TWiV Special: Measles Mini Symposium

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

0:10Hello, measles virus mini symposium.

0:19It's not bad for the first try. Very good. I thought you'd be subdued because you're negative, right? I should not. From Microbe TV, this is TWIV, This Week in Virology, a special episode recorded on July 26th, 2026. I'm Vincent Racaniello, and you're listening to the podcast all about viruses. Today we are in Rochester, Minnesota. We are at the Mayo Clinic where I'm attending the eighth Measles Virus Mini Symposium. About 70 people all working in the field. Fascinating research. And I have two guests today from the meeting. We're going to talk about their careers and their science. on my left, he's a former branch chief in the Division of Viral Diseases at the US CDC, Paul Rhoda.

1:17Welcome to TWIV. Hi, Vincent. This is not a rotavirus meeting. No. You get the joke all the time, right? Pretty much, yeah. Sorry. Well, our listeners don't know anything about it. And over in the other seat from the University of Iowa, Patrick Sinn. Welcome to TWIV. Yeah, thanks for inviting me to talk. You're welcome. And we have a nice little audience here, as you heard. They're eating their lunch. So no matter what we say, they'll be happy. Right? If you enjoy these programs, we have This Week in Virology. We have many others. We have nine other science-based programs with hosts who are scientists.

1:53We'd love your support. MicrobTV is a non-profit. We don't do ads on our program. We depend on your donations to do our program. So you can go to microb.tv slash contribute for different ways that you can help us out. Before we talk about your science, I want to talk a little bit about you and your training. So let's start with you, Paul. Tell us as far back as you can remember. So where are you from and what kind of training you got to end up at your job? Okay. Yeah. I'm originally from New Jersey. Wow. Still am I. Okay. I graduated from Rutgers College with a VA in microbiology and then went to Michigan State where I got a PhD in microbiology working with herpes viruses, feline herpes viruses.

2:46And as I was nearing the end of my graduate training, I became aware of a fellowship program at the CDC. It's a brand new fellowship program that was being run by the National Research Council. So I was able to contact a few people and secured a postdoc in the influenza branch at CDC, where I started doing vaccinia vectoring. So that was pretty far back because that was a very popular technique in the early to mid-1980s. After a couple of years in flu, we were experiencing, the U.S. experienced a big resurgence of measles from 1989 to 1991. We had about over 50 ,000 cases and 100 and greater than 140 deaths.

3:37Before that resurgence, CDC really didn't have a measles lab. And so the CDC tapped my colleague, Bill Bellini, who had worked on measles at NIH for a long time before he moved to CDC to start a measles lab. And Bill made me an offer I couldn't refuse. And so I eventually joined the measles lab in 1991 and stayed working there. And the measles lab eventually, through all various reorganizations that happened in the government, we eventually became a branch, which was now responsible for measles, mumps, varicella, rubella, and human papillomaviruses, including both a big lab component and the programmatic component.

4:29And so that's where I left it. A big part of my career at CDC was also working to build global lab capacity for measles and rubella. So I worked quite closely with our major colleague, the World Health Organization, to support a network of over 700 labs serving 191 countries. So heavily involved in methods development, training, things like that. Remember why you decided to go into science in the first place? Why I decided to go into it? Well, it may sound corny, but I read a book about Jonas Salk. I don't remember. I pulled it out of the library, and I thought, wow, that was really cool. I was really fascinated with vaccines and infectious diseases.

5:17When I went to Rutgers, I was telling my advisors I wanted to study infectious diseases, and they said, no, don't bother. We have antibiotics. What are you worried about? This was before toxic shock syndrome and HIV and everything else. Yeah, famously in the 60s, someone said infectious diseases are over. We've got it handled, right? Yeah, I'm not sure we have it handled yet. No, of course not. So where in New Jersey did you grow up? I grew up in Bergen County. So did I. Oh, good, in Milford. I was born in Patterson, though. Oh, Patterson. So is anyone from New Jersey? Wow, look at that. Yeah, Andy is from New Jersey.

5:56Yeah, Linden, New Jersey, I know. Cool. How about you, Patrick? Where are you from and how did you train? Actually, I went to high school in Waseca, Minnesota, which is a short distance from here. There's a couple of towns over, a little town. And then I did my undergraduate work at the University of Minnesota Morris, which is a small little arts college here in Minnesota. And then I did grad school in Iowa. So I've been in Iowa ever since. I have a degree in physiology and biophysics from Iowa. And after I finished my degree, which was mostly looking at human renal gene, hypertension genes in mice, I went across the hall and I got a postdoc in Paul McCray's lab in pediatrics.

6:45And we were interested in gene therapy for airway diseases. I'm still working on that today as well. And I got my toes wet in the measles field one year in early 2000s, very early 2000s when I had a meeting. We were looking for glycoproteins that we could pseudocyte lentivirus to target the apical side of airway cells for gene therapy. And that's when we met Roberto. And at the time, everyone knew that measles comes in from the apical side. And so he sent us some virus and we applied it to the apical and the basilateral side of airway epithelial cells and found that it came in exclusively basilateral.

7:32And so it was the opposite of what we wanted. We wanted things to come in from the apical, so it was basically a failed experiment that led to basically all of my research in measles since then. So science doesn't do what you want it to do, right? Yeah, exactly. So do you remember why you wanted to do science in the first place? So when I was in high school, I think in my simplistic high school mind, I thought you either had to go into business or you had to go into science. Because those are the only kind of classes they taught at mine. And I had no interest in business. And I had a physics teacher who I really loved.

8:13And so I went to college thinking I was going to be a physicist. And then I quickly found out that after Calc 2, I hit my max of what I could possibly comprehend. But I really enjoyed my biology classes in college. And so I switched my major into biology. And that was my research focus ever since. I like what you said. I went across the hall and did a postdoc. That's the way it used to be. You worked in one place. All right, let's talk about some science. Paul, you spent 40 years at CDC. That's correct, 40? Yeah, that's correct. So give us an overview of what you did there, and then we'll talk a little more in detail.

8:54Well, I started as a postdoc in the influenza group. Back then, the flu group was 15 people, maybe, working with Nancy Cox, who unfortunately passed away a couple months ago. But I was looking at host cell variation in flu, you know, and I was focusing on influenza type B. So there was not a lot of work done on flu B. So I was doing some work on host cell variation. And then I started to do some phylogenetic analysis of some of the different strains to look at the different lineages. I was able to show at one time that there were two co-circulating lineages of flu B, the B. Yamagata and the, what was the other one?

9:40Oh, B. Victoria. And that actually caused them to include two strains, two B strains in the flu vaccine for a while. Then working to measles, we had this gigantic resurgence of measles in the U.S., and we were trying to understand why. Was there a change in the virus? Was something going on? we were seeing a lot of cases in younger adults and things like that. And so there had been not much work done for characterization of measles viruses, at least doing the sequencing of wild type viruses. And there was a handful of labs working on that. So we started to try to sequence some of the viruses and look at some of the antigenic properties to see if there was some kind of a drift, which we really didn't find anything.

10:29You know, coming from flu, I always had an antigenic shift and drift on my mind, right? So that was the first thing we were looking at. I mean, it turns out the real reason for that resurgence was really that the vaccination rates plummeted in just like inner cities and things that rates were below 50%. And that's, you know, but we were trying to cover all our bases. And so we moved forward with the genetic characterization. And this resurgence at that time was not just the United States. It was global. So we were getting lots of calls from labs who wanted to help set up some methods and things like that.

11:13So we started initially, this was 1990, right? So we were really focused on developing IgM assays using baculovirus express, nuclear protein, and things like that. So trying to get a good, high-quality IgM capture. assay, which was because we really didn't think that we could, at that point, doing something like PCR in a global lab setting was going to be very, very difficult. Very few labs had molecular training and capacity. So we ended up doing quite a bit of training. We had international scientists visit us from the get-go. Probably in the early 1990s, we were having workshops just for focusing on IgM detection as well as virus isolation.

12:04And in those days, we were using something called B95A cells, which is an EBV transformed marmoset cell line that we were using that we were able to make viral isolates in. But it took a little bit of practice to teach people how to use that cell line. It was a little bit complicated. And then gradually, we moved into more molecular methods. And sequencing, as more and more labs started sequencing, these all evolved independently. And at one point, we were reading, there were maybe five or six different nomenclatures for genotyping. We were all describing the same thing. But one lab was calling it genotype 1, 2, 3.

12:50The other ones were calling it ABC. And so we did what we always do. We had a meeting at WHO, and we said, look, let's all call this everything the same. And so we were able in 1997 to standardize the genotyping strategy for measles virus. At that point, we were looking at, it seems, you know, not very much right now by today's standards, trying to sequence the 450 nucleotide hypervariable region that codes for the carboxyterminus of N. And that was a big lift back then for a lot of labs to do just that because we were working on Sanger sequencing, usually sequencing either directly from RNA. And so gradually we started to introduce molecular methods.

13:36And, you know, most of the labs now are doing real-time RT-PCR. And still, IgM serology, though, on the global level is still the first test because there are commercial tests that you can buy. So basically, technology revolutionized measles, epidemiology, screening, and so forth, right? Yeah, I think there was, you know, that we were able to, I think, getting better methods out. Before the 1990s, I think the ability to do lab confirmation was not so great. I mean, and there was a handful of labs doing it, but they were all doing different methods. And as you know, there's a lot of other rash illnesses that cause fever and rash.

14:22And so it's really critical to have lab confirmation for measles. Yeah, we have measles in like 190 countries worldwide, right? That sound right? In 1990, it was probably everywhere. So are they all able to track it by PCR and sequencing, or is there differences in lab capacity? There's huge gaps still in surveillance. I mean, it's not just the lab. The labs are in many cases, are pretty good. And remember, all of these labs a couple of years ago had shifted almost seamlessly into doing PCR for SARS, right? But a lot of the places you're competing, it's really the surveillance parts that are competing to try to get the specimens back.

15:09And that's really the hard part, is getting the sample back into the lab and getting it tested. because that relies on someone out in the field to go and collect a proper specimen. If you want to do PCR, you need a throat swab and everything that requires having a swab to begin with and having some way of transporting it back to the lab. So if you think about countries, low-income countries, think of countries in the African region, it's really a challenge for them to get all the different specimens. And so if you look at the global sequence database, even though most of the measles infections are occurring in sub-Saharan Africa, there's only a handful of sequences in there.

15:50Where on the other hand, there are from the Americas, from Western Europe, and even some of the Western Pacific countries, there are thousands of sequences because the labs are much more. The surveillance is much better at getting the specimens. So in the U.S., in the old days, if there were a measles outbreak, how would the CDC respond to that? Well, I mean, the first line of response has always been the state and local capacity. So they would investigate the outbreak. They would call us on the phone. As far as I know, that's still the practice. You know, you would think we'd have, and it's not a red phone.

16:30It's just a regular phone. So they call us on the phone and say they had cases. We discuss it. So a lot of the work that CDC was doing was what we call jurisdictional support. Talk to the state and local labs. See if they had any questions about contact tracing and source attribution and things like that. And oftentimes there were questions about laboratory testing. Where should they send the specimens? What did the test results mean? And so we would try to talk them through all of that. If things got really bad, then sometimes we would send a team out to help with some of the investigations. As far as you know, is this still happening in the current CDC?

17:13Yeah, I think obviously capacity is limited because we've seen unprecedented numbers of measles. So when we were having 100 cases a year, this is different than that. So what CDC does is, you know, we had, even when the cases were very low, we developed an outbreak response plan that described very clearly some trigger points from when we're going to escalate into what we call a response mode. So in January 2025, when the outbreak started in Texas, we saw that this was going to be serious. We were starting to get pings in other states. So we escalated into what we call a response. But what that does is it brings in people from all over CDC to work on the response because our team was good when things are routine.

18:06But when you start having lots of outbreaks and now when they're having concurrent outbreaks, very large ones, you need to bring in capacity. And this is something that we learned to do very well during COVID is form all these response units. So you're able to bring in staff from other places to rotate through. You develop things like a communication strategies. We bring in communications experts to develop, you know, appropriate communication materials for whatever population we're dealing with. I was there in charge of the lab team until I retired. But, you know, so coordinating work with the labs, we work closely with APHL and always the state public health labs.

18:51So back in 2000, measles was declared eliminated in the U.S. How was that achieved back then? Well, I think, you know, following the resurgence, I think we realized that we had a problem with vaccine availability. And so the U.S. government took a lot of steps, increased the vaccines for children and some congressional initiatives to provide vaccines in the public sector. This was backed at a very high level by Rosalind Carter and Betty Bumpers, who was the wife of Senator Dale Bumpers. So vaccines were available. They also strengthen the school entry requirements in all of the states. So by 2000, we had all 50 states having a two-dose requirement for school entry.

19:44I remember we were all celebrating when the last state, which will remain nameless, finally passed the law. And then, you know, the availability of vaccines, especially for, you know, inner city and things like that, where the availability wasn't so bad was much better. Is surveillance important at keeping measles down in the U.S.? Yeah, surveillance is critical. I mean, you have to, I mean, you have to, then the state, I must admit that the folks who are in the ones dealing with these outbreaks of the state and the county folks are really on top of this. Even though they, you know, you look at a state health department or even a county health department, they got a lot to do, right?

20:30and they've been chronically understaffed forever, but they really jump on measles. We usually find, you know, we would get a phone call within a few hours, and, you know, we wait for the lab results to come back. And then, you know, depending on the situation, if it's in a school, they have to go in and review the vaccination records. They have to decide where it came from. Lots of times it involved international travel. Sometimes if the person was infectious while they were on the plane, and they would have to do contact tracing with everybody who was on those flights. So that's a lot of work.

21:03So now we have a resurgence in the U.S., more cases since 2000. What's the cause of this in your view? Well, the virus is persisting in under-vaccinated populations, to put it mildly. But, you know, and there's, if you look at the, you know, the case distribution, it's hitting, you know, it's hitting the very young kids, but also you're seeing, you know, 40 something percent of the cases in the 5 to 19 year olds. So it's in some of the older age groups. So it's, the virus has gotten into some groups that are under vaccinated by choice, you know, tend to not be too cooperative with public health.

21:46and so the virus got in. Unfortunately for some of these, we had probably multiple rounds of transmission before it was reported to the state. You know, typically if you get a case in an emergency room here in Rochester, some child comes into the emergency room, they would go into overdrive, they would find it, they would try to decipher in with the court. The source was, you know, maybe this person was traveling with their family in a measles endemic country. But, you know, the populations that was in now, particularly like West Texas, they were, you know, kind of off the grid. They were off the grid and extremely mobile.

22:31So they drove to Mexico, they drove to Canada. And so we really, and, you know, it's really been a regional outbreak because there's 12 ,000 cases in Mexico and over 4 ,000 to 5 ,000 in Canada. So we're detecting the same viral sequences and same what we call distinct sequence ID in all of these outbreaks. So looking at the outbreak now and having spent 40 years in measles, does this frustrate you to see these cases arising? Absolutely, yeah. I mean, we worked really hard to achieve the elimination in 2000. That was a major effort. And it's frustrating to see that it's been harder to control these outbreaks.

23:22And it's frustrating because we want to try to get in there and control the outbreaks with vaccinating. There hasn't been a lot of vaccine uptake in some of these populations, which is the best way to end an outbreak. Well, I think in the past, the CDC was vocal in messaging about vaccination, and now it no longer is because of the leadership, right? Yeah, I mean, there's been a real lack of communication from CDC, which has been unfortunate. And, you know, in the past, CDC was always in the front trying to communicate. I mean, on the outbreak response side, I mean, in the communications with the local staff, I think the jurisdictional communication, I think that's still working pretty well.

24:04But we're not putting out national, CDC was not putting out national messages and things like that. Yeah. And so national immunization rates are dropping, right? And part of it is that people seem to be, some people seem to be afraid of MMR vaccine, and other people seem to think measles is a mild disease. So how does, in the old days, how did CDC deal with it? How did you message? Yeah, well, I think, you know, we have constantly messaged that it's not a mild disease. And if you look, the hospitalization rate for 2026 is a little bit lower. It's around 6%, but it typically runs 11 % to 12%. So that means you have a 1 in 10 chance of having a child in, say, the ICU for a while for, you know, just dehydration or other types of things.

24:54So it is a severe disease.

24:59And then we have lots of messaging on vaccine safety. I think it's been more of a challenge now to work with the vaccine-hesitant populations because you really have to develop effective messaging. And maybe the messaging that we were using back in 2000 is not relevant. I mean, you have to look at your different target audiences. So, yeah, we've seen this drop in the vaccination rates among kindergartners. It's down like 2 % or something like that. We're down to like 92%, which is still not terrible. But if you look at the map, it's a big country. So 92 % on average means that there are some counties in the low 80s, and that's not good.

25:49So it seems likely that the U.S. will lose elimination status, right? Is that something you can get back? Oh, yeah, you can get it back. So, you know, the meeting to decide on the elimination status will occur in November. That's a committee that's run by the Pan American Health Organization. It's the Regional Verification Committee. They'll look at all of the data. I haven't seen the latest. You know, it's been a year and a half since I really had firsthand contact with the information. I would say that it doesn't look good for maintaining elimination status. And so if that happens, then you have to, it's the same criteria apply to re-verify.

26:39You have to show lack of sustained transmission for three years. And, you know, earlier a couple of years, you know, like Brazil lost elimination status, then they regained it. Brazil so far has been pretty good with measles in the last couple of years. Same thing happened with Venezuela. They lost their elimination status there and they regained it. Countries in Europe are up and down. The UK has been lost their status and they regained it and they lost it again this year. Do you think that globally measles could be eliminated? I mean, I think it could be. I mean, all of the regions have a measles elimination goal targeting through vaccination.

27:29So it's going to take, you know, a lot of resources to do that. I think, you know, I think the current vaccine would be able to help us achieve elimination. I think what some of the focus is now are on better delivery systems for vaccines. So just a little plug here. One of the things I worked on at CDC for quite some time is this microneedle patch vaccine. They like to call it a microarray patch, but I think it's really a needle. It's a lot of little tiny needles. But that looked really good in the first clinical trial a couple years ago. and there's another clinical trial plan for 2027. So I think if you think about ways of somebody being able to carry these basically Band-Aids to help get vaccines into areas where it's hard to get good coverage, that might help improve some of these gaps in vaccination.

28:29Yes, you don't need a health care specialist to put the patch on you, right? Yeah, you just have to put a Band-Aid on. It's maybe a little more complicated than that, So you were loyal to CDC for 40 years. What made you leave in the end? It was time. 40 years, I think, being the underlying.

28:55So there obviously have been massive changes at CDC. A lot of people have left. Do you think it can be fixed in another administration? situation?

29:08Yeah, I hope so. I think we need a strong CDC, a strong, you know, I think we need an agency like CDC to do the kind of things that CDC does. And I think there is still a good group of people there that are getting the work done. I think they're going to have to rebuild some things. Certainly going to have to rebuild the leadership structure because that was really affected. Well, so not just the U.S., but other countries are helped enormously in the past by CDC and outbreaks, right? Yeah. I mean, for measles, I was kind of on the domestic side. We also had a global health group that was working to support immunization campaigns and outbreak responses all around the world for measles.

29:59But I mean, you know, CDC also was heavily involved in, you know, the President's Malaria Initiative, PEPFAR, a number of these things that have all been seen some serious cuts. Yeah. Okay, Patrick, let's add a little bit. You had a nice article in Plas, I think it was Pathogens, three paradoxical paradigms of measles virus. It's a good way to frame our discussion. So let's touch on those briefly. One is, despite being an RNA virus, measles remains antigenically stable. Right. So most RNA viruses, you think of influenza or something like that, where every year you need a new vaccine, because it's constantly changing and changing because the plum race is very error-prone, whereas with measles, it seems to be very stable over time.

31:00We've been using essentially the same vaccine since it's created minor modifications. And yeah, so we talked about it a little bit in this meeting too, where it seems like you need multiple mutations. And for it to become evading the vaccines, you have to have multiple mutations in F and H and other. And it just doesn't seem to be mathematically possible to get all those mutations at once in one variant. Okay, number two is measles infection is immunosuppressive but leads to lifelong immunity against itself. Yeah, the concept is really amazing, right? Where you get measles and you have this immunosuppression and you have immune amnesia of a lot of other types of viruses.

32:03Whereas if you get measles, you establish a lifelong immunity. it's pretty really interesting how it can do that where it probably knocks down your memory T cells and B cells against other viral pathogens but it replicates in those populations to change the distribution of viruses or change the distribution of antibodies away from all those other pathogens you've seen and so now you've replace them with antibodies against measles specifically. Okay. And number three, which you've kind of touched on, measles is a respiratory virus with a respiratory epithelia, or not the initial site of infection?

32:50Yeah. Yeah. I think that's pretty interesting. It's a respiratory virus. Certainly, everybody knows it's a respiratory virus, but it initially infects through immune cells, potentially dendritic cells that are sampling the lumen and then it carries it back to the lymph nodes where it amplifies and then spreads systemically and then ends up in the respiratory epithelial and that's where it leaves from. Okay. So I want to talk a little bit about you use immortalized human airway epithelial cells and you make air liquid interfaces. I guess you can use immortalized or primary tissues as well, right?

33:36Yeah, you can. For most of our work, we use primary way epithelia from human donors. So we have a donor network. So there's a few places around the country where people donate their lungs either after transplant or after they die. And then we get their lungs and we collect epithelia by enzymatic dissociation from the trachea and the main stem bronchi. And then we seed those right on the filters and expand them. So these are P0, unpassaged primary airway epithelia that polarize an air-liquid interface and they form basal cells, ciliated cells, secretory cells. They have mucus, they have ciliary beating.

34:27Yeah, they recapitulate the conducting airways really well. And you could do that with other immortalized cells as well where you can get them to polarize. But we found that the concept of EMT, this epithelial mesocondonal transition, is really important in recapitulating how measles infects epithelial cells. And it's kind of a spectrum from epithelial to mesenchymal, and we think it's pretty important for it to be skewed pretty far towards the epithelial side. And it seems like the P0 primary epithelial are best at most epithelial like. We'll talk about that in a minute. I wanted to talk about mitochondrial targeting.

35:16Was that done in these interface cells, the mitochondrial targeting? Yes? Yeah, that's right. So mitochondrial targeting by measles and protein, how did you get interested in looking at that in the first place? So a lot of that I can credit my graduate student, Laurel and Durnell, who kind of figured that out. It's already been reported that it's something we observed in these cultures as well. So if you look at the cells under electromicroscopy, the mitochondrial will either, you see some mitophagy, you see some of these weird mitochondrial phenotypes. And that's something we observed a long time ago.

36:02In infected cells. Yeah, in infected cells specifically. And that's been observed by other groups as well. And so we were kind of following up on this mitochondrial phenotype. So we also found that in these airway epithelial cells, we don't see a lot of interferon-stimulated. We don't see any interferon so much being activated. These cells make a lot of interferon beta and lambda. But when you infect with measles, we don't really see a lot. But we do see some interferon-stimulated genes being upregulated. And so based on some other stuff in the literature, we observed that the measles gets in and causes C gas activation.

36:48And C gas is typically activated by double-stranded DNA in the cytoplasm. And what we found is that the mitochondria were, based on the mitophagy, a lot of this mitochondrial DNA was being released into the cytoplasm and it was the mitochondrial DNA that was activating C gas. And that was leading to the interferon-stimulated genes being upregulated even though the interferon itself wasn't. And so then she plugged the sequence, and she plugged basically the sequences from measles into this online predictor of mild-targeting peptides and found that the C-terminal domain of N protein had a pretty strong mitochondrial targeting motif.

37:49And so then we started following up on that, looking at different deletions, and we put the N portion of N protein on the GFP, so I was targeting mitochondria. So that sequence will direct proteins into the mitochondria. Yeah, yeah. Do you see that in infected cells as well? Yeah. So yeah, that's important. And so if you...interestingly, you know, I was like, how did this not get discovered a long time ago? But if you just take the full-length N protein expressed by a plasmid and transfect it into cells, it seems to go to the nucleus. And that was discovered, I think, by Roberto and others back in the 80s.

38:30But if you just take the N portion of N and put it on GFP, it goes to the mitochondria. So there seems to be conflicting targeting motifs perhaps in N in different places. And it's also thought that P involvement in binding N will also chaperone it, keep it out of the nucleus. Not sure about that research. So, yeah. And just to follow up on that, we, in collaboration with Virgo and Calpina, We made a couple different mutations. We thought of a key residue in protein and found...we mutated it into two different types of amino acids, one which completely made it an opposite charge and one that made it a neutral charge.

39:23We got two different phenotypes where one of them targeted to the mitochondria better and one of them targeted worse. And we predicted that both of them would be worse. So it was a completely unexpected result that we're still trying to figure out now. So can you make a virus that does not target the mitochondria? Yeah, we can. And it is attenuated a bit, and we're still trying to figure that out. So you have no idea why N would go into the mitochondria then? Yeah, well, it could be. That's kind of where it seems like it's very counterintuitive. Why would a virus want to go to a mitochondria and disrupt its energy source that it needs for replication?

40:12And, yeah, I don't really have a good answer for that except for maybe it's going there because that's where the energy is. And it's taking advantage of that region to enhance its own replication. Maybe it's going there. to it. If we think of it's budding intracellular, maybe it's taking advantage of mitochondrial membranes to do something as well. Because you say in the paper that the replication centers of the virus are actually adjacent to mitochondria, right? Yeah. So in primary and polarized, there with epithelial cells, all the mitochondria kind of cluster at the top of it. So it's very reproducible.

40:48And that's where we see coincidentally or perhaps coincidentally or non-coincidentally, that's where the measles is assembling its replication as well in the same region. So maybe it's deriving energy that way? Yeah, maybe. And maybe in doing so, it's damaging the mitochondria. That's why you get this ISG response. Exactly. But really, how could you get ISGs without interferon, though? Yeah, I've gotten that question before, too. I don't know. It seems like if you're getting the ISG upregulation, you'd start seeing more interferon production as well. And we hand-wavingly respond to that by, you know, V protein is also perhaps tamping down.

41:32So does this happen in related viruses, this N-targeting and the replication around mitochondria? Not that we've seen. It's measles-specific. So far. Okay. I'd like to know if it is something that's seen in NEPA or any sort of related viruses, but we don't know. Many RNA viruses do damage mitochondria, like I'm thinking of dengue, and then the DNA gets sensed, and that initiates an interferon response. But I don't know about the reason for being so close. It's interesting. So the other paper I wanted to discuss, you mentioned before, epithelial to mesenchymal transition and live cell extrusion contribute to measles virus release.

42:18So you mentioned in the paper that we thought about measles. So on the way out, measles does leave the respiratory epithelium, right, in contrast to how it gets in. And you say, we thought it budded, but maybe it doesn't bud. Yeah, at least in this model system, we see the, instead of classically, when you think of a respiratory virus, it comes in through the apical side and replicates and buds and infects it. But measles comes in basolateral, spreads cell to cell, And then these entire infectious centers peel off the epithelium. And we hypothesize that it's a combination of cell-associated and cell-free virus that you cough out.

43:01That could be partially explained why it's more contagious than other respiratory viruses, that it's both aerosolized as well as potentially contaminating surfaces for longer periods of time. So you're saying pieces of the sheep basically break off. Do you expel those? Yeah. Well, it hasn't been detected, but nobody's really looked carefully. I mean, that's something I'd like to look at more carefully. But in work done by others in experimentally infected primates, they detected both free and cell-associated virus. So there is still some free virus. you're breathing out obviously, right? So is that released from cells by budding or?

43:49Yeah, it's normal. Most of the virus comes out intact within the

43:57excreted cells, perhaps during the coughing process or some other process it breaks open. So this paper was done in these air liquid interface cultures, right? So when a piece extrudes, how many cells are typically in that? It's very variable. It could be

44:19hundreds of cells. It could be a few dozen cells. So is this a normal process that happens in respiratory epithelia that is independent of virus infection? Not so much. We don't really observe this with... Maybe if you had a chronic infection or some other pneumonia or something. But under normal circumstances, this wouldn't... Okay. So this is a virus-induced process. Yeah. And we specifically think it's a measles. We haven't really seen a sort of large-scale cell extrusion in any other virus that we've looked at. Okay. So it's presumably induced and facilitates dispersal, right? That would be the idea.

44:59So what is epithelial to mesenchymal transition? What does that mean? Yeah, that's a good question. It's typically associated with cancers where certain cancers you'll see transition from epithelial to mesenchymal. there are two different types of cell types or it's mysic mesocom or squamous there's yeah EMT is one of those things where once you hear about it you can start seeing it in association to all kinds of things and it's a pretty yeah there's no way I can possibly sum it up it's so complicated and it's There's a lot of different proteins that are involved in it. And there's... But if you do a search for it, it's most commonly associated with cancers.

46:01In researching it, I came to appreciate how much overlap sometimes there is between viral infections and cancers and how the cells respond to these kind of things. Does this happen in, say, gastrointestinal epithelial? It could, yeah. All right, so then there's another phenomenon. It's called live cell extrusion. Is that the same or is it distinct from the other? Yeah, so that happens all of the time where you'll have an epithelia and it'll detect something wrong with a certain cell. It could be precancerous. It could be some other thing. And the cells around it will react and extrude that cell.

46:42And normally it's a single cell that it will dislodge from the epithelium. But with ours, it's an entire group of cells that it will dislodge in response to measles. But it seems to be the same type of, based on single cell sequencing, it seems to be the same type of overall response that we're seeing. So you probably never did this, but if you take a person with measles who's expelling virus, can you find clumps of cells in what they're expelling? Yeah. We like to do that. We have a protocol in place, so we just need a big outbreak in Iowa to answer. I'm sure you'll get it. Yeah. Yeah. Yeah. We've looked the...

47:36There was a big outbreak in South Carolina, and our vaccination rates in Iowa are probably a little bit worse than South Carolina, so it could be a matter of time. So do you know how infection induces this extrusion? We know some of the proteins that are involved, but some of the pathways are still the black blocks. Because I know you mentioned sphingosine kinase. Is that a kinase that's involved in the extrusion process? Yeah, yeah. Yeah, it's...

48:06Yeah, it's a signaling process in which involves a lipid that can communicate to a neighboring cell. And is that induced by infection? Yes, exactly. Okay. So in that, you don't know how that happens, the induction of the enzyme, right? Not down to the granule level, yeah. So you'd like to, because that might provide some information on what the virus is doing, right? Mm-hmm. So does this answer the question of why measles virus is more contagious? Yeah, well, I think it contributes. That definitely is a mantra that we have in my mind, where we think that these formation of infectious centers are really important for explaining the contagious nature of measles.

48:50It may not be the only explanation. It's probably multiple things, but yeah. And you said this is measles-specific, right? Well, I heard and I've known before, so canine distemper is a good model in animals. Does that happen there, for example? In primary cultures, so if we take CDV and infect human airway epithelial cells, you don't see infectious centers because we don't see any infection. But if you infect ferret airway epithelial cells, you do see infectious centers. Oh, you see release of clumps like this. Yeah, we haven't done a lot of work with CDV and ferret epithelia, But the one experiment we've done, you see a similar phenotype.

49:31So these two things that we've talked about today, the mitochondrial targeting and the release of clumps of cells, are these things that you want to keep working on and understanding in your lab? Yeah, you bet. It's something we're understanding how the virus sets even spread cell to cell is still some key questions that we're trying to figure out. All right, so I have two more questions for each of you to wrap this up. So this one, I did a podcast at a university in front of a group of high school students, and one of the high school students asked me this question. I thought it was so good that I'm going to ask you.

50:12So, Paul, if you could message the whole world, if everyone could listen to you at the same time, and you wanted to message them about viruses, what would you tell them? This is a tough question, right? It's a tough question, yeah. Give them one message and they're going to listen to you. Eight billion people or whatever it is, right? Well, I think viruses are something we have to keep an eye on. They have a tremendous amount of potential to cause disease. and they're kind of, you know, for me, viruses are kind of things you love to hate because they can cause a tremendous amount of disease, but like at the meeting, just listening to these presentations, the way they work is just fascinating.

51:07Okay. Patrick, what about you? Well, I guess the first thing that jumped to mind was get vaccinated and get your children vaccinated. an important message, I think, for her.

51:21Yeah, and call your congressman and get the NIH more money. Yeah, well, we're talking about the whole world here, but that would work in the U.S., yeah. Okay, one more. Paul, what would you have done if you hadn't been a scientist? Yeah, I don't know. I was briefly in a pre-med track. That didn't go well. Once I started studying micro, I think I decided that's where I wanted to be. I don't know what else I would have done, to be honest with you. Okay, that's a good answer. It's fine. What about you, Patrick? I guess I'd be a woodworker. Woodworker? It's a hobby of mine. Yeah, I've had people say that, too.

52:08In fact, one of my former podcasters, Dixon Depommier, was a woodworker. He used to build cabinets. He was really good at it, too. Okay, so that's a TWIV at the Measles Minivirus Symposium here in Rochester, Minnesota. You can find the episode at microbe.tv slash TWIV. You can send questions or comments to TWIV at microbe.tv. and if you enjoy our programming, we'd love your support, microbe.tv slash contribute. I'd like to thank Roberto Catanio for having TWIV at this meeting. Really appreciate it. My guest today, Paul Roda, former CDC. Thank you so much for joining me today. It was a pleasure.

52:53And Patrick Sin at the University of Iowa. Thank you, Patrick. Thank you. 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. Thank you.