095: The Evolution of Virulence with Andrew Read

Coming up on this episode of Meet the Microbiologist. These vaccines with Marix disease are really not sterilizing. They make the bird healthier, so they stop the production losses in the death. But they do not stop the action of the virus. It's still able to replicate inside the birds and then transmit on. So what we would say is that they're very good anti-disease vaccines. But from the point of view of the virus, they're leaking the vaccines. They let the virus get through vaccinated hosts. Welcome to Meet the Microbiologist, a podcast that features the people behind the scientific discoveries. I'm your host, Julie Wolfe of the American Society for Microbiology. And I'm joined today by Andrew Reed, the Evan Pugh University professor of biology and entomology and the Eberly Professor in Biotechnology at Penn State University. Welcome to the show, Andrew. Hi, Julie. Nice to have you here to discuss a very interesting topic, the Evolution of Varylince. Our listeners may appreciate that Varylince is not a static trait, but can change over time. And the theory of optimal Varylince is one that posits that pathogenic microbes evolve with their hosts toward a commensal state, or at least one that has maybe a lower degree of disease. But today we're going to be talking about a disease that has greatly increased in severity over the past several decades. And the role that vaccines have played or may have played in this increase. Why don't you start by telling us, Andrew, about the disease that we'll be discussing called Marix disease, which is a disease of chickens caused by the Herpes virus, Marix disease virus. Yeah, it's a really interesting virus. As you say, it's a Herpes virus, which means it's a large DNA virus. And it's a very serious issue in the poultry industry. So these days, every commercial bird in the world was vaccinated against Marix disease. And when it was first discovered or described in the 1920s and onwards, it caused mild paralysis in the birds and wasn't much of an issue. But as the industry intensified in the 1950s and 60s, it became a significant cause of production losses, especially in broilers. Those are the birds that we eat and also to some extent in the layaboods. So when a chicken contracts this disease, what does it look like in the chicken? How does it die immediately? Does it have like a foul stench or how does it affect the bird? Yeah, well, so the ancestral case of the sort of 1950s virus, it infects the birds through the track-eth, through the breath of the dust in, feather dust in, which contains the virus. And then it invades the body and spreads around the body. And in the next few weeks and months, it would go on to cause small tumours. So it takes over the host cells and starts to cause them to replicate. And in some of the more severe strains, that tumour-causing process can lead to the death of the birds. Well, in any case, the way that the transmission works is that the virus gets into the feather follicle, so the base of the feathers, and it causes the cells there to replicate. And as there's a shed, they're full of virus. And that is what's spread into the world. So if you shake an infected bird, the virus drops out in all that dust, which means in an commercial operation, the dust, which is just everywhere, can contain millions, billions, perps, trillions of copies of the virus. And that's what's breathed in by the next bird. And so I guess this is kind of a strange question, but if the birds are dying of the tumours, let's say in their airway, does it affect the muscle, the meat of the boiler? Yeah, so actually the tumours can be everywhere, so they're not so much in the actual airways that they, but they are often in many of the other internal organs. So the bird can die of the tumours themselves. All the tumours can cause the birds to be what's called condemned. Every bird in the US, every commercial breeder, is inspected by a USDA vet. And if there are obvious tumours in organs, then the bird is not allowed into the food chain. So the losses from a production point of view can be the direct death or disfigured birds that don't make it to the supermarket. I see. So if a farmer discovers an outbreak of Merrick's disease among his or her flocks, does that mean that the entire flock has to be cold? Yes, it depends on how far advanced it is, but it can lead to the loss of the entire flock. And these days, that happens most often when something goes wrong with anisobundry or the vaccination. And in those cases, you can have the loss of an entire group of birds. And that can be maybe up to 100,000 birds in any one cohort. So that's a very, very big deal. That might be worth an order of a million dollars. Yeah, easily. So we're talking about the US, but I imagine that this is a disease that might be a found around the world since chickens are eaten around the world. That's right. It's almost ubiquitous. I don't know anywhere where there isn't Merrick's disease. There's some very isolated operations, but it's a very big deal. And Europe, the very big deal in the States, is big deal in India, Australia. Yeah, no question. And these birds in all these different countries are just about universally vaccinated the same way. That's right. If you don't vaccinate or the vaccine fails, so it's a cold chain vaccine. I see. And if the cold chain doesn't work, then the vaccine is not effective. Yes, then you can lose the entire flux. So let's start talking about the vaccine. Since that is the subject of today's talk, the first vaccine, the first iteration of the Merrick's disease virus vaccine was introduced in 1970, I believe, which halted outbreaks for about a decade before the disease resurfaced after which there was a second iteration of the vaccine. And yet again, the disease resurfaced. And so I wonder how the disease may have differed prior to introduction of the vaccine. You already mentioned in the 50s, it had a slightly different pathology and how the cycle of virus adaptation and vaccine iteration may have affected the size of outbreaks or the age of the affected birds. Yeah, so we need to be careful here about how we use the terminologies here. To me, this, the observation is that after about 10 years of vaccination with the first generation vaccine production loss has started to climb again. In other words, the virus was causing a sufficient disease in vaccinated flocks that it was starting to impact yield. So either the survival of the birds or the weight of the birds or the number that were condemned. So we got what was we would call that I think reasonably uncontroversially vaccine resistance by analogy to drug resistance. The virus was able to replicate and shed from vaccinated birds and start to cause significant disease. That's the second say second the birds enough that it was causing issues. I think it's important at this point, maybe to say that the vaccines themselves are live attenuated viruses. So the first generation vaccine was a strain of a related herpes virus that comes from turkeys. It's called HVT and it worked very effectively, but only for 10 years or so. So in the second generation vaccine was a two strain vaccine, which was the HVT. So the original one plus a new one, which came from a second serotype. But again, it was alive strain. Both of those generations of vaccines, they produce virus that is actually shed into environments. So vaccinated bird does shed vaccine strain virus. So there's turkey strains or turkey viruses where they modified in some way before they were used as vaccines. No, no, no, very interesting. Just people just tried different strains of HVT, a turkey virus that they've used one called HVT work just fine. Okay. There's actually quite a triumph because for those days, because really there was huge losses going on in the industry. Some people say between 4% and 10% of broaders were not making it to the food chain. And so, you know, very large amounts of money involved and very quickly these vaccine strains were found and the vaccines were rolled out across the US and Europe. And the problem was essentially sold for as I say a decade or so before the problem started to come back. And I imagine when the problem started to come back that everyone was like double checking that cold chain to make sure that there wasn't something wrong with perhaps the virus delivery system or some other factor other than the fact that the vaccine was not conferring protection. Yeah, that's right. And in fact, it wasn't really until the 1990s that there was to my eyes, exiled data to show that it was the evolution of the virus, the wild type virus that was causing these losses. That was suspected reasonably early on. So by the mid to late 70s, when people started to see this, they were suspecting that these were vaccine break strains that were starting to spread in these vaccinated flocks. And in the 90s, that was demonstrated experimentally by injecting wild type strains into vaccinated birds and looking at the, you know, what the outcomes were. And no question that the virus itself, the natural virus had evolved to be able to get around this vaccine induced immunity. Oh, how interesting. And so the timeline would be approximately 10 years after the first generation. And was it about 10 years after the second generation where those two strains were used as a vaccine? That's right. So for a while, it was starting to look like it was going to be every 10 years of the vaccine would need to be replaced. But the third generation vaccine has been holding now since the 90s and it is the reason that the vaccine has been holding now since the 90s. chicken nuggets are so cheap because it allows the production on the scale. It's difficult for people who are not familiar with the poultry industry to appreciate that there's serious challenges to raising birds which are you know the same age, the same immune status, same parental genotypes, raising 30,000 of them in a big barn and maybe in four or five barns on the same farm. So you get a hundred thousand birds that are more or less identical and you know virus gets loose in that sort of setting and you know bad really bad things can happen. So the only way you can have an industry this intense is by vaccination and good hygiene. And so in the 90s what was that third generation vaccine? Is that yet another strain that's been added to the two previous ones? It is and it's called a RISPEN strain. It was discovered in the field in the Netherlands and it's the same strain as the wild type virus. So this was the first time of vaccine from the same serotype was used and we now know that this particular strain works very well because it's got a big deletion in the genome which has removed several genes which are responsible for some of the virulence away in which the birds get sick and so it produces a live infection that's a very benign infection and so the birds get immunized by this strain and subsequent infections of wild type virus are not nearly as nasty. So if the birds are infected by this attenuated strain and are shutting it does that help to produce protection within the flock as well? It does although actually it's a little bit unclear. We know that the wild type viruses get shed even from vaccinated birds and so the virus is that's true for all three of the vaccine generations. The viruses can get through vaccinated birds and shed into the wild but the viruses seem to be inducing so that means they're not inducing sterilizing immunity like we get with for example smallpox or polio or measles or something where when we get out of response to those vaccines if they work and we we do matter response then we don't we can't get infected with those viruses again and if we do we certainly they don't go anywhere and they certainly don't transmit through us. So once you've had measles for example you can't transmit measles in the future you don't get it again and you're out of response but that's sterilizing immunity. These vaccines with Marix disease are really not sterilizing in that sense at all. They do protect make the bird healthier so they stop the production losses and the death but they do not stop the action of the virus it's still able to replicate inside the birds and transmit on. So basically it keeps the bird from having any symptoms or disease from being infected but the bird is still itself infected. That's right so what we would say is that it's a very good anti disease vaccines. I see but from the point of view of the virus they're leaking the vaccines they let the virus get through the vaccine. So it sounds as though there might be specific merit disease viruses or similar types of viruses for every either foul or perhaps types of birds that exist. Is that the case or are there other I guess my question is is there a reservoir in the wild for this particular virus? No so Marix disease virus has been detected in wildlife geese for example it can get into some turkeys so far as I know only turkeys on poultry operations. The wildlife side of it is probably just carriage and it might not be very well supported. No I think this is really a virus of the poultry industry. I see. We don't know very much about what it whether it's present where it came from and we don't know whether it's present in ancestors of domestic chickens which are jungle failed Southeast Asia but these days the virus is definitely a problem in the poultry industry and is not known to be a significant issue anywhere else. I see. So during those times very few outbreaks are there any hypotheses about where the virus went or how it was contracted again if for example it was such a great vaccine in the beginning. Yeah so actually we don't know too much about that because the vaccine was the vaccines were generated the first ones were worked on in the 1960s and so we went into widespread use as you said in the early 1970s late 60s and that meant we had wild type virus and vaccine type virus coming out of the same birds and so nobody knew nobody in those days could distinguish the viruses. Right right and so it wasn't until a molecular era and in fact in the last 10 or so years it's become possible to distinguish the virus that's from the vaccine from the virus that's from the you know the naturally circulating virus. I see. The folklore in the 1960s was that the virus was everywhere and then after the vaccines came in the assumption was that it was still everywhere but just the birds weren't getting sick but nobody actually knew and we and a few others have been doing big surveillance projects looking for where the virus is and at least now we know in central Pennsylvania at least that it's on about 30% of the farms at any one time and on some farms it's continuously present so month after month co-hortabirds after co-hortabirds for several years we find it there all the time and on other farms it seems to die out at least we can't detect it and then it's reintroduced maybe a month or three or maybe six months later so it looks like some mixture of dying out on farms and reintroduced introductions and some mixture of some farms that might be the reservoir farms. I see perhaps they have the taffy-dmarie of chickens that are on those those farms. So it might be that although in our hands in our area it looks more like it's the farms that are not quite so well maintained. Oh I see so perhaps something to do with cleaning or decontaminating between flocks or something like that. That and how the birds are being housed during the flock time so there's variation in humidity and air flow and so forth and if the house is what seems to us to be slightly more unclean and that's really meaning reduced air flows and high levels of ammonia and things in the bedding then we seem to have this persistence. If the company is maintaining the farms at the best possible conditions that's where we see them the virus dying out and being reintroduced. I see and the virus you said it can be found in the feather dust it's it's shed by the chickens. How long does it last in the environment? Is it stable for a long time? Yeah that's one of the most amazing things about this forest. It is we don't know how long it lasts but if you take a bunch of dust for an affected farm and just leave the dust sitting on the lab shelf for a year it's still infectious. So it's completely stable even without being frozen and this is probably because the virus particles are inside the characterized cells that are being shed off the birds so they're really wrapped up inside this very hard layer. So the only really good way to get rid of it in the from a farm is to bulldoze out all the dust and then and the bedding and so forth and then really steam clean the place and get rid of the dust you can get it down but you know you can imagine the situation 30,000 birds generates a lot of dust. Yeah and they're not always cleaned out between co-words it depends on where on the world but the economics are such that often times in the next co-order birds just comes into the bedding that was left from the last coat. Right of course that's very interesting that they are thought that they might survive inside of the epithelial cells because a herpes virus I believe it's an envelope virus right? It is and it's very interesting nobody really fully understands how it's working it is a very interesting question that the persistent sort of it. Yeah. Have you filled out our survey yet? If not I'd really appreciate getting your feedback. Special thanks to those of you who already have filled out the survey but we need even more data so that the MTM audience is truly represented. You can tell us what you like but you don't like or what parts you want to hear more of in our listener survey. You can even fill out the surveys you listen to the rest of this interview. You can use whatever device you're currently using be it phone, tablet or computer by visiting ASM.oritz. Org/MTMpool. That's asm.org/MTMPOL. A big thanks in advance. I really appreciate it. So previous to your work with Marex disease even before the disease that we're talking about you had predicted that an imperfect virus such as one that does not confer sterility might facilitate virus evolution using mathematical modeling. So how is Marex disease and chickens a good case to test some of your models? You're right. The theory came first and the thinking here was that if you ask why is a virus or any other pathogen not more nasty. You know why doesn't it kill more host as quicker? And in the limit the answer has to be that if it kills its host it kills itself and therefore it's got no evolutionary future. You know at some level that has to be true. And so if you buy the argument that that is the main reason why super hot strings of anything don't exist or at least don't persist then the question becomes well what happens when you vaccinate a host and keep it alive now? So if the host would have died from the super hot strain but you now keep it alive then that means that the super hot strain is not wiping itself out it's able to persist and transmit on. And so in the late 1990s and early 2000s we developed mathematical models of that process. What would it be like if instead of these super hot strains killing the host and therefore themselves instead of that they were vaccinated and they couldn't kill their host but they could still transmit what would happen and those mathematical models showed the essentially you would get the evolution of more virulent pathogens in vaccinated hosts and that means that they would those hot strains would do more damage to a host that hadn't seen the vaccine. That's right. So that theory was, you know, we laid that out. I was very controversial. And then oddly, I was on a grant panel in London in England at the time, every six months we would meet. And I was talking to one of the other panel members about this work before it was published. And he said, "That's what happened in Marrix disease. This was in the late 90s, so there's no internet type things going on there." And so I wrote that down and went to have a look at it and what we used them, which was a zoological abstract, a big book that you'd look through. And I couldn't find anything. And then the next month later, I said to my car, funny, "What virus is it? Turn that, I'd spelt it wrong." Oh, so you were looking it up in the abstract car? Exactly. And we went over and went back. It's sure enough. People had said, some vets had suggested that the Marrix disease virus was becoming more virulent in vaccinated forks. And at that point, I did an exhaustive literature search, so that was around 2001. It looked to me consistent without hypothesis and so consistent with our predictions. But I couldn't persuade myself completely of that. So in the first theory paper, we just had a single sentence saying this may happen in Marrix disease. But then I got out of the back of that single sentence. I got invited to give a talk at one of the Marrix disease congresses, which was in Oxford, thinking 2002 or 2003. As an outsider, they just wanted an outside perspective. And so I finished up my talk by saying, "If I was you guys, the experiments I'd be doing are the following." And I laid them out. And then somebody got up at the end of the meeting and said, "I'm from one of the UK funding agencies. If you work with that guy over there, we'll fund you to do that work." And so obviously, I went and talked to that guy over there and that's everything flowed from that. Actually, the funding agency, that particular one, couldn't afford the experiments. They turned it to be way more expensive than anyone thought. So we had to get the money through regular means. But in any case, that's how we got work on Marrix disease. And so, you know, whatever that is, 15 years later, we're still going. I see. I see. It's almost like you give a grant talk and not just an invited outsider's perspective talk. That's right. That's why you should always give your ideas, really. Well, especially with that Marrix disease community, I have to say, has been one of, was very, very welcoming to outsiders and outside perspectives. And they are very broad-minded group of people. But I think that comes from the fact that many of them are vets. And vets have to do with many diseases and they have many problems. So they don't sort of feel ownership about things and perhaps some other ways that Medics do. Right. Right. Really looking for a solution, especially with something that affects the industry in such a major way. That's right. And there's also no Nobel Prizes in veterinary diseases. So that's right. The ego is a smaller in the applied issues are bigger. So your next step was to do the series of experiments that you would lead out. You care to explain, Embry, what those were? Sure. They actually took us an enormous little long time to do them in the order of 10 years to get things right. Because you have to work with these different strains in isolators or in very contained facilities. And so there's not very many places in the world where you can do these experiments. The person I was talking with was a Venu Nair who is a virologist working in the Institute of Animal Health in England. And he had the access to these facilities that we still had to do a lot of work to develop them. So essentially what we did was we took a handful of strains that defeated their virulence. And so the nicest strain we had was isolated in the 1960s. And it did kill about half the unvaccinated birds. But it took several months to do that. And then the other end of the spectrum was a highly virulent strain that killed every bird in 10 days, every unvaccinated bird in 10 days. And so that more or less captures the virulent spectrum. 50% case fatality rate after two months versus 100% case fatality rate after 10 days. And just to put that in perspective, there are very few human diseases that have anything like that case fatality rate. And as SARS was 15% or so, malaria is maybe 1% or so. You know, bad year for flu can climb a bit up about in a few percent or so. But there's almost nothing that is as nasty as these strains. Right. Rabies can kill 100% but it's a spillover from other areas. Yeah, it's not contagious between individuals. Yeah. So these are very lethal strains by any imagined sense of imagination. But the actual spectrum is, you know, from what we would call a nicer strain through to these high pathogenic strains we call them. And so then that we, what we wanted to know first off was whether or not, you know, what would happen to those strains in vaccinated hosts. So we used the first generation vaccine. And the vaccine is completely protective against the lower end of the virulent spectrum. And then again, it's the higher end of the virulent spectrum, we would start to see some vaccinated birds dying. But that gave us a spectrum which showed that the vaccines were work. The first generation vaccine was working, but not quite so well in the with the very hot strains. And then we, with these isolators, we collected dust and looked at the amount of virus being shed. And what we found was that in unvaccinated hosts, the nicest strains, the least nasty strains, they shed the most. And that's because the hot strains would kill every bird in 10 days. And then there would be no more virus shed because the birds would, whereas the nicer strain would keep the birds alive for 60 days or so. And so the birds would shed virus for, you know, two months, which wasn't happening with these hot strains. And that difference was billions of viruses. So it's enormously big difference in the amount of virus shed into the world by a bird when it's not vaccinated, which it's got too hot a strain, it just simply is not trying, you know, it's not shedding. Right, because it's not alive. That's not alive. Exactly. And then when you vaccinate the birds, the virus happens. So the vaccine bears down disproportionately hard on the viral shedding of the less virulent strains. So they really get the shedding into the wild, it's really suppressed by the vaccination. Whereas the hot strains, those vaccines keep the birds alive. And then they shed this hot virus for, you know, two months. And so it turns it completely on its head. If you vaccinate, then what you do is you give the world over to the hot strains. In an unvaccinated world, but the hot strains can't spread because they're simply too hot. They kill their host too fast. And so there's no transmission. Whereas in a vaccinated world, the vaccines keep those hot strains going. And the birds alive. And so then they shed millions, billions more copies of virus into the environment. And so those hot strains are making as many progenies if they weren't actually not quite as virulent. Yes, exactly. And so we went on to do actual transmission experiments. One of the great things about chickens is that the chickens themselves are super cheap and you can do lots of transmission experiments with them. So long as you've got the right facilities within which to do them. Right, right. Yeah. So what we would do, we co-house birds that were infected with sentinel birds and look at the fate of the sentinel birds. And what we found was that in direct, those direct transmission experiments that the super hot strains just didn't transmit at all except if they were in a vaccinated bird. If they weren't in a vaccinated bird, they just killed their host so fast there was no transmission. But if they were in a vaccinated bird, then they could spread from that vaccinated bird into the unvaccinated birds. And those birds then died. So one way putting that is that if you vaccinate the birds, that kills the unvaccinated. It makes it possible for the transmission to take place. So in other words, the vaccination allowed these super hot strains to spread in a way that would not have been possible otherwise. Right. It sounds like they would have to be naturally circulating both these hot and perhaps not quite as hot strains. Yeah, that's right. Infect the vaccinated bird, but given that even both strains, both of those strains infected a vaccinated bird, the hot strains would have the advantage in that vaccine. That's exactly animal. And a vaccinated, our experiment showed that in a vaccinated world, the hot strains, these hyper pathogenic strains can spread and they wouldn't be able to spread in an unvaccinated world because they're simply too lethal. And I should bring up again that we're talking about a chicken vaccine that is non-steralizing. And you mentioned already that most human vaccines are sterilizing. Yes, that's right. I think it's really important to realize that the reason you can get virus evolution in these settings is because the viruses are able to replicate and spread from vaccinated birds. So that makes evolution possible. If they were, the vaccine was better and it was sterilizing like it is for measles or mumps or whatever in humans, then there wouldn't be any evolution because any host is just any vaccinated host is just a dead end. There's no spread from it. So this is really a feature or something that you might expect to happen in settings where the vaccine isn't good enough to stop onward transmission of wild type pathogen. And so I don't think this, any of this has any relevance to many of the human vaccines that we think of, polio and smallpox and measles. And so for the classic childhood diseases of humans, this is not relevant because they're also sterilizing. Right. The issue is about non-steralizing vaccines. And we are now in the business of trying those out and so there are non-steralizing malaria vaccines, there are non-steralizing typhoid fever vaccines, eating hoop and cough, patussis, those vaccines now that we use seem to have a waning immunity. So they're not sterilizing in the long run. So the question is in my head, and that's why I'm so attracted to this Marix disease system is what can it tell us about what happens when we use vaccines that are non-steralizing? And that's to me is why this is a general interest. So I happen to say now that I know a lot more about the poultry industry. It is a very cool and exciting business in its own right. Oh certainly. Now you mentioned already that this was a somewhat controversial idea. And even some of your ideas have been met with people who say, well, what about cases of human non-steralizing vaccines such as as the Salk polio vaccine. Do you think that that would be a different case because there are so many other factors including two vaccines being used at once? - I think that's exactly what I think. And also the situation with polio is slightly different anyway 'cause the virulence comes about from the invasion of neural tissue from which there's no transmission. So it's not obvious that polio virulence is under the same selective forces as many of these other things where the disease symptoms are part of the transmission process as they are in something like where you sneeze and cough and so forth. Those things aid transmission. Whereas polio is a bit different I think from that. But I do think that we have something to say about what's happening with pertussis with hoop and cough. And I think there's quite good evidence that the pertussis bacteria that causes hoop and cough is evolving and some of the in response to vaccination and some of that evolution is taking place in genes, bacterial genes that are associated with producing bacterial toxins. So I mean, there's a lot of way from understanding that story and it's complicated 'cause there are many genes evolving. But I do think that there is things going on that we minimally we should be looking out for in those settings. - The pertussis example is a good one. Why don't you explain that a little bit more how the vaccine changed not to be a non-sturalizing vaccine but to be a subunit vaccine and that may have affected evolution of the bacterium itself. - Yeah, so we used to have the hoop and cough that pertussis vaccines used to be killed whole cells. So the bacteria were killed and injected into people. And that mean you could got thousands of antigens and every bit molecule that was exposed and that dead bacteria was antibody responses were raised to those. But they also had issues with adverse effects. And so in order to try to give vaccine which had fewer side effects, there's a movement to the acetylivaxine which is made up of a subset of antigens and is much more purified. And that vaccine does not seem to be as immunizing. In other words, the immunity does not seem to last as long, doesn't give the lifelong immunity that you get from pertussis natural pertussis infections or from the whole cell vaccine. And so when you first get the acetylivaxine that does seem to be sterilizing but over time and that's in the order of a decade or so or bit more, it seems to move to being less sterilizing. And so that's one of the issues that we see these days that you can get outbreaks even vaccinated populations because the people are returning to being susceptible again. And in that process, we've got leaky vaccination happening. So that is a setting where I do think some of these things can go on. And I was very controversial about what the evolution of the bacteria of the pertussis bacteria in this setting actually means. But there are some situations where some of the antigens that are in the vaccine now are being lost by the pertussis, the wild type pertussis bacteria. So there's evolution going on of that sort to make one of the most interesting parts of the story is the pertussis toxin, which is toxin that's produced by the bug and the toxin seems to be good at downregulating immunity as immune cells come up to the bacteria. It inactivates those. And it also seems to be quite helpful from the digestion of host tissue. So it starts to break host tissue down the bacteria and access nutrients and things. So the toxin is what one of the things that makes us sick. And if it's an immunosuppressing toxin, it's pretty interesting question, well, what would natural selection favor when the bug is not being killed by the immunity and it's producing a toxin, which can help it survive the immunity more, or maybe it should produce more toxin. And so if it did that, then you'd have a bug that was producing more of the thing that makes us sick. And there is some evidence from Australia that that's what's happened. There's controversial evidence. I don't think there's any doubt that there's evolution taking place. The fact that the strains might be more virulent than previous generations, that's a bit controversial. And I think this is one of those situations where we need to watch what's going on. Right, right. None of this incidentally is an argument against vaccination because obviously you personally, if any of this episode has happened, you personally are much better off if you've vaccinated. Right. In any of these situations. On a chicken farm especially, you know, the bird doesn't get vaccinated and gets exposed to these hot Australians in its inter-interimals situation. So this is a strong argument in favor of individual vaccination. You know, you just should do it. And so this is definitely not an issue to be misinterpreted by some of the vaccine resistors that, you know, we've got on the wrong end of some of that, the websites and stuff. Sure, sure. But it's definitely not an argument against vaccination. It's an argument that we need to understand what's going on and try to either make better vaccines or try other approaches to, you know, hygiene or whatever, to try to deal with some of these things. Absolutely. Yeah. So I always constantly amazes me. And it's especially true with these birds. You know, the 30,000 birds get injected just before the eggs have to inject into the eggs. They inject a little colorless fluid. And then that keeps the birds alive in the fuzz of these diseases. It's just amazing, you know. Really nice. Absolutely. So we've been talking about vaccine resistance. But you also brought up earlier antibiotic resistance or antimicrobial drug resistance, which I'm sure many of our listeners are not less familiar with. But they vary quite a bit, for example, in terms of when you would give those therapies, right? And a vaccine usually comes before infection. Drug therapy comes after. So genetic diversity of the infecting microbial population would also differ. And so I wonder if you could talk a little bit about how these different types of resistance vary based on the types of a selection that they might affect. Yes. So it's really interesting if you look at the drug resistance problem, antimicrobial drug resistance, pretty much every bug that gets attacked by our drugs has evolved resistance and often quite quickly. So sometimes, you see the first drug resistant bugs within a few months of the release of a drug. But that's very rare in vaccines. And the vaccine resistance where you see this replication of the target pathogen in a treated host, that's rare. And that's one of the reasons why I find the Marrix disease situation so interesting, because it is in a case where that happens. And it's generally not the case in other bugs. And that seems to me to be very interesting. And as you say, I think the difference there, the reason we see lots of drug resistance and we don't see much vaccine resistance is-- well, several reasons. But one of the issues that you're talking about that the vaccine induces a protection which attacks the very first few particles of a pathogen that are getting into a body. And so that can even be a handful of particles. Whereas when we hit them with drugs, that's typically when we're sick. And we might have, I don't know, even a billion parasites in a human body while the time they start taking antimilarial drugs. And so there's just a huge amount more of genetic variation to act on in that case. And the other thing is that even if a semi-resistant vaccine resistant bug gets into a vaccinated person, it's often cleared by immunity long before it's had time to get up to transmissible densities. So I think the vaccines, because of their prophylactic nature, have quite a lot of advantages in terms of being evolution proof compared to antimicrobial drugs. So you work on other aspects of virulence in many different types of microorganisms. And I would love to be able to talk about all of them, but we unfortunately don't have time. But I would like to talk briefly about your work with mixoma virus. This is apox virus that infects rabbits and was isolated from South American rabbits, I believe, but released on purpose to control the European rabbit population in Australia. And this was several decades ago in the 50s. And this was almost uniformly lethal for the European rabbit population at first. And then over the years, decreased virulence selected for less virulent isolates. Is that the story more or less in a nutshell? Yeah, that's right. So the virus, the mixoma virus, was discovered in South America. And it was lethal to European rabbits. So if you take it out of the rabbits that it's found in South America, which is a different genus, succident to European rabbits, then 100% of them die. And in the 1920s and 30s, Australia, especially but some extent Europe as well, was being eaten out by rabbits. And so the Australians got very agitated about finding a biological control solution and released this virus in the end of 1950, actually to escape from quarantine facilities. But then it spread over the next six months over about a million square miles. And so all of the southeast of Australia and estimates vary in the number of rabbits that die. But it was somewhere between 10 million and 100 million rabbits that died in that first six months. And it's very hard to concede. I think that's probably the biggest die off of any disease, at least of a vertebrate, in that short space of time. And the virus itself that was released, that killed 99.99% of rabbits. If you can believe it. And over the subsequent decade or so, it evolved to be less virulent. And you can easily see why. I mean, it was like these extremely hot Marrix disease strains. It was killing almost all of the rabbits. And so any virus that was a little bit more benign would allow the rabbit to stay alive long enough for the transmission to happen more effectively. And it's a mosquito and fleaborn disease. And so keeping the host alive a bit longer, had some advantages. And Frank Fener, who was an Australian and a microbiologist, he, as soon as this thing escaped, he got. very interested in this and set up, switched his whole career to work on the POX viruses and set up experiments to look at what was happening to both the virus and the rabbit. And he showed that the virus was evolving lower virulence and he showed that that was because the strains that were less virulent and had longer transmission windows. But he also showed that if they were too benign, then they didn't get high enough densities in the rabbit and wouldn't be transmitted either. And so it's become a textbook example of evolution of virulence. But if you're too hot, you kill your host and therefore no transition. And if you're not hot enough, the host is all over you and you don't ever get high enough densities to be transmitted. It's a real classic piece of microbiology is beautifully demonstrated by Fender in the 50s and 60s. But he also showed that rabbits became more resistant to the virus. And you can imagine if 99.99% of rabbits die, the 1.1% or 0.01% that survives is going to do what rabbits do and breed like crazy. And then you're going to have resistant rabbits really fast. And that is what happened. And Fender demonstrated that brilliantly. I knew all about this story because it's a textbook classic. And while I went over to the States about 10 years ago, a number of us were sitting around in the coffee area discussing this case. And it was all happening before all that work happened before the genomic era. So the question was, well, what genes changed in the virus? What evolution happened in the virus? And we teamed up with Peter Kair who is one of the last people in Australia working on Mixomatosis virus. And he had a whole bunch of Fender's original isolates and a whole lot of others that he collected in the field. And so we sequenced these things and then we worked for the last few years. And incredibly, we still don't know what genes are responsible. So even now we can't tell this dramatic changes in virulence. Even though we know they're in the viral genes and you can see the string differences in the lab, we still can't find the genes. And that's because it looks like there's many different ways that the virus has modified its genome and every virus seems to have its own story. So it wasn't just one gene that spread across Australia. It seems to have been one mutation. It seems to have been different stories in different places. And some of those might have been in response to the changing rabbit genome that perhaps wasn't saved. And that's right. And although Peter did some very nice work in the late 90s to show that identify some of the genes and the rabbits that had risen in frequency and the rabbit has mounted an increasingly strong innate response. So it's able to kill wild type viruses more effectively just as they're entering the rabbit from the mosquito or the flea. And so does this the next summer virus, does this similar to Merrick's disease virus cause 100% death, why not infects rabbits? They are. So it did when they released it. And now in European rabbits, it still does that. But in the field, because the field rabbits have become more resistant, then the death rates are lower now. And the virus typically persists in a rabbit for longer. It is still a very bad disease if you're a rabbit, but it's not as bad as it was in 1950. I see. I guess my question is, does it cause asymptomatic infection that anyone has detected? Yeah, no, it's never become that nice. And so in order to transmit it still needs to make lesions and poxaws and so forth. And so if it's symptomless, it never hits high enough viral titers in the bloodstream to be transmitted. What we discovered accidentally during all this work was that the strains have actually become actually nastier than ever before. So if you assay viruses in a lab rabbit in the 1950s when they released that virus, it killed all the rabbits, but it took in the order of two weeks or so to kill all those rabbits. Now it kills all the rabbits, but it does it much faster. And so the strains that have come from Australia and indeed Europe in the last decade or two, those strains kill rabbits faster than the original ones. So it's become even more lethal. And the reason, well, what we find associated with that is the rabbits aren't dying of a mix of a classic mix of a toses anymore. They're not dying of lesions and all sorts of things associated with the virus. By dying what looks like toxic shock. And it looks like the viruses are so immunosuppressive. As soon as they get into a European lab rabbit, they shut down the immune response of the rabbit and all the bacteria in the rabbit go crazy and the rabbit dies from just gross invasion of bacteria and draw sorts of organs. Yeah, we were really shocked. The first time we saw it was actually, yeah, was a very big, very, very big surprise. And in fact, it was some shock that maybe there's some other virus that got into the animal house. But we now know that it's these strains from the 1990s and 2000s are super, super immunosuppressive. So we think our interpretation of that is that as the rabbit became more resistant, the virus fought back by becoming more immunosuppressive. And so we have an escalating arms race with more resistant rabbit being overcome by more immunosuppressive virus and then you need a more resistant rabbit and so forth. And so one of the long term questions is where is this arms race, this escalating arm race going to finish? How's it going to stop? Because it's not the observation you see in the field. You know, the world is not filled with highly lethal pathogens killing everything all the time. My speculation is that at some point the rabbit's going to come up with a solution, an immunological solution, which is going to stop the virus because there's always going to be selection for resistance in the rabbit because this thing has to be pretty nasty to transmit. So there's always going to be strong selection for resistance and the virus is going to have to be stopped to end up with a situation that might be an odd in end to the arms race. And maybe that's what's happened in South America with the natural host. Maybe the natural host has contained the virus and now it's at a very low grade systemic thing which doesn't cause many symptoms. Yeah, that would make an interesting parallel study. We'd also love to be able to take these viruses, these super hot viruses and find out what they did in the natural host in South America because it may be that they have got solutions to the European rabbit but the South American rabbit has. It can outdo them. Sure. The very fast generation time of both the virus but also the rabbits has allowed all these really amazing studies. Exactly and I do think that's one of the terrific things. I think the other side of it is that because it's a wildlife disease and in the poultry case, a farm animal, the experiments we can do where you are inoculating animals, vaccinated or not or wild type or not, with virus, those are possible and we can't do those studies obviously in humans. Right. You know what the rabbit study makes me think of almost as Ebola virus. It's not a perfect parallel but it reminds me of the virus jumping from one distantly related species to establish itself in a new population as Ebola does when it jumps into people. I think that's a very interesting question and Ebola at the moment we managed to stamp it out. There's some suggestion from the very big outbreak a couple of years ago that there were viral, there was virus evolution going on towards increased virulence. That's controversial and it was really only one mutation but it's a question to ask. So for example when SARS popped into the human population it was killing 15% of people and the question is if it had stayed there so if we hadn't been able to stamp it out, would it have become more or less virulent? And I don't think we know the answer to that and there's no reason to think it would have become necessarily less virulent. That question is being asked of HIV these days, so HIV jumped into humans and there are people working on the evolution of virulence and then HIV that's the time to AIDS. And there are people making the same sorts of arguments that I've just made for if it's too nasty so that means the viral set point is too high, it'll progress to AIDS too quickly and then there'll be fewer secondary infections, per infection than if the thing is a bit nicer. But if it's too nice then the host will be able to contain it properly and you'd never get any transmission. So there are people making the same argument in that. I do think it's a fascinating question when pathogens jump species boundaries, what happens next and obviously that's a highly important question for many settings and agriculture and in human health. Yeah, absolutely. So Andrew, you're originally from New Zealand making a wild gas here based on your accent but also I did some research and I saw that you had studied zoology which also emphasizes evolutionary relationships. So I wonder when your interests shifted from evolution of macro organisms to that of microorganisms? Yeah, when you're in New Zealand it's not interested in evolution because all the animals are so different from everywhere else in the world and so it's like an evolutionary laboratory. It is. I went to do my PhD on bird evolution pretty much prompted by that question about how the birds evolve. While I was doing my PhD I became interested in sexual selection which is essentially the question of why would something as stupid as a peacock's tail evolve. Obviously it's putting a bird at a bigger risk of being eaten by a predator and Darwin's answer was that females prefer those big sexy tails and then the question is why are they sexy? Why does she prefer them? And the going theory in those days was that it was a way of indicating health. Females want mates who are going to be healthy so that they offspring are healthy and we used to in those days measure bird health by looking at blood parasites in the birds and many birds including around here. American Robins for example, about half of them have a malaria like parasite. And so I was very interested in that side of things and then it dawned on me towards in my PhD that nobody was working in those days on the evolution of the actual pathogen even though in the malaria context that it was people and happens really fast and so you can see it in the laboratory and you can work with it and you can't do any of that with the bird evolution but you sure can with the parasite evolution. Yeah, absolutely. So that's where I got to pathogen evolution. Now I can't imagine why anybody does anything else. Yeah, well there's so many interesting questions to ask in this field. Yeah, I mean it's really deeply interesting evolutionary biology and it's many of the phenotype, stroke resistance, and vaccine escape and things, make a lot of people sick. So we have a lot of early career scientists and people who are interested in entering the field listening to this podcast. I wonder if you have any advice before they enter their careers? I guess I would say that the number one thing to do from the point of view of career development is make sure you get into a good lab and there are good labs all around the world in this space and that's a really important thing to do. I mean find the right place. For me I would say I took a while to get to some of the applied issues even because I used to think that they were mostly under control by people that clinical microbiologists and so forth. But actually I think there's a lot of evolutionary biologists have to offer in this space. And so it's worth the effort of trying to figure out the veterinary terminology or the medical terminology. It's worth trying to learn another field in order to see the evolution of ever work with them. Hopefully the aim of this game is to try to change the evolution going forward. And I think it's worth the interdisciplinary effort that it takes to get your head around several fields at once. It's absolutely worth it. But you do have to be in for the long haul and you do have to be pretty enthusiastic about it to start with. Absolutely. All right well I'd like to thank you so much for taking time to speak with me today. This has been a wonderful conversation. Thank you very much for having me. Isn't it great when a guest naturally introduces the topic for a history of microbiology tidbit? This happens so frequently in our interviews since microbiology has really been building on generations of discoveries. And many of our guests are very quick to acknowledge the contributions of previous scientists. The story of the mixoma virus and rabbits in Australia that Andrew Reid told during our interview rang a bell in the far corners of my mind. But I'll admit that the name Frank Fenner was not one that I was familiar with. So in today's history of microbiology tidbit I want to highlight Frank Fenner and his place in microbiology. Fenner was born in 1914 in Australia, where he was educated and lived most of his life. He trained as a medical doctor in surgeon and joined the Australian Army Medical Corps during the Second World War, fighting malaria in Papa New Guinea. After the war he returned to Australia. He was recruited by Frank Burnett, who himself is no microbiology slouch. Burnett won the Nobel Prize in 1960 and is the scientist who developed the system for growing influenza in Hens eggs, which we still use today to produce most of the influenza vaccine. However, Fenner returned to Australia to set up his own lab at Australian National University, where he studied a number of different subjects. Among them, using the mixoma virus as a means to control the invasive rabbit population. And this rabbit population had been a large problem for a number of decades. And biological control of this invasive pests using infectious agents had been studied by a number of different scientists for decades, even by Louis Pasteur, who had entered a contest offered by the Australian government to find some sort of infectious disease that would kill off their rabbits. Pasteur had suggested using the bacterium that we now call Pasteuralla multiceeda, but this had proved unsuccessful. Fenner collected mixoma virus strains from South America and studied the virulence of these strains and different lab-raised rabbits before the virus was eventually released. Fenner followed the virus and host evolution as Andrew described during our conversation. But in addition to this really fundamental work in mixoma virus and pathogen evolution, Fenner played an important role in the smallpox eradication campaign that was run by the World Health Organization. He worked with D.A. Anderson, who was the scientist overseeing the eradication campaign. And Fenner's role was to evaluate monkeypox and otherpox-like diseases to determine if these would affect the success of the eradication campaign. Fenner was actually the scientist who was able to proclaim to the World Health Assembly in Geneva that the world and its peoples have won freedom from smallpox, which he did in May 1980, signifying one of humanity's greatest achievements in the fight against infectious disease. Fenner passed away in 2010, but he wrote a summary of some of his work in a FEMS microbiology reviews piece. We'll link to this piece and add links to other things we discussed in today's conversation on our show notes page, which you can find at asm.org/mtm. While you're checking out links on the internet, don't forget to take our poll at asm.org/mtmpol or mtmpol. The average time it takes to complete is only three minutes, and we appreciate everyone who's taken three short minutes to contribute feedback about our show. That's it for today's Meet the Microbiologist. I'd like to thank our podcast producer, Ray Ortega, and music composer, Ronald Jinkies. I'll be back next time with a new guest for you to meet the microbiologist. (upbeat music)