- Welcome everyone to Wednesday Nite @ The Lab. I am Tom Zinnen, I work here at the UW-Madison Biotechnology Center. I also work for UW-Extension Cooperative Extension, and on behalf of those folks and our other co-organizers, Wisconsin Public Television, the Wisconsin Alumni Association, and the UW-Madison Science Alliance, thanks again for coming to Wednesday Nite @ The Lab. We do this every Wednesday night, 50 times a year. Tonight it's my pleasure to introduce to you three extraordinary researchers in influenza. This is a centennial of the pandemic of 1918/1919 and it's going to be a time to think about what that pandemic meant to the world. It killed about 50 million people, and what it's meant since then in how we look at public health and in the field of virology. We'll have three speakers, the third speaker will be Professor Yoshi Kawaoka. He was born in Kobe, Japan, and went to Hokkaido University for his undergraduate degree, and his master's, and his PhD. He came to UW-Madison in 1997. Our second speaker will be Professor Chris Olsen, who was born in Saratoga Springs in Upstate New York. He went to Saratoga Springs High School. He went to St. Lawrence University, and then got his DVM and his PhD at Cornell, and in 1992 came here for a three-year postdoc,

and never left. 
(audience laughs)
 That's how he said it. I liked it.

And our first speaker is

one of the remarkable people here on this campus,

is Bernard Easterday. Barney was born in Hillsdale, Michigan, and went to Hillsdale Michigan High School. Then he went to Michigan State University, where he was a pre-vet student for one year, and then he went right into the DVM program, got his DVM at Michigan State, and then he came here to do an MS and a PhD, then he went into the Army for two years, and he was in East Africa working on livestock diseases that had the potential to be bioweapons. We're going to have to have him come back and talk about that. Then he came here in 1961 to be on the faculty and most extraordinarily, he was the founding Dean of the School of Veterinary Medicine which opened in 1979. He is the only person I know who has a creek named after him, and that is Easterday Creek, right here between the School of Veterinary Medicine and the Biotron. And someday I hope I got a creek named after me but I'm going to have to work harder at it. So tonight we're going to talk about 100 years of influenza research here at UW Madison. Please join me in welcoming Emeritus Dean Barney Easterday to Wednesday Nite @ The Lab. 
(audience applauding)
- 
[Tom]
 Just want to make sure you get through the valley of death.

- Yeah. 
(coughs)

- Good.- So thank you very much Tom, and good evening everybody and I'll try to keep you awake so you can listen to the other two guys, is what this is. So you can get the real poop about this. So 100 years ago, right now in October of 1918, Madison was not a pleasant place to be.

Nor was Wisconsin.

Nor was the United States. Nor was a lot of places in the world, because of this devastating disease that was taking place, and the big surge of this was starting rightly in September, going up in October. And all of this along with getting close to the end of World War I. So a lot of things were happening during that time.

And speaking of World War I, it is known that there were many more military people who died with influenza than died in military action. So it was again, another example of how devastating this thing was. And this will give you some-- This is not a very good photo and there will be a better one later on, but this is at a military post in Kansas, and there're lots of pictures like this where there were so many sick people, they used big places like this full of cots to take care of the people, and as Tom indicated, 50 million people, you'll hear anywhere from 20 to 100 million people who, throughout the world, who died with this disease. It was, indeed, devastating.

At the same time that this happened in 1918, a new disease appeared in hogs, in swine, and particularly in the Midwest. Iowa was the big place where it began to appear and then it spread throughout the area. And so you can see what was happening, they were coughing. Clinically, it was a disease very similar to the disease in human beings, but one of the major differences was that in the hogs, the mortality rate was much lower than in human beings. But nevertheless, it was a severe economic burden because of what it did to these pigs along the way.

So this disease appeared along with it in 1918 and people kept talking about the disease in pigs and finally there was a US Department of Agriculture veterinarian in 1920 who gave it the name swine influenza which we have until this day.

So that gets us started a little bit. And then it was a few years later, and the--

In 1930, Dr. Shope recovered a virus from pigs and it was in 1930 that the first influenza was isolated from pigs and then three years later, in 1933 in England, the influenza virus was demonstrated in human beings. So that took a little while to happen, you think from 1918 until 15 years before the cause was really known.

So this may be difficult to see, but it gives you a little sense of what we're here in 1918, but it shows you that before that time, down through here, there were a lot of influenza epidemics and pandemics that were known about. If you go online you can find several days' worth of reading that will show influenza way before then, and it's reported that Hippocrates described what was influenza in human beings 400 years BC. So it's been with us for a while. So, there we are and if you look on this, there's a little bit of gap from 1918 up until 1946 and '50 when there are other sorts of significant occurrences of influenza in human beings.

The virus is a swine influenza in 1930, a human influenza in 1933, so there was a disease a long time ago, back in 1878, described in Italy, and other places in Europe. It was a highly infectious and fatal disease of particularly chickens and other fowl. And over the years, it was finally determined that it was not caused by bacteria, it was caused by a filterable virus in 1900.

Well, it was only 55 years later that in Germany, Professor Shaffer was able to show that this disease was an influenza virus. And so this was our first real understanding of an avian influenza.

In the next year, in 1956, simultaneously in England and in Czechoslovakia, a respiratory disease of ducks was described, and from them, those two places, the influenza virus was isolated. And notice this duck with respiratory disease but with substantial sinusitis here.

And so 1955, first time we knew that there was an avian influenza and then bingo, 1956 in England and in Czechoslovakia in ducks. And then also in 1956, there was a respiratory disease in horses in Czechoslovakia, and that was finally determined to be caused by an influenza virus. So all of a sudden, animals are playing a big role with their influenza.

So we're now in 1956. Well in 1957, was the Asian influenza pandemic. And this is the World Health headquarters in Geneva, Switzerland, and the director of veterinary public health there, a Dr. Martin Kaplan, a veterinarian, who had a lot of connections with the University of Wisconsin, decided that during that pandemic, it might be appropriate to look at animals from various places in the world to see whether they might have had influenza. So they collected-- He had people from various places collect serum from those animals and it was tested to see if they had influenza antibody. And sure enough, they did. A variety of animals. So this is now 1957.

So we jump two years, 1959, in northern Scotland, then bingo, there was an outbreak of disease in chickens, widespread, that was highly fatal disease to the chickens, and it was eventually determined that that was caused by an influenza virus. And then we jump two years to the tip of South Africa and you see a dark place down there, and the bird flying is a tern. And there was mass mortality of terns along the South African coast and it was eventually determined that that was caused by an influenza virus. It just happened to be similar to the influenza in chickens in northern Scotland, which is a place that terns migrated to. So they see more and more species are being involved with this.

So as more and more species

began to be involved,

Dr. Kaplan here decided that maybe the World Health Organization should start an international program on animal influenzas, and see what's really going on in the world. And he established this in the early 1960s and these are some of the people, not all of the people, who participated in the program, but across there we'll see people from Switzerland, Germany, England, Hong Kong, France, USA, England, Australia, New Zealand, Soviet Union, Czechoslovakia, USA, and there were others I know from Italy, Hungary, Israel, et cetera, whole bunch of places. And over the years of about 25 years, this group of people, international group of people, were able to find many different kinds of influenza viruses in many different species.

So in the mid-- Along with the time that that program was started in the mid-1960s, influenza viruses were reported in turkeys. Now you may know that northwestern Wisconsin is a very active, big-time turkey production area. This happens to be a turkey house in northwestern Wisconsin. And it's nice when they all look like this. But then, something may happen and some how they don't look so good anymore if you look at this, they're kind of ruffled, and you see some dead ones lying on the floor, and give it a couple more days and it might look like this. So with virtually the whole flock being wiped out by an influenza virus. So this happened in several places in the United States in the mid-1960s, started in England, Ontario, Massachusetts, Wisconsin, California, et cetera, lots of it. And you can imagine when you look at that, go back to that first slide of turkeys, and this slide of turkeys, what an extraordinary economic loss this was, caused by that influenza virus. And then a little later on in the '60s, it hit some chicken flocks in various places, and here is a dump truck full of chickens,

most of which had died, but the rest of them would have been eradicated just to try to stop the disease, so again if you look at that you get a sense of what a fantastic economic loss this was.

With all this happening, people began to wonder, well, I wonder about all of our experience except the tern, are domestic birds, what about wild birds? And the wildlife disease people in Wisconsin had collected serum from several different waterfowl in locations marked here on the map, and so we're able to get some of those sera and test to see whether they might have antibody in their serum which would indicate they had been infected, and sure enough, there were.

So this sort of sparked here, and in other places, to begin to look at wild birds, and particularly migratory waterfowl, to see what might be the situation. So it was very convenient here in Wisconsin during the duck hunting season to go over to the Mississippi River and wait for the duck hunters to come off the river with their ducks and say, sir, could we sample your duck? Well I'm not sure about the duck but you can sample my friend. 
(audience laughs)
 So, 
(laughs)
 but anyway, so when sampling ducks there for several years, many different influenza viruses were recovered there.

And so many other places in the world began sampling to see what they might find on migratory waterfowl and sure enough, there it was. Lots of it. And then the question was, well, what about seabirds? And so we had an opportunity to go to the Pribilof Islands on the Saint Paul Island where thousands of various kinds of seabirds nested on these cliffs. We were able to net those birds, sample 'em, and sure enough, there was influenza virus there too. So just more and more of these things going on. So turns out, yeah, avian influenza is indeed a global problem and continues to be.

In 1976, at Fort Dix, New Jersey,

there were a few military people who became ill and when they were taken care of, a swine influenza virus was recovered from those GIs. And what was interesting there, but with those people who were involved, they could make no connection to swine. None of them had been in contact with swine at all. But by that time, there was considerable evidence that people who had been in contact with pigs

had been infected because when they were tested they had antibody in their

serum which indicated yes, they had been infected. And a very dear colleague and friend of mine, Dr. Dean Pawlisch in Broadhead, Wisconsin, said to me at that time, he said, "You know, I think I've seen in my practice, "when I've had a case of swine influenza, "that the farmer, the farm help, "have been sick." So I said, well, when it happens again, let's see whether we might find it. And not long after that, I got the call, and he says, "I have sick pigs and a sick farmer." We collected the samples and sure enough,

both the pigs and the farmer had that. So that was the first evidence that there was indeed a direct transmission. Well, that was reported to CDC, the communicable disease center, and that brought upon a deluge of national media to Broadhead, with national TV, et cetera, et cetera. And a lot of the local people weren't very pleased with that, and the local newspaper then said, "Here's the reaction to the case of flu. "It's a bunch of hogwash." 
(audience laughs)
 And so with that hogwash now,

I'd like to pass on an unfinished story of discovery to my colleague, Dr. Olsen.

(audience applauding)
- Thank you very much. Just switch presentations here quickly.

So it's really a pleasure to be here and thanks so much for so many people coming out.

You've probably all heard the expression that something or other will happen when pigs fly. Well, I guess the past tense of that would be when swine flu. 
(audience laughs)
 I'm sorry, it's bad, I realize. 
(audience laughs)
 But I'm going to try to, in 15 minutes, summarize 27 years of research that I've been doing here at UW and to just try to give you a sense of kind of the progression of influenza research that's happened on this campus. And really the theme that I'm going to cycle through my comments will be this idea of what we call zoonotic swine influenza. A zoonosis is an infection that is caused by an agent that can be either transmitted from animals to people or shared by animals and people, and we're going to keep coming back to that.

You know, science is a very iterative process. Everything builds on what has come before, and I am extraordinarily lucky to have stood on the shoulders of really two giants in influenza research here at UW-Madison. And the first is Dr. Easterday, who you've just heard from, a valued mentor for me and an even more valued friend.

And Barney was smart enough to hire this woman, Dr. Ginger Hinshaw.

When did Ginger come to UW, 1980...

- Eight, I think.

- '88 or so? And at about that same time, I was a graduate student at Cornell, and she came and gave a guest lecture in one of my graduate virology courses, and talked about this idea of influenza viruses moving between animals and people. And I was hooked, I knew this is what I wanted to do for the rest of my career. And so I am, as I said, extraordinarily grateful for the base upon which I was able to then contribute a little bit more.

So going back to the story that Dr. Easterday told you and the original isolation of a swine influenza virus from a human being in Broadhead,

jump forward now about 30 years from there, 35 years, and I worked with Greg Gray and one of his graduate students, then at the University of Iowa. And basically what we wanted to do was look back through the literature and say, well exactly how many times do we think this has actually happened?

And the answer was, over a period of almost 50 years, we were able to document 50 cases. A bunch of those were from that Fort Dix outbreak in 1976, the other were among civilians. And when you go through that literature, you find that people who have swine influenza look an awful lot like people who have normal human influenza. It is typically not a clinically remarkable disease. There were a few cases of mortality events in people, but these were primarily in individuals who had some comorbidity, some other underlying disease, that may have made them more susceptible to having a serious outcome.

But then we really started thinking, and saying, you know again, thinking back to the work that Dr. Easterday had done, if you consider how many pigs there are in the United States and how many people there are who work with those pigs in the United States, surely these infections must be crossing from pigs to people more commonly than those clinically-defined cases would suggest. And so working with, again, Greg Gray and colleagues at the University of Iowa and working with colleagues from the CDC and elsewhere, we did a series of studies to essentially build on what Dr. Easterday had done and look at farmers, farm family members, farm workers, in Wisconsin and in Iowa, who were working with pigs, and follow them to look for the presence of antibodies in their serum against swine influenza viruses. And we did two studies in which we looked backwards, retrospective serologic surveys, to say, you know, do we see evidence of past infection, and then we did two studies in which we looked prospectively and we enrolled individuals, in one case, hundreds of people, and followed them for clinical illness consistent with influenza and for seroconversion, for evidence that they had developed antibodies in their serum. And to cut a very, very long story short, I will just say, there was very clear evidence that swine influenza viruses move from pigs to people on quite a regular basis when we're talking about people who are regularly, repeatedly, daily in most cases, exposed to pigs.

So I think we can all agree 
(audience laughing)
 on any of a number of levels, that this is just not a good idea. 
(audience laughing)
 I, of course, look at this and I'm only thinking about transmission of influenza viruses.

Now, when we look at those cases, we find that the vast majority of them are what we call epidemiologically, dead ends. Meaning that there is transmission from a pig to a person, person seroconverts, they may or may not get sick, but they typically don't transmit it on to someone else, who transmits it to someone else, to someone else, as we see in community outbreaks of regular human influenza. That's a good thing. But that story changed dramatically in 2009.

Because in 2009, we saw a pandemic of influenza throughout the world that was ultimately shown to be due to infection with an influenza virus that had originated in pigs. And so this was not a series of one-off infections, this was a virus that had acquired the ability to move effectively from person to person to person to person and spread throughout the world.

Over 200 countries and territories were affected.

It's really unclear what the total impact of that pandemic was. The official count was a little over 18,000 deaths from the World Health Organization numbers, but in fact, estimates in the United States suggest that nearly that many people died in the United States alone. And so it's very likely that this outbreak was much more widespread with much greater morbidity and mortality than what the actual reported WHO numbers would suggest.

So what was special about this virus? What was different about this virus than all of the other instances where a virus had moved from a pig to a person and that was as far as it went? Well, to try to explain that, I need to take you back to a couple of basics. One is the story that Dr. Easterday already alluded to, the idea that influenza A viruses infect a wide variety of animals in the natural setting and as well as domesticated animals, and I point out that we have this lovely picture of a duck in the center because we know that all around the world, migratory waterfowl are the primary reservoir for influenza viruses of all kinds.

And then periodically, either over evolutionary time, or in real time, periodically viruses move out of that natural reservoir in waterfowl to infect other animals, to infect poultry, to infect pigs, maybe to directly infect people, to infect marine mammals. There's an outbreak going on off the Massachusetts coast right now in real time today. It appears to involve both influenza and a virus related to distemper in dogs. To horses, we've seen instances then of movement of influenza viruses from horses to dogs, the origin at least of the original canine influenza virus a number of years ago. And we've seen some of the highly virulent avian influenza viruses move into cats. Both domestic cats and the large cats.

So that's one thing that I want you to just keep in the back of your mind. The other thing is I need to tell you a little bit about what influenza virus looks like as an entity, okay? So, very accurate representation of an influenza virion.

Two large proteins on the surface of the virus are called the hemagglutinin and the neuraminidase and we abbreviate them H and N. And it turns out that hemagglutinins come in 18 different flavors, neuraminidases come in 11 different flavors, and any given strain of influenza, when we talk about it being H1N1, H5N1, H3N2, all we're referring to is the form of those two proteins that the strain of virus carries. And then there's a lot of other proteins that we're not going to worry about, and the other thing I want you to notice is that influenza viruses are unique because instead of encoding all of their proteins on one long piece of DNA or RNA, they encode their proteins separately on individual pieces of RNA.

And that becomes very important. Because what that means is that if a host is infected with two different influenza viruses at the same time, then as each of those viruses makes millions of new copies of itself, sometimes it makes mistakes, and so in this example instead of getting all viruses with red genes out, or all viruses with black genes out, we get new combinations. And if you're quick with the math, you know that two raised to the eighth power is 256. Which means you can get the two original parent viruses back or you can get 254 new combinations. And so this is a very powerful way of generating genetic diversity and the potential for new influenza viruses to appear.

So that process is called genetic reassortment. Now we're going to talk about that over the next few minutes. So let's go back to the pandemics that we've known about. 1918 pandemic was an H1N1 virus. The next pandemic was in 1957, and it was an H2N2 virus. And how did that new virus come about? Well, if you take that original 1918 virus and you reassort it with a virus from a duck, and that duck virus provides a new HA, a new NA, and a new polymerase protein. Abracadabra, watch the diagram on the left, you have created a new virus, and in this case, a virus that had pandemic potential. The ability to spread throughout the world.

Part of the ability to cause a pandemic is that immunity against any one hemagglutinin type does not confer to another type.

1957 was H2N2, that was the so-called Asian flu. In 1968 the so-called Hong Kong flu, that was an H3N2 virus. You're probably starting to get the idea here. You take that virus, you reassort it again with another duck virus, and voila, we now have another generation of reassortment happening.

And guess what, that's exactly how the 2009 pandemic virus came about as well, although a very complicated reassortment because this virus has genes from four different genetic lineages of influenza viruses. The classical swine viruses that we've been talking about, a human virus, avian viruses, and then this unique Eurasian swine virus which is actually an avian virus that has evolved over time in pigs.

So a complicated reassortant with genes from multiple places. That was 2009. Guess what? In 1998, my lab and two other labs almost simultaneously isolated a unique virus from pigs. It was an H3N2 virus and basically we hadn't seen H3N2 viruses in pigs. Since 1930, we had seen nothing but classical H1N1 viruses. And I remember when a gentleman in my lab named Dr. Alexander Karasin first isolated this. I sent him back to the bench and said, nah, that must be a mistake. We don't see those kinds of viruses in pigs. And in fact, this proved to be very much not a mistake.

These viruses, we have come to call triple reassortant viruses because they have genes from human influenza viruses, from the classical historical swine influenza viruses, and from avian influenza viruses.

This backbone is the basis of the 2009 pandemic virus. If you wonder how we do this, how do we decide where these different genes come from? We do it by what is now a very rapid process of sequencing every gene in the virus, and putting it into a computer program that spits out a tree like this, this is a phylogenetic tree, and basically it tells you who's related to who. And you can then do that for each gene in the virus and say, well, these genes are related to human viruses, these genes are related to swine, these genes are related to avian, et cetera.

And that H3N2 triple reassortant in 1998 was the beginning of a flood of novel reassortant viruses in pigs. We isolated and characterized H3N2 reassortants, H1N2 reassortants, H1N1 reassortants. We characterized H3N3, H1N1, H4N6 avian viruses in pigs. The world of swine influenza was exploding in the late '90s through the early 2000s.

And to spring this back to this idea of transmission from pigs to people, many of these viruses that we initially characterized have now been shown to infect human beings. H3N2 reassortants, H1N2, and H1N1. And now there are hundreds of isolations of these viruses from human beings. Remember that first study we did from 1958 until the early 2000s, we could find 50 cases over more than 50 years. Now we're finding hundreds of cases in a given year.

Something has changed, and what has changed is this triple reassortant combination of genes that has given these viruses a tremendous replication advantage to be able to infect and spread. So the last thing I want to tell you is I've made this sound like this is something really easy for the virus to do, and in fact, it's probably not, if you'll allow me to be a little anthropomorphic. Because we know that although we can have viruses moving around this triangle of waterfowl, pigs, people, and throw in poultry on the other side, we also know that it doesn't happen on a routine basis because there is a barrier, what we call a species barrier, to the movement of influenza viruses, particularly from avian species into mammalian species. And the basis for that is the kinds of proteins and sugars on the surface of our cells and pig cells and bird cells that influenza virus likes to bind to and use. And so I had the pleasure-- These are called sialic acids. I had the pleasure of working with some great collaborators, Jim Gern from University Hospital here at UW, as well as colleagues in England and in Russia to develop a system of taking cells from the trachea and bronchus of a person or a pig, learning how to grow them in culture and then learning how to drive them to actually grow on a piece of glass to look like a respiratory epithelium, with all of the different cell types with cilia that you could watch waving at you in a microscope, producing mucus, doing all the things that your trachea or a pig's trachea does. And then using those cells as a model to study influenza viruses, now in very realistic situation. And we could then look at the types of sialic acid receptors that all of the different cells possess. We could look at them from humans, we could look at them from pigs, and in doing that we've been able to really define what's important from the perspective of the virus, and what's important from the perspective of the cell to allow these types of interspecies jumps, if you will.

Sorry, another bad pig cartoon. 
(audience laughs)
 The reason we could do that is because of a technique that was developed by our next speaker, Dr. Kawaoka, and his colleague, Gabriele Neumann, a system called reverse genetics, which allows us now to make targeted mutations and then put it back into a virus and say, what does the virus do when we change these things? And that is a process that has absolutely revolutionized influenza virus research, and really, virus research in many ways, in general. And so I'm thankful that you developed that because it made a huge difference for me, and again it's this idea of iterative science and building one on another. So thank you all very much. 
(audience applauding)
- Okay, thank you very much, Chris. Look a lot younger than now. 
(laughs)
 
(audience laughing)
 All right, so what I'm going to do today is that I'm going to discuss some of our work.

I'm going to talk about something that we did on the Spanish influenza viruses. And Spanish influenza virus did not exist because back in 1918, there was no method to isolate influenza viruses.

At least, at that time we didn't even know Spanish flu was the influenza virus.

But Jeffery Taubenberger first PCR amplified the gene from the lung tissue embedded in paraffin block. And then they went to Alaska and dig out the victims of 1918 outbreak and obtained the lung buried under the permafrost. Then amplified the gene and then sequenced entire genome of the 1918 virus.

And as Chris Olsen described,

back in 1999, Gaby Neumann in our group, established method to generate influenza virus from plasmid. It's a very simple technique, simply introducing these plasmids into cells, you get influenza virus. Even high school students in my lab was working and produced viruses, very simple. That actually create the problem later on. 
(laughs)
 
(audience laughing)
 All right, so because the sequence now is available and what we did is that we simply synthesized entire Spanish flu genome and put it into plasmid and then generated the virus.

Then we tested its pathogenicity in non-human primates. We wanted to know why 1918 virus was so pathogenic.

Now because this virus caused major outbreak back in 1918, we wanted to be very careful, so we went to BSL-4 facility which is the highest containment laboratory. We collaborated with Dr. Heinz Feldmann.

He is the Ebola virologist, so he works with the Ebola virus under this condition. So we went there.

So people from my group, Darwyn Kobasa, and Hideki Ebihara went there and then generated 1918 virus.

So as a control, we tested the seasonal 2001 virus, just plain human influenza virus.

So we infected seven non-human primates with 1918 virus, and three non-human primates with the 2001 human influenza virus.

On day three,

all the animals in 1918 group lost appetite.

And no difference in 2001 group.

We took two animals from 1918 group and one animal for seasonal influenza group for pathological and virological studies.

On day six, one of the animals died, the other four animals got very sick, and no difference in the seasonal influenza group.

Took these animals for pathological analysis, and remaining three animals in 1918 group all died. We are supposed to watch these animals for two weeks, but they all died by day eight. Now this is 1918 virus, is the only influenza virus that kills non-human primates. Later on I'm going to discuss other, the highly pathogenic avian influenza viruses, bird flu, they don't kill non-human primates. This is the only influenza virus that we know kills non-human primates.

All right, then we looked at the tissue, lung. So this is a lung section, and this white area is where the air is filled and exchange oxygen. So this is 2001 seasonal influenza virus, almost no pathology.

But when you look at lung of 1918 group,

this white area is filled with fluid and the immunocyte.

So essentially, animals drown to their death.

So couldn't exchange the oxygen.

These brown cells are virus-infected cells, so virus do replicate in lungs.

And this pathology is essentially the same as what we see in humans who died in 1918.

Now, as we heard,

there are lots of avian influenza viruses out there, and 1918 virus likely originated from avian influenza virus. Now Chris Olsen described two, 1957, 1968, pandemic viruses are reassortants. But 1918 Spanish outbreak was caused by entire avian influenza virus.

And as Barney's already mentioned, waterfowl serve as a reservoir of influenza viruses, there are so many avian influenza viruses out there.

So the question is, does the gene pool that can lead to the emergence of a virus similar to 1918 virus still exist in nature?

So we wanted to experimentally test this question.

So as Chris Olsen mentioned, there are eight gene segments of influenza viruses. And there are lots of sequence information in a database. So what we did is that we compared amino acid sequence of this particular protein, PA protein, of the 1918 virus, and all the sequence in the database, and we found this virus contains PA protein that differ on the nine amino acid, compared with Spanish influenza PA protein. PA protein has about 750 amino acid residues. Out of 750, only nine differences.

So then we did essentially the same thing for all the genes. Now HA and NA, they differ about 5% compared with Spanish influenza viruses, but all the rest is very few, less than 10 amino acid residues.

So what we did is that we synthesized these genes

and then created a virus.

And we called it 1918-like avian influenza virus.

Now for virus to cause pandemic, the virus has to transmit airborne, okay, through the respiratory droplets. Now of course we cannot use humans for experiments, so we used ferrets. We used ferrets because when we infect ferrets, ferrets show symptoms like humans, like runny nose, high fever, and sneezing.

So this is experimental setting. We infect ferret with the virus,

and then next day we place a cage containing uninfected ferret but there's a gap between the two cages about two inches.

For the virus to be transmitted from infected to uninfected is only the via respiratory droplet. There's no touching between the two ferrets.

So under this condition we tested the transmission using authentic avian influenza viruses. Avian influenza viruses do not transmit between ferrets.

1918 virus under this condition we saw transmission in two pairs out of three pairs tested.

Now the 1918-like avian virus that we created based on the sequence similarity,

this virus did not transmit.

However, with only seven amino acid changes,

we saw transmission in two pairs out of three pairs.

So avian influenza viruses similar to the 1918 virus can become transmissible, in fact, with only seven amino acid was change.

Now when you think about potential of a pandemic with new viruses, there are several viruses we need to watch. One of them is H5N1 bird flu virus.

This is a virus that was first identified in 1996 in Hong Kong. Infected 18 people and six of them died. And since then they spread Asian country, and 2005 spread to Europe, Africa, and 2013 this virus introduced into United States.

So we tested transmissibility of this H5N1 virus in ferrets, and did not transmit. And in fact, this virus is not transmitting among humans.

But we found that with only four amino acid changes, this become transmissible in ferrets.

So when we tried to report these data, and our colleague in Holland have similar data and tried to report data, and there was a major discussion up there. 
(audience laughing)
 So, New York Times called it engineered doomsdays.

But I'm going to tell you this,

nature created much more worse the virus, that is the H7N9 virus. This virus was first identified in 2013. And since then, every winter this virus infected humans.

More than 1,600 people got infected and more than 600 people died.

Now this virus when first identified in 2013, this virus was non-lethal in chickens. So what was happening is that when chickens got infected with this virus, chickens do not show any symptoms, and in China people go to live poultry markets and buy live chickens. And they're not showing any signs of disease, so people don't know whether they have viruses or not. And then people get infected. But this virus became highly lethal in 2016, start killing chickens.

And people have been infected with this highly pathogenic form of the H7N9 viruses, and some of these people died.

So we tested transmissibility of these H7N9 viruses under the condition that I described

to test the pandemic potential of these viruses.

And this work was done in collaboration with many people, including Masato Hatta in our lab.

And so 2009 pandemic virus that Chris Olsen described, this virus caused pandemic, and when you test the transmissibility in this model, it transmits very well. We see transmission three out of three pairs.

Now the described H5N1 bird flu virus...

does not transmit if they don't have any mutations.

Now low pathogenic H7N9 virus that first identified in 2013, we saw transmission in one out of three ferret pairs, so without any mutation, this virus has ability to transmit in ferrets.

How 'about the highly pathogenic H7N9 virus that appeared in 2016? We tested in four pairs,

and we found, out of four pairs, we saw transmission in three pairs.

In addition, these two ferret died.

Also, these two ferret died.

And then this virus actually goes to the brain.

So this virus transmits well in ferrets via respiratory droplets, and ferrets die when exposed to even a very small amount of the virus in respiratory droplet and die.

So this is virus that is out there we need to watch.

Luckily China decided to vaccinate chickens,

the fall of 2017. Since then, number of human cases dropped, but because they used vaccines, the virus is out there, still circulating. So we need to watch this virus.

So in summary, Spanish influenza virus is lethal to macaques, and virus genes similar to those of Spanish influenza virus exist in nature, and H7N9 virus has a pandemic potential. Thank you very much. 
(audience applauding)