- Welcome everyone to Wednesday Nite @ 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 cool organizers, Wisconsin Public Television, Wisconsin Alumni Association and the UW-Madison Science Alliance, thanks again for coming to Wednesday Nite @ the Lab. We do this every Wednesday night, 50 times a year. Tonight it's my great pleasure to introduce to you Tom German of the Department of Entomology and the Department of Plant Pathology. I first met Tom when he was a Postdoc, back in about 1983 in Gus de Zoeten's lab and he's an extraordinary addition to this University. Plus he can tell us great stories, some of which he will share tonight.

He was born in Aurora, Illinois but as he said, he escaped as an infant 
(crowd laughs)
 to Eau Claire, Wisconsin where he graduated from Eau Claire High School and then he came here to UW-Madison to study zoology. Then he got his master's degree in biology at Michigan State. I forgot to ask you this, but what years did you teach high school?

- 
(Tom)
 Just before that.

- Just before that. 
(crowd laughs)
 And it's an amazing thing, having researchers, a constellation of researchers here that have taught high school and there is something remarkable about that experience.

Then he came to UW-Madison to get a PhD in plant pathology and and plant virus work. He did several postdocs and then he went to the University of Hawaii in 1984 and got tired of the lousy ice fishing and came back to UW-Madison in 1990, where he has been ever since. He lives in, outside Hollandale in Iowa County, is that correct? It's a great part of the state. Tonight, he's going to be talking with us about exploring how insects transmit viruses, potential for biorational control. Would you please join me in welcoming Tom German to Wednesday Nite @ the Lab. 
(applause)
- Wow, thank you for that introduction Tom, but let me say right away-- I've told all my handlers here this-- I don't hear well myself, so it's hard for me to know how loud I am talking. So if you can't hear me, please raise your hand. I'd hate to stand here and talk for 40 minutes and then have you say, "What?" 
(audience chuckles)
 And thank you for coming out on such a beautiful fall night which Tom alluded to. Mosquitoes are finally going away. It's my favorite time of year.

I have a big task before me tonight. I want to talk about virus-vector. I'm getting the "hi" sign already here. Virus-vector relationships.

And you can easily teach a five credit semester course on this subject, so I'm course not going to go deep into things. I want to sort of skim some highlights so you get the idea about what's going on. So I thought, maybe there'd be nobody here. 
(crowd laughs)
 And I thought that because it seems to me that when you think about insect transmission of viruses you might say, "Well, what the heck is there to say about that? "They stick their mouth parts in an animal "and get a blood meal that has viruses in it "and they act like a flying syringe "and inoculate somebody else. "Or in a plant, get plant sap and do the same thing." But if nothing else, I would like you to disavow that idea right now. It's a very specific process and I'll try and explain that to ya as best as I can.

So what do I mean by transmission of viruses? Well, obviously it involves a plant or an animal pathogen, an arthropod vector and some host. And the process of-- Clearly, it's this three-sided thing here. And recently people have been taking note of the fact that the gut flora of the insects actually might be involved in the transmission process. You see a lot about the microbium these days in humans, how it affects your health and all kinds of things. It's an up and coming important field so it's actually four, at least four parts are involved. So my goal is to tell you something, some of you may know more about any of these topics than I do, but the general topics I want to cover, the viruses involved, something about the insects involved and the hosts that are involved. What we know about the interaction between these three things, and significantly, how we know these things.

Tom rarely tells anybody but he has a PhD in the same thing that I do from the same department that I got mine in here at the University of Wisconsin, roughly 10 years apart. You can guess which way that 10 years goes probably. And we talk often about the process of figuring these things out and how incredibly,

what, magnificent it is that the people that came before, even me and Tom, who figured out all kinds of biology about this process without the benefit of cloning and sequencing, modern genomics and bioinformatics. You can study things at the molecular level. And the hypotheses they came up with that describe these interactions were often times exactly correct and are now being validated by molecular techniques. It's really quite remarkable.

And I want to tell you something that's the process of discovery but I mean that in another sense of the word. What kinds of experiments are done to figure these things out? Because I think, to me, that's the beauty of doing this. The clever experiments that people think of and the interesting tools that can be brought to bear this subject. And finally, if we get there before my bedtime, how we might apply what is known about those processes to develop-- buzzword I hate to use-- biorational controls for insects and the diseases they transmit. By which I mean, they're not environmentally harmful and they're not toxic to humans or other things and they're specific. Insecticides have a way of killing everything and not just what you want to kill and we all know why that's bad. Okay, so let's begin. Probably the best place to begin is at the beginning.

This is an old subject. In the 1880's, we figured out that Rice Dwarf Phytoreovirus-- it's an RNA plant virus-- was transmitted by leafhoppers, maybe obvious now that that's going on but then it was not obvious at all. They put leafhoppers that had been raised in places where they couldn't become infected on diseased looking plants, then put them on healthy plants and voila! Something's being moved between plants. 1906 Beet Curly Top, another plant virus was discovered and Cucumber mosaic virus in 1916 is aphid-transmitted and that's a very important one because it's a very important virus and aphids are one of the most important vectors of plant virus diseases.

And of course, not of course, but in fact in 1901, we all learned in school about Walter Reed and Yellow fever which is an important disease

that is epidemic and endemic periodically from time to time. Right now it turns out there's yellow fever outbreak in Brazil. There are Yellow fever outbreaks in this country where thousands of people died in the southern part of the United States. They thought it was going to be impossible to inhabit that part of the country because of this disease. But, it's no longer the case.

And here's another example of an important insect vector-virus relationship. I'm prejudice because this is what we work on in my lab. In 1916, in Australia, a disease called Yellow Spotted Tomato was described. Nobody knew how it got from plant to plant, but it turns out that thrips, tiny little insects transmit this virus from plant to plant.

And in 1932 the interesting phenomenon of a biological, developmental barrier to the insect being able to get the virus in and back out again was described because this virus has little tiny first instar larva, then second instar larva, then adults. Unless the insect feeds as a first instar larva on an infected plant, it will never transmit the virus. In other words if you just put adults on the plant, they eat the plant material, but then never transmit the virus. So there's a barrier to transmission somewhere that became a very important way of looking at these things, transmission barriers.

Okay, and then I'm pleased to say in 1993 in my laboratory, we showed that the virus actually multiplies in the insect. So the virus infects the insect, and when you think about that it's quite remarkable that a virus that lives in plants can also replicate in an animal. Might be a scary thought but there are no plant viruses that infect humans.

There's a picture on the bottom. There's a thrips on the lower left and a tomato with typical yellow discoloring and there's and an electron micrograph of the membrane-bound virus particle, little proteins sticking out of the surface.

Okay, so why do we care at all about this?

Well, partly because of what I just told you, but on the left is a list of viruses of humans and other animals that you've all probably heard something about.

The Zika virus pandemic in Brazil basically freaked us all out because of the horrible problems that is has to newborn children.

That has subsided substantially in the last few years but we still pay a lot of attention to it. Yellow fever virus, I already mentioned and Dengue virus was discovered, it came from New York to here a few years ago. It infects birds and other mammals and sometimes makes humans sick. Doesn't make them very sick usually, but in old people or immuno-suppressed people or people who aren't basically strong to start with, it can be a devastating disease. Lacrosse virus is a bunyavirus, which I'll explain in a minute,

found in La Crosse, Wisconsin, transmitted by mosquitoes. Recently, Jamestown Canyon virus has been found in Wisconsin. Chikungunya, Riff Valley fever virus in Africa and Asia causes serious diseases of ruminant cattle and is a huge problem in that part of the country, Mosquito transmitted. West Nile virus and Equine encephalitis virus are mosquito-born viruses. Equine encephalitis is the major cause of human viral encephalitis.

So, what can you do about these things? In the case of the animal viruses, you can vaccinate people, you can leave, get out of where those are and you can control the vectors, of course. But, we are lucky here in Wisconsin because recently

Susan Paskewitz and Lyric Bartholomay-- Susan Paskewitz is the chair of Entomology and Lyric Bartholomay is associate professor in the School of Veterinary Medicine-- got a 10-million-dollar grant to establish one of several centers of excellence for vector born disease. So their job, their goal, is to do surveillance. They are the ones who just found Jamestown Canyon virus recently introduced into this state. But more importantly, or equally importantly, they train people how to do these things. They've been training people and sending them to Brazil to help with the Zika epidemic. It's remarkably difficult to get people to go into this and we need to train more people to do it, so I'm really glad that's going on. Now, what's on the right over there are a list of plant viruses. I dare say that you are less familiar with plant viruses. People, sometimes I say I'm a plant virologist and they often say, "Really? "I didn't think plants got viruses." Well, indeed they do. Tomato spotted wilt virus, I've already mentioned.

I have to say that plant viruses are poorly named. They're usually named for the first host they were found in, but tomato spotted wilt virus infects over 1,000 species of plants. In fact, trying to control that virus and its' vector, thrips, costs over a billion dollars a year and has a side effect of being an environmental problem because insecticides are used to control the vectors. It doesn't work. They become resistant. So what's the logical solution these days? Spray more pesticide.

You can see from the names of those viruses over there, Cassava mosaic virus, Barley yellow dwarf virus, maize mosaic, soybean, rice, citrus, bean. Almost every kind of important crop plant has some virus that it can get that's spread by insect vectors and the consequences can be devastating. So that's partly why we care and at the very end I'm going to come back to why we care in a more general sense.

Okay, the viruses.

That's a chart of the shapes of plant viruses over there. A chart of the shapes of animal viruses would look very similar that that. Another important thing about that chart, it tells you what is the genome of the virus. Our genome is DNA and our nucleus has the information for who we are. We have 10's of 1,000's of genes. These viruses have five, six, seven or eight genes.

They can get away with that because they live in a host that has 10's of 1,000's of genes and they live very intimately associated with the cells and they use the host machinery to change their DNA or RNA to make more virus, to make proteins to build the shells that they live in and to move from place to place in their host and to ward off host defense mechanisms.

They have a high degree of similarity with animal viruses and they've evolved unique genes to function or facilitate infection. So, there are single stranded RNA viruses are the largest group of viruses there are. But notice this virus right here. It's in the family Rhabdoviridae. There are rhabdoviruses that infect plants. We've almost all heard of Rabies virus. These viruses are in the same family.

This virus up here is called a Bunyaviridae, that's a plant virus, tomato spotted wilt virus, but La Crosse encephalitis is a very similar virus to that, okay?

The viruses can have rigid rod-shaped structures, flexuous rod-shaped structures and some of them are quite long compared to the others. Some of them have their genome spread out between more than one component. These are protein shells with the nucleic acid that you don't make nucleic acid inside of. So, they're quite diverse in their variety.

Okay, but this is the important thing for what I want to talk to you about tonight.

The top is a model of tobacco mosaic virus, which in fact, is not insect transmitted, which is quite remarkable. I often start out when I'm teaching this stuff, I say when I'm trying to get people to think of what I said about well what's there to say? They're just flying syringes. Well tobacco mosaic virus is one of the most stable plant virus there is and it is not insect transmitted. And when people start falling asleep in the class, I say, why isn't HIV, the virus that causes AIDs transmitted by mosquitoes? And aren't you glad it's not? It's a blood born virus. Mosquito takes a blood meal, doesn't transmit the virus. But these other viruses, this is a virus here that's a member of the poliovirus family. It's actually an icosahedron, looks like a soccer ball with faces and there is protein in those faces.

This is my favorite virus, tomato spotted wilt virus and a Bunyaviridae, like La Crosse. It has three RNAs inside of it and a membrane on the outside of it. And on that membrane there are glycoproteins, molecules that stick out of the surface. Obviously, I'm telling you that because these are the features that will ultimately interact with the insect vector making it a specific process. Here is a long flexuous rod-shaped virus, I put the TMV in there to show you how the red is the nucleic acid, just a single stranded RNA, the coat proteins wrap around that very tightly in association and protect it from degradation, and the same for these long flexuous ones. The genomic nucleic acid, which is RNA, is inside this structure that's coated with many copies of a single protein sequence. In fact, in this virus, there is 6,400 of them.

And this, just to remind you, we have to talk about nucleic acid a little bit. That's a model of could be, your DNA, it's a double helix. Keep in mind that the two sides, one side dictates what the other one will be. If you take that molecule and heat it up and melt it, they come apart. If you cool it back down, they come back together. It's called complementarity. So that looks like the DNA in your cells, the DNA in the genome of double stranded DNA viruses. One of those strands would look like the DNA in a single stranded DNA virus.

Or if it was RNA it would be like the RNA in a single stranded virus. So it's all the same basic machinery. That's why they can use your-- if you get a virus infection-- your machinery to do what they need to do. That's why it's nearly impossible to come up with good pharmacological anecdotes to virus infections because it's inside your cell, using the stuff that you use to make protein, using the stuff that you use to make DNA, so if you try and do a strategic strike, guess who you get? You! So it's very difficult, but there are ways.

Okay, so let's talk about vectors because that's what we came here to do. The majority of plant viruses are transmitted by arthropod vectors.

Most of them are hemipterans, like aphids and leafhoppers, 51%. 20% of them have no insect vector at all. Notice that fungi and nematodes also transmit plant viruses. Cause the important difference between plant and animal viruses is that animal viruses get inside cells by receptors. They attach to a cell surface structure and are taken inside the cell. Plant viruses get inside the cells by being poked in through a hole.

Think about a plant cell wall, looks huge compared to the size of a virus. So something, such as an insect, pokes a hole in there and delivers the virus. That's why they can also be delivered mechanically. You can take an infected plant, put something scratchy, like carborundum on a healthy plant, rub it on there, make little holes, virus goes in and voila! Infection.

The other important difference between plant and animal viruses is that plants are sessile. They can't run out of the room if somebody starts coughing over here. To be ludicrous, they can't leave the field if their partners are starting. That's why insect transmission is so important.

Okay, this is just a chart showing what I just said. Most of the viruses are hemipterans, 27% of them. Thysanoptera, thrips, which I talked about is only 4%. So there are diverse vectors and it's important to note that some insects transmit some viruses. Other insects transmit others. But, there's a very high degree of specificity between the vectors and the viruses. Notice, just pick one example, white flies.

115 of the total viruses transmitted by white flies are all DNA viruses and they're all transmitted by white flies. No thrips transmit the same viruses as white flies transmit. So there's both specificity to the insects and to the plants that they infect.

Again, making the flying syringe hypothesis not right.

Okay, so what is an insect vector.

Transmission describes an entire process. Acquisition. 
(slurp)
 Taking up the virus. Retention. Holding on to it long enough to go somewhere and deliver it. And inoculation, delivering it to a new host.

Those are important steps in the process.

A good vector of a virus also has to have the ability to disperse, host relationships that overlap with the pathogen. If an insect comes in contact with the virus and it doesn't feed on a host for that virus, there will be no transmission. The behavior, feeding and physiology that enhances all the steps in the process have to be right.

And it's good from the viruses' point of view, they have a high reproductive potential in large populations, which many insects do.

Here is one of the most

important vectors of plant viruses. That's an aphid giving birth to an aphid. The fecundity of these things, or that is to say their reproductive capacity, is mind boggling.

That aphid's delivering an aphid and that aphid that it's delivering could be pregnant. 
(Tom and audience laugh)
 I've seen pictures of aphids delivering aphids with little aphids inside them. That's reproduction! 
(audience chuckles)
 So, you get numbers like this, so controlling these things. You could see how fast they could just rip through some field and it happens.

Okay, so the lifecycle of these things-- I won't belabor this much-- is you start in the winter, the eggs over winter on some specific hosts, or a few specific hosts. This is an example of the soybean aphid, which over winters on buckthorn. It's really hard to find out there. We've had, in the last seven or eight years, we've had a big influx of soybean aphids and now it's going back down and it caused big problems with spreading viruses in potatoes, believe it or not. Viruses in potatoes are a big problem because they're vegetatively propagated. So if you take a potato plant that's got a virus and you take the tubers from that plant, which will have the virus, how do you plant them? You cut them into little pieces.

All those plants have that virus. So there are programs in place. I used to run one called the Wisconsin Seed Potatoes Certification Program, to keep that from happening. Basically, starting with clean material and keeping it clean by keeping the aphids that are transmitting the viruses out of the fields where the seed are grown, the plants that product seed potatoes. Okay, the other thing that these insects are remarkably designed for transmission. This is the picture of a face-- not to be to technical-- of an aphid cut off like that. In the center is a food canal.

On the two sides are ducts and two sides outside that. This is a little tube that moves independently in a sheath and it can move its way down inside the plant. The aphid actually seals its face to the surface of a leaf so that it can suck plant material in and out and it delivers saliva when it does that and it has a little thing called a cibarial pump. So it's got sort of like a toilet plunger. It's going... 
(mouth gurgling and sucking)
 Perfect vector.

Here's a picture of the labrum, which is that part of the aphid I was talking about, sliding that stylet and that down through the cells, those are cells, down into the center where the phloem, the tubes that take carbohydrate and water up and down the center.

So it's going in there to get the goodies. When it does that, it makes a sheath of saliva that gets hard so it makes itself a little tube to take advantage of that plant.

That's another scanning electron micrograph over there on the other side showing the parts.

The point is, it's wonderfully designed for the transmission of plant viruses. They feed down in the phloem like that, but when they first land on a plant, they want to decide if that's a place they want to be, so they take little meals out of the epidermal cells, not the tubular cells down low, and see if the plant is suitable for them. Taste a little, "No, I don't think so. "Ehh, I don't think so." Well doing that, they keep taking little bites, so they-- bites is the wrong word-- but they take sampling the epidermal cells, so if there are viruses that live in the epidermal cells they'll get those. Then they say, oh I do like it here. They spend quite a bit of time getting down into that juicy part in the center. And if there are viruses that live there, and there are. They get those.

I just said that, they move over the surface and feed then they can do that and they fly a long ways and when they find a suitable host they'll stay for a long time. Okay. So, what I was talking about how people figured this out without using molecular techniques or electron microscopes or actually seeing what was going on.

A bunch of terminology came to be and that terminology led to hypotheses about the relationship of viruses and their vectors. And as I said at the beginning, they're now being evaluated with the modern tools and molecular biology and informatics and genomics.

Here are those terms, I've said them before but, acquisition, inoculation and latent period. How do you measure that? The acquisition access period is the time it takes to acquire the virus. You measure that very painstakingly. So let's say you have some aphids, you have an infected plant. You put X amount of aphids on that plant. You leave them on there for one second, put them on a healthy plant. Leave them on there for one minute, put them on a healthy plant. Leave them on there for five minutes. So you can, by doing that, you can figure out what the minimum time it is for them to become infectious and it's important. Inoculation access period is the reverse of that. You take, after you figure out how long you have to leave them there, so you know they're infected. You put them on a plant, oops, take them right off. Do it again, wait about how long does it take them to deliver that virus. And the latent period, what if there's a time lag? They get the virus, you put them on a plant. They can't deliver it for a long time. Then all of a sudden, they can, even though they haven't revisited the infected plant. That's a latent period. Something's happening between the initial acquisition and their ability to deliver that virus, important concept.

Okay, that terminology led to the naming of several kinds of transmission. Non-persistent, where the acquisition is in seconds or minutes. Retention is only for minutes. If you take them off the plant and put them on another plant, they are only infectious for a short time. There's no latent period and it doesn't multiply in the vector. And I'm already running behind time, but I'll tell ya how you can apply that kind of information.

If it's true that the retention time is minutes and the acquisition time is seconds, if you have a field over here with viruses in it that aphids transmit, you have a field over there that you don't want them in. You can plant a barrier around that field of host plants.

These insects will now go, "Oh, I am going over there." They land on those first plants and those rows out there and they deliver their virus.

If you can put plants in there that you don't care about. And now they go in board to your crop. Guess what? No more virus, because they are not infectious that long. That's a good non-chemical control. Like most things like that, it doesn't work in isolation, but it helps, that make sense? 
(audience says yes)
 Okay. So, here is a little nuance about this. Here comes the specificity in the molecular interactions.

If you feed-- Rockow in the 50's figured out that you could take a tube and put parafilm or a screen over it and put virus in there and put insects on there, they would acquire the virus from your little apparatus, okay? In vitro. So if you feed purified virus

to an aphid, a certain kind of virus and then put it on a plant, you don't get any transmission. They cannot acquire the purified virus.

But if you feed on infected plants, you do get transmission. So purified virus is not transmissible.

Here's what he did. If you feed the plant-- I'm repeating myself-- if you feed it a virus, feed it on a healthy plant extract, you don't get transmission. If you feed it on a healthy plant extract and then on virus, no transmission.

But if you feed it on a plant extract from a plant that was infected but it no longer has any virus in it, just take my word for it, you can do that. You can spin the virus out of there actually. So it's the sap from an infected plant which no longer has virus in it. Then you feed it on a virus.

Then you can transmit.

Now if this was a class, I would say, what's going on? How many of you think you know what's going on?

Okay, good cause I can tell you. 
(audience laughs)
 In that infected plant extract, there are viral proteins that are made during the replication of the virus that are not a part of the virus particle.

That are required for transmission.

They wouldn't be in healthy plant sap and they wouldn't be in the virus, 'cause the virus is made of what we call structural proteins. Just for the sake of argument, say virus infects a plant and it makes 10 proteins and three of them end up in a virus capsule, well there's seven more in the plant.

One of those is required for transmission.

That's called a helper component.

And it was shown eventually that if you take virus and make it radioactive, that it will stick to the very tip of the stylet.

See that in B, the little hairy stuff. In C, I told you that that maxillary stylet opens up and the middle tube comes out. That is the receptor for that virus, only in that spot and it will only happen if that viral protein in that infected material is there. On a purified virus, you do that, that won't happen.

So eventually, these proteins will purify. They were expressed in laboratory, you can manipulate them, you can stick, you can make the protein and attach to it something that won't go all green, GFP, and when you do that, guess what happens? That is a picture of an aphid stylet with a helper component from a virus, a single protein that was engineered to glow green when you shine UV light on it and look where it sticks. It doesn't stick anywhere else on that aphid.

It's at the tip of the stylet and if you put some virus in there that's a different virus than the one that made that helper component, it won't be transmitted. And this one has to be there for the whole virus to be transmitted. The other pictures are just treatments to try and figure out what it was. It was treated with various proteases and various glucanases and things to see what the nature of that spot is and it turns out to be a protein of some kind. So, I think you can see that if that's the spot where the virus sticks and it sticks there by virtue of an interconnecting protein, that you can make a plant that expresses a protein that will stick there, but won't allow the virus to stick there, and block virus transmission.

Okay, here's another kind of transmission, semi-persistent.

Acquisition time in hours, retention time is much longer.

No latent period, so they figured maybe this virus goes farther up in there. It takes a while for it to get in there. Well, here's an electron micrograph of such a virus.

If you look at the top left picture you can see that that virus was treated with an antibody with little gold particles on it and those little gold particles stick to that virus.

Another antibody, made to a different viral protein only sticks to the end of the virus. So there's two kinds of proteins in that virus, gold protein in that virus shell.

Turns out that if you feed that protein on the very tip of the virus, that was made the same way as the one I told you about, namely fused to something to make it glow, then you look in the head of the insect, you can see right there that up higher in the insect head is where that protein sticks. So it's the same phenomenon in a different place and it's called semi-persistent 'cause it lasts a little longer but still not real long.

And if you treat that with an antibody that prevents that protein from binding there, you will prevent transmission of that virus.

So they went on and on doing this. There's a kind called persistent circulative. It actually goes in the insect, cross the gut wall, into the hemocele. The hemocele is like your peritoneal cavity in an insect. You have your gut and a space. Well, that space is a hemocele in an insect. The acquisition time is hours.

Why would it be hours? Because it has to go way down in the plant and phloem to get these viruses. The retention time is days. So they say, "Well, maybe it's going, "spending a lot of time in there going all around." And, they were right.

They further went to show that these kinds of viruses show extreme specificity.

Down the left-hand column are different aphid species.

Across the top are different virus species all in the same family. The top aphid can transmit only RPV and maybe PAV a little bit, so it's not completely specific. R. maidis, an aphid can transmit RMV. S. avenae, MAV, so there's specificity between the aphids and certain viruses, because of these molecular interactions that have to happen.

Okay, this was figured out by Fred Gildow, who looked in the electron microscope and he saw that these different viruses go to different places in the insect because there are different barriers. Some can get across the gut, go into the hemocele and get to the salivary glands and come back out. Others can go into the hemocele but they don't get in the salivary glands. Some go right straight through.

The specificity, whether or not it can get across these various barriers is, of course, determined by the coat protein.

The blue virus and the blue genome make the blue coat protein and the blue aphid can transmit it. The yellow virus with the yellow genome and the yellow coat protein can transmit that one.

But if you put the yellow RNA in the blue capsule, now you can transmit that RNA with the other aphid because it's the outside that determines the vector specificity.

You can even do this. Put both coats, if you will, on the same particle and both insects can transmit it. Pretty clear proof that the molecular interaction between the outside of the virus and some specific spot in the insect determines this specificity.

So here's what's going on. We have three different kinds of viruses with different sets of tissue barrier specificity. Some of them go in and they can't get across the gut. So what happens?

Poop, out they come.

Others can actually cross the-- This is the inside of the gut chamber, this is the hemocele out here. They actually can cross at some specific point and get into the hemocele and then get into the salivary glands and then go back into the new plant. So all levels of specificity occur here. That's called circulative transmission, for obvious reasons. But these viruses do not replicate. They're just passively moving around in there.

All right, oh, this has been used in the most clever way.

That's a paper that we wrote in my lab. We did not do this, we were asked to write a popular article about this. So a woman by the name of Bonning,

decided-- Here's a cartoon of what happens. She knew that it was one of the capsid proteins that carried this virus across the gut barrier. So she made fusion proteins, that is to say hybrid proteins, between one of the capsid proteins and a spider toxin. Spider toxin is not toxic to mammals. It's not even toxic to the insect if you feed it. It's only toxic if it gets in the hemocele. So, if you're an insect, you can eat it, be in your gut no problem, but if it got in your perineal cavity, you're toast.

Same thing here. So she fused that to the coat protein, which she had already figured out carries the virus into the hemocele. So it used it to transport a cargo into the hemocele.

I won't go through all this data, but this right here, she made a fusion between the cargo carrying protein in GFP. And as you can see, this is the hind end of a gut. The hemocele of that gut is bright green because the protein carried the green fluorescent protein into the hemocele. That's all you really need to know about that. Very clever and it works.

This is percent mortality. The white bars are insects that were fed in vitro with this fusion protein between the caps of the virus carrying the cargo into the hemocele. It killed almost all of them. The others are various controls, just to show that feeding them foreign protein isn't killing them or feeding them GFP isn't killing them. And she even made a fusion between a coat protein and a mutant toxin that was no longer toxic.

Didn't kill anybody. So it really works.

Furthermore, if you make transgenic plants. You deliver this from the chromosome of a plant

and then you score median number of aphids per plant and you look at this one.

This is the toxin fused to the coat protein, not so many. All the controls, lots of aphids.

Here's a control with no toxin. Here's a control, very few. And if you look at it over time. Here's the plant making the toxin, here is the controls, number of aphids per plant. Seems to work. I like it because it's specific for insects.

Not for insects. For a few insects.

Okay. I'm going to skip that.

So now we're going to try one more kind of transmission. Persistent propagative transmission.

Takes hours or days to acquire. Retention time is days or weeks and there is a latent period. And the reason for that latent period is because the insect becomes infected and even if it's not feeding on an infected plant, the virus is multiplying in the plant, so it takes time for that to happen and it does multiply in the vector. So it's kind of a circulative, where they're just going around and round and round, adding that it actually multiplies in the vector.

Okay, this was work done in my lab so,

selfishly, I'm going to go a little deeper into this. Probably because I know most about it. Here's a picture of tomato plants infected with tomato spotted wilt virus or healthy plants, pretty devastating. This virus causes a billion dollars of damage a year.

It has a wide host range and thrips are extremely difficult to control because if you use chemical pesticides, they become resistant very fast because their fecundity is enormous.

Okay, their lifecycle. They start out with eggs in a leaf. They hatch into first instar larva. As I said, this is the stage that has to acquire the virus. Then they become second instars. Then they become pupil stages. Then they become adults where the female is larger than the male and they can transmit the virus if they acquired it in this stage only.

The idea that we had about that was that there must be a receptor because it's acting like an animal virus now, that will take that into the cells of the insect. And it must only be in that first instar larva 'cause if it was in the others, they could become infected with it. So we did electron microscopy and you can see virus particles. This is the lumen of the gut. These little things sticking up are like the villi in your own gut, little fingers. And you can see the virus sitting on those. Made us think, that's where there was a receptor.

Where would that receptor-- What would bind to that receptor by analogy to those other viruses. Something on the outside. This is a model of TSWV with a membrane-like protein sticking out of it. Most likely, that's where they are. But we had other clues to that too. You can see-- Here's an electron micrograph of the virus, you can kind of imagine them sticking out of it. We knew that in mutants of this virus that are not insect transmissible, those mutations were in the genes that code for those proteins sticking out of the surface, logically so.

Okay.

So you can actually see this guy Montero-Astúa, down here, and Ullman and a student of mine, Anna Whitfield and I published this paper. The green material is a virus. This is the primary salivary gland. It has two lobes, so it had to get to the salivary gland to be delivered. You can see this green stuff is virus. Blue stuff are nuclei and here is the lumen of a salivary gland. So we know the virus is going through the insect and multiplying in those tissues.

And here shows you that larva become infected very fast.

Whereas the fore gut and the mid gut and the hind gut aren't infected in the larva. But in adults they're all infected, so the whole insect becomes infected.

So we reasoned the following. If we made a little piece of that protein that sticks out of the surface of that virus, that will stick to the same place that the whole virus does...

and feed it to the insect. It should block transmission, shouldn't it? It's sort of like a lock and key, right? If the place where the virus sticks takes-- And the surface of the virus is a key that goes in the lock and then it gets taken up. If you take a key and stick it in the lock and break it off, now it can't be taken up anymore. So we tested that hypothesis in the following way. Here's a model of what I just said. The little curly lines represent the receptor for the virus. TSWV represents the virus. And the little cylinder sticking out of it represent that little piece of that protein only, absent the virus, sitting on the receptor. Now, it's blocked. No transmission.

Well, here's my favorite picture of all time. It was actually on the cover of this journal. If you feed them just protein from the store, BSA.

And stain them with a red material that stains the muscle fibers in the gut, you see this.

If you feed them just virus and look at their gut, the blue stuff is virus in their gut. This is a confocal microscopy image of viruses tagged with an antibody with the blue flora on it. So all this blue stuff is virus in the gut, but if you feed them that little busted off key, that piece of the protein from the surface of the virus at the same time as you feed them virus... Voila! No virus in their gut.

So it's blocking the attachment of the virus to the insect gut. Oh, I liked it so much I put it in there twice by accident. 
(audience laughs)
 So, can we inhibit transmission of the virus that way?

So if you feed the thrips on virus alone or on that little piece of protein and you rear them to adulthood. Then you feed them on a leaf disk and then you test those leaf disks for virus transmission, what you see is, if you feed them just on buffer, no virus, you get no virus transmission, fortunately. If you feed them on just virus, you get about 60% of them transmitting. But if you feed them on that little piece of protein, and the virus, a much smaller percent transmits. So it's being blocked. So how could you deploy this? Well, if you make again transgenic plants that make that little piece of protein, you can see...

You measure the frequency of transmission after acquiring virus from those plants, the non-transgenic plants have a pretty high transmission rate. The transgenic plants have a much lower transmission rate. It's not perfect because nothing in biology ever is, but you'd rather have this situation than that situation.

Should have showed you this the other way around, but in fact the reason for what I just said is 'cause the thrips themselves, if you look at the tighter, the amount of virus in the insect, it's much lower on the transgenic than the non-transgenic plants.

So this seems to work. Okay, so let me just summarize what I've said here for a minute. We have capsid strategy, where the virus just sticks

to a receptor and circulates through. We have a strategy where there's a helper component, sort of an adapter for the virus to the mouthparts of the insect. And we have flexuous grab virus particles, where there's a unique piece of the protein that attached. They're all different, but the net result is the same. That's a mosquito over there, which I haven't said much about because there's rift between the animal virologists and the plant virologists, I think. Animal virologists say there's no such thing as transmission of a virus that doesn't replicate in the insect. They have identified none of these receptors for non-replicating viruses. They just say, "yeah, it's a flying syringe."

Dare I say, I don't think so.

All right, I want to tell you, rapidly here I hope, one more strategy that I think you should know about. I really mean it. This is a technology that's coming online. It's called RNA interference. Don't tune me out if I talk a little molecular biology here. I only say that because people tend to do that. This is very straightforward.

There's a process in cells where they can get rid of messenger RNA. Messenger RNA is made from your genome and it codes for proteins. This process is called RNA silencing. It's a way to cause messenger RNA to be degraded. Why should that happen? It happens because all of your cells have the same DNA in them, right? They all, potentially all your cells should be the same, but some things are expressed in some cells and not others. So you say, why is a skin cell different from a muscle cell? Well, because muscle cell makes things that the skin cell doesn't. It's also true that things are not made in A versus B. So, to be not made, you have to control that. So it works like this. There's genomic DNA. This is a normal process of transcription. Makes a messenger RNA that codes for amino acids that make a protein.

DNA, RNA, Protein. We all know that litany. But over here in a different place on the genome, RNA is made that's a hairpin structure. So this side of it is complimentary to this side of it. It's also complimentary to this. So there's an enzyme called Dicer

that cuts that up into little pieces.

They look like that.

Those two pieces are complimentary to each other.

One side of it, is by definition complimentary to this guy over here. It sits on there and now what does it look like?

That piece looks like this piece. Complimentary double stranded RNA and dicer now chops that up and you don't make that protein. Here's an example that's very tractable.

It's called RNA interference. You can add that RNA anyway you want. It doesn't have to come from the genome. You can stick it in the cell by transgenically, by injection, by bacterial or viral expression, by feeding it to insects or by spraying it on plants.

Here's an example. Here is a normal looking plant that was infected with a virus that makes a double stranded RNA, like those little ones I showed you. Complimentary to a gene that makes a protein called phytoene desaturase. Its job is to keep that plant from photo-bleaching in the sun. So it's been degraded. Now, it photo-bleaches. Make sense? You got rid of the protecting protein by putting double stranded RNA complimentary to the messenger RNA for that protein. Here's another example here. This plant is expressing green fluorescent protein at a high level. If you inject double stranded RNA into that leaf, complimentary to that, it gets rid of the messenger RNA and it now turns like this. This is what a plant looks like in UV light without GFP. This is with GFP. Now, you've turned it backwards by destroying the message for the GFP.

How can we use that? If you inject this double stranded RNA to an important gene V-ATPase, in this case, into a leafhopper, it causes them to be deformed. This isn't the clearest thing in the world, but these wings are deformed and these wings are deformed and these are control. They are not deformed.

In fact, if you do that the fecundity of these insects is greatly reduced. Here's the control, double stranded RNA to a gene, double stranded RNA to a different gene. So you've shut off the synthesis of an important protein to the lifestyle of these insects.

I forgot what that is.

No I didn't. 
(Tom chuckles)
 This is the same thing if you do this in thrips. The vectors of Tomato spotted wilt virus.

This shows the amount of knockdown of the messenger RNA. Double stranded to the same gene as the other insect, you can see here that much less of that RNA is made than in the controls. It's not much less enough for my satisfaction, but it does knock it down.

It does knock it down enough to have an effect. These were injected using a syringe. Inject the double stranded RNA as a proof of concept. I'll just look at this right here.

This is the number of viable offspring from the population. It is much reduced in the injected insects, okay? And this is female mortality. The injected insects have a much higher female mortality.

Okay, so another way to get this double stranded RNA in there is to feed them bacteria that are making the double stranded RNA.

Bacteria had been fed to this insect that are expressing, not double stranded RNA, but just GFP to show that you can feed thrips

an endosymbiotic bacteria that they harbor. Take the bacteria out of the insect, transform is so it makes GFP, feed it back. They'll take it up, the insect will multiply and it will live in their guts. It's a normal endosymbiotic bacteria.

If you engineer those bacteria to make double stranded RNA to an important gene called tubulin--

Here's the control and here's the amount of tubulin-- You knocked down the amount of that tubulin greatly. And if you do the same thing now, you look at the effect of that and you can see this is double stranded RNA in the bacteria, directed toward the gene called tubulin, which is a cellular structural component.

This is the number of dead adults, this blue. I'll just use this as an example, there's more dead adults than in the heat killed bacteria which aren't making it anymore and even less in the control. And this is the big one right here. These are dead larvae

fed bacteria making double stranded RNA to tubulin. So it works pretty well, but not well enough.

But, here's the example that works very well.

This is double stranded RNA sprayed on plants by Russ Groves in Entomology for formally Monsanto, now Bayer.

Just sprayed on the plants. Hard to believe you can make that much double stranded RNA and spray it on plants, but you can.

This is the control with no double stranded RNA. If it was dark you could see that these plants are pretty eaten up. I hope you can see that. And here's the plants that are sprayed with the double stranded RNA. They look fine cause these Colorado potato beetles that are defoliating these plants ate the double stranded RNA and knocked out a gene that they need and it killed them. And it's very specific. It knocks out a gene presumably that's only in those Colorado potato beetles. You're not killing pollinators, you're not killing predators of the Colorado potato beetle, which helps some. It's a new technology. This is actually now commercially available. It's called BioDirect. There's another one for the corn rootworm.

So I tell you that because I think it's important that you weigh in on decisions. This is a completely new idea in pest management.

Going back to finish up in the next few minutes, but why I think these things are important, is kind of--

If you followed me, technically you can see that they're unusual, but they are ways that could work to reduce insects and the diseases they transmit.

Consider that the world population is pushing nine billion by 2050.

Agriculture production needs to go up hugely.

We have to do this in safe ways. I'm really concerned about the use of pesticides. So the mission that we have in these programs is to develop new knowledge of high priority plant diseases with the goal to translating basic findings into new kinds of applied disease control. It turns out that 47% of the new emerging diseases on plants are viral.

And most of those are insect transmitted.

The other component of this problem, well there are many, but one that sticks out in my mind is global warming.

This is a paper in a very recent Science that says, "The results show that insects "will cause significantly increased grain losses "among many regions of the warmer world." Why might that be? Insects, of course, are not warm-blooded animals, as the temperature goes up, their fecundity goes up and their geographical range will change.

And as globalization increases, pathogens will be spread around the world. Things are going to get worse not better.

A specific example of that is this paper "Water deficit enhances the transmission "of plant viruses by insect vectors".

"The evidence that infected plants subjected "to drought stress are much better sources "for insect vectors may have extensive consequences "for viral epidemiology and should be investigated."

And this is what concerns me the most. The ways I've described to you, whether you understand the finer details or not, I hope you understand that they are different than this.

And it's been going on for so long. That truck is about a 50's truck isn't it? Look at that. That picture down on the bottom is contemporary. The middle of the last century, we don't seem to be getting better.

Here are some facts. 1.8 billion people engage in agriculture. One billion pounds of pesticides are used every year in the U.S. A billion pounds! It's not all in agriculture, you know, some of it's for getting the ants out of your house and other things, but a lot of it's from agriculture and going on food. That's 5.6 billion pounds per year in the world.

Five million agricultural workers are unintentionally poisoned each year and I am getting to be an old man, but I am newly concerned about the effects of organophosphates on children. Makes sense to me that things like hyperactivity syndrome. They're neurotoxins. 33 million pounds of organophosphates are used every year in the U.S.

Wouldn't you like to do something about that? So, the reason why I keep bothering to talk about these new strategies. They're not shelf ready, but it's in the right direction.

And not that you're probably need plenty, but you should understand them, so that you can vote for people who will use logic in the deployment of these kinds of things and try and change things for the better. And so that you can understand them yourself and weigh in on the argument when people just go,

"No, because I said so!" Right, you know what I mean. The imbalance between logic and outcomes seems to be so prevalent these days. We need to know about these things and stand up for them. I'm not saying you should stand up positively even. But being informed, I believe is critical.

So what do we want to do? Exploit the specificity of virus-vector interactions in ways that I've just described. Block virus acquisition by using viral proteins to inhibit virus attachment or entry. I think I gave you an example of that. Target insect receptors or other proteins. Directly target essential vector genes using RNA interference and breed conventionally for resistance to viruses and their vectors.

None of it's a silver bullet, but a combination of things can work and to end on a favorable note. Here is an example of potatoes. These potatoes over here are expressing from the chromosome. They're transgenic for BT, a common bacterially-made insecticide. Even organic growers use it. It's very benign. It kills Colorado potato beetles. This works really well. This is the control.

Here are papaya.

My colleague and friend, Dennis Gonzales, made transgenic papaya in the 80's that expressed a little piece of a viral genome that are resistant called papaya ringspot virus. Here's the non-transgenic one, here's the transgenic ones. It works remarkably well. And in fact, if you eat such a papaya, you're eating less viral RNA than if you ate an infected one. Which most of them would be if it wasn't for this.

But they're tying to withdraw it from the market, even in Hawaii where this was done.

This is the double stranded RNA I just told you about.

This is with double stranded RNA, this is without. This was sprayed on here three times in the summer

to control the Colorado potato beetle.

So, I as always, would be remiss if I didn't thank a lot of people for the work that I presented from my own lab. Particularly Anna Whitfield, one of my students who left the lab a few years ago and is carrying this work on.

Done wonderful things on that project. All those people down there, to Tim Carlson, are students of mine who now mostly have faculty positions somewhere and they're carrying on this work. I put Mauricio down there on the bottom, he was never in my lab, but he was a student of my student, Anna Whitfield and he made those remarkable

confocal micrograph images of the thrips in the vector that were essential for us to do what we wanted to do. My colleague, Diane Ullman, is from UC-Davis. She just visited me, I met her in Hawaii in 1985 and we have collaborated on this project for the last 30 some years. D. Kyle Willis and a faculty member who has left was a source of inspiration to me. Russ Groves helps me understanding the real world of entomology. And Ranjit Dasqupta and Hao Wei Teh are current members of my laboratory. I'm still, sort of, plugging along at a slow rate and working with them on the bacteria delivery of double stranded RNA to thrips. So, even though it's past my bedtime, I thank you and ask any questions you might have. 
(audience applauds)