9UNP0146SD_Allen Bateman and Christina Carlson_WordPress

- All right, can everybody hear me? All right, well thank you for coming and welcome. I'd like to start just by introducing myself and letting you all know where I come from. I'm a Wisconsin native. Born and raised. I grew up in a small town in northwestern Wisconsin called Balsam Lake. Graduated from Unity High School there in the year 2000, and then went on to undergraduate at the University of Wisconsin- Stevens Point, where I studied biology and chemistry. Then for the last three years, I've been here at the University of Wisconsin-Madison in the cell and molecular biology training program. And I've been actively studying avian influenza viruses. 

So my talk today will focus on human infection by these avian viruses. And I also want to note that Al Bateman, my partner in crime will be talking about swine influenza after I've finished. You guys will kind of get two sides of the story of influenza today. 
(inaudible)
 
- The lab is located actually on the University of Wisconsin campus. And yes, that's another thing I want to note. If you guys have questions at anytime throughout the talk, feel free to just ask. So my first slide here is really just to give you a sampling of all the known different pathogens, as we like to call them. You're probably more familiar with calling them germs, or bugs that are able to cause disease within humans. 

Inarguably, bacteria and viruses are probably the most common pathogens in humans. And I'm sure all of you can name at least one or two members from each of these groups. But in addition to bacteria and viruses, we also know of fungal pathogens. For example, there are many different Candida species that are able to cause yeast infections. We also know of histoplasmosis, which is a fungal infection of the lungs and respiratory tract. Parasites are another common human pathogen. And they're far more prevalent in the developing world than here in the developed world. But in the United States, we still do suffer from several different round worm infections every year. Protists are known as single-celled bugs. These involve, or include rather, the commonly known giardia and cryptosporidium species, which are able to induce very severe gastrointestinal distress. 

And finally, perhaps one of the most interesting and yet perplexing pathogens that have come to human attention over the last 20 years are what we call prions. Prions are really nothing more than just proteins that are able to replicate themselves and transmit from organism to organism, causing communicable infectious disease. And we as scientists, still really don't fully understand how prions work. But I'm sure as all of you know, they include chronic wasting disease, which we have here in Wisconsin in our deer population, as well as the human counterpart the Creutzfeldt-Jakob disease, and mad cow disease. So just to survey the audience, which one of these pathogens here represents influenza? And if you've been paying close attention so far, you already know the answer to that. Anybody? 
- The virus. 
- The virus, of course. So influenza is indeed a virus. 

And viruses are probably one of the most simple pathogens out there. They're really nothing more than a protein coat with some nucleic acid, either DNA or RNA, inside. And viruses aren't really even classified as truly living organisms, yet they can cause some of the most devastating and deadly diseases in some of the world's most complex organisms. So we ask, how is it that influenza viruses can make us sick? Well, we know that flu is a respiratory pathogen, so that means it can be transmitted through the respiratory route. How this works, is the infected individual will cough or sneeze up infectious flu particles, aerosolizing them in the immediate air environment. If you're close enough, you can breathe in these infectious particles which will make their way down to your lungs, which is the primary site of influenza infection. The lung provides the optimal environment for the virus to replicate itself producing more virus. Here we see an artistic representation of what happens at the cellular level down deep in your lungs during influenza virus replication. So in your lungs, individual flu virus particles will come in contact with the surface of your cells, actually gain entry to your cells. 

And at this point, they kind of start to take over the cellular processes, reorienting them so that your cells are driven to become nothing more than viral factories, producing progeny virus particles that go on to infect the next neighboring cell. This continues to happen, and the viral replication process actually ends up killing your lung cells. This, in part, is one of the reasons you feel so sick when you get a flu infection. Once enough virus has accumulated within your lungs. you will end up coughing or sneezing up the virus and passing it on to the next person. This in essence is how the human influenza virus perpetuates itself in the human population. We can actually see evidence of flu virus replication in infected cells by using highly-powered microscopes. This is an image taken with what we call an electron microscope. And what we see, is here's the edge of the infected cell. This cell is releasing tons and tons of infectious progeny flu virus particles. 

So when I say that your cells become virus factories, I mean it in a very literal sense. So how many of you have had the flu before? Okay, pretty much almost all of you. Are any of you think you've had it, but you're not too sure? No, you're all pretty sure. That's because the symptoms associated with flu are pretty bad. They're bad enough that if you've had it once, you're going to remember it for the rest of your life. These symptoms include a high spiking fever, which is usually the cardinal symptom of flu. A fever anywhere from 100 to 104 degrees Fahrenheit. Headache, malaise, nausea, a loss of appetite, respiratory secretions and muscle ache, a dry cough, and sore throat. 

How about the stomach flu? How many of you have had the stomach flu before? Well, that's funny, because there's actually no such thing as the stomach flu. The stomach flu was a general term that kind of arose to describe gastrointestinal problems that people came to associate with influenza virus infections. In fact, one of the problems with flu, is it's often misdiagnosed as many other conditions, because there are many other bacterial and viral infections that mimic what we call flu like symptoms. However, you should never experience gastroenterologic symptoms during flu infection unless you are a duck or a goose. Avian species can get the flu actually, except they present it as a gastrointestinal disease, whereas every other mammal that gets flu presents it as a respiratory disease. If you think you have the flu, but you're not totally sure, the best thing you can do is go to your doctor, get tested, get on anti-virals, if necessary. For most people, flu is an acute infection. It last roughly one to two weeks, usually shorter than that, and then it will resolve itself. 

However, there are certain groups of people who are at high risk for contracting and getting complications from influenza. And so this leads us to ask, who can get the flu. Well, I like to say that everybody can get the flu. In fact, even your pet dog or cat can get the flu. And I'll talk a little bit later about how that happens. But in the United States, every year, roughly five to 20% of our population is affected one way or another by influenza virus infections. And these infections cause about 200,000 hospitalizations and roughly 36,000 deaths every year. Now, these hospitalizations and deaths occur mainly in high risk groups. And these include people such as the very young and very old, immuno-compromised individuals such as HIV patients, pregnant women or healthcare workers who will be in constant contact with people who may be ill with the flu. Additionally, agricultural workers, especially those in constant contact with animals who can carry the flu, such as poultry or swine workers, are at increased risk for getting influenza. 

So the very best thing that these people can do to prevent themselves from getting the flu every year is to get their annual flu vaccine. In fact, the best thing you and I can do to prevent ourselves from getting the flu is to get our flu vaccine. Now I'm sure many of you are pretty familiar with the thoughts and feelings associated with this image shown up on the screen. But how many of you got your flu vaccine this year? Okay, good pretty much everybody. And how many of you are happy that you got your flu vaccine this year? Well, I'm glad to hear that, because even though most of you probably know that the flu vaccine was not nearly as efficacious this year as it should have been, it's still a very good idea to get it. And I'll tell you why in a moment. But first one more question. Anyone know why we have to get the flu vaccine every year? 

It's kind of a pain. Yep? 
(inaudible)
 
- Yes, that's exactly right. The influenza virus is constantly changing. And every year, new strains of influenza emerge that are different from the strains you were vaccinated against last year. So just because you got the vaccine last year, doesn't mean you're going to the protected from the circulating strains that are in the human population this year. We all know that influenza is a seasonal disease. The virus visits us every year around the peak months of December through March. 

What I'm showing on the screen here is the influenza incidence rate for the last four flu seasons in the United States. And in fact, I do have the information for this year's flu season, for just Wisconsin cases alone. Wisconsin actually has one of the best influenza surveillance systems in the entire country. And what we see for this year's 2007-2008 flu season, that we're just now starting to come down off of peak flu season, which for Wisconsin, usually occurs right at the end of February into the beginning of March. But back to this graph. If we go back and look at the last three flu seasons, we notice that of all the samples that were tested, the percent positive rate is between 25 and 30% over the last three flu seasons. But if we go back to the 2003-2004 influenza season, we see that the prevalence of flu that year was much higher. Does anyone remember why that was? Nope? Well, the reason is that the influenza vaccine that was administered in the 2003-2004 season was actually not a perfect match to the circulating viruses that were in humans that year. 

So the influenza vaccine is what we call a trivalent vaccine. There are three killed whole influenza viruses contained in that vaccine given to you to stimulate your immune response, so that should you actually come face to face with the real thing, you'll be prepared and you'll be protected. Now, every year, well I should say the efficacy of the vaccine depends on how well matched the vaccine strains are to the circulating strains. And every year, the World Health Organization right around spring time, has to make a guess, or an educated guess or prediction on what flu strains to contain in the vaccine to protect from the upcoming flu season. From the time that prediction is made by the World Health Organization to the time that the flu season actually hits, six months will have past. And those six months is enough time for a new strain of influenza to emerge, and to become dominate within the human population, and be different enough from the vaccine strain so that you won't be protected. Again, this is what happened in the 2003-2004 season. And this is actually what happened in this flu season. Of the three viruses of this year's flu vaccine, only one was well matched. So this year we had a roughly 40% effectiveness to our vaccine when normally we can benefit from a 75% to 95% effectiveness rate in our vaccines. 

However, like I said, it's still a good idea to get your flu vaccine even if the match isn't perfect. There's a couple of reasons for this. Number one, you're still going to be protected against the strains that are well matched. And number two, there's some level of cross protection that can occur. So even if the viruses aren't perfectly matched, if they're kind of close enough, you still get some protection. So it's better than no vaccine at all. But every once in a great, great while, an influenza virus will emerge for which the human population has never seen before, doesn't have any previous immunity to, and for which the vaccine doesn't protect against. And this is when influenza viruses can cause what we call pandemics. And a pandemic is a worldwide episode of disease that affects a majority of the world's population. And of course, the most notorious influenza pandemic was that of the 1918 Spanish Flu Pandemic. 

This virus that caused this disease was so deadly that it's estimated that 40 to 100 million people died worldwide in the roughly one year that this virus circled the planet. This disease affected almost every aspect of everyday life for the people who lived during this time. Schools and shops had to be shut down. University buildings were converted into large mass clinics, like the one seen here taken from the University of Iowa during 1918, to try and care for the mass number of people who were coming down with this illness. Normal funerals couldn't even been held, because not enough caskets could be made for the number of people dying. And normal activities, like just spitting on the ground became criminalized, because people were so scared of contracting and spreading the disease. Actually, World War I was going on at the same time that this pandemic was hitting the world. And it's been confirmed that more deaths were due in the war to the influenza virus than they were to actual casualties of war. In fact, this virus was so deadly that during 1918 and 1919 it actually dropped the average life expectancy for humans in the United States roughly ten years. And we see it as this big dip on this life expectancy chart from 1900 to 2001. 

However, I want to emphasize that viruses like this are extremely rare. We hardly ever see disease of this magnitude and spread. And in fact, a lot of the infections and deaths that occurred because of the 1918 and 1919 flu were due to secondary bacterial infections, which at that time there were no antibiotics to combat them with. Which now, of course, we have the antibiotics, and we experience a much lower mortality rate from influenza. The 20th century saw four major flu pandemics. Yes, question? 
- What made it go away? I mean, how come it didn't hang around after the major pandemic? Why didn't it hang around? 
- We're not entirely sure. 

There are some reasons that are speculated why the 1918 flu just kind of stopped. Number one, the virus seemed to strangely target very young people. Well, I shouldn't say very young people. Normally, influenza seasonal viruses target the very old and the very young. But this virus targeted people who were in the prime of their life, who had strong, healthy immune systems. And it's suggested that perhaps so many of those people ended up dying, the virus kind of ran out of targets in the human population. Another idea is that this virus was actually an avian virus. It wasn't a human specific virus. So maybe it wasn't well matched to the human system. And so, again it just wasn't, the humans weren't the right target for this avian specific virus. 

But I think the idea of there not being enough humans, because so many people ended up dying from it, that the virus ran out of kind of substance to work with. So as I was saying, there were four major flu pandemics in the 20th century. Of course, it started with the 1918 Spanish flu pandemic. In 1957, we saw the emergence of the Asian flu, which was not nearly so deadly, but did end up affecting people worldwide, as well. The 1968 Hong Kong flu was the next one. And then in 1977, we had kind of sort of a pseudo influenza pandemic, which we called the Russian flu, because it was speculated that this virus was leaked accidentally from a Russian laboratory and introduced into the human population. And what we see here is that with the roughly ten to 20 year frequency, we had been experiencing influenza pandemics pretty regularly throughout the 20th century. And after the '70s, these pandemic strains of flu kind of went silent in the human population. We're not entirely sure why, but this has led a lot of scientists to predict that we are currently overdue for an influenza pandemic. And that leads us to the emergence of the H5N1 Avian influenza in 1997. 

So far, this virus has killed only 236 individuals, which doesn't seem like a whole lot, but its mortality rate is currently at roughly 60%. This virus is what many epidemiologists, researchers, as well as healthcare workers, have predicted will be the next causative agent of an influenza pandemic. So I kind of want to go now into taking a closer look at these H5N1 viruses, and looking at what makes them special and what gives them potential to cause a pandemic. So what's so worrisome about H5N1 viruses? One of the big things is that these guys cause an increased severity of disease in people they happen to infect. We already talked about the symptoms that seasonal influenza causes in people, but when we look at patients who end up coming down with H5N1 avian influenza they experience something far worse. These people get things like acute respiratory distress syndrome, viral pneumonia, multiple organ failure. In rare cases, we see people come down with diarrhea and encephalitis. What's really interesting about H5N1 infection in humans and other mammals is that we see the virus has an increased ability to replicate faster in human hosts, and also to spread to areas outside the respiratory tract. So in animal models, we've actually isolated H5N1 viruses from the brains of mice, from the kidneys, as well as from the lungs. 

That's not what we see normally with seasonal influenza. Perhaps the most alarming thing again about these viruses is that they're currently presenting a roughly 60% mortality rate in people. This is in contrast to the normal seasonal influenza mortality rate, which hovers around .1%. So in addition to the worsened clinical manifestations of H5N1 influenza, these viruses are also exhibiting an expanding geographical range. H5N1 arose in Hong Kong in 1997, and then from '97 to about 2002-2003 the virus stayed mainly in the Guangdong region of China. After 2003, it started moving largely throughout Southeast Asia. But not until really 2005 or 2006, did we see the virus start to disseminate greatly over the planet. And so here in 2006, which is what this maps represents, we see H5N1 geographical distribution in wild birds and poultry, which is indicated by the light red countries. The dark red countries indicate those countries that had recorded cases of H5N1 in wild birds, poultry and in humans. We see the virus in 2006 had moved largely throughout Asia, gone into the Middle East and into Europe, and had just started to show up in Africa. 

If we move ahead now to the current situation in 2008, we see that the geographical distribution has widened even more. Pretty much, Asia has been dominated by this virus. Europe, it's pretty much everywhere there, but only in bird populations. The Middle East, most countries have started to report H5N1 cases. And now Africa has human, as well as avian cases showing up there, too. One thing I want to note is that we have yet to see this virus in the western hemisphere at all. Although some scientists are predicting it's only a matter of time before birds that migrate between Asia and North America may bring the virus over. 
- Is it seasonal, as it is in the United States? 
- Yes, H5N1 has a seasonality, just like seasonal flu does. Okay, so one of the other concerns with H5N1 influenza is that this was the first time we had ever really seen a truly avian virus jump directly from an avian host into a human host. 

What we know about influenza viruses is that they're very species specific. So for example, an avian specific virus normally usually only infects bird species, whereas human viruses normally only infect human species. We do know however, that all influenza subtypes can be found within the natural zoonotic reservoir, which are aquatic birds. And aquatic birds can actually distribute the virus to all different species of animals. However, the transmission of avian viruses to humans is not a direct event. Rather, for an avian specific virus to move into a human it first usually has to go through an intermediate species. And oftentimes, this is the pig. And like I said, Al will talk a little bit more about pig infections after I'm done. But the pig is able to adapt the avian viruses to the human system, and allow infection of the human host. And so, in 1997, when the H5N1 viruses emerged, they completely bypassed this intermediate species and moved directly into the human host causing disease. 

We think that the avian viruses that did this were not well adapted to the human host, and as a result, this may be part of the reason why they are so virulent and cause such horrible disease. So how exactly did these bird viruses get into people? Well, the biggest factor is probably the very close association between birds and people in Asia. Asia boasts probably the world's highest density of humans, as well as some of the highest densities of birds, too. And people live with their birds in their home, they sleep with them, they bring them everyday to their live birds markets. In some cases, some of these images look almost ridiculous to us, but it's how they live. Bird farming is very much a way of life for these people. So not only is this virus devastating them in these parts of world, because humans are getting sick from it there, it's also devastating them because their way of life is being taken away when these birds come down with H5N1, and they have to be massively culled or killed to try and contain the virus. So in addition to an expanding geographical range to H5N1 influenza, the virus is also presenting an expanded host range. We know that H5N1 can infect wild birds, domestic poultry as well as humans, but more recent research and surveillance efforts have found that H5N1 viruses can also move into other species as well. 

These include dogs and cats, including large cats like leopards and tigers, as well as what we call palm civets, which is a raccoon-type animal found in Asia. So the increased host range also gives this virus the increased possibility to become a pandemic virus. So to kind of put into perspective the major events that have occurred since H5N1 influenza, I have a timeline here. Humans first came into contact with H5N1 in 1996 when it was isolated from a farmed goose in China. But it didn't show up in the human population until 1997, when we had the first indexed human cases in Hong Kong. 18 people got sick from it, six ended up dying. Yes? 
- How did they find it in '96, I mean, were they looking for it? 
- Right, no there are constant influenza-- Because influenza is a disease that's been affecting humans for thousands of years, we've been getting sick from it for a very long time. And so, there have been more severe and less severe influenza cases throughout humanity's interaction with the virus. 

So, constantly within the last, probably, since the virus was identified in the 1930s, there have been surveillance efforts going on to monitor what kind of viruses circulate in the human populations, and then upon discovery that the virus was also harbored within animal hosts as well, looking within animals and within humans to see what kinds of viruses are continually circulated in these hosts. I think it was probably just a common surveillance effort that found this new subtype that had emerged. After 1997, H5N1 kind of disappeared from the human population for a while. Until February of 2003, when two more humans came down from it in Hong Kong again. They had traveled to China and returned to Hong Kong. And it was believed that they picked up the virus in China and brought it back with them. One ended up dying. Later in 2003, the virus demonstrated its ability to move into other species of animals, infecting two tigers and two leopards that actually died from it. And these animals were fed infected chicken carcasses. This also demonstrated and suggested that H5N1 could be passed or transmitted through food products. 

In 2004, the virus spread widely throughout Asia, and we saw our first account of human-to-human transmission in Thailand. And this kind of started to scare people, thinking that now the virus had acquired the ability to move easily throughout the human population, and it was just a matter of time before we had a pandemic on our hands. But it didn't happen. In 2005, the role of wild birds was kind of confirmed in H5N1 transmission at Quinghai Lake in China, where over 6,000 birds died massively in one big event. And prior to that, it was uncertain whether wild birds were able to carry and transmit the virus to domestic poultry or not. From 2005 until the current day, H5N1 continues to spread throughout Asia, the Pacific Islands, the Middle East, Africa and Europe. Currently, we have experienced 373 human cases and 236 deaths. Those numbers are still tallying up. Where exactly this virus is going to lead us in the future, we're very uncertain. But I kind of want to summarize the current status of H5N1 influenza worldwide. 

Like I said, we have roughly a 60% mortality rate in humans. Most of these human cases are occurring still in Southeast Asia, with the exception of now African countries like Egypt, which has had 47 human cases in the last year. We see an expanding geographical range. However, I want to emphasize again, we haven't yet detected H5N1 in the western hemisphere. The virus has an expanding host range, but again very limited human-to-human transmission has been observed. And this leads us to conclude that H5N1 was not yet fulfilled the prerequisites to becoming a pandemic virus. What do I mean by prerequisites? Well, for influenza viruses to become pandemic, there are kind of three conditions that have to be met in order for that to happen. Number one, a novel influenza virus must emerge into the human population, for which we have little or no immunity against. Second, the virus must be able to replicate in humans and cause serious illness. 

And three, the virus must be able to establish efficient human-to-human transmission. Now, H5N1 has fulfilled the first two conditions. But, of course, we have not yet seen it go easily from human-to-human. And in fact, recent research in the influenza field has suggested that in order for H5N1 viruses to acquire efficient human-to-human transmission, they would be making a great trade-off in virulence. So in other words if we were able to start transmitting this virus to each other, the virus would probably not be nearly as deadly as what we're experiencing right now. The question, probably at the forefront of most of your minds is should we be worrying about H5N1 influenza? And my advice is I think we should be vigilant of the worldwide situation, but I don't think we should be losing sleep or getting pandemic fever just yet. Consider reported statistics carefully. We're reporting a 60% mortality rate to this virus, which is higher than any flu virus we've ever seen in the history of the virus, but in reality they're probably far more bird flu case cases out there than what is actually getting reported to medical institutions. So the actual mortality and morbidity rates may be a lot lower or even higher than what we know of. 

Also, we haven't yet found it in the western world, so we don't need to really lose sleep on that until we first detect it. And this is in face of very vigorous surveillance efforts. Currently, we have people all over North America who are trapping and swabbing wild birds to see exactly when H5N1 might actually come into our country. And again, the virus cannot yet transmit person-to-person. And also, some countries have eradicated H5N1 on their soil. How have they done it? Well, for example, Japan had H5N1 detected in their country. And what they did was massive vaccination efforts. They vaccinated not only their people against obviously, not H5N1, but they got cross protection from other normal seasonal flu vaccines. And also, they ended up doing vaccination efforts of their poultry as well. 

On top of it, once H5N1 was detected in their poultry flocks they ended up killing those flocks off very quickly to try and contain the virus. So, if H5N1 were to occur and show up here in North America, it's not like we would have a doomsday. We would be able to potentially contain the virus. 
- How is it transmitted? I mean, you talked about the two leopards and tigers from being fed. 
- Right. 
- So was the transmission-- It must be raw? 
- Yep, they were raw chicken carcasses. It wasn't cooked, they were raw. And so, the virus hadn't been cooked. 

If you actually cook or heat it, the virus it dies, it's done. So if you eat raw poultry meat, you're at risk for getting influenza. 
- So it isn't transmitted respiratorily? 
- Not necessarily. It doesn't necessarily have to be. It's respiratory most of the time, but remember that birds actually present influenza as a gastrointestinal disease. And so they can actually shed it through their feces in the fecal oral route. And we can pick it up through their feces, actually, in this case. Also, the FDA last year, just last year approved an H5N1 vaccine. This is not commercially available. 

You can't go out and buy it, but it's currently being stockpiled in our country in the event that an H5N1 outbreak would occur. So we have made strides on this. This was not available until very recently. So the $65 million question is what is being done about H5N1 worldwide. And I really kind of just want to talk briefly about what's being done right here in Wisconsin, because of think a lot of the efforts that are going on here in Wisconsin kind of reflect the global efforts. The biggest thing that's going on right now, and the biggest thing that's stressed is very, very strict and rigorous surveillance of birds, wild birds, poultry and even human influenza cases, so that we can be aware of an H5N1 case the minute that it would come into North America. So here in Wisconsin, actually right here in Madison, we have the Wisconsin State Lab of Hygiene. And this institution conducts statewide influenza surveillance and diagnostic tests in humans. So all the samples, all the specimens of influenza illness are sent to the State Lab of Hygiene. They test these samples, find out what kinds of viruses people are having this year. 

That information is sent, ultimately, to the World Health Organization. It helps to decide what the vaccine selections will be for a particular year. The Wisconsin Veterinary Diagnostic Laboratory does very much the same what the Wisconsin State Lab of Hygiene does, except in animals. So for example, if you have a backyard flock of birds, and you find that some of them are kind of dying off, you could call these guys. They come pick up your bird, test it for you and let you know whether you have bird flu in it or not. In addition to doing surveillance and diagnostics in animals, the Wisconsin Veterinary Diagnostic Laboratory also does training programs abroad in countries that are most hard hit by H5N1 influenza. So for example, they go out to Thailand and Indonesia and train the people, the medical personnel over there, how to use the most up-to-date and cutting-edge diagnostic tools to help better the surveillance efforts in those countries. And again this is right here on the UW campus. The U.S. geological survey is part of the United States Department of the Interior. And we have a branch here in Madison, who has people, always up in Alaska again, taking samples from wild birds to see if any of those birds that have migrated into Asia will be bringing H5N1 influenza into North America. 

And we haven't seen anything yet. Then finally, I don't believe that good counter measures to seasonal influenza, or even H5N1 influenza, can be developed without basic research that looks at how the virus replicates, how the virus interacts with its host, the immune response to the virus and so on and so forth. Here at the University of Wisconsin, we have at least three major influenza labs that do basic and applied influenza research. And our lab is actually one of those. I work on campus here in the laboratory of Dr. Stacey Schultz-Cherry. We are a World Health Organization collaborating lab. And our research focus is on influenza pathogenesis. What that means is how the virus actually makes you sick. And we do work with bird flu here on campus. 

This is actually a picture of my lab-mate Jeremy Jones in our high containment facility, where we do all of our work with bird flu viruses. So we are particularly interested in the cellular and viral factors involved in flu infection. My personal thesis project is focused on one particular aspect of the immune response to influenza infection and H5N1 infection. We've been successful in developing at least one anti-viral peptide that has effectively prevented H5N1 flu from infecting mammalian cells. Then finally, we collaborate with area biotechnology companies to develop better diagnostic tests for seasonal flu as well as H5N1 flu. And so with that, I would like to thank all the people who let me come and speak today. My lab and especially my PI, Stacey Schultz-Cherry. I'd like to thank Al for asking me to come do this with him again. It's always a lot of fun. The University of Wisconsin Biology Outreach Program. 

And, of course, the Plato Frontiers in Life Sciences Seminar Series. And to all of you for coming and letting me talk to you for the last half hour. So without any further adieu, I'll take any questions that you guys may have. Thank you. 
- That second to last slide that you had on about the immune...? 
- Yeah. 
- Could you elaborate a little bit about your own particular thesis on immune response? 
- Yeah, I work with a protein in the body that's called transforming growth factor beta, or TGF beta. And what we know about TGF beta is it's a very important regulatory molecule in the human immune response. We found that influenza viruses are able to differentially modulate TGF beta. 

What's interesting is that we found all influenza viruses are actually able to activate TGF beta, which serves as kind of a warning response to the host to say, hey, you're infected, start implementing immune responses to get rid of this. So all flu viruses activate it except for the lethal H5N1 viruses. They don't trigger the sensor. And we're trying to figure out why exactly that is. What's different about the bird flu viruses that they don't alert the host as well as the normal seasonal viruses. And how can that play out in the outcome of infection? And so, we've found that animals who-- Actually, if we deplete TGF beta from animals and give them a non-lethal influenza virus, that infection becomes lethal, much like the H5N1 viruses. And additionally, if we infect animals with lethal influenza viruses, and we add back TGF beta, we find that now animal survival is prolonged. So it seems that TGF beta is playing a very critical role in modulating the host immune response, and actually playing a role in influencing the outcome of influenza infection. So that's kind of my personal project and what I'm working on. 

Yep? 
- A couple questions. The flu shot has three different viruses. 
- Yep. 
- When flu hits the country, how many different strains really are going on? Is the three an arbitrary number or diminishing return of some sort in the choice. 
- Yeah, I actually don't know how many are circulating in the human population at any one given time. What we do know is that the reason that three is chosen is they're kind of a representative of the three major subtypes. So when I say H5N1, that's a specific subtype of influenza. There are multiple known subtypes. 

The H actually stands for one of the proteins on the surface of the virus. The N stands for another protein. There are 16 known H's, nine known N's. And you can have any combination of those. So that gives you an idea of how many subtypes are out there. Not all those infect people, but we choose three for the vaccine because they represent the three major subtypes that are known to infect humans, which is an H1N1, an H3N2 and a B type influenza virus. And so, those are the common circulating human strains, and have been for a long time. Not to say that other strains can't move into the human population, but that's what the vaccine's based on. 
- A second question, is SARS related to this in any way? 
- Yeah, SARS actually presents with very similar clinical manifestations as influenza. 

Actually, the viruses are similar in a lot of ways, but they're primarily very different. Influenza is what we call a negative stranded RNA virus. That's based on the nucleic acid that's inside that virus. SARS is actually a positive stranded RNA virus. So they're basically very different. They're transmitted very much the same through the respiratory route, but SARS is principally a human pathogen. Although, I think it can be found in civets, as well. But, no, at the very basic level they are very different viruses. And you shouldn't expect to be protected from SARS if, for example, you got the flu vaccine. There would be no cross protection there. 

Yep? 
- What factors, or how did they determine the forecast of which viruses they're going to choose for the vaccines in advance? 
- Right, so what happens is I think there are, I forget the number, four or nine influenza major surveillance institutions throughout the world. There's one in Australia. There's a couple of them in Asia. One here in the United States, which is related to the CDC or the Centers for Disease Control and prevention. Throughout the year and throughout the influenza season, these institutions will do surveillance. And this is collecting samples on a massive basis, analyzing those samples and finding, really trends and what the dominate influenza viruses in the human population are. So based on what those surveillance efforts tell us, that's how the World Health Organization collects all that data in one centralized location, reviews it and says, okay, it looks like this A Wisconsin type H3N2 virus looks like it's the dominate strain this year for the flu season, that's what we're going to put in our vaccine. Or, you know, this H1N1 is the dominate type. 

It's the result of the whole last flu season year's worth of collecting data, analyzing it and seeing what the trends are in influenza circulation in humans. 
- So are there patterns in the way flu travels around the world? 
- Yeah, and it seems that, you know, people always ask why influenza is seasonal, why it always happens to show up in December? Some groups have suggested that there is a relation to when it starts in Asia, and when it moves over. I think it's more of the dry and cold weather keeps people close together, and that's why it tends to be a seasonal virus. 
- One thing to add about the choosing the different vaccine types. Often at the end of the season, there's a little blip where the viruses actually infect more people for a little bit before it disappears. And that little blip, often the virus has changed a little bit. And that's pretty similar to what the virus will be like the next year when it comes around. So that's also pretty helpful, sometimes. 

- Yep? 
- With the large goose population we have, and the gastrointestinal possibilities, is anyone monitoring them in particular? 
- Yeah, and actually again this goes back to the U.S. Department of the Interior's efforts up in Canada, as well as in Alaska, because those sites would be the first predicted sites of where H5N1 would enter North America or our country. What they do, is they have people who go out in the field every day. They trap these birds and take what they call a swab, because it's a gastrointestinal disease for the birds, and then they culture that swab and end up sequencing the viruses to determine what the subtypes are. So yes, there's a very vigorous surveillance effort going on in the northern part of North America. Okay, well if there's no more questions, I'll let Al take it over. Thank you very much. 
(applause)

- All right, thanks Christina. Can you hear me all right? Okay. My name is Al Bateman. I'm working in Dr. Chris Olson's laboratory also here at UW. I'm also part of the CMB, Cellular and Molecular Biology graduate program. And, oh, yeah, and little bit about me, sorry my background. I'm also from Wisconsin. I grew up near West Salem. 

It's near La Crosse, near the Mississippi. I went to undergrad at St. Olaf College in Minnesota. I majored in chemistry and Norwegian, actually. So, I put those two together. 
(laughter)
 A little random, but it was a good combination though. I actually put those two together for a year, and I studied in Norway for a year. I worked in a laboratory there. After that, I've come back, and for the last two and a half years now I've been at the UW studying swine influenza. 

So I'll tell you a little bit background about what exactly it is, and then I'll actually go into a little bit of the research that I do, and some of the new data that we have. So with that, first of all, for my talk I'll start out like I said, with the basic about what swine influenza is. Basic information about viruses, influenza viruses, some of the different species that influenza viruses infect. This might be a little bit of a repeat from Christina, but it's a little different angle, I think. I'll specifically talk about swine influenza for a while, and then I'll go on to why swine influenza is important. It's a pig pathogen. There is a zoonotic potential, so there's a possibility for pig viruses to infect humans. And also I'll talk about the role that pigs can play in possibly generating pandemic influenza viruses. Oh, yeah, I don't know if this comes up very well, but I just included this picture because I'm kind of proud of it. It's one of the viruses that I work on in the lab. 

And we just got that picture recently, so I decided I'd include it. So viruses are defined as obligate intracellular parasites, which means they cannot replicate by themselves. But when they're inside of a cell, as Christina said, they take over the cell and use it to make more copies of themselves. Viruses infect all different types of cells from humans and other mammalian species to insects and even bacteria. Basically, if there's a type of cell, then there's at least one type of virus that infects it. Viruses are also very heterogeneous. They vary in their size, shape, and their nucleic acid. It's either the DNA or the RNA that they have inside them. Shown here is just a depiction of a bunch of different viruses. Right here, is a tobacco mosaic virus. 

It infects plants. This one infects bacteria, and right here's an influenza. So they're very heterogeneous. In terms of their size, they're very small, but just for a little comparison here, here's an influenza virus. When influenza infects humans we obviously get very sick. So consider, if you as a person if you were the pathogen, if you were the virus, how big do you think your host would be that you would infect? Do you have any idea? 
- As big as the earth? 
- He's on top of it, wow. That's roughly how big the pathogen that you would have to infect would be. 

So, to me it's just amazes me how small viruses actually are, and how they can still cause so much disease. It's quite impressive and a little scary, too. 
- Do all viruses really look like prickly? 
- Not all of them do. Like here are some of what the other viruses look like. But influenza is-- 
- They're all fuzzy. 
(laughter)
 
- Some of them are fuzzy, yes. 

In terms of the cell, viruses are about roughly a thousand times smaller than even one single cell. And Christina went over this a little bit, but the virus, in the infection cycle, it binds to a cellular receptor on the surface of the cell. It then gets endocytosed, it's called, when it enters into the cell. This is really complicated, but basically this is all the replication steps influenza has to undergo. This is maybe not even all of them, but some of them. To produce more infectious progeny virus, it then exits to infect nearby cells. So influenza viruses, there are three types. A, B and C. Type A is the most serious burden, both economically and health wise. I'll just be talking about influenza A viruses from now on. 

But Christina briefly mentioned that B also does infect humans on a yearly basis. Influenza viruses are segmented. This means that they contain their genes on eight separate segments of RNA. And this allows for the virus to reassort. And this means that if two different viruses infect the same cell, these different segments can kind of just play a mix and match, and can reassort to produce a new virus with a different combination of the segments. And I'll talk a little bit about that more in a little bit. The two main proteins on the outside of the virus are the hemagglutinin or the HA, and the neuraminidase or the NA. And as Christina had alluded to, there are many different types of the both the HA and the NA. All of these types are present in the wild water foul population. And the subtypes are based on these two, so humans are commonly infected by H1N1 and H3N2 viruses. 

Whereas the possible avian influenza that Christina was talking about is H5N1. Not all combinations have been seen. I think there are a possibility of 256 combinations of different subtypes, but quite a few of them have been seen. Again, this is the same sort of wheel of misfortune of the different species that influenza viruses can infect. We have all of them present in the wild bird populations and the ones that we are most interested in the Olson Lab, is the swine influenza, and so the interspecies transmission between pigs and poultry, wild birds and humans. So swine influenza actually is constituted of many different subtypes of influenza. I'll just talk about the different subtypes in North America, because they're different in different areas of the world. For most of the 20th century, swine influenza is mainly just caused by the H1N1 virus. But then in the late 1990s, a new type of virus called H3N2 virus emerged. And this combined again by the reassortment I just talked about. 

Some of the H1N1 segments, and some segments from avian and some segments from human viruses. Because it was from three different species, we call these triple reassortment viruses. And since this time, they've spread throughout the swine population in North America. But as you can probably tell by the white on the outside there, the story continues. There's been more reassortment from in between the H5N1 and the triple reassortments. We call these second generation reassortment viruses. Basically, most of the genes from here, except either the H1 the N1, or both of them, from these will be called classical swine viruses. So now we have H1N2, H3N1 and these R for reassortment, rH1N1. And so all of these types of viruses have been isolated from pigs in the past few years in North America. Most recently, there's even another one that's been isolated. 

This is an H2N3, which is a reassortment of one of these viruses with the hemagglutinin and the neuraminidase from the avian virus. So basically the take-home message is swine influenza is not one particular virus, there are many different subtypes. But yet they only constitute the H1 and the H3, and also the N1 and the N2. That's mainly what circulates in the swine population. However, regardless of the different subtype of influenza the clinical signs, once the influenza virus infects pigs, are pretty similar. There's a sudden onset of disease and the pigs are quite inactive, they huddle together. They often sneeze and cough. They have a pretty high body temperature. They have rhinitis, and they lose weight. But then about a week after the onset of the disease, they just suddenly recover. 

So it's a sudden onset and sudden recovery. And influenza in pigs has a high morbidity, meaning that when a swine farm is infected, almost 100% of the pigs on that farm become infected and become sick. But also, in general, it has low mortality, meaning that of all the pigs that actually get sick, less than 1% actually die from the virus. So these clinical signs might seem pretty similar, might sound quite familiar, because they are actually pretty similar to the symptoms that humans get when they have typical seasonal influenza. There are actually many other similarities between swine and human influenza. In addition to the clinical signs and symptoms, they are the same main subtypes. As I said before, swine influenza is mainly H1 and H3, and N1 and N2, and different combinations of that. And the two main subtypes that circulate in humans are H1N1 and H3N2. The virus replicates in the respiratory tract of both humans and swine, whereas it replicates in the gastrointestinal tract of most bird species. And while influenza viruses are mainly species specific, relatively, to other species, or relative to all the different directions that influenza can cross species, the transmission from pigs to humans and from humans to pigs is relatively common. 

And in our lab, we've shown that some viruses have pretty similar infectivity in both human and swine cells. I'll get back to that again in a little bit. Yep? 
- Is the transmission, again, respiratory, or like if you have a sick pig and you ate the meat, would you get it? 
- That's never been documented. It's strictly respiratory. Yep. But it's still a good idea to cook your meat. So, on to the importance of swine influenza. First, it is a pig pathogen so it is a pretty serious pathogen. 

It causes them to lose weight and get pretty sick. It's a pretty big economic burden on the swine farmers, because it takes more time to get the pigs up to market weight, and it's a pretty big economic burden. The other two I will talk about now. Zoonosis comes from the Greek words of zoon and nosos. It's defined as an infectious disease that's able to be transmitted from animals, this is either wild or domestic animals, into humans. And just for a couple recent examples are SARS, the Ebola virus, or HIV. And these are all actually viruses too. Swine influenza also has zoonotic potential when it infects humans. There are quite a few isolated incidents in the past 50 years. They're mainly when a swine influenza virus infects one person. 

And this is usually an animal caretaker like a swine farmer. They haven't been shown to transmit from human to human very often. But there was one incidence in 1976 in New Jersey, where an H1N1 swine virus spread and infected quite a few of the soldiers there. So there is the zoonotic potential of swine influenza. As for the ways that pigs could be involved in the generation of pandemic viruses, shown here are three possibilities. I said before, all 16 subtypes of influenza are found in the wild bird population. And right now it's only the H1 and the H3 that are circulating in humans. So if any of these other 14 are able to infect humans and transmit among humans, that could be the next pandemic. Christina mainly talked about the direct transmission from a bird into humans, but also there's this adaptation hypothesis, where an avian influenza virus could infect pigs. And upon replication in pigs could mutate to become more infectious for humans. 

And the third possibility is reassortment, where an avian virus and a human virus could co-infect a pig, because pigs are susceptible to both types of viruses. And if they infect the same cell of that pig, this is the reassortment, again, that the viruses could undergo and produce a new reassortment virus that could then infect humans. I'm going to talk a little bit about the adaptation hypothesis, and some of the work that we've done in our lab. And this is specifically talking about H4N6 viruses. It's just another type of virus. H4N6 have mainly been found in the bird population. Actually, have always been found just in birds, not in any other species until a couple years ago when our lab and another collaborating lab isolated them from pigs. So normally they're just an avian virus. But the interesting thing about the one isolated from pigs is that it had two mutations. And these mutations were in the HA protein, the hemagglutinin. 

And they were at positions 226 and 228. They were actually at amino acids 226 and 228. Amino acids are the building blocks of proteins. So just different parts of the proteins. Since the virus that we isolated had these two mutations, we wondered if maybe it was already undergoing this mutation, to possibly become more infectious for pigs, and more infectious for humans. So like I said, this is what we isolated from pigs. And so, to try to mimic this in the lab to see if there was any sort of selection pressure for this mutation, we took an avian virus and we infected swine cells. We have a system to grow swine cells, so we don't have to infect pigs all the time. It's a nice model system. We put it on swine cells. 

We then took it back off, took the progeny virus, the virus that's produced from the pig cells and put it on a new set of pig cells. And we did this ten times to see if we would get these same mutations that we saw. So, to do the comparison here, the avian, again, has positions 226 and 228, has a Q and a G, whereas the mutant that we found out of pigs had an L and an S. And so there's a passage from P0 down to P10. And we found that 228 did not change at all after the ten passages. But the 226 at P3 started to change from Q to L, and by P4 it was completely changed to L. And then it stayed L the whole way until the P10. So we think this is the same mutation from the virus that we had isolated. So we thought that this 226 might be pretty important. So to test, we then infected the cells, these pig cells, with either the virus that we started with or the mutant that we got out of pigs. 

Red is infected cells here. This is just the amino acid at position 226, either the Q or the L. So we had the avian and then we had the double one. But we also had a single one that just changed the 226, and we had a single one that just changed the 228. And basically, you can see this correlation between the amino acid at position 226 and the infectivity. Whereas, if it's Q, the viruses have quite low infectivity. And if it's an L, the viruses have a much higher infectivity in these cells. So yes, this amino acid at 226 does definitely have-- This mutation does have effects for infectivity. What about this last part here, to be able to infect humans? Well, recently we also have an in vitro system with human cells, so we don't have to infect humans, obviously. 

So we did the same thing. Here are the swine cell infections again, and here are the human infections. And it's looks quite similar where you have the same high-low, high-low infectivity. The infectivity in human cells also is determined by the amino acid at position 226. So it does lend credence to the possibility of this adaptation hypothesis of a new pandemic virus. But I don't want to completely scare you guys. You don't have to worry too much. These H4N6 viruses are not highly pathogenic, unlike the H5N1 that have infected humans. They're not highly pathogenic. There are possibilities that the pigs that these viruses were isolated from were also infected with other viruses or bacteria that could have possibly together made the pigs sick, and that's why we noticed this outbreak. 

None of the pigs on the infected farm died. And the viruses did not spread beyond the initially infected farm. So there's not a huge risk here. But it is still a possibility. One last thing. The H3N2 viruses commonly circulate in humans. And so most of us have, either by becoming infected, or from being vaccinated, we have antibodies against the H3 virus. And the closest of the 16 to H3 is actually H4. So there's actually been some cross reactivity with the antibodies that our immune system makes, that they could cross react with the H4 viruses also. So even if these H4N6 viruses could infect humans, and even spread it among humans, it likely wouldn't be nearly as devastating. 

It would likely be similar to the seasonal influenza that we see on a yearly basis. But even so, this still might not be the best idea. 
(laughter)
 As much as you like pigs. 
(laughter)
 So with that, a quick summary to say that swine influenza does definitely infect pigs. It's a major respiratory pathogen in pigs, and that also does infect people both in terms of the zoonotic potential and the potential for the generation of a pandemic virus. There are many different subtypes. So, as Christina had talked about, the surveillance of swine influenza viruses is also very important both to detect new viruses in the pig population and also for possibilities that those viruses could then transmit into humans. And yep, as far as the generating pandemic viruses, it could be by reassortment, adaptation or possibly a combination of the two. 

I thought I'd throw this out. This is the type of jargon that we as scientists use just to be very specific. So amino acid 226 in the HA of H4N6 influenza viruses determines infectivity in swine and human cells. And hopefully now you have some sort of idea of what that means. So thanks to the other members of the Olson Lab, my mentor Chris, Jordy, Mark and Sasha. Also to this Plato Seminar Series, to the Biology Outreach Club at UW, and to Christina Carlson and you for listening. So with that I'm going to put this up because it's priceless. And I'll take any questions. Yes? 
- In addition to swine and birds as hosts, is there this kind of study going on with rabbits, and squirrels, and horses, and dogs? 

- That's a very good question. Horses, yes. Equine influenza is quite common and has been known for decades. And there are a couple different subtypes there. There are some people starting to look at other wildlife species like raccoons, to see if they get... Yep, exactly. I'd say definitely the main ones are avian influenza. And the other thing with avian influenza is we tend to lump it all together, but there are many different species of birds, obviously. So to pick apart the ducks versus the geese, versus the swans, and what the differences are between the different bird species is also very important. I'd say one other thing about swine, the reason why there's been quite a bit of interest is because they are susceptible to both human and avian influenza viruses. 

So there's just a lot of possibilities for this cross species transmission to happen. Any other questions? 
- Where are your labs located? Christina's and yours? 
- They're both on campus. I'm in the Veterinary School. 
- Down beyond the Ag barn? 
- Yep, down beyond the Ag barn. 
(inaudible)

- That lab, is that in the new building? 
- We will be moving there shortly. Currently, we are in the Medical Sciences Center. By the end of the semester hopefully we'll be in our new building. 
- Okay, thanks a lot. 
- Thank you. 
(applause)