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May 22, 2019 – Dr. Kelly Diehl talks about equine herpes virus with Dr. Tracy Stokol, Professor of Clinical Pathology at Cornell University. The two cover this devastating disease, as well as Dr. Stokol’s recent Morris Animal Foundation-funded research into certain white blood cells, called monocytes, to help horses fight equine herpesvirus type 1 (EHV1) infections.

00:16 Dr. Kelly Diehl: Welcome to Fresh Scoop, Episode Eight: Understanding Equine Herpesvirus. I'm your host, Dr. Kelly Diehl, Morris Animal Foundation's Interim Vice-President of Scientific Programs, and we hope you're learning some things with our episodes. For those of you who might be new, this is the monthly podcast of Morris Animal Foundation, one of the largest non-profit foundations in the world dedicated to funding studies to find solutions to serious health threats to animals. Founded in 1948 by Dr. Mark Morris Sr., a veterinarian, we've invested more than $126 million in more than 2,600 studies that have improved and protected the health of companion animals like cats, dogs, and horses, as well as wildlife.

01:04 DD: In each episode, we'll feature one of the researchers we fund, or one of our staff members, discussing their work in advancing animal health. Whether you're a practicing veterinarian, technician or student, or just an animal-loving science geek, Fresh Scoop is the podcast for you. You can learn more about us at morrisanimalfoundation.org. Okay, on to today's show. Today we welcome Dr. Tracy Stokol, Professor in clinical pathology at Cornell University. Dr. Stokol is a Foundation-funded researcher as well as a former member of our Large Animal Scientific Advisory Board. Dr. Stokol got her undergraduate and veterinary degrees from Melbourne University before coming to the United States to join the faculty at Cornell. She also did a post-doctoral fellowship at Brigham and Women's Hospital at the prestigious Harvard University. And, Tracy, thanks for joining us today.

01:57 Dr. Tracy Stokol: Well, thank you very much for inviting me, I'm very happy to be here.

02:01 DD: As I mentioned, I know we wanted to talk about equine herpesvirus 1, but before we start, can you tell us a bit about some of your other research interests?

02:09 DS: Yeah. So I guess my research is focused mostly on hemostasis, and that's kind of what I did my PhD on all those years ago. And so anything related to hemostasis, I'm kind of interested in, as well as I work on cancer. And so if I can link the hemostasis to cancer, that's great, and that's what I try to do with a lot of the research. Mostly dogs, just because dogs tend to get a lot more cancers than most of the other species. And I'm also particularly interested in hematopoietic cancer, such as the leukemias. But the other aspect which is not related to hemostasis is that I'm a clinical pathologist, and because animals can't speak for themselves, we often have to take blood from them and let their blood talk for them and tell us what diseases might be there. And so as a clinical pathologist, I look for blood markers or what's called "biomarkers" of disease, and so I'm also interested in trying to find new biomarkers to tell us that an animal may have cancer in the body, and we may not be aware of it. Kind of like those sniffer dogs that you may have read about, that they can smell your breath, and they can potentially detect cancer, well, we kind of use blood to do that. So that's the other... My interest is to find new biomarkers for cancer.

03:37 DD: Oh, great. So I know we're going to talk about Equine Herpesvirus, and in particular, your study was interested in looking at white blood cells and the virus. But before we start, can you tell us a little bit about equine herpesvirus for our listeners who might not be familiar with that disease?

03:56 DS: Yes. So equine herpesvirus is in the family of Herpesviridae and is actually one group of that family, which is an Alpha herpesvirus, which, your listeners may be most familiar with Herpes Simplex Virus, or the virus that causes cold sores, so it's very similar to Herpes Simplex, it's in the same group of viruses. But in horses, it does very different diseases, and it behaves very differently in the horse than Herpes Simplex affects humans and how it causes disease in humans. So with herpesvirus, the disease is mostly discharged in the horse, the EHV-1. And I probably should back up here and say that there's multiple types of herpesvirus, and the one that I work on is the Type 1 or EHV-1 is how I’ll refer to it in the rest of the podcast. And how that virus is shared is through respiratory secretions or nasal secretions. So essentially, like when we sneeze and cough and transfer the cold virus, that's how horses transfer herpesvirus from one to the other.

05:09 DS: And when a susceptible horse gets it, it infects the cells in the nose, it replicates within them, and it causes, just like when we get a cold, a nasal discharge and can actually cause pneumonia like you can get when you get a very severe cold, you can get a secondary pneumonia. But in some horses, the virus actually gets into the bloodstream, and it's carried there by white blood cells, as you mentioned. And when it gets into the bloodstream, it can go systemically, and then it can go to the placenta in pregnant mares, particularly in the late stages or later stages of pregnancy and can cause abortion, which is pretty awful for the owner as well as for the mare. And then in some horses, it also goes to the spinal cord, and it causes neurologic disease which is called herpes myeloencephalopathy, which is, again, a pretty devastating disease for owners and trainers, etcetera, because the horses, they can become paralyzed. And these are very big animals, to not be able to use their hind legs, or to even be weak on their hind legs, they can collapse, and so it's a pretty devastating disease. And obviously, because it's a virus, it's transmissible from one horse to the other, so you can get these big outbreaks. And some horses have to be euthanized or put down, because of the severity of the illness.

06:42 DD: So when you were talking about the ease of spread between horses, can you... Do you have any idea how prevalent this is in our horse population, in general, and how widespread the virus is, globally?

06:55 DS: So the disease is fairly widespread globally, although I can't answer exactly which countries don't have it, except that I know EHV-1 has not been reported in Iceland, and so I actually have a colleague here, Dr. Besin Wagner, who works on the HV1 as well, and she actually has brought over horses from Iceland, because they've never been exposed to it, and they represent a completely naive population, which provides a great opportunity to study their antibody or immune responses to the virus, but in the United States, for instance, and in Europe, such as in Germany or even in India, because I was reading an article about EHV-1 in India. So, it's pretty world-wide where the virus is, it's been estimated that 90% of horses have been exposed to the virus at some point in their life and often soon after birth. The problem is, the virus, even if they're exposed to it, most horses don't get permanent immunity to it, and that's why they're susceptible to getting re-exposed to the virus.

08:06 DD: We think of herpesvirus, and you mentioned cold sores in people or feline herpesvirus. Do these guys have latent infections that then can crop up later in a time of stress? We think of that, especially for feline herpesvirus. Is it similar in horses?

08:25 DS: The belief currently known is, yes, that it is. So some previous studies by other researchers on EHV-1 have been able to take blood cells from horses, particularly cells called t-lymphocytes, and have been able to get virus out of them in horses that were not ill at the time. So that's why, EHV-1 is thought to be latent. And we know that it can be latent in T-cells, but there's a recent study showing, because some of the belief is is it always new exposure to the virus, is the virus that is latent now coming back out again, if the horse is stressed? And there's another... One of the biggest stresses you can do in animals experimentally is to give corticosteroids. And there's a study showing that corticosteroids do not cause EHV-1 to come out again. So there's a lot bit of controversy about latency. And actually I was at a recent Dorothy Havameyer conference on EHV-1 that gets held every couple of years or so, and it gets together all EHV-1 researchers that are working in the field and they come up with what additional studies and what new directions do we go in, and one of the things that came out on is we need to answer the question of latency. Where is the virus actually latent, as you know, herpes simplex virus is latent in peripheral nerves or the ganglia's nerves, but we don't really know fully is a disease in this horse or this new outbreak due to coming out of latency or is it because of exposure to new infection.

10:15 DD: Okay, so you mentioned, since you mentioned T-cells, why don't we go on to your research, since your research was looking at monocytes, and can you summarize for all of us again the basics of your study and why you chose to look at that cell population for this virus?

10:35 DS: Yeah, so it's kind of funny, because as you said, science nerds or clinical pathologist we're generally geeks and nerds, and we have this kind of, we have these group meetings with clinical pathologists around the world, and this one person threw up this PowerPoint and said, "Of all the blood cells in the body, which would you choose to be?" And I actually picked the monocytes. And then I was thinking now why did I pick the monocytes. I picked the monocytes because it's one of the longest lymph cells in the body, and it does an awful lot, and it really controls things... And maybe I'm a bit of a control freak, but it really controls things. So the monocyte is, even though it itself may not be responsible for killing the virus, which we don't think it is, but it actually regulates how the other cells in the body, and that's like the normal B- and T-cells or regular immune cells and lymphocytes, and actually regulates how they respond to the virus. So, I consider them like the master regulators.

11:45 DS: And so that's kind of why I was interested in them, because there's lots of studies now showing that monocytes are not one type of cell, but they can be very versatile, and there's actually sub-populations of them in the blood of humans, in mice, and in horses, as I discovered. So the pilot project that was funded by the Morris Animal Foundation, I'm very appreciative of them for funding it, was looking at the question of sub-population of monocytes in the context of EHV-1, because we know that monocytes are one of the cell types that is infected by the virus that we're not really sure how the virus uses the monocytes or if the monocyte is actually good, or if the virus uses it, hijacking it, to help it get around the body, which is kind of one of my theories.

12:42 DD: Okay, so what were your results from your pilot study?

12:46 DS: Okay, so essentially, what I did is, I discovered markers on the surface of the cells. In normal horses, I took their blood from them, and you can get a good amount of blood from a normal horse, just like you can from a normal human. And what I did is, I identified particular markers on the surface of the cell that could separate them into different populations, and one of these populations in other species is supposed to be an anti-inflammatory cell that secretes these proteins that dampen the immune response. Which is a good thing because you don't want to get over response to everything, because then you get really sick if your immune response is too reactive.

13:34 DS: And the other one is the pro-inflammatory response, a pro-inflammatory monocyte and that's a good thing, because if you want some degree of an inflammatory response to a pathogen like HB1, otherwise you can't clear it from the body. So what I did is, I was able to separate these two cells from the blood, using techniques in the labs. I take the blood and add anti-bodies to those markers, and I got them into two populations. And my ultimate question is, is a virus affecting these cells differently? And is it making the pro-inflammatory cells secret more inflammatory markers, so the over-stimulated, or is it doing something to the anti-inflammatory cells? And that's kind of where I wanted to go with this project. How does the virus affect these sub-populations of monocytes which are important immune cells in blood? But before I could really answer that question, you have to answer the question is, does the virus equally infect these two populations? Because if you have the virus preferentially affecting one cell type over the other, any differences you may see in downstream effects, such as where proteins are secreting, could be entirely just a consequence that it likes to be in one cell more than the other.

15:02 DS: And so that was really what the pilot project was about. It was showing, can the virus affect these two sub-populations, equally? because if it couldn't, that would be a very interesting question in itself. And can it replicate in both of these efficiently when I exposed blood? These two populations that I have isolated in the lab, and then expose them to the virus in the test tube. And the answer I got is that the virus does infect them equally, which is good from the point of view that now I can go on to say, "Well, okay, now what does it do to the cells, or what does it turn the cells into, if anything?" And that I'm still in the process of trying to answer.

15:46 DD: Okay. Did that surprise you, that result, or were you expecting something different?

15:51 DS: So, okay, so, yeah. So the hypothesis was that it would actually equally, that there would be equally susceptible, but... So what you want to do is you want to get support for your theory or hypothesis that you then test. And actually, I could have tested all kinds of hypotheses. I could have said that it would infect the pro-inflammatory cell population more than the anti-inflammatory cell population, because there's evidence to support both from other viruses in the literature. So this is a fairly new field with viruses and monocytes in these normal publications coming out. And there was evidence to support that both theories could be correct. So my initial hypothesis is that they would be equally susceptible. And I proved that, which is great, but I could have turned out to be wrong, but I didn't in this case.

16:49 DD: So if you were talking to equine veterinarians and horse owners, what do you think the take-home message is from your research, and what we're learning about, sort of the role of monocytes in this particular viral infection?

17:09 DS: And I'm going to be perfectly honest here, is that the research that I'm doing here is asking very basic questions on immunology or the mean reaction to a virus. And is this actual grant going to relate to how veterinarians should treat a horse or anything now? Absolutely not, but what I'm hoping it's going to do, is increase that knowledge of how that virus works in the animal, and then we can use that knowledge to design better vaccines, and I don't think we can design a better vaccine that induces better immunity without understanding how the horse's immune cells actually respond to the virus and currently we know very little about it. But my research is not going to have like an impact tomorrow to change the way the owners treat their horses or look at their horses or and try and protected horses from the EHV-1. And maybe we'll make a difference in 10-15 years time. Then we have to have that knowledge in order to do better with trying to protect horses or treat horses. And I'm hoping that my research contributes to that knowledge base. Does that make sense?

18:27 DD: Yeah, that does make sense. And that actually brings me... That makes me think about another question which is... And you alluded a little to this, where do you go next? Where do you want to go next? Now that you've got some basic information that's important and new, and what would you like to do next?

18:49 DS: Now that I know that the virus infects both these cells equally, so they're both equally susceptible to infection, I want to ask, does this matter, and does it actually change their responses, and how does it influence specific proteins that can affect inflammation? So what I'd like to do is actually look at what proteins are being produced by each of these subsets after they've been exposed to the virus.

19:18 DD: For those equine practitioners out there, we know that horses are a little different in their inflammatory responses. And do you think this... What you're seeing with the monocytes, and focusing on the monocytes, will maybe have applicability to other species, or do you think this is really unique to the horse?

19:41 DS: So that's a great question. And actually, there's a lot of people studying monocyte responses and these sub-populations and how they react to different pathogens, not just viruses, also bacteria, and how the bacteria actually or viruses manipulate the monocytes into doing their bidding and help them replicate. So people are looking at these sub-populations in more than just the horse. Yeah.

20:08 DD: Well, that's great. Well, thanks Tracy for joining us today and telling us about your research and equine herpesvirus. And particularly, I was really fascinated by again talking about the monocytes and the role of monocytes, because I think you're right, I think it's a cell we don't think of as being very sexy in immune system sometimes, but they're really important. And I'm really looking forward to hearing more about your work on this topic in the future. So again, thanks for joining us.

20:45 DS: Well, thank you very much. It's good that people are interested in work on EHV-1 and supporting the Morris Animal Foundation with supporting the great research that is done by lots of researchers on horse problems Thank you.

21:01 DD: Well, thanks a bunch, Tracy. Take care. That does it for this episode of Fresh Scoop. Once again, thanks to Dr. Tracy Stokol for joining us. We'll be back with another episode next month that we hope you'll find just as informative. The science of animal health is ever-changing, and veterinarians need cutting-edge research information to give their patients the best possible care, and that's why we're here. You can find us on iTunesSpotifyGoogle Play Music, and Stitcher. To learn more about Morris Animal Foundations work, again, go to morrisanimalfoundation.org. There you'll see just how we bridge science and resources to advance the health of animals. You can also follow us on FacebookTwitter, and Instagram. I'm Dr. Kelly Diehl, and we'll talk soon.