9UNP0164SD_Steve Marshall_WordPress 

- Welcome to Wednesday Nite at the Lab. I'm Tom Zinnen. I work here at the Biotechnology Center at UW-Madison. I also work for UW-Extension Cooperative Extension. And on behalf of Wisconsin Alumni Association, who also is a co-organizer of Wednesday Nite at the Lab, thanks for coming tonight. We do this every Wednesday night, 50 times a year. Delighted to have you. 

Tonight, we have Steve Marshall, who's with the State Lab of Hygiene. He'll be talking about "Pyro-Sequencing and Influenza Anti-Viral Resistance." 

Steve is a native of Cedar Rapids, Iowa. 

He got his undergraduate degree at Iowa State University in Ames, and then got his master’s degree in Epidemiology at the University oif Iowa. That's where our football coach is from. And then, he came to the State Department of Health for several years. Two years ago, he moved up here, two doors up on Henry Mall, to the State Lab of Hygiene. I don't know if any of you remember the Monkey Pox outbreak here in Wisconsin. He was instrumental in that. I don't think he caused it. I think he probably diagnosed it. 
(laughter)

 So would you please join me in welcoming Steve Marshall to Wednesday Nite at the Lab. 
(applause)

- Thanks, Tom. 

Monkey Pox. I didn't have too much to do with Monkey Pox, but I was there. As Tom said, I come from Iowa. I am a Hawkeye. I apologize. But I was at the University of Iowa doing clinical microbiology research for about eight years. 

We were looking at resistant pathogens from, it started from all over the nation, and then it moved to all over the world. This was a really big interest to me. I was really into the surveillance part of it. So I decided-- Excuse me? 
(inaudible)

 Sure, is this not picking up good? How's that? Is that much better? Okay. Am I going to be adjusted here? 

Okay, good. Thank you. 

Where was I? At the University of Iowa. Thank you. 

And I really got into the surveillance work there. So I decided to go back and get my masters in epidemiology. After that, my wife and I decided, let's go somewhere else. We've been in Iowa all our lives. So we made the big leap to Madison, Wisconsin. 
(laughter)

 It's huge. We actually went down to CDC and we had just started to have kids. And decided we could not raise kids in Atlanta. So Madison was the perfect choice for us. I actually got hired there under some bioterrorism preparedness money. 

This is pre-9/11, when the Feds were just starting to look at this. I was the only person hired at the State Health Department as an epidemiologist under those funds. I was enjoying doing a little outbreak investigation, looking at some E. coli. 

That was fun. And then 9/11 happened. Then the Anthrax attacks after that. That's when the Feds threw a lot of money at all the different states to start preparing and putting forward their plans, and doing exercises, and a myriad of other things on getting prepared for bioterrorism and chemical terrorism. And since I was the one there being paid under bioterrorism, they said, Steve, go do this. 

So we sat down with the state health officer at the time, the chief medical officers. 

We came up with our plan. We implemented our plan. But one of the biggest lessons of many lessons that I learned there is that the federal government, they're not the most proactive organization. They're much more reactive. 

As all the states moved forward with their plans, and had a good idea of where they were going, what they were doing, the federal government would start throwing curve balls. 

The first one was Small Pox vaccinations. Does everybody remember this? They decided that there was some intelligence out there that some Small Pox stocks from the Soviet Union were still out there. And so, we really needed to get all of our healthcare workers, to start with, vaccinated against Small Pox. So this was big. 

And our illustrious governor, Tommy Thompson, who was the head of HHS at the time, had this move forward. 
(inaudible)

 Sure. You want me to move this up? 

No, okay. Don't touch the thing, all right. I'll speak a little bit louder. 

So what happened was, basically, all of the states had to stop what they were doing and ramp up to go out and vaccinate, the plan was, half a million healthcare workers against Small Pox. That was Phase One. Phase Two was to open it up to all first responders, police, fire, what have you, and probably several million people to get vaccinated. That didn't quite happen. 

Most folks were more scared of actually getting the Small Pox vaccine and the complications that come with that than the actual risk of contracting the disease, which was a complete unknown at the time. A lot of people did get that vaccine. Most people did not. 

That was one of the first curve balls that was thrown at us. We recovered from that, started moving again, working again. 

Then we got another curve ball. This was called CRI, or Cities Readiness Initiative. And what happened here was there was some intelligence out of Afghanistan that there was some Anthrax stocks that were potentially going to be used in a large city. We were ordered to basically stop what we were doing and make sure we had everything in place to deliver antibiotics to an entire city, in our case, Milwaukee and all its suburbs, within 24 hours. That was what we spent a lot of time doing. 

After that was done, we started moving back into what we were doing to begin with. 

Then came the third curve ball. This was the obvious one. 

This was Hurricane Katrina. After Hurricane Katrina, and all the dismal things that happened to the special populations in that area, all of the states were ordered to look at their special populations. Make sure that everything we're doing is going to take all those folks into account. Luckily, in Wisconsin, we had already written that into our original plan. We had already made all those contacts and made those plans. But a lot of other states had to stop what they were doing yet again, retool and move forward. 

The first thing, and that will bring us to our story tonight, is pandemic influenza. 

Pandemic influenza, and most scientists agree, is going to happen. We don't know when it's going to happen. We don't know how big it's going to be, if it's going to be on the level of the 1918 Spanish flu or not. 

We've had a lot of advancements in medicine. We have a lot of antibiotics that can take care of the pneumonia, which is the secondary infection that comes from influenza in the pandemic flu situation. 

But this was the fourth thing that we were then told, stop what you're doing, now go work on pandemic influenza. What came out of that, and this is something that all the states were struggling now, is federal guidance that everybody should have a stockpile of antiviral medications, vaccines, so that if a pandemic flu does happen, we will have something to combat it with. 

There's a very big debate in the community and the scientific community which many of you, I'm sure, are aware of, that are these antivirals actually effective. And are they going to be effective against a pandemic influenza strain. 
(inaudible)

 2018? 

Oh, 1918, right. 
(inaudible)

 Right, and so, in every so many years, it depends on the situation, that there is a pandemic influenza. We've had, I believe, two or three since 1918. And they have been significant, but not on the scale of 1918. There's a lot of fear out there right now, with the H5N1 Avian flu out there, that this could be the next 1918 influenza. We don't know that for sure. We don't know that at all. We don't even know if the next pandemic influenza is going to be the H5N1. It could be a completely different strain. 
(inaudible)

 No, they have no idea. 

They have as much of a good idea as we do. But all the states were ordered to look at stockpiling antivirals. Antivirals are very effective in situations, for instance, in a nursing home, if there's an outbreak of influenza, when trying to prophylaxis or give people antivirals to keep them from getting sick from flu while other people in that institution are sick. It's a very effective tool. Whether it's very effective in the general population is also up for debate. You have to get those antivirals in your system within about 24 hours, 48 at the most, for it to have any effect at all. If you miss that window, they're pretty ineffective. 

Now, I've heard from several people, and it's anecdotal evidence, that have the influenza then got those antivirals, and they will swear by them. And so, they're out there to stay. What the best it will do is cut off a day or two, three at best, of your symptoms that you're suffering from influenza. 

It is by far not a cure-all. You're not going to instantly be cured from influenza. It doesn't work that way. 

So will it be effective in stockpiling and using against a pandemic influenza. A lot of research going on. A lot of modeling going on. But the development of resistance to those antivirals is a big concern. And most of the models are showing that yes, if you use antivirals in a pandemic situation, they will rapidly develop resistance. They may be effective in the first wave of pandemic influenza for helping. But after that, there's usually a second wave that follows in a pandemic situation. 

It will most likely be completely resistant and useless at that time. 

So this kind of gets me to our story tonight. And before I start, I want to say that coming to the University of Wisconsin, now being at the State Lab, which is a wonderful place to work, I feel very honored to be here to be doing any kind of influenza research at all, because I'm a bacteriologist. When I came here two years ago, my boss, this was all starting to play out with the pyro-sequencing with the antiviral resistance, and he said, hey, do you want to take this on, do you want to run with this and work with it. And I said, yeah, pyro-sequencing, that sounds cool. 
(laughs)

 There's music. There's music when I joined. 
(laughter)

 And I thought, pyrosequencing, you know, I used to be a Boy Scout, which means you're a pyromaniac if you're a Boy Scout. 

I thought this has to be cool. I can't pass this up. 

It has nothing to do with fire, whatsoever. But to be here at the University of Wisconsin is a special thing, because right now, there's so much great research going on in influenza. You have researchers doing some fantastic research, looking at the 1918 strain, looking at H5N1, looking at the mechanisms and pathogenesis of that. Fantastic research going on. You have Chris Olson in Vet Med doing swine influenza research. You have Pete Schult here at the State Lab doing fantastic surveillance of influenza from all over the state. You have Stacey Shultz-Cherry at Med Micro doing some fantastic research looking at diagnostic methods for influenza. She's working with nanotechnology companies, trying to use new diagnostic technology. 

It's really special stuff. It's Stacey, that actually recently has pulled together all the different influenza researchers on campus and is having a monthly influenza research group getting together and sharing all of the advances that we're working on. 

And I would say that arguably, we're one of the epicenters of influenza research in the world right now, if not one of the top couple. Yes? 

- Recently reporting not to worry about bird flu, that is the latest thing to come out. You know, I know we scared you, now it's not nearly as bad as we thought it would be. 

- Wow, the question was, why is the media now saying don't be so worried about bird flu. We scared the hell out of you, and now we're telling you don't worry about it. I haven't heard too much of what you're talking about. But my guess is it's the flavor of the month has gone stale. 

And something else will be filling those shoes soon. The danger is always out there. 

It's always been out there. It will continue to be out there. The risk is no greater now than it was ten or 20 years ago. We have no idea if the bird flu that's out there is going to become the next pandemic influenza strain. We are in an elevated, if you look at the pandemic phases that move through, the pandemic, we are in an elevated stage right now. But there's still not been sustained human to human contact. And until that occurs, that's when you're going to see a lot antsy-ness and a lot of people jockeying. So I guess don't worry about, yes, carry on. 

It's the same threat that's always been out there. 

So let me jump right in to where this all began. 

Our story starts in 2005 when, and I hope you are able to see these. I'm a data geek, and I love this stuff. And I'm hoping you are too. I get excited about it. But hopefully, you'll be able to see what I'm talking about. 

What happened is there's a fellow at CDC named Rick Bright, was doing some research and looking at using pyro-sequencing as a rapid method to detect the existence of antivirals. Some researchers up in Marshfield Clinics here in Wisconsin were also doing some influenza research. They identified a lot of influenza strains that were showing up resistant to the most commonly used antiviral. 

I'll share a little bit about that with you in a minute. 

What they were seeing was a big leap. 

Let me see if I can use this pointer. For many years, it was less than one percent resistance to the Influenza A. I'm not sure how detailed I need to get. Influenza comes in a couple different forms, Influenza A and Influenza B. Influenza A is usually what the pandemic strains are. They're never Influenza B. Influenza A is further divided into subtypes. The current ones that are out there circulating in the community right now is Influenza A H3N2, and I'll explain what those numbers mean in a minute, and Influenza H1N1. 

Those are the three trivalent pieces that are in the influenza vaccine. The H3, the H1 and a flu B. 

So those are the three strains that are in there. The vaccine didn't cover everything that greatly this year. It wasn't a perfect fit. That's a whole other lecture that I won't get into. 

But the point being here, is where these influenza A H3s, there's been a less than one percent resistant rate for many years, hovering at about one or two. Then we get into the '04-'05 season, and it spikes up to about 14% or 15% resistance. The following year, we came up with 98% resistance. It's basically made that antiviral completely useless against influenza. 

Rick Bright at the CDC had done some fantastic work with this. He was an LTE down at CDC, not really a full employee. 

After this discovery, it hit JAMA, it hit all the journals. It was a pretty big deal. He actually went back to CDC and said, hey, I'd like to be a full employee, if you don't mind. I just made this discovery for you all here. They said, sure, at our pace, will get you to be a full-time employee, in two years or something like that. He left. He went to Novavax, a private vaccine company. Since then, they've hired a couple people to do the job that he was doing. 

They're really great people, and they've been really good to work with. But that was very unfortunate. 

Now, this should've been no surprise, and it wasn't any surprise. Because what this graph shows is these are different countries in Asia that saw this same spike of resistance starting in 2002, 2003, 2004. This is China, Hong Kong, South Korea, Singapore, and then Japan is in there somewhere. In Japan, they eat antivirals like candy. They just, everybody takes them. They think they're great. In Asia, they use them, they feed them to chickens, to their flocks, to keep the influenza down. So it's no surprise that we saw this resistance emerge there first. 

What was also no surprise is this now came into the United States and the western hemisphere. 

The CDC then made a health alert notice, which basically said stop using this type of antiviral. 

You should all be using this kind, which is a whole different class of antivirals. I'll talk about that in a second as well. This had implications right away. This is before the pandemic stockpiling started to occur. It was right around the same time when people were starting already to look at, do we load up with one kind of antivirals, because they're cheap, they're usable, or do we go with the Tamiflu. Most everybody has now gone with the Tamiflu, and here's why. 

These are the two different classes of antivirals. There are M2 inhibitors, and there are neuraminidase inhibitors. 

The M2 is the Adamantanes, two types: Amantadines and Rimantadine. Amantadine has been around since the '60s. 

It's the mainstay antiviral. We've had it forever. It's now a generic, it's been out on the market so long. 	00:19:33:27	It's dirt cheap, $1.60 for a whole treatment. It is only effective against Influenza A, as is Rimantadine. I'll show you that one in a second. Rimantadine was developed, Flumadine, late in the '90s, because there are some psychotropic effects of Amantadine. There can be some hallucinations, that kind of thing, as a side effect. 

Rimantadine was supposed to solve those. Rimantadine is also used for Parkinsons disease. 

For folks who have the shakes or Parkinsons disease, this is one of the drugs they can take that will suppress the shakes. Michael J. Fox, or others, this is what they're taking before they go and do a shoot or a movie or something. So it's still a useful drug and it's being widely used today, but not for treatment of influenza. 

The neuraminidase inhibitors, Oseltamivir and Zanamivir. This is Tamiflu and Relenza. Most of you, I'm sure, have probably heard about Tamiflu and Relenza. They are effective against both Influenza A and B. 

The problem is, they're not dirt cheap. They're very expensive. 

Roche is making a tidy profit off the stockpiling of Tamiflu, not only in this country, but in countries all over the world. This is a five-day treatment regimen for $63. That's not cheap. What the federal government said with their stockpiling recommendations is states will pay this much for your stockpile. And I forget what it is, if it's 25%, or such a percentage. If you go out and pay for the rest of the stockpile. That has put all of the states in an awkward position of they're still having to debate whether they should even be stockpiling at all. Now all of a sudden, they look really bad if they don't start stockpiling, and the federal government is giving them money to stockpile, and they need to pony up their own money to start doing this as well. 

It puts all the states in an awkward position. And they don't have much time to debate it. 

I believe in Wisconsin, and luckily this happened after I left, they are doing some stockpiling, but not to the maximum. I think most states are going to end up doing that. Frankly, politically, they can't just say no, we're not going to stockpile. It's something they just can't do. Question? 

- What's the shelf life? 

- That's an excellent question. I don't have an answer. 

I don't know how long you can keep them. Oseltamivir is an oral antiviral. 

It, I imagine, has quite a long shelf life. That's why it's being used to stockpile. Amantadine and Rimantadine are as well. Zanamivir is a mist inhaler. It's a spray. You have to actually buy an inhaler with it and then spray it and breathe that in. I don't know if it has the same shelf life as the Tamiflu. I suspect not, but I don't know that for sure. 

But you see Relenza is actually a little cheaper than Tamiflu. That does not factor in the cost of you having to buy the inhaler. 
(inaudible)

 Are they a synthetic compound? 
(inaudible)

 That, again, that's another question. I believe that Oseltamivir is a synthetic. But again, I don't know that for sure. Tom? 

- I just looked it up. 

The shelf life of Tamiflu is ten years. 

- So that's going to be good for ten years. But ten years goes by fairly quickly. And if we don't have a pandemic in ten years, we're going to have to re-do this all over again. 

So, let's hope for a pandemic right away, right? 
(laughter)

 Okay. All right, I've got some great work to do! Okay, let's move on. 

I'm going to do a little virus 101 for you here, so stick with me. 

This is a typical, rendering of an Influenza A virus. You have your RNA genetic material on the inside. You have a lipid membrane all around the outside. You also have surface proteins. You have a hemoglutinen, which is the H in H3 or H1. 

You have your neuraminidase, which is the N in N2 or N1. That's where your nomenclature comes from. The hemoglutinen is there. It's a receptor to attach to a host cell when the virus gets into your body. It attracts a host cell. 

It penetrates that cell, goes inside that cell, replicates itself 100 times, 1000 times. And then it releases itself from that cell and goes on to infect more cells. That's how the virus operates. The neuraminidase is a receptor that once that virus has gone and replicated and is ready to leave the cell, it lets it go away from the cell. Without those two receptors, we would have no viruses, which would be great, wouldn't it? 

Also on here is an M2 protein. And there comes in the M2 antiviral inhibitors. This is a protein that allows ions to go in and out of the cell. This is the mechanism of action for the Adamantanes. And why these are only found on Influenza A, and that's why only Adamantanes are effective on Influenza A and not on B. 

This is a very difficult slide. I apologize ahead of time. This is a rendering of what happens when a virus enters into a host cell. So here's a virus here. It penetrates into the membrane of a host cell. 

It gets into the cell. It then uncoats and releases all of its genetic material. The genetic material is carried into the nucleus. It's replicated there, many, many times. It's then carried out of the nucleus, where it's restructured again into new virus particles. 

Those viruses then head out of the membrane, using the host cell's own membrane to become its new membrane. Then it's released on to infect new cells. 

What happens with the Adamantanes is the virus gets in here. And here's a rendering now of that M2 protein, where there's a pH change. The hydrogen ions are able to flow into the virus. 

And that pH change is what triggers it to then uncoat and release all of its genetic material. What Adamantane does, is it gets right into this M2 protein and blocks it. It does not allow those hydrogen protons to go in and do its business. With no pH change, therefore the virus gets in the cell, but it doesn't do anything. It just sits there. 

That's the end of it. 

So how do we detect the resistance that we're seeing in viruses. There's basically two different ways. There's phenotypic assays and there's genotypic assays. The phenotypic is, you basically take a virus and grow it up in a cell culture, and you grow it up without the antiviral. 

And you put antiviral in another one. And you see, does it grow, yes, it grows here. Did it grow in the antiviral? If it doesn't, it's inhibited. If it grows, you know that antiviral is resistant to that antiviral. 

That can take from seven to ten days to do that. Growing virus in culture is a very long process. Not too many people are doing it anymore. There's only a handful of virus labs in the state. Most of those are going away from culture. 

We still do the culture at the State Lab, but we're moving rapidly to all molecular methods. We'll always do culture, because we are the State Lab, and we always have to have that method on hand. But molecular is the wave of the future. That's, again, a whole other lecture. 

So the genotypic assay, that is where you actually look at the genetic material that is responsible for causing that resistance. 

So you can locate the DNA where that change has occurred that's causing resistance. Two different ways to do that right now. There's is conventional sequencing, the traditional method. And there's now pyrosequencing. What Rick Bright did at CDC, was he showed that there was 100% correlation between pyrosequencing and conventional sequencing. 

Pyrosequencing is a lot quicker than conventional sequencing, as well see right now. Question? 

- What does, the virus is adapting in the M2 protein is common to all viruses. What's the change that keeps the M2 inhibitors from blocking that channel? 

- There's a point mutation, one amino acid change that's allowed for that. 

That's an important point. Because without there being just one resistant mechanism, it would be nearly impossible for us to do surveillance. When this was occurring, there's probably a handful of different resistant mechanisms for Adamantanes out there that can occur. The one that took off was this one S31N mutation. Because it's the same mutation that we're seeing all over the world, it's very easy for us to pinpoint and detect that one mutation. 

So the traditional Sanger cycle sequencing and the pyrosequencing are compared here. Both of them require a PCR step, where you have to amplify the virus DNA. You isolate the virus DNA, you amplify it. Now you have something to work with. Then you go and sequence it. 

With the Sanger method, you have to run that out on a gel, by electrophoresis. So you actually separate the genetic material you just amplified onto a gel. You then have to clean that up. That means literally taking a knife and cutting it out of that gel so that you have that product. Then, you go through and you do your cycle sequencing reaction. 

You have to do another further clean-up step on that. I'm not that familiar with cycle sequencing, because I don't have to do it. I just do the pyrosequencing. Then you actually pull down the sequencing. This, after doing the sequencing, which can take a half a day or a day, you now have to do the data analysis. 

And looking at the information that comes out of cycle sequencing is very complex. And it takes quite a bit of know how and it's almost an art form in some cases to do the data analysis. This all takes at least two days to perform. 

With pyrosequencing, you take that same genetic material that you just amplified. You do a preparation step, where you actually anneal to your PCR products a sequencing primer. 

You put it on the power sequencer. It runs for about an hour. And then it automatically does the data analysis in less than five minutes. So it's a fantastic thing. 

Now the problem that CDC was saying early on was, that even though they do all of this PCR, they need to culture the virus before they could even do that PCR stuff. 

And that, again, can take several days, up to seven days. 

What we did at the State Lab was we looked and compared it using direct clinical specimens, taking those specimens as soon as they come in the door. Amplifying them and then testing them on the pyrosequencing. There's a certain point where some of them just don't work. But when you're just looking at surveillance, you don't have to look at everything. 

You just want to get a good sizeable number to look at. So we determined it was okay if there are some that weren't going to work at a certain point. And it's okay to use all the other ones. Basically, we're missing maybe ten percent of what we wanted to look at. So we've got 90% of what we're looking at. 

That's pretty good. That allows us to do this directly. We can do this entire reaction for somebody, if we get a sample that morning, we can have the PCR result before the end of the day pretty easily. That is going to be important, especially in these nursing home type outbreaks, where we have resistant strains circulate. 

So I'm not trying to do a commercial for the pyrosequencer, but this is the machine. 

It is faster. It's much simpler than doing the cycle sequencing. You don't have to worry about gels. It's less expensive, as long as you're doing large volumes. This is a 96-well plate format. 

You can do 96 reactions in one hour. As long as you're doing a sizable number, you're covering your reagent cost, you're covering your tech's time for doing this. It's much more cost effective. 
(laughs)

 Okay, here's another graphic about the preparation step. There actually is a vacuum tool to pick up all the PCR product out of your 96-well plate. 

You then go through several wash steps. You put it into another 96-well sequencing plate that anneals the sequencing primer to your PCR product. You're good to go. You're ready for your actual sequencing. 

Here's a picture of a tech actually putting a reaction onto the machine. Loading it into the machine takes about ten minutes. You have to put in a little cartridge. All of your nucleotides, your A's, T's, C's, G's, some enzymes, substrates. And again, you're good to go. What the reaction is, is a systematic cycle of dumping in nucleotides. 

So you have four nucleotides. And what we're doing here is we're actually creating, synthesizing our own RNA. So you dump in, for instance, a cytozine, the C, is dumped into the reaction, in these little, tiny well, these little, tiny reaction wells. If there is a corresponding G for that cytozine, it binds. 

That binding reaction releases a pyrophosphate, hence the name pyrosequencing. It had nothing to do with fire. I was incredibly disappointed. 

Sulfurase is an enzyme that's in the reaction that converts that pyrophosphate into an ATP. Luciferase, another enzyme, converts that ATP into energy in the form of a burst of light. The machine measures that burst of light. 

If there is another G waiting, then another C will insert, and another burst of light will be generated, and so on. If there is not another G, all of these C's that were dumped into the reaction are eaten up, gobbled up, by this apayrase enzyme. Then the next nucleotide is dumped in, say a T. Same thing, if there's a match, you generate a burst of light, and so forth and so forth. 

To go through one of those steps, dumping each of those four nucleotides takes about one minute. Then we do about 60 cycles, so we get about 60 of them, which is pretty optimal for this. It's one hour to generate this whole thing, which to me, is just incredible. 

What is generated is a pyrogram. They must have patented these names, because they're very cool. The pyrogram actually gives you the sequence. 

Here is the sequence that is generated. How you read this, this is susceptible wildtype that's out there and this is the resistant mutation that we're looking for. You get a burst of light that's about that high. That registers the A. 

Another burst of light registers the G. And then this one registers the T. The mutation is two A's and one T. There's a burst of light and then there's a second burst of light, the first going to a higher peak. You can see that over here. As an example, there's three C's in a row. 

This is three bursts of light. That's basically how you read that. The computer automatically generates this. It automatically evaluates the quality of each one of these pyrograms, so that if there's something that's wrong, you can know right away. 

Again, that takes about five minutes to do. 

So what have we been seeing, now that we've been looking for these? Well, for the first year we looked at this, we saw a lot of resistance in the H3. Remember, it was 90% resistant when we started looking at this. We saw about 75% in Wisconsin. The CDC saw about 99%. 

They were looking a lot at the southern states. We're looking more at the northern states. We're partnering with the New York State Health Department to try and form a network of state public health laboratories that have this technology. The CDC, how we got the pyrosequencer at the State Lab in the first place, the CDC actually called us up. 

Because, remember the original research was being done at Marshfield. They were starting to panic, because that was when the H5N1 was being seen. They thought that if this does turn into the next pandemic influenza, we are going to be inundated with requests for looking at antiviral resistance. There was about two people down there doing that work at that time. They asked us to get a hold of one of these pyrosequencers and start doing this work in case they needed some relief. We actually, with our state health department, used some of that pandemic influenza money that the feds were giving us, to actually buy the pyrosequencer, and get a hold of that. 

Right now, Wisconsin and New York are the only ones doing this testing. But several other states, Minnesota, Michigan, California, Connecticut have got the technology to bring it onboard. So they'll all be a part of this network, hopefully by this time next year. But right now, Wisconsin and New York are going to publish some of this data. 

Question? 

- Why in the pyrosequencing technique do you get different amplitudes of the pulse on each nucleotide? 

- Let me go back. Why do we get different pulses? So why are we getting one of these as opposed to one of these? Okay. 

Let me go over my example one more time. If there are two A's here in a row, okay, that means there's going to be two bursts of light. There's one burst, and then another burst right on top of it. So it gives a higher peak. 

The next thing that comes, there is no T in the next reaction. There is no G. But then there were three G's in a row. One, two, three. One of the limitations of doing pyrosequencing is having homopolymaric regions. That means when you have lots of areas where you have multiple same nucleotides in a row. 

When you get five, six or seven in a row, you're going to have a peak that's off the chart. And it's going to be difficult for the software and the computer to be able to handle that. So we only want to look at segments that have not too many repeating nucleotides. 

The other limitations I was talking about in pyrosequencing is you only get these short reads. 

Optimal is about 50/60, okay. They're working on the technology to get this to read out up to 100 or 200, base --, but that's for sure going to be the absolute limits of the technology. If you want to look at long, long regions, you need to do full sequencing, and you need to do Sanger sequencing. 

- Did someone say cycle sequencing is still the gold standard? 

- Yes, absolutely. When we first got this technology, I got a hold of Yoshi and said, hey, we've got some cool technology. 

Yoshi has a great lab and he's got a lot at his disposal. And he's not in a situation where he needs something right away. He wants to have the best information possible. So he sends his out to the North Carolina facility and gets all of his sequencing done there for the top dollar. 

So in our situation, we're looking for surveillance, something we need to turn around right away. This is going to be more primo for us, because it's also going to be less expensive. So it all depends on what you're trying to do with your research. Question? 

- What is the implication-- the reagents and the reaction? 

- Exactly, the reaction size, the technology just isn't there. 

I couldn't go into a lot of the detail about why that is, but people much smarter than I am have told me that that's the case. Okay, does that answer your question? 

So, what we saw the first year we started looking is, again, lots of resistance in the H3. We saw nothing in the H1. 

CDC saw a little tiny bit. They were looking more in the southern hemisphere, where this was actually starting to look at some of the places like Puerto Rico, that kind of thing. They've had some of these H1 resistance. This year, a different story. Again, everything is resistant with the H3. We had hoped that removing the use of Adamantane into the community, that susceptibility would return and we'd be able to use that drug again. 

That doesn't appear to be happening, at least over the last two years. That may happen in the future, but I certainly wouldn't hold my breath on that one. 

The H1, however, are a different story. We started seeing them here. 

The CDC has seen a lot more. The one that we saw here didn't actually occur here. This was from a person who had traveled to Nepal and got sick in Nepal and brought it back here. We actually tested him right away because we thought this might have been H5N1. It wasn't the case. It was an H1 that was resistant to Adamantanes. 

But it wasn't really a threat here. The CDC is, however, starting to see this in the southern hemisphere. So I expect we'll see more of that next year. 

Now what about Tamiflu? 

Adamantanes now are kind of off the table. Everybody is putting their eggs in this basket. What's the picture look like here? Again, looking back, it looks kind of similar to what we were looking at on that first slide. Less than one percent resistant. Less than one percent resistant. 

These things in red here, these are high percentages. These are resistant variances that are shed during or after Tamiflu therapy. So for instance, in Vietnam with the H5N1, they're treating people with Tamiflu, trying to save them, trying to get that infection down. When you put a lot of that antiviral into that system, you pressure the virus to throw out resistant mutants in the hopes that they're going to survive. 

And that's what happens. You expect to see this. But you see it in quite significant rates. The same thing with the H3 and H1 in Japan. This basically points out that resistance is definitely possible. Whether it's sustainable or not, is a whole different issue. 

So I'm going to bore you again. That's the Tamiflu mechanism of resistance. As I said before, those viruses get into the cell, they replicate. They make new cells. 

And then they go to leave. When they leave, that neuraminidase holds onto the hemoglutinen. The Neuraminidase actually reaches over and chemically cleans off that receptor. It releases the virus so it can go on and infect other cells. What the neuraminidase inhibitors do is they basically put a cap on those neuraminidases, so they can't reach over and they can't work. So all these viruses get all dressed up and they've got nowhere to go. 

They can't be released. So that's the end of it. 

The difference that I want to point out here is between Tamiflu and Relenza. The molecular structure of Tamiflu is not a very good fit for the receptor. 

So when it goes to block that receptor, it's a tough fit. And if there is a mutation that can cause that resistance, it happens pretty easily. And so, Tamiflu can get resistant. Zanamivir, or Relenza, is a much better structural fit. So we don't see very much resistance with Relenza. And we are now starting to see some resistance out there in the community to Tamiflu, but not to Relenza. 

There are many different resistant mutations that can happen. One that is starting to happen right now is this H274Y. That, again, is a dominant mutation. And we've kind of been waiting for Tamiflu to start throwing off, what's going to be that sustainable mutation that's going to cause resistance. 

Because again, it's very difficult to do surveillance if there's five different mutations out there. But when one kicks up, then game on. 

Okay, pyrosequencing influenza in the NA gene. This is the same thing, pyrosequencing, as we did with the Adamantanes. What we're looking for here in the susceptible is the CAT. And the resistant is the TAT. 

This is a difficult one to read for pyrosequencing. It starts right off with four T's right in a row, like what we were just talking about. So you get these nice, big spikes right off the bat. But it's right at the leading edge of the sequence. 

It's not too bad to read. 

So what are we seeing? We just did our last run of the season. Influenza season has peaked. It's now on the decline. In another couple weeks, it'll be completely over. 

So we did our final run. We do one at the very beginning of the season to see what's out there. We do one in the middle of the season. And then we do one at the end of the season. 

We just did another one yesterday. One of our EID Fellows has been doing some testing. 

In Wisconsin, we did not look at Tamiflu resistance at all. New York was doing the full sequencing, the Sanger sequencing. They hadn't gotten their pyrosequencer yet. So they were working. 

The CDC is looking. They had seen a little bit here and there. But really, not much to speak of. 

This year, that changed. 

The H1N1, we started seeing right off the bat early in the season. New York saw it. We saw it. The CDC saw it. All at about 9% resistance. The H3, no problem. 

No resistance there whatsoever. The Adamantanes, you have resistance in the H3. Tamiflu, you have resistance in H1. 

We just did our last run and we found a whole bunch of resistant ones. 

We, in Wisconsin, are now at 17% resistance. The CDC, nationally, what they're testing there is about 10%. That reminds me so much of the Adamantane the year before, when it was about 15% resistant. And the next year, it shot up to 90%. So we're going to hold our breath till next year. If this happens, that puts the whole stockpiling of Tamiflu into question. 

And again, that doesn't guarantee anything. There could be a pandemic flu strain that could come along that could remain completely susceptible to all these drugs. We just really have no idea. It's just a bad indicator for us. 

So what are the implications of this resistance? Those stockpiles could become somewhat ineffective. That would be a lot of money unfortunately down the toilet. Increased dosage and duration. In treating those Avian Influenza cases in Asia right now, it doesn't take a typical regimen of Tamiflu to do the trick. You have to double the dose. 

You have to double the duration you're giving it to the patient. And even then, in many cases, it doesn't help much. So even though we have these stockpiles, and we're planning how far those stockpiles go, how many people can be treated, who's going to get treated first, what the priority is going to be, one of the things that hasn't consistently been factored in is, we're going to have to use twice as much of it, twice as long. The whole point of an antiviral in a nursing home environment is prophylactic, to keep the people that are not sick from getting sick. 

You can't use that in pandemic situations. You can't keep a population healthy by putting them on Tamiflu for the duration of the pandemic. That's just not going to work. So there's a lot of issues there. 

This could mean more problematic nursing home outbreaks. As we see some influenza, there would be resistant H1's. 

We saw this year, the CDC is now investigating a nursing home outbreak in Illinois that had, all the strains were resistant to Tamiflu. They were able to use Adamantanes, however, because it's in H1, and remained susceptible to Adamantanes. So this is going to be something that's also going to be complex to manage. 

Subtype specific rapid test kits. 

This is yet again another lecture. Many physician offices have rapid tests to tell if you have influenza or not. You can do a test that can say this is Influenza A or this is Influenza B. And you can give them an antiviral right off the bat. The problem is, those things won't work at all unless you're right at the peak of the flu season, so you know that you're positive predictive value is high. If it's at the beginning or the end, it's a crap shoot. 

You don't know if the result you're getting is real or not. But what we're trying to do at the State Lab is have a surveillance network. We're having physician's offices send in their specimens whenever they get a positive, so we can confirm that. And they like that, because then they know what a good time is that they can rely on these handheld instruments and when it's not. 

What's coming down the pike is the molecular revolution and these handheld devices. Right now, these are fluorescent antigen type kits. These are all going to be handheld molecular devices. They will be able to tell whether it's Influenza A, whether it's B, whether it's H3N2, whether it's H1N1, whether it's H5N1. You name it, they'll be able to know. That has big implications for us, because those physician offices are now our laboratory. 

And we need to make sure that what's going on in those "laboratories" is accurate. So that's why we're doing this surveillance. 

That's going to mean, you know, it puts a lot of complexity and power into the hands of the physician. Especially if you have a situation where you have an H3 that's resistant to one antiviral, but susceptible to another. 

Or an H1, which has the same situation. That's a good thing for the physician office. They'll be able to look and say you have H3, you get this drug. So this is going to come whether we want it to or not. It's certainly going to come. 

And then the other boon to this is probably for the drug companies. 

They are new investigational antivirals out there, different classes, that they're looking at. I haven't delved into this enough to know what those differences are. But if they get one that's going to work well, that could be the next stockpiling agent, and the next profit margin for that drug company. Question? 

- In your list there, why is there just nursing home outbreaks? What about daycare outbreaks? What about outbreaks for other populations? 

- Same thing. That's an excellent point. The key one is the nursing homes, because you have an elderly population that can be very susceptible to this. 

You can get the same thing in a school. You can get the same thing in a daycare. We just haven't seen that as much as in a nursing home. That's been the big one that we have to deal with. 

- The 1918 epidemic was actually after people in the middle aged groups. It was more severe in them than in the elderly and young. 

- The comment was that in 1918, it was the middle aged folks that were most affected. The people from 20 to 40, who you'd think would be the most healthy. But they would get this disease, they would stay home for a couple days. They would start feeling better. 

They would go back to work. They would get pneumonia and they would die. That's part of it. There's another part that Yoshi has been working on, how this actually affected so many people and why. 

And where it infected them, in their lungs, that made it so disastrous for this age group. 

You know, again, preparing for pandemics is a whole other lecture. But by far, the most important thing that's going to happen in a pandemic is just doing social distancing. Just the same things. Washing your hands. Being smart about it. 

There is not a heck of a lot we can do when a pandemic is going to hit. And we can throw antivirals at it and they may do some good, and that's important. But by and large, we're going to have to live through it when it happens. And hopefully live through it, and hope it's not going to be as bad. 

There's a lot we can try to do, but not much that's going to be a cure-all. Question? 

- Did any of you have anything to do with how they developed the vaccine for the coming year? 

- I do work at a project with another doctor, who is at UW Family Practice, family medicine. We're doing some research with him because he does his own influenza research. He does some fantastic analogies looking at the time when Lake Mendota freezes and thaws in relation to the influenza season. 

And it correlates quite a bit well. 
(laughter)

 But he is actually on the American Influenza Practitioners group that actually votes and decides on what that next vaccine make-up is going to look like. So they're already identifying, I think, now, what that vaccine is going to be. 

It's going to be different next year than it was this year, because we had a vaccine slight failure this year. Question? 

- Why is sequencing more accurate in the middle of an epidemic than at the beginning or the end? 

- It's got a sensitivity and a specificity. I'm not sure if you're familiar with that. But that's basically, if it's a true positive, is it really a true positive. Or is it telling you it's positive when it's not. 

Or is it a negative, and it's telling you it's positive when it's really negative. It's, I believe the specificity for a lot of these is about 80%, 70%, 60% in some cases. So when there's not a lot of flu in the community, if you do one of these in the summertime, when there's no flu and you get a positive, odds are it's a false positive. 

But if you're in the middle of the flu season, you're more apt to be testing people that are coming in that have influenza. Then it's a much higher predictive value. It makes my head hurt. 
(laughs)

So the take home message is get your flu shot. This is the most effective thing you can do, even in a year when the flu vaccine does not match as well as did. It's still the most effective thing you can do. I want to thank the folks at the CDC, the State Lab, the Division of Public Health, and more importantly, the Virology Labs around the state, the rapid test sites with the health departments who are all the ones out there getting us these specimens. 

We wouldn't have any surveillance program if it wasn't for them. 

More questions? 
(applause)

 Thank you. We've got one here. 

- Why do these viruses originate in Asia? 

- Hm, why do these viruses originate in Asia. Well, I'm not qualified to answer that. But like an idiot, I will try to. 
(laughs)

 My understanding is that there's a large avian population. 

Some theories are those new strains come out of the avian population. 

There's also a large swine population. Those can come out of the swine industry. They don't have as many safety checks and take as many precautions for controlling disease as we do in the United States and in Europe. So that increases that risk. 

There is a big theory that the 1918 influenza strain did not come from Spain. Everybody calls it the Spanish flu. They called it that because all the soldiers were coming back from World War I, a lot of ships from Spain were coming in, so they thought this must have come from Spain. There was a predominant theory right now that that actually originated in the United States, that that's where that disease started. I don't know if I can totally answer your question. 

- Are these seasonals? 

The Hong Kong and Singapore? 

- The question is why is influenza seasonal. Is that right? Why is it seasonal? There is another large debate in the community. That's one of the big unanswered questions. Several theories abound. Some is that we have all those viruses and they lay dormant in us over the wintertime. And it's until we're stressed with the winter that the virus then goes back out into the environment and is infecting people. 

There's another theory that is predominant, I think, right now. 

And it just kind of came out in the last year or two about, this is very temperature sensitive. The flu virus can survive much better and be passed person to person in a colder environment. And that makes sense, because you don't see that kind of level of activity in the warmer climate states. There's several other theories, some of them kookie, several are not. I really can't answer your question. 

- There's more close contact. 

- That's one of the other ones, too. You know, you're indoors more. You're close. 

You shake each other's hands, touch doorknobs, that kind of thing. 

There's the whole theory of, you know, your mother always says put your coat on or you're going to catch your death of cold. And you figure out, it could be a combination of all of these. 

- Is there any correlation between the release of antivirals and the resistance? In other words, you see that quick peak. Is that because of studying those situations, there's a high level of use in Japan, and so forth, so it took off? The following question is, does that have any implications? You know, 1918, the reason that didn't come back was because everybody died. But is it similar to antibiotics, where resistance problems that will actually perpetrate a supervirus? 

- The question is, is there a correlation between use of antivirals and the development of antiviral resistance. 

Very much like you have the use of antibiotics and the development of antibiotic resistance, which used to be my former life. 

And that was a much more clear cut case. You would apply these drugs in a hospital setting. Those resistant strains would develop. You could watch that move across the country. There was a very clear relationship. 

Not as clear with the antivirals, at least in my opinion. We saw the spike in Asia, Hong Kong and Japan, where they use antivirals openly and freely. And we thought we would see a similar thing with the H1 resistance to Oseltamivir, the Tamiflu. We're not seeing that. 

It originated, the first one that we saw was in Norway, and then in Europe. 

And now, it's being seen here. And now it's being seen in China and Japan. But it certainly didn't come out of there. They're looking the hardest of anybody, because they have so much antivirals. They have a surveillance system you wouldn't believe looking for resistance. So if it would've been there, they would have found it. So that, the relationship is not that clear. It could just be blind luck. Question? 

- Wisconsin has an extremely large prison population. 

Does the state of Wisconsin have any plan in place to help these folks if we have a pandemic? 

- It does, yeah. They're actually part of the special populations I was talking about earlier. So, as far as I know, the Division of Public Health has worked with the Department of Corrections to be able to dispense whatever antivirals or antibiotics they need to get. They get medical supplies. The federal government has a whole stockpile that they can either fly in on an airplane or drive in at a moment's notice. So they have built in that those special populations, including prisons are not going to be forgotten. Again, it's been a couple years since I've been over there working on that stuff, so I'm not sure what their stated plans are right now. But we were planning on that when I was there. 

- How much does the machine cost? 

- How much does the machine cost and how much do tests cost? 
(inaudible)

 No, not really. The actual pyrosequencer itself, I believe costs about $80,000. Many of the equipments we have at the State Lab can be very pricey. A friend of mine likes to say if you can put it on a bench, it's probably worth about as much as a brand new car, high end. If it's on the floor, it's probably as much as a brand new house. And as far as doing the test, the reagents are not that expensive. It's the time for the technician to do the test that is really the limiting factor. 

- Are there any newer pyrosequencers? 

- There are new generations coming out. 

This comes out of a company from Sweden. And they are trying to expand the uses and the reads of this. 

And I was anticipating this question. Other applications for pyrosequencing, and this is really a minor application for pyrosequencing. The big money in pyrosequencing is this bottom one, cancer research. DNA methylation and gene expression is going to be a huge wave of the future. And identifying what those genetic variabilites are in people, if you can do that rapidly and cheaply, it's going to be very big. So they're actually planning on working with some of the folks in our State Lab. 

And then, infectious disease, communicable disease, working with screening folks, cytogenetics, to get them to use some of our technology. We hate having a nice machine that sits there waiting for flu season. 

We want to use it for a lot of other things as well. 

Michigan is looking at identifying E. coli with this. They just came out with a publication on that. 

Some other common practices are identifying microbacteria, non-TB tuberculosis, and then other molecular epidemiology, and other resistance mechanisms. 
(inaudible)

 How long ours was? Only about 50 or 60, not that many. 

And again, we just need to see that one point mutation. So everything else is kind of irrelevant to us. 

- That's not the entire sequence? 

- No, no. 

- How long does the protection last for the flu shot? 

- Oh, boy. Like I said, I'm not a virologist. 
(laughs)

 But how long does the protection last from a flu shot, anybody? 

- I presume you ask about a length of time, but the problem is that the virus strain is different than next year, so you have to have a different immunization. 

- Did you hear that? Let me repeat that. The answer to the lady's question, which is how long does the immunity last from the flu shot. Basically, the answer is it lasts quite a while, but the strains of the viruses change so often that you need to continually get a new flu shot. 

So it's not a situation where you can get just one flu shot and be done for ten years. You need a new one every year. Thank you. 
(inaudible)

 Oh, that I don't know. 

- The big one. 

- The big one. 

- You don't keep that? 

- No, no. We, for a long time, at the State Lab, would use the Sanger over here for doing all of our sequencing that we needed to do. They just recently got a full sequencer over there. 

I have not been involved in working with that. 
(inaudible)

 I don't know if that's the version they got or not. Over here? 

- This may be inappropriate, but doctors usually report any suspected flu. 

If they have the handheld moleculars, will it have a chip in it to send the information to you right away? 

- Oh, good God, no. 
(laughter)

 We wouldn't be able to do that. That would be great. Influenza is not actually reportable in Wisconsin. There's a movement afoot to get it reportable. 

Any unique strain is reportable. So, anything that might be an H5 or anything else that's out of the norm is reportable. Most, however, sort of the physicians don't actually have to report those, even if they have the handhelds. 

But we've set up a surveillance network so they send in their specimens so we can confirm that. So we have that data without it being in the statute and being required. That may change in the future. That's going to be up to the Department of Health and Family Services, what happens there. 

As far as actually collecting data electronically, we have a whole electronic laboratory for a reporting system that is trying to be implemented right now. All the states are trying to do that. It's very difficult, trying to link up with hospital databases and their laboratories, and give us their data. 

There's whole translational issues that go along with that. We've got several sites already up and active. The plan is to get all the big ones in the state reporting to us electronically. 

And then all the small ones, we can't expect them to foot the costs to actually do that. We're setting up a Web site where they can actually go in and enter this data, so it directly comes. Right now, they just fax it to us, or they mail it to us. But it would be so much nicer to have it put in through a Web-based interface that would just drop it into the database. 
(inaudible)

 Oh, yeah, they're fantastic. Not every state lab has a really good working relationship with their hospitals and clinics, but Carol at our state lab has just done a fantastic job going out working. She has regional meetings. 

They're just ramping those up this week, as a matter of fact. Every year, they go around the state and have meetings, bring all the laboratories together, bring all the local public health departments together. 

They all get a chance to talk and network, and discuss with each other. They get training. They give us surveillance isolates. It's a win-win situation. And we're not coming in there as a state regulator. We're trying to be a partner with them, and they know that. That isn't the case in every state. Many state labs are put down upon and really shunned by the hospital environment. 

But we've got a really great system here. We're actually doing a grant right now where we're trying to educate every state lab in how to do the same thing. 

Hopefully, that will have some effect. 

- I have a question about the ten percent of isolates that don't work. Do you have to have a certain amount of sequence for your primer to work? 

- The question is, the ten percent that don't actually work on the pyrosequencer-- 
(inaudible)

 Hmm? Well, let me explain that. What we do, is we actually do a PCR to identify is this is a flu A or H3 or N1. Part of that is we do an extraction of the RNA from the direct clinical specimen. So we get a specimen, we extract that RNA. 

We can tell off that PCR step, because we have realtime PCR, there's a crossing threshold that we can actually see. It's called the CT value. If there's a low value, that means there's a lot of RNA in that sample. If it's a high value, that means there's not much RNA there. 

To do the pyrosequencing step, you need a lot of RNA in there. So we can tell by that PCR step which ones are not going to work well in pyrosequencing and which ones are going to work. And the cut off seems to be at about 26 at the CT value. Anything above that, we don't even bother testing. Now, we could culture that and get a lot of RNA, and then find out. But again, for surveillance purposes, it's not necessary that we have every isolate. 

We just want to get the picture. 

- Is there any chance you're looking at a novel isolate, where if your PCR is not working? 

- Oh, absolutely. Again, we're looking at just one mutation. There's a whole handful of other mutations. Right now, it's the predominant one that's out there. When the CDC is looking at it, we're looking at it, New York, across the ocean. We're all looking at it. That's the one that's out there right now. If people start seeing something else, maybe we're going to shift and start looking for that one. 

We just got some new primers from CDC this morning, as a matter of fact, for other resistant mutations for Tamiflu. We'll play with those over this summer and be ready to use them if we need to. But right now, there's no point in doing that testing. It would just cost money and detect nothing. 

But there's always, that's the whole issue, the molecular revolution versus growing things up in culture. If we're just looking for a certain thing with a molecular test, we're not going to see anything new. We've got to keep doing the culture, so that if something new comes up, we'll catch it. So we'll always be doing that. One more? 
(inaudible)

 I don't know enough about that. 
(inaudible)

- One of the advantages of the pyrosequencing, presumably, is that you can do a large number of samples in a short time, which is really different than other situations where you might just have a few samples that you're trying to analyze, and you can afford something that's more extensive. 

- The thought was that one of the advantages of pyrosequencing is that you can do a large volume of samples for relatively cheap. It all depends on what you're looking for and how hard you're looking for it. 
(inaudible)

 Sure, the question or comment was that one of the advantages of pyrosequencing is that you can do a large volume of testing for relatively cheap, so it really kind of just depends on what it is you're looking for. If it's something that you want to see a lot of and you want to do it quickly, pyrosequencing is a great tool. 
(inaudible)

 As many runs as you can do in a day. If you have a 96-well plate, again, it takes about an hour to an hour and a half to do that. You could probably do several runs, and do several hundred in one day. Now, of course, we could have a night shift and we could really crank things out. But I don't think they're going to go for that over there. 
(inaudible)

 For what we're looking at, in our surveillance system, we get, maybe for the entire influenza season, 400-500 strains. So that's really, we can test all of these pretty rapidly. Typically, when we look in the beginning of the season, the middle and the end, we only look for about 20 to 30, sometimes 40 isolates, depends on what's going on. If there's nothing going on, we're not going to look any harder. 

That costs money. And we're on a tight budget. If there's something interesting, then we'll ramp it up and we'll put our resources there and do some more testing. 
(inaudible)

 If it doesn't fall in this slot, we miss it. We don't see it. So we're hoping that someone else is seeing it. 

Again, the odds of something popping up like that unique in Wisconsin are pretty remote. It'd probably be seen elsewhere. But a big reason why we have this technology is, if something big does happen with pandemic flu, CDC is going to be overwhelmed, and we're going to get a lot of their samples to do a lot of their testing. 

That's kind of the real benefit for CDC and for us, for having this relationship. 

- Thank you very much. 

- Thank you all for coming. 
(applause)