infection? Because you only see them, they're only immunogenic. They happen all the time, but they're only immunogenic in infected cells, right?

1:21:49They will only stimulate RIG-I in infected cells. Well, it seems like they will only stimulate RIG-I in the absence of NP or in the presence of NP plus mystery protein. Well, I think if you extract the RNA from NP in an infected cell, it will activate the promoter, right? Okay. Because they have triphosphates, and that's why NP is binding them, because they're immunogenic, right? You got that? Yeah, I got that. Okay. So that's the story. Self-sensing is a key to antiviral response to influenza virus, maybe other viruses too, and then the NP has evolved to block that, which you had to do. I was thinking, so it took me a while to get my brain around this.

1:22:38Yeah, of course. Self-sensing is part of the antiviral response. Normally, I hadn't thought of it that way. Yeah, of course, yes. I kind of conceptualized this as being like something in electronics called a bias voltage, where you have a transistor that's going to amplify some sound wave, let's say. And you apply a little bit of a DC voltage to it to bias it, to make it, put it into its right sensitivity range. And that bias voltage is not what you want to amplify, but it's something that you do to kind of get the system at the right set point. And it seemed to me that maybe this rig eye binding to these PAL3 transcripts is that bias voltage.

1:23:24It's turning things up a little bit so that there's this, it's more easily pushed over the threshold to stimulate the immune response. Yeah, and that's used in other places in immunology, that sort of idea. Yeah, I kind of was thinking of it as it's training on itself as to how to deal with when the virus comes in. Right. And itself has made all these small RNAs. So that's how it... I think it's just that there's so many small RNAs in a cell that this is how RIG-I evolved to be able to distinguish. I don't know. Yeah. The thing that I found was interesting in the very end of the discussion, They talked about how there's so much excess NP relative to viral genome that it makes sense that NP is doing something else.

1:24:15Yes. Yeah. Yeah, that's an interesting view. So they make the point that all negative strand RNA viruses have an NP or an N protein. And even at one plus strand RNA, the coronaviruses or the nidoviruses that bind the RNA in a sequence agnostic fashion. So maybe they have similar functions worth looking at. Maybe it's a universal strategy, right? Yeah. Okay. That's pretty cool, I thought. Let's do a couple of emails. Kathy, can you take the first one? All right. Lisa writes, I'm a Twiv fan and Provax. I live in Florida and belong to a Facebook moms group. Here's a recent post, and it's not the first I've seen something like this.

1:25:05What's wrong with people? Original question to the group. Looking for recommendations for a pediatrician that does house calls. I think my 11-month-old has RSV, and her pediatrician can't see her until Monday morning. Mom number one response. RSV is extremely serious. If you suspect that, don't wait. Go to the ER. My daughter had it and had to be hospitalized for several days. Mom number two response. response, Wolfson's Children's ER. Been there with two of my own. Mom number three response, my 19-month-old had it a few weeks ago. He went downhill rapidly. I suggest Wolfson's. We went to Town Center and they were on the verge of admitting him because they couldn't get his stats up, but luckily by the end of the night he was responding to treatments and were able to discharge us.

1:25:50Mom number four response, I definitely wouldn't wait. Take your baby to Wolfson's now at Town Center. It's convenient and they are great. And so on. Why doesn't anyone, including me, say there's a vaccine for that? Once you have it, is there a point to being vaccinated? Is the potential backlash just too much? Am I just afraid to speak up because crazy people will bite my head off? Why are they letting their children suffer with this? Thanks for all you do, Lisa. What is the age for the RSV vaccine? Let's see. So you can give moms globulin, so it will protect them for six months, and then at six months you can – Six months, okay.

1:26:32Yeah. I sort of feel like in this situation, I don't know if my first answer would be there's a vaccine for that because I'm not sure if the sick 11-month-old – Yeah. Like a vaccine is not going to be acutely useful for that sick 11-month-old. Right. And in some ways looking at the mom and being like, hmm, sad for you. you could have protected yourself for this might not be the like thing to do when that kid is acutely sick yeah but you could do it oh you know for the future by the way there is a vaccine that your children can get at six months but as for whether you get should get vaccinated if you um i think you should because infection doesn't give you good immunity yeah uh the vaccine does So far, it's been a couple of years that it's been out.

1:27:19And so. I think you should wait until your kid is out of the emergency room. Well, of course. Yes. I think that you should give your kid the vaccine before, like way before they go to the emergency room to make sure it doesn't happen. Yes. You can hear from these folks how serious it was for their children and they're having to go to the ER. And that's great reason why you should get the vaccine. But maybe when you're in the acute phase, it's not the time. But I take this as Lisa asking, why doesn't somebody suggest something about the vaccine? Which is why I would say she could say, for the future, you might want to consider that your child could get this vaccine and prevent this horrible disease.

1:27:59For others reading this thread, you know, you can potentially avoid this. One could. You could change the year. One could, yes. But notice that Lisa says, why didn't I suggest this? Because you're going to get a backlash. That's why. And you don't want to. And I understand people are going to say, oh, you know, all this stuff. Yeah. No, both Kathy and Alan's points here are like, yes, those are all awesome things to say. Brianne, can you take the next one? Sure. Toby writes, Dear Twiv, my name is Toby and I'm a third year biomedicine student in Australia. I started listening to your show in the first year of my degree in 2024 and I had to wait until my final semester to take the virology subject.

1:28:41but next week I will be sitting my first virology exam and finishing this stage of my degree before hopefully moving into a career of virology research. Aside from thanking TWIV for cementing my interest in viruses and research more broadly, I wanted to offer some timely sentiments from Australia given your recent and our upcoming elections. At the beginning of COVID in 2020, Australia, like much of the world, looked to America for leadership, whilst imperfect, in some ways I think it was found. I maintain that Operation Warp Speed was the greatest success of the first Trump administration. The Herculean effort it must have taken to develop safe and effective vaccines in such a short timeframe represents, in my opinion, the greatest public health success story in my memory and the best of a government that took action to protect its people.

1:29:30I have linked below to a speech that Trump delivered touting the success of Operation Warp Speed and it astounds me that these words come from the same person who has overseen systematic dismantling of public health with implications far beyond American borders. Over the past two years, my peers, educators, and I have been absolutely dumbfounded by the public health decision-making coming from America, amongst other things. How could someone who championed a vaccine drive just six years ago be using health care as a wedge to divide? I feared that health care was seeping into our politics in Australia too, I spoke to my family about what I could do if it became impossible to pursue my dream career because of an anti-science wave.

1:30:15Your midterm elections have restored my hope that people won't stand for the destruction of health and science infrastructure. Aside from high gas and food prices, corruption, war and cruelty, healthcare was a big part of campaigning in America. I am pleased to see several people who campaigned on healthcare as a cornerstone of their policy elected to your Senate. The day after Americans voted for change, surging right-wing political parties in Australia removed all references to Trump from their website, and I hope they too will see healthcare and science as the apolitical entity it ought to be.

1:30:48I also hope that America holds its newly elected to the promises they made. The same changes that have set us back so far have given us the opportunity to make a system that works better for everyone, And I hope that the weight of this opportunity isn't lost. Best wishes from Australia. Great points. Thank you, Toby. It's great. Really well said. I love it. Yeah. How can someone? Yeah. He said it perfectly. Just amazing. Yeah. We hope that things change now, at least in science and healthcare. Alan. Jerry writes, hello again. This is mostly for Rich. Thanks for reading my letter on Feynman and names and listers.

1:31:32My wife and son love birds, so I follow them as well. There is a move in the ornithology world to change the names of birds away from who discovered them, for example, Ridgeway's hawk, to a more characteristics-based form, possibly the Hispaniola hawk, as it's only found there. Neither I nor the birds could care less, but many birders have strong opinions. I understand the importance of naming in science for clarity and relationships. At the same time, a certain level of humility and humor is important, since the naming goalposts are always moving as we learn more. I am not sure how to feel with the current discussion of whether we are descended from comb jellies rather than the current consensus of it being sponges, for example, a.k.a.

1:32:16who branched from whom. Again, it is our human understanding coded into names, terms, and taxonomy, not to be confused with reality. I didn't explain the idea of listers well in my last email. These people live for adding species to their life list. It is best summed up in a short story from a recent trip to Brazil. We ran into a couple where the man was a serious birder. He saw a short sighting of a red and green macaw and was excited. A couple of days later, there was a group of about 10 of these birds soaring against red sandstone cliffs. It was one of those magical moments where neither words nor pictures can convey how free and beautiful they were.

1:32:57We ran into the couple again, and I exclaimed how beautiful the macaws were. He flatly said, I've seen them already. I could see those birds every day and still be awed and enchanted. That's why I'm not a lister. This doesn't do justice to them, but here is a hint at how beautiful they are, probably taken the same place we saw them and provides a link to a picture. The worst story I ever heard was in Britain, where they are known to have crazy twitchers. There was a rare bird reported in a marsh area, and many twitchers descended on the marsh to see it. Somehow, one of the people managed to step on and kill the bird.

1:33:34All for the chance to get a look at a fairly drab bird a long way from its normal home and add it to their list. Thank you, Jerry. Now I get it. Yeah. I didn't understand the listers, but yeah. Oh, I've seen it already. I don't care.

1:33:51Wes writes, Hi, Vincent Allen Rich. I've been a fan of your podcast for quite some time. So I really appreciated hearing you say on this week's episode that you're against raising and killing animals for their fur. I was naturally excited to hear PETA mention too. But to clear up a misconception that you mentioned, we at PETA stick to splashing paint on ourselves in colorful protests. Thanks so much for your kind words here. And if we can ever serve as a resource for you, please don't hesitate to contact us. We would love to hear from you anytime. And Wes is a press officer for PETA.

1:34:28Let's finish these up. We only have two. Who went first? No, Kathy went. Go ahead, Kathy. Mary writes, Dear Twivers, as a former dairy farmer, I was interested to hear your discussion of bird flu in mink, TWIV 1371, and the relationship, if any, to bird flu in dairy cattle. One characteristic of commercial dairy farming that might be important to your discussion is financing. Milk producers can lease their cows. This allows them to focus their efforts on milk production as opposed to breeding replacement stock. The way it works is farmers called heifer operations buy female calves from dairy farmers soon after they are born.

1:35:08These operations then raise the heifers up to breeding age and, using artificial insemination, impregnate them. A leasing company buys the bred heifers, which are then leased to milk producers when they are ready to calve. As you noted, a producing dairy cow only lasts three or so years in a high-production milking facility. As her production declines, she is replaced by the leasing company and goes to slaughter. Hopefully this helps eliminate how many opportunities there are for various people interacting with the animals and the animals to be transferred from farm to farm. Perhaps they also move a virus along the way.

1:35:45My information is dated and limited to the states of Washington and Oregon. Heifers might be tested now to ensure they don't carry bird flu from their mothers, and I just don't know. If they are, it would be a significant regulatory change for my time in the industry. Thanks for all you do. Mary. That's really interesting. I had no idea. Yeah, I didn't know. But I don't think there's any testing as far as I understand in cows. Nope. And that would certainly explain the amount of cow movement that we've been talking about in terms of spreading. Yeah, sure. Wow. Brianne, last one. Anthony writes, and Anthony gives a link, and I believe the title of the thing that's linked is Measles in Fiji, 1875, What a Naive Population Might Expect.

1:36:37And a quote from this, before leaving the South Seas, we must glance at Fiji, the island state annexed to the British Empire. contrary to our usual maxims of late in 1873 the unhappy inhabitants of which received as their first gain from our rule a fearful epidemic of measles carrying off 50 000 inhabitants out of a population of 150 000 the british annual register two again and this is an ignoring sequelae And so that is a rather dramatic situation where if there were 150 ,000 in Fiji, that a third of them died, not counting other issues. So, yes, if we are not getting good immune responses to measles, it can spread quite well.

1:37:29Yes. This article linked to is by David Morins. Measles in Fiji, thoughts on the history of emerging infectious diseases Cool, thank you And there's a Jack London short story collection South Sea Tales From the early 1900s There are a couple of stories where measles comes up there as well With sometimes it being deliberately introduced to an island To cow the local population yeah okay let us do some picks of the week Brienne what do you have for us so as I've mentioned a couple of times I have sort of picked up a new hobby over the past few months and my new hobby of crocheting and when that happened I remember seeing some links about the benefits of what they called in that situation, grandma hobbies.

1:38:32And as time has gone on, and I have enjoyed learning this hobby, I have thought back to some of those same articles, as I've thought about, you know, this is actually really good for me. I do really enjoy this. I do think I'm getting something out of this hobby. And so I've gone back to try to find some of those original articles that I read when I started talking about the reasons why, as these articles note, grandma hobbies, I had actually heard of them as grandma core hobbies, tend to seem to actually have some positive benefits. And I like these articles because they do have some links to some papers that try to talk about this.

1:39:18But there is some idea that these sorts of hobbies can be good for different types of mental health issues. They may be good for burnout. They may strengthen neural pathways. They can provide sort of healthy stress from the brain and improve memory and attention. And like I said, as I do them, I sort of have noticed what a calming effect I feel and how much I just feel better. But I also really liked thinking about this because I think that a lot of us in the lab actually have come up with similar types of things. So in the part of this article where they point out that it could prevent burnout, the paper that they link talks about the importance of repetitive handwork to signal that your nervous system that it's safe.

1:40:08And as soon as I read that, I remembered a time where I had a student who was very stressed about some exams and who came by my office and said, do you have anything I can aliquot? I would really just like to do some repetitive pipetting. And I know that same kind of the zen of repetitive pipetting that you sort of get into. And it's the same thing. It's repetitive handwork. And so, in fact, I think that this gets into some of the tasks that we as scientists do all the time. And thinking about all of this and the ways that these were beneficial and sort of the science of the benefit also reminded me of some of the long discussions we would have with Dixon about art and science and all of the interesting interactions.

1:40:52Very cool. We used to rack pipette tips too. Sure, exactly. Yeah. And I can just think of, you know, you could always aliquot PCR quality water. Yeah, exactly. You know, because then you could just use them once, throw them away. You know, that's why we would do that. So, yeah. Yeah. So I think that we, that many people in molecular biology labs may actually be tapping into some of these same benefits with some of our tasks, though it may not be as cute as something like, you know, a little bat. That's really good. Yes. Good. That's very cool. Kathy, what do you have for us? Well, so I'm not a birder.

1:41:30I'm not a bird lister. But I did recently start getting an email from somebody, Ben Tonelli, called Bird of the Week. They're very short. They come once a week with about four to five facts. They're fun, easy to read in less than a minute. And so I put the link in here and you can just sign up and subscribe. And so I'll just read you about the mountain chickadee. You may be familiar with the black cap chickadee, one of the quintessential backyard birds of North America. The mountain chickadee is its more rugged cousin with sweet racing stripes on its head. The mountain chickadee is adapted to rugged alpine environments in North America's Mountain West, where it meticulously prepares for the lean season by stashing conifer seeds.

1:42:11Because of this caching behavior, this bird is one of the few species that can stick around these high alpine environments year-round. When food is scarce, the more dominant birds will push the less dominant ones out of these forests where they head to lower elevations to look for food. These birds are pretty social, hanging out and mating among small groups, with about four to six adults and their offspring. Although pairs often mate for life, sometimes there are divorces when an attractive chickadee joins the group. so I've sent it to several people who are birders and several people who are not and everybody pretty much likes just getting them and you know you get it and you read it and it's over in a minute and it's just fun.

1:42:53That's delightful. Sounds great. I'd love to do that. Yes. Chickadees. Alan what do you have for us? I have a book pick. The Dark Frontier by Jeffrey Marlow. Jeffrey Marlow is a marine biologist and policy wonk. Interesting combination. He's been on a bunch of deep sea expeditions going down in the Alvin Submersible and studying various organisms at deep sea vents. And he has written a really good book about, you know, essentially different kinds of darkness. There's the darkness of the deep ocean where you can't have anything growing photosynthetically because there's no light at all. And what has evolved there, and we've all heard about the hydrothermal vents and the cool stuff there.

1:43:47But there are several different types that he goes through, and he tracks the series of discoveries historically that have been made there. there's also just the scientific darkness about these environments that we know a tiny tiny fraction of what's down there and every time somebody goes on another dive they seem to find more stuff like we've really not even tapped into the the edges of this and whole new types of vents and that kind of thing and there's also kind of a dark theme to the book, especially toward the end, where he starts talking about what's going on now with powerful industrial interests who want to strip mine the deep ocean on the thinking that there are all these resources there that we can just pick up and there's no life down there.

1:44:38So who would care and what could it possibly hurt? And he's got some thoughts on that. It was a fascinating book. Cool. Love it. All right. My pick is a YouTube video called Where Do Photons Actually Go When You Turn Off the Light? So I just stumbled on this. I was looking at something in YouTube and I saw this. And it's a physics channel called Carter Studios Physics. And, oh, my gosh, I didn't know anything about photons before. But, you know, when a light generates photons, right? They are massless, tiny packets of energy. And when you heat the filament in a bulb, LEDs are slightly different the way they make photons.

1:45:28You're making a kind of a photon, an infrared photon comes off in the form of heat and also light photons. And they fill your room. And when you turn the light off, it gets black because they go so fast. They all impact into the walls and go out the window when you turn. because they're going at the speed of light, these photons, right? C. And so this video talks all about it. And then where do they go when they go in the wall? They're not destroyed. They hit other atoms in the wall and excite them, and those atoms give off infrared photons, which heat up the wall just a tiny bit, right? And then those go out into space because they can penetrate through the walls and go out into space, and they probably travel unimpeded in space.

1:46:13There are very few molecules for them to run into. And so they can travel forever and ever. And the sun, of course, photons are generated in the core by the fusion reaction. It takes them over 100 ,000 years to get out because they keep bumping into other molecules on the way out. I've been to parties like that. Once they reach the surface, then it's only nine minutes to get to Earth. It is how fast they go is just amazing. And they're trying so hard. They have all of those years to get from the core to the outside and then nine minutes. And then they're almost going to hit the Earth and then they hit you and make your shadow.

1:46:50Yeah, that's right. And that's why the cities on Earth have a glow because of all the photons coming. And they are spilling out into space. If they didn't, the Earth would heat up terribly if the photons all stayed in the atmosphere. But they go out and that's why we don't want to have a restrictive atmosphere. But this is fascinating. And then it got me on a rabbit hole of looking into photons. And it's just, I mean, they are, so why do they have no mass? I thought that couldn't happen. And that's why they have to travel at the speed of light because they have no mass. It's the only thing that can, so lots of cool stuff.

1:47:26And this channel also has other cool videos as well that you might want to check out. But the photons are just, I mean, we're all bathed in photons now, right? And then if we turn off, the light gets dark because the photons go so fast. And some of these are impacting me and they're warming up my skin a bit, right? Holy cow. I never knew this because I was so focused on viruses my whole life, right? But now that I, I don't know why, but I'm just interested in a lot of things now. I'm also reading a book on neuropsychiatry, which is what you talked about, Brianne. We know exactly what part of the brain that is tapping into when you do that stuff.

1:48:07Oh, it's so cool. All right. That's TWIV 1373. You can find the show notes at microbe.tv slash TWIV. You can send us questions and comments or picks. Send us your picks of the week. We'd love to hear what you want to share. That can go to TWIV at microbe.tv. And if you enjoy these programs, we would love your support. microbe.tv slash contribute. Kathy Spindler is an emerita professor, university or professor emerita. I guess you could say it both ways. Is that right? Yeah. University of Michigan in Ann Arbor. Thank you, Kathy. Thanks. This is a lot of fun. Brian Barker is at Drew University Bioprof Barker on Blue Sky.

1:48:49Thank you, Brian. Thanks. I learned a lot. Alan Dove is at alandove.com, turbidplac.com. Thank you, Alan. Thank you. It's always a pleasure. I'm Vincent Racaniello. You can find me at microbe.tv. I'd like to thank the American Society for Virology and the American Society for Microbiology for their support of TWIV Ronald Jenkes for the music and Jolene Ramsey for the timestamps. You've been listening to This Week in Virology. Thanks for joining us. We'll be back next week. Another TWIV is viral.