University Place 723 Wednesday Nite at the Lab  Holly Basta ANtiviral Drugs and Vaccines  May 16,2012 CC >> Welcome, everyone, to Wednesday Nite at the Lab. I'm 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 partners, Wisconsin Public Television, the Wisconsin Alumni Association, and the Science Alliance, thanks for coming to Wednesday Nite at the Lab. We do every Wednesday night, 50 times a year. It's not always this gorgeous out, however, so I really appreciate you coming tonight. Tonight I'm delighted to be able to introduce to you Holly Basta. She's currently a fourth year PhD candidate in the cell and molecular biology program here at UW Madison. She's a research assistant in Ann Palmenberg's lab, and in that lab they study picornaviruses, pico standing for small and rna standing for RNA. These are the small RNA viruses that you'll get to hear more about during Holly's talk. 

Holly was born in Great Falls, Montana. If you've never been to Great Falls, I highly recommend it. It's got a great Lewis and Clark museum, and the Great Falls are still pretty great. She completed her undergraduate degree at Montana State University in Bozeman in 2008. She just told me that she graduated, she got married, and got in a car and moved here to Madison. So it's all been on continuous, enjoyable series of research. Tonight, she's going to talk about Antiviral Drugs and Vaccines: Insights From the Model Structure in the Rhinovirus C. Please join me in welcoming Holly to Wednesday Nite at the Lab. [APPLAUSE]  >> Can everyone hear me okay? Alright. Thank you all for coming to see my talk today. 

I'm going to put in a plug for vaccines because of a lot of things that have been going on in the news lately, and then I'll get into my research. I'm excited to talk to you today. So the central dogma of molecular biology is that DNA is made into RNA that's made into protein. So if you leave with anything from this talk, you will know the central dogma of molecular biology. So DNA goes to RNA through a process called transcription, and then RNA leave the nucleus of the cell and is then made into protein by a process called translation. So most of the things I'll be talking about today are either RNAs or proteins. So to get you, to introduce you to the jargon I'm going to be using today, so when we're talking about the host, which in the case of rhinovirus is humans, the DNA is called the genome, the host genome. RNA is typically messenger RNA, and then that's translated into protein. When I'm talking about my rhinoviruses, their genetic information is actually stored in RNA that completely skip the DNA step, and that puts them a step ahead of the host. So when I talk about their genomes, I'm talking about RNA, and then when I talk about the capsid, that is a protein that's made by their RNA. 

So what is a virus? Like I said, a virus can have either RNA or DNA, and that's where all of their genetic material is stored. And then they have to have something to carry around their genetic material in, and that's called the capsid, and that's just this shell of protein. What I'm showing you here is the capsid cut in half. So it's basically a hollow globe full of their genetic material. And throughout the talk I'm going to introduce these misconceptions in hopes that you guys come away with better understanding of what viruses are. So the first misconception I've run into is that viruses are a type of bacteria, and that is absolutely not true. Viruses are a completely different thing. Bacteria are actually much more complex, but viruses are just this: a protein shell and RNA or DNA. So another misconception that people have is that viruses are nearly as big as human cells. What I'm showing you here is a scale of size. 

So here's a human, a small human, about one meter. Down here are things that you can see. Thickness of human hair. Then visible light is here, and viruses are actually smaller than that. And that's what make them so difficult to study because you can't actually look at them underneath the light microscope. And they're pretty much only bigger than pieces of cells, and then atoms are down here. So I'm going to show you a cool little schematic here. We got closed down. Let's try this way.  We're actually going to skip that one because it's being difficult. 

Alright. So, this is a video. >> So let's say that this guys has the flu. >> And I'm just going to let them go. >> And here's a droplet from his sneeze containing, if we move in and take a really close look you see each one of those little purple things is a virus, and there are a lot of viruses floating through the air, some of which go inevitably up this unfortunate man's nose. How did that guy feel you ripped off half of his face? >> It was interesting because we did it while he was sleeping. [LAUGHTER] >> Okay. I am talking with medical illustrator David Bolinsky who designed this video for a research company. So here comes the virus, and it's going to land on one of this guy's throat cells. So notice it's covered with little yellow knobby things. 

You all these key. >> Those are the keys. >> This is a key. This is a key. This is a key. Okay, if the keys on this virus happen to fit the locks, which are the little purply stick-uppy things on the surface of the cell, if there is a match, the cell, watch this, welcomes the virus in. And what's this? >> This is the welcoming committee. They all interlock with each other, and they pull this membrane down into the cell. >> And down it goes deeper and deeper, and that welcoming structure disperses and the virus capsule bursts and out comes the secret recipe for how to make more viruses, those little noodly things. So this unsuspecting cell has been tricked into guiding these virus recipes right into its own command center, the nucleus. 

So in they go. >> And they are immediately recognized by this big pink molecule which his a mini factory. >> What is it doing? >> It threads the nuclear material, the instruction code of the virus, through one hole, and out another hole comes a brand new instruction set. >> So it's a copying machine making copy after copy after copy of virus recipes which then go out of the nucleus. The little chefs, those blue peanutty things, they cook up proteins that go back into the nucleus where they are reassembled into baby viruses, and then out they go. They get covered up and head to the surface where they get new keys and then boom. Here they come. This is an eruption of virus after virus after virus after only virus entered this cell, but how many came out? Well, millions. Millions. So if one virus can produce a million babies and do it again and again and again, how come this guy doesn't just drop dead? [LAUGHTER] >> Well, because you have about a hundred trillion cells. 

>> So a million viruses is just a drop in the bucket when you have a hundred trillion cells. Remember, you do have your own immune system which when it sees a virus usually... [LAUGHTER] So while the virus does multiply fast, with any luck your immune system will work just a little faster. So yes viruses, all viruses, want to spread. That's what they do. But most of the time we do keep them in check. Most of the time.  >> Alright, so here's the thing I was going to show you before this. So this is actually a really cool website. It's called learn.genetics.Utah.edu, and this has a little scrolling bar, our movie is still making noises. 

So there's a little scrolling bar. Now I restarted. I'm sorry. Alright, so there's a little scrolling bar, and as you scroll it gets smaller and smaller. So here's a coffee bean and a grain of rice. Sesame seed. Grain of salt. And as you go in, now we're getting to the microscopic things. Amoebas, paramecium, there's a human egg and sperm. A photoreceptor in your eye. 

Red blood cell. Baker's yeast. X chromosome. E.coli. Mitochondria. Now we're starting to see the viruses. There's measles. There's HIV. There's influenza. And all the way down here is rhinovirus. 

So it's really, really small, and it's even small for a virus. And there's a ribosome, which they showed you in the movie was making the proteins, and then as you get smaller and smaller there's an antibody all the way down to a single amino acid and then a carbon atom. So that should give you some indication of how tiny viruses are and how amazing they are to cause so many problems. Alright, so we watched that. So there are two ways that we treat viruses. Either antiviral drugs or vaccines. Both treatments are good in their own ways. Drugs you're only given once you're sick, and they're specific to the specific virus that you have. The picture here is for Tamiflu, which is for the influenza virus. They usually inhibit viral development, and they're typically small molecules. 

So just little chains of carbons, usually, and they help only you versus vaccines which help you and help others in that you're not infecting other people. So vaccines are given before you're sick, as you know. They're specific to a certain virus, and how they work is they tip off the immune system. They say this is what this virus is going to look like. When you see it, kill it. And so vaccines are typically made from pieces or whole viruses. So there's many different types of vaccines, and they range in their safeness and effectivity. So not all vaccines are just dead versions of viruses. So these attenuated and inactivated viruses are, yes, dead versions of viruses. Attenuation, they take out a piece of the virus that it needs to be a full replicating virus. 

And the vaccines that you may have had that are attenuated viruses are the MMR, chicken pox, or the nasal flu spray. Inactivated viruses, typically what they do is they shoot them with UV or they put them in a really highly acidic condition, and then the virus is dead or inactivated. One of the polio vaccines was made this way, and the flu shot is made this way. Fractionation, they essentially just chop up a virus, and then you see little bits of the virus but it never forms into a fully infectious virus. The newer methods in making vaccine are actually much safer because they take a piece of the virus and then they put it into a bacteria's DNA, and the bacteria actually makes the viral proteins, and then you purify them out, and in that way there's never a full virus ever in contact with the vaccine that you get. So one of the vaccines you may have heard of that are made like this are hepatitis B vaccine and the HPV vaccine. So the problems with these kinds of vaccines is that you never know what the best part of the virus for your immune system to recognize is. So HPV requires three boosters because it's not a really potent vaccine. So here's the vaccine schedule that the CDC recommends. It's been a while since a lot of us probably got vaccines, but these are the ones that you get up until you're six years old. 

So a major misconception is that most vaccines are new, and therefore untested, and what I'm showing you here is that most of these vaccines were around when I was a kid. A lot of them were around when you were kids. So influenza started in 1945. The Varicella is from 1974. So a lot of these vaccines have been around for a long, long time, and there's really strong data about how safe they are. So I don't know if any of you heard of any of the news about vaccines. Can anyone call out something they've heard about a vaccine? [INAUDIBLE] Autism. Autism is a big. So I'm going to show you the actual data about vaccines and autism, and then you can make an educated decision about vaccines and autism. So some of the other things that people have concerns about are that vaccines contain mercury, and that actually was true but none of the current vaccines contain mercury. 

In the past, vaccines had trace amounts of mercury, and there were never any negative effects noticed from them. But currently no vaccines have mercury in them. Other ones that I have heard are that doctors get paid to vaccinate. That's false. Unless you know some dirty doctors that I don't know. I don't think there's any biotech plots to get people vaccinated. So let's talk about vaccines and autism. So how they determine if something causes something else is, essentially, you divide the people who got, in this case autism, who had the vaccine by the people who got autism that didn't have the vaccine. And this is how you calculate relative risk. Now relative risk of one means there's no difference between the two groups. 

So the same amount of people who got the vaccine got autism as the people who didn't get the vaccines. So when they tested the MMR vaccine, which is the one that was said to be associated with autism, actually slightly more people who didn't have the vaccine got autism, and then within 24 months it was pretty much equal. And to put this into context, the relative risk of lung cancer in smokers is over 20. So this says pretty much vaccine or no, you have even chances of getting autism. >> What about five years or ten years later? >> So the association, he asked what about five or ten years later. The association was said to be within weeks or months after the shot happened because many times you get the vaccine at age two or three, I believe, and that's when autism symptoms cropped up. And that's what made them originally go after this vaccine for that reason. So it just happened to be a coincidence that kids would start developing autism around the same time they would get the vaccine. So, by later ages, typically kids have shown to be autistic or not. So then other myths are there's no risk to not getting vaccinated and that vaccines can wait. 

So is it okay to not get vaccinated? So here are the MMR and DTaP vaccines, and here's the risk of getting them. So one in a million people can get encephalitis or have a severe allergic reaction versus these are the three diseases that you are immunized against. Measles, one in 300 people die. Mumps, one in 300 get encephalitis. And rubella, one in four can get congenital rubella syndrome. And the numbers are pretty similar for the DTaP. There's been no proven deaths from it, but one in 20 people die from diphtheria. And it's easy to forget that these diseases were a big deal, but actually now because a lot of people are opting not to vaccinate, a lot of these illnesses are making a comeback. So these are pretty recent news articles. This one says there's been 640 cases of whooping cough as of March 31st compared to 94 during the same time period last year. 

And this one is an outbreak of a meningococcal disease, and in a single school district there were five cases and two students actually died. And last year the CDC reported that the measles cases were at a 15-year high despite vaccines being available. >> Can you speak to the whooping cough epidemic at the moment going on? >> So, yes, there is a whooping cough epidemic going on at the moment. There is a combination of issues between children get vaccinated very young for whooping cough and sometimes the vaccines wear off, but that's also being perpetuated by not being vaccinated. So I'm just going to talk about herd immunity right now. You lead me right into it. So there's certain number of people that need to be immunized to actually protect a population from having a disease, and it's different for different viruses. So, essentially, if 83% of people in a population are immunized to small pox, small pox won't be able to spread. And so the problem right now is that these numbers are dipping below, number of immunized people are dipping below these numbers, and we're losing our herd immunity. And then another misconception, since we're talking about polio, is a lot of people think polio is eradicated and there's no need to be vaccinated anymore, but that's not true. 

In a lot of places it's impossible to get the polio vaccine. It requires a cold chain. So the vaccine always has to stay cool, and there's places in the world that you just can't bring something cool to them. So there's actually polio still cropping up in many places. So we still need a vaccine. >> What about small pox? >> Small is eradicated Small pox hasn't cropped up again. So the only thing they're worried about small pox is if it's in somebody's freezer somewhere because small pox, we were lucky in that it doesn't have an animal host so there's nowhere for it to come from except for other people and nobody has it anymore. So, yes, small pox is officially eradicated. >> But if it should come back, they have not been giving small pox vaccines for quite a while. >> That's correct. 

We haven't been vaccinated for small pox in many years. So why aren't there vaccines for everything? I just told you that vaccines were awesome and everybody should get them. So there's big problem with safety, and particularly with the rhinovirus, which I'll talk about later, it definitely can't have side effects that are worse than the virus. So the common cold isn't so bad in the spectrum of viruses, so it has to have very, very minimal side effects to be considered. So to get a safe vaccine, you have to make sure that it actually does what it's supposed to and that it alerts your immune system to a virus. It has to be biologically stable, like I was talking about with the polio vaccine. It has to be able to be delivered to people in Third World countries and have a practical delivery system. So an impractical delivery system would be 10 shots over three months. Nobody is going to go in for 10 booster shots. And the public must see more benefit than risk, which has actually become the biggest issue in recent years because when I said vaccines, you guys said autism and we all saw the news story linking vaccines and autism but I don't know that anybody saw that data that refuted it. 

And there's also been a lot of issues with the HPV vaccine because people have moral issues with it. That's all I'm going to say. And then to kind of lighten the mood, I have heard that people believe there's a zombie virus, and actually there isn't just so you know. Not yet. [LAUGHTER] So major challenges in making vaccines. Viruses have a very high mutation rate. So, to put into context, you and I may have a couple kids over our lifetime and they may be slightly genetically different from us, but a virus, like you saw on the video, can have billions of offspring, and each of them can have different mutations, and they replicate within hours, and in that way they're evolutionary time is much shorter than ours. DNA viruses can integrate and associated with the host genome, and so rhinoviruses don't do this but some DNA viruses can actually shove themselves into your DNA so your host machinery makes the virus right along with your own proteins. >> Which ones are those? >> Retroviruses do that, like HIV. So viruses can also infect immune privileged regions of the host, and what I mean by that is your nervous system and your brain don't have hardly any immune response whatsoever. 

Places like you airway and your intestinal tract have great immune responses because things from the outside come in and you need to be protected from that. But many times viruses can find their way to your nervous system which have not a very good immune system at all. And then viruses have ways to get around the immune system by directly shutting down parts of your immune system or sneaking around it. This is a major issue. With rhinoviruses there's multiple serotypes, and the same thing with influenza. So the reason you have to get an influenza vaccine every year is it's a cocktail of many different serotypes of influenza. And then it's very expensive and time consuming to develop vaccines. Years and years and millions and millions of dollars. So now we're going to talk about my research. Rhinoviruses are the main cause of the common cold. 

There's over a hundred unique serotypes, and they're divided into three species. They're really small, simple viruses like I showed you. They're much smaller than HIV or small pox. So why don't we have a vaccine for rhinoviruses? We all get them. People are studying them. So rhinoviruses actually have a lot issues with developing the vaccines that I told you about. They have a very high mutation rate, and that's because they have the RNA genome that allows them to be really fast and mutate quickly. They target the immune system, there's a lot of serotypes, and the cost and time involved in developing the vaccines becomes an issue because there's not a lot of people that die from rhinoviruses. So the money goes into things like HIV which a lot of people die of. So here's the schematic of the rhinovirus genome. 

And remember your central dogma. This is the RNA. It gets translated and these are proteins. And then the proteins get chopped up into smaller proteins, and then they form into a capsid. So here's a picture of a rhinovirus capsid, and we kind of look at this like a 3-D map of the virus, and there's specific landmarks that we know about. So here's, this triangle right here is blown up into a cartoon here. So all rhinoviruses have a canyon. So kind of a dip. And then there's actually a hole in them, and most of the antiviral drugs are targeted to go into this hole. And how they work is they essentially stick that hole closed and then the virus is trapped in its own capsid. 

So when it gets into the cell, it's just stuck in the capsid. It can't release its genetic material, and it can never make more viruses. So then this is that same triangle thing but these are what the proteins actually look like. So the capsid is composed of a VP1 which stands for viral protein 1, 2, and 3 and then the VP4 is inside the capsid. So these are all color coded. This blue is this blue over here. So they have a five-fold, three-fold, two-fold, and another three-fold axis. So this triangle is essentially repeated around into this capsid. Because they only have four proteins that make up the capsid, it's repetitive. So that's how they make their capsid. 

So what this is is a phylogenetic tree, and we use these a lot to tell how closely things are related to each other. So these are all the known serotypes of rhinoviruses. So these ones right here are rhinovirus As. These right here are Cs, and these here are Bs. So these, the As, Bs, and Cs are all very different from each other and make it a real challenge to make a single vaccine or a single antiviral drug that will work on all of them. And all of these strains are circulating right now. Most of these were actually isolated at the hospital here. So the rhinovirus C species is the one I'm interested in, and the reason for that is it was just recently discovered in 2007, but they found it associated with almost half of rhinovirus infections in young children. The reason they didn't find it before is it can't be propagated in cell culture, and how they grow it now is they have to get primary cells from patients. So if you have an operation on your nose, they can take a snippet of those cells and then put rhinovirus on there and grow it, but it's a really inefficient way to do it because you have to count on people having surgeries and signing off on their cells being used for things. 

And then, oftentimes, those people are already infected with a rhinovirus so it convolutes the results. And then to grow these are any length, you need an air liquid interface system. So the reason for that is these rhinoviruses grow in your airway and those cells like to be partly in air and partly in your mucus, essentially. So they're very difficult to grow. So what we normally use for cell culture are HeLa cells, and here's what they look like. And we grow them in little dishes like this in serum. And then how you tell if the virus has infected them is you cover them with this overlay and dead cells will appear as holes. So these are cells that have been killed by the virus. So this is how we normally study viruses, but because rhinoviruses can't infect these cells, we weren't able to study them. So the rhinovirus Cs are really interesting because they actually have a higher propensity to cause more severe illnesses. 

So these are actually patients from UW Hospital, and they ranked how severe illness was with which rhinovirus different patients had, and they found that rhinovirus C in red and rhinovirus A in black usually caused more severe illnesses than the ones in green, which are rhinovirus Bs. And this separate group found that when they studied 128 children with asthma, they found that rhinovirus C really exacerbated the children's asthma much more than other rhinoviruses. So these viruses are very, very medically relevant, but we can't grow them so it's very difficult to study them. So why won't they grow? So in the video we saw, remember they had the lock and the key? So the lock they were talking about are actually host cell receptors, and how these work is your cell essentially says I need something from the environment, I'm going to put out bait for it. And then when it picks up whatever it gets, it brings it into the cell, into those balls that you saw in the movie, but the viruses pretend like they're something else, and the cell says, oh, I have what I'm fishing for, and they pull it in thinking they have what they want, and it's actually a virus. But we don't know what bait the rhinovirus Cs take, and we know it's not present in any of the common cell culture lines we use. It's in your airway. We don't know what it is. So there's no way to get enough virus to figure out this actual 3-D structure of what the Cs look like. So what I did was using computational methods, and we knew the 3-D structures of other rhinoviruses and a lot of other proteins, I made the hypothesis that I could use the known structures and computational methods to predict the structure of rhinovirus Cs. 

And the method I used for that is called 3-D protein threading. And so how that works is you take the protein sequence of whatever protein you want and you force it into different confirmations. These are different confirmations of protein. And then with equations you find out which is the lowest energy structure that sequence could fit into. So I did that with the rhinovirus Cs, and then when I overlaid it with the real structures of Bs and As, it overlapped pretty well. The only differences were in this region right here which is the five-fold axis. And so here's the full structures when I made them into a capsid. So here's real experimentally solved structures from A and B, and here's the model I made. So they're pretty similar looking. But when we went in and looked closer, so right here, these are the five-fold axis and you can tell because that has five little points, when we zoom in on that and cut it in half, we see that the rhinovirus Cs have a very short five-fold axis. 

And then when I make these road map projections, so how these work is it's just like a topographical map. So red would be a mountain, and blue would be a valley. So here's the As, here's the Bs, and here's the Cs. So right here at this five-fold axis where there's normally a hill, there's actually no hill in the Cs. And that's very interesting that they're so drastically different from these other types of rhinoviruses. And this is very interesting when you take into account that they don't use the same receptor as the As and Bs. So this is the five-fold axis and this is the five-fold axis. Both different types of receptors that rhinoviruses use involve the five-fold axis. And the rhinovirus Cs just don't have it. So they must be interacting with their receptor at somewhere else on their capsid. 

So can we make a vaccine for the rhinovirus Cs? So how you test for potential vaccines is that you find the regions that are most likely to confirm mutant protection after vaccination or natural infection. And these have been mapped for the rhinoviruses. I don't think you can see it very well here. So here's these road mapped projections. And these are the regions that your body sees the rhinovirus. So right here, a lot of them are at the five-fold axis. There's other regions that your body sees, but the major one is at the five-fold axis, and the Cs are just missing those. And how the virus deals with this is it makes loops of variable regions. So it says, oh, you're looking at me here? Well this is going to be the region where I vary the most and where I mutate the most. But the rhinovirus Cs just don't have this spot to mutate. 

So there might be a possibility that a vaccine would work better because they actually lack this evolutionary space. So major focus of my work was to see if current antiviral drugs would work on the rhinovirus Cs. So, like I told you before, drugs are typically designed for this region here. It's called the hydrophobic pocket. It has an open pore and a closed toe. And normally in nature there are pocket factors, so just pretty much long carbon chains that sit in there, and they don't bind very tightly and allow the virus to open up and release their genetic information. But drugs mimic these but bind much more tightly and glue the capsid together. So here's the five-fold axis, and here's it zoomed in, and here is the antiviral drug sitting in the pocket. So that's where they hang out is pretty much right up in here. So there are two antiviral drugs that have come out of research that look promising. 

So there's pleconaril, and it was actually evaluated in two large phase three clinical trials, and it was found to work. It shortened the duration of common cold virus and the window that you spread the virus. It wasn't ultimately approved by the FDA, and I heard that it somehow interfered with birth control, and that's why it was canned. But the next one that's coming down the pike is called BTA. It inhibits 56 rhinovirus strains, and an analog to this drug right here is actually in clinical trials here at the hospital. So there were two drugs I was interested in because they have been the most effective so far on rhinoviruses. So we know which amino acids line the inside of the hydrophobic pocket, and this is a representation of those amino acids. So all of the rhinoviruses besides the Cs fall into one drug group or another. One group of drugs works on one group of rhinoviruses and one group of drugs work on another. So I wanted to know which group the rhinovirus Cs fit into. 

So these are representation of their sequences, and at lots of places they're the same. So they all have the same amino acid here, but at some places it differs a lot. So this amino acid is in these two drug groups and it's not in these ones. So I then asked, well, which of these is actually important? All of these amino acids are in the hydrophobic pocket, which ones are most important for the drug binding? So I analyzed all 27 of the structures that have been solved for different rhinoviruses and found that in all these cases most, the drug was within four angstroms of this residue. So what that means is these particular amino acids were close to where the drugs was suggesting they were interacting with the drug. And then the ones in gray are the ones that have been found experimentally to bind pleconaril. So in this way I weighted which amino acids were important for drug binding. So then I modeled in pleconaril into the different rhinoviruses, and what I found was pleconaril fit just fine into the rhinovirus Cs making me think that it would probably work on them. So then through a collaboration with a group at the hospital, they went and tested pleconaril and this BTA 188, and they found that neither of these drugs significantly decreased rhinovirus C, which was interesting to us because my modeling had suggested that these drugs would work. So then I went and looked a little closer and found that the hole to the drug binding pocket is actually smaller in the Cs than it is in the As or Bs. 

So this is the As and this is the Bs, and they all have an alanine which is a very small amino acid. All of the rhinovirus Cs, however, have an isoleucine or a similarly large amino acid. So when I simulated the alanine being mutated to an isoleucine, you can see that it makes the pore smaller. So what that made me think was that yes maybe the drugs could fit once you forced them in, but they probably couldn't get in in the first place. And this particular amino acid at the pore residue is very interesting because it's conserved within groups of enteroviruses. So rhinovirus is a part of a bigger group of viruses called enteroviruses, and all the different groups either have the small alanine or something bigger and it's conserved by the group. So all bovine viruses don't have an alanine, but all human enterovirus Cs have an alanine. So I thought that that was an indication of how biologically relevant this particular residue was. So what does this all mean for rhinovirus C treatment? Maybe a vaccine is possible. Probably not. 

They are only that one immunogenic site so probably they would still be able to mutate away from vaccines. But we now know a lot more about what type of drugs would fit into the rhinovirus Cs, and it might be that we need to develop drugs specifically for the rhinovirus Cs. None of the drugs that work on the As or Bs will work for the Cs. So the good news is that we now have an idea what the rhinovirus Cs look like, and we didn't have to grow them in anybody's primary cells. And we can steer the antiviral testing and we can also suggest receptors, and we've done that a lot with the collaboration at the hospital. Oh, we missed misconception. You can catch computer viruses. [LAUGHTER] I looked online and some people think you can catch computer viruses. So if you learn nothing else from this talk, you can't give your computer a virus and it can't give you a virus. So you should feel safe. 

So I'd like to acknowledge the people that helped me with this work. My lab is the Palmenberg lab, and we collaborated with -- for the computational stuff and the Jim Gern lab at the hospital helped us with testing the drugs. And I'm funded by the NIH, and thank you all for listening. [APPLAUSE]