good morning and thank you first I want to start by thanking Martin and the organizers for the invitation to come and participate in this meeting as you can tell from the title of my talk we'll be talking about enteric viruses in poultry so for our time together this morning first like to just give a short introduction to in diseases of poultry then I'll talk about the pathogenesis of enteric viral disease talk about some of the enteric viruses that were most interested as they relate to disease syndromes in the field challenges that we've experienced in diagnosing these diseases and then just to illustrate an example of some applied research that we've done with my group at UGA how we identified an etiology for running and stenting syndrome and then I'll end by just sharing with you some of the recent advances that we've been fortunate enough to experience in the field of enteric viruses so we've this is probably not anything new to this group we've been looking at the avian digestive tract for a day now but there are a couple of aspects of the GI tract of poultry that I think are extremely important especially as we start to look at viral diseases first we know that the rapid growth rate that occurs during the first week of age tremendous the GI tract grows at a rate five times greater than that of the rest of the body just during this first week the duodenum is the primary site of nutrient digestion and absorption and so any impairment to the GI tract can certainly affect the intake of these nutrients during this critical growth time in the chick in the small intestine the Phylis length doubles in the first two weeks of life and this enables these growing birds to digest increasingly a variety of feed ingredients and the GI tract doesn't relie reach maturity until two to three weeks of age so when we think about diseases of the GI tract one of the common outcomes is the appearance of undigested feed and the feces it's a common indicator that something is not going well in the bird so enteric diseases as we've learned over the past day are difficult to manage and we have a lot of limitations in our diagnostic procedures but also we have many contributing factors we have genetics that can contribute to the disease disposition of birds nutrition management the environments and of course infectious diseases we've heard a lot about the bacteria and in their contributions to disease and management we'll hear more about the contributions or a role of protozoa this afternoon but as a clinical avian biologist we'll talk this morning about viruses that play a role in some of these diseases as you'll see from this laundry list here of enteric viral diseases the identification is limited to clinical descriptives so we have a lot of syndromes listed here rather than specific etiology in Turkey's we have pol enteritis and mortality syndrome or PEM's we have turkey viral enteritis spiking mortality of turkeys and pulled enter itis complex all very complicated polymicrobial diseases in turkeys and chickens infectious stunning syndrome malabsorption syndrome pale bird syndrome and wrenching and stunting syndrome there are some common features that all of these syndromes share one of the primary features is that most of our enteric viral infections actually occurred during the first couple of weeks of age so there's definitely an age-related susceptibility the younger the bird the more susceptible they are to clinical disease many of these diseases share similar clinical signs and gross lesions so it's very hard to look at a clinical presentation in the field and know for certainty that a particular virus is the causative agent common signs or diarrhea and depression we see lack of uniformity and again very difficult to associate a specific virus to what we see in the field this is a very economically important group of viruses since the GI tract is responsible for uptake of nutrients and conversion to protein and meat any assault on the GI tract can result in permanent damage caused causing a delay in growth we might see some mortality or the need to call before birds go to the processing plants and certainly an increase susceptibility to secondary agents if we look at the pathogenesis of viral and iritis once a virus infects the host these viruses generally infect cells in the intestinal villi they cause damage to the intestinal mucosa and this leads to several events first we have a malabsorption mal digestion that's manifested as an osmotic diarrhea so we get an increase water and electrolytes in the GI tract we also have secondary bacterial and protozoa infections we have immune dysfunction because we do have quite a large component of the immune system in the GI tract of poultry and an inflammatory response that inflammatory response can result to damages to the intestinal mucosa so taken together this is a situation that can cause permanent damage to the GI tract we think about the viruses that are most commonly associated with enteric viral diseases in poultry we have members of the Rio Verde family real virus and rotaviruses adenovirus --is corona viruses and then we have a group of viruses that kind of get characterized together as small round viruses this is based on their appearance by electron microscopy this is just an image that doctors Peter Wilcock and Shiva Prasad put together showing many of these viruses and these small round viruses or the name is pretty descriptive they're small they're round they don't necessarily have any structural features that when you see them through an electron microscope you wouldn't know that it was a corner virus versus a khaleesi virus it's a very generic description of viruses here but again this is the list of usual suspects we have a lot of challenges in diagnosing and teurac viral diseases it's very difficult one historically electron microscopy has been used to identify these viruses from fecal preparations of affected Birds unfortunately you need a lot of virus to be able to visualize this by e/m somewhere on the order of 10 to the 5 to 10 to the 6 virus particles per ml is the minimum to be able to observe them by e/m we have difficulties propagating many of these viruses either they're very difficult to grow or we just don't have a system that we can grow them in so it's not possible to isolate some of these viruses and we don't have many tools for detecting the unknowns advances in our molecular techniques such as PCR and rt-pcr or we can amplify large quantities of nucleic acid has certainly helped us but those essays really enable us to detect things that we already know in order to develop those assays we have to have the knowledge of their sequence and so they're they're very useful but but they have limitations just because we detect a virus does not necessarily indicate that that virus is the causative agent many of the viruses on this list here we can detect and chickens that are healthy so mere detection does not implicate a virus and disease and lastly on the list of challenges is that it's very difficult to reproduce some of these enteric diseases experimentally so it's difficult to prove Koch's postulates which is what we generally expect to do when we're trying to implicate a particular pathogen as a causative agent but very difficult to do that with some of these syndromes I've already mentioned electron microscopy but we can also make attempts to isolate viruses if we're trying to isolate chicken viruses we can try to isolate those viruses and chicken embryos or chicken cells when it comes to the enteric viruses of turkeys those viruses really have a preference for growing in a turkey so so you need to try to find turkey embryos or prepare so cell cultures from turkeys it's very difficult to find specific pathogen free turkey embryos in the u.s. so that's not a readily available substrate for us to test in many times we find ourselves trying to isolate or culture these viruses in the host sometimes that's easier than working with embryos or cell culture in addition to PCR s we can do immunohistochemistry to try to detect antigen in tissues from these affected Birds we also have the tool of RNA or DNA probes and we can actually perform in situ hybridization again in the tissues from affected animals and we compare these results with the pathological changes that are observed in tissues biological characterization again this is an important component because as I mentioned many of these viruses are found in healthy animals are detected in healthy animals so there's variability and the viruses and they don't all cause or contribute to clinical disease so as an example of how we utilized laboratory systems for diagnosing and identifying the etiology of running and stunning syndrome I'd just like to share with you some of our approaches and results that that we generated so running extending syndrome is not a new disease this is an enteric disease of chickens primarily meat type birds are broilers causes a decrease in body weight an increase in our feed conversion ratio most of the time we see this in very young chickens but certainly by the first or second week of life it's visually apparent when you walk into an affected poultry house you'll see hatch mates that are drastically different in size the picture that I have here is from an experimental reproduction of RSS both of these chicks are hatched mates you can see the one on your left is far smaller than the one on the right and this chick will never catch up in body weights with its hatch mate another common feature are thin pale and test ins with watery contents that the intestines have lost their tone you can actually see gas formation and the liquid contents of these intestines and then finally we have a cystic Interop a--they this is probably the most consistent histopathologic finding in RSS affected Birds many of these affected birds have severe cystic enteritis and you can see these large cyst formations highlighted by these arrows we can see in many cases an abundance of necrotic cells and the lumen of these cysts the villi become atrophied and blunted there's also crypt hyperplasia and when we look at different sites of the gastrointestinal tract for the presence of these cystic lesions we find in most cases more lesions in the duodenum compared to other locations so certainly it these this virus has a predilection for that region of the gastrointestinal tract people have been trying to determine the etiology of RSS for decades this is as I mentioned not a new disease descriptions date back more than 30 years there have been multiple ideologies suggested all of the small round non-enveloped viruses have been implicated over the course of the past 30 years however when those viruses were put back into chickens as single virus inoculum we could not reproduce the disease this wasn't a situation where our laboratory wasn't able to do it but many investigators have tried reproducing the disease with these single virus and Occulus generally will see weight suppression because many of these viruses do cause body weight suppression but we don't see the body weight suppression paired with the Cystic Interop a--they in addition to the small round viruses certainly riyo viruses and rotaviruses have also been implicated so our question was what else is in the GI tract what else could be causing this disease just to give you an idea of the chronology of lesions in the field first it's important to state that in the US we raise our broilers on built up litter this is generally these are pine shavings that are put into a poultry house and over the course of the next year sometimes a year plus those shavings stay in the house and new placements of broilers are put in on top of the use shavings occasionally that litter will get top dress with clean pine shavings but we're putting day-old chicks on this used litter for our SS within four days after placement of day of hot chicks we can see the first intestinal legions forming the peak of these growths and microscopic lesions occur between seven and twelve days and then after twelve days of age we see this lymphocytic enteritis and then from nine to thirty five days we have the Cystic enter Interop a--they and enteritis so one of our initial studies was to confirm that what we were seeing in the field was in fact infectious so we harvested the gastrointestinal tract from affected boilers and we inoculated day of hatched chicks with a filtered homogenate and with an unfiltered homogeneous I what I was showing here we have negative controls represented by the orange bar and this is the average body weight at 12 days 12 days of age 12 days post placement average body weight of broilers that received the filtered intestinal homogenate and then the unfiltered homogeneous so you can see that we definitely had body weight suppression in in both groups filtered and unfiltered and then we also collected intestines and in both of those groups we saw the presence of these cystic lesions in the duodenum so from this study we were able to conclude that we were dealing with an infectious disease that viruses alone were capable of reproducing the disease but that the addition of bacteria and and possibly protozoa would certainly make this disease more severe from there we did pursue the usual virus isolation attempts where we took material from infected birds and put them into embryos and into cells we isolated many viruses we characterized these viruses using PCR and sequencing and aside from finding some variant reowww viruses nothing really stood out as potential etiologic agent so then we pursued some metagenomic studies we infected SPF meat type chicks with our filtered intestinal material and alongside of those chicks reared uninfected chicks and then harvested the GI tracts we isolated DNA and RNA from those intestines and performed metagenomic analysis basically we wanted to see if there were sequences and the RSS and oculi of chicks that were not present in the unenacted chicks in addition to finding many sequences that were identified as unknowns we did see a high degree of representation for astro verde in picornaviridae present in the RSS infected chickens and they were absent in our unchallenged controls so this kind of gave us a little bit more direction rather than trying to look at everything we focused our efforts on trying to determine the role of out Ribeira day and parvo Verde in RSS we also at around the same time there were some publications about a chicken parvovirus from michael day at the US department of agriculture and they identified that as a possible novel and teurac virus associated with RSS we were able to amplify sequences from our viruses from RSS affected chickens and one of our PhD students dr. King yo King was able to do full-length sequencing of this Oscar virus and based on its comparison with other avian astra viruses it was a variant it was novel and would be considered possibly a new member of the genus of the a vaster of iris genus and the family of astro vieira de at this time this was back in 2012 high throughput sequencing was still quite expensive and so he was able to achieve the or obtain the full length sequence through primer walking and amplification of the five Prime and three prime ends I think nowadays we could sequence this in easily but it took him a couple of months was to highlight some of the progress that we've made so our question was is novel chicken Astro virus the causative agent of RSS and what I'm going to share with you is how we developed the diagnostic reagents that we needed to perform these studies to answer this question what attempts or how we were able to isolate the chicken Astro virus and then utilize this virus and the diagnostic tools to examine the pathogenesis in an experimental model and then methods for control so first when you're dealing with pathogen that people haven't really dealt with very much you quickly realize that you don't have many diagnostic tools aside from the PCR that was really the only tool that we had to use to test tissues from the field so it felt like we really needed to generate some specific serum so that we could look at the tissues look for staining of antigen and tissues from affected Birds and and study the pathogenesis so we were able to amplify the capsid the gene that codes for the capsid protein for several chicken astra viruses we have the novel chicken astra virus and then two additional chicken astra virus is called a b and nephritis virus one and two and then we also amplified the chicken parvo virus capsid these were cloned into a baklava race expression system and we were able to purify the protein all four of these proteins and use those proteins to immunize rabbits so that we could get mono specific hyper immune serum to use in other studies these capsid proteins were also very useful because we could and we did develop Eliza's that we used to look for antibodies specific for the oscar viruses and the parvo virus and then we were also able to use the capsid protein as a recombinant vaccine other diagnostic tools that we developed were aimed at looking at sites of viral replication so we made Ryba probes targeting regions of these four viruses and these rival probes enabled us to perform in situ hybridization on our formalin-fixed paraffin-embedded tissues we had many of them from our clinical submissions so we were able to look for replicating Ostrow viruses and parvo virus in these tissues and one of the studies that we did we wanted to look at experimentally infected birds and look at the progression of or the appearance of these viruses in the GI tract so we inoculated our day of hot chicks with our RSS filtrate and then we started collecting tissues at 6 hour intervals and looked at those tissues with our Ryba probes what we saw was an immediate presence of Ostrow viruses these were chicken aster virus and the avian nephritis virus one and two in the duodenum in our RSS RSS exposed chickens we also saw that there were co-infections with multiple astra viruses so what we're showing here on the top panel this is a cross-section from our uninfected chickens and then on the bottom panel we have a cross these are the duodenal cross-sections from our RSS exposed chickens and we can see that the chicken astra virus pro is staining in the intestinal villi we have avian nephritis virus one also chicken astra virus staining and to a lesser degree may be nephritis virus - in our studies we didn't detect any chicken parvo virus at any of the time points we also use these tools to determine if any of these viruses were present in this lesion that's most commonly associated with RSS which is that cystic formation in the crypt of libra [ __ ] and using an oral challenge model with the RSS filtrate we did detect the chicken astra virus here and the Crypt and this is just a close-up of that and this was by five days post inoculation so very quickly after the chick was infected we saw this replication of the chicken astra virus in the site of this lesion and that was the first time that he had a direct association between the most common histologic finding and a particular virus that was at five days post infection we also wanted to look at other primary lymphoid organs as well as the pancreas to determine whether or not the virus was replicating in tissues outside of the intestinal tract as you can see here again we were looking at both uninfected and RSS exposed chickens we only saw staining for these chicken observe irises and the duodenum not in the pancreas the hard area gland thymus or Bursa so this was an implication that these Ostrow viruses do have a selective tropism for intestinal tissues which was originally what we thought but we needed to to confirm that while we were doing those studies we were also trying to isolate the chicken Astro viruses and as I mentioned earlier this can be a difficult process some of these services don't grow well or at all in our lab systems but nonetheless we wanted to do our best to try to isolate this virus so we homogenized the intestinal tract from RSS affected chickens that homogenate was filtered through a 0.2 micron filter we incubated that with REO virus and rotavirus antisera to try to reduce the possibility that we would also isolate those two viruses which at least REO viruses are commonly found in the GI tract of poultry and then we inoculated several cell lines we have the canine kidney cell line we have a quail fibroblast cell line a chicken fibroblast cell line bureaux cells our primate epithelial cell line and on an avian liver cell line and we looked at the cells daily for any kind of morphological changes or cytopathic effects and then when we weren't seeing cytopathic effect we decided that we needed to look at these cells with immunofluorescence an indirect immunofluorescence assay because it was possible that we had virus replicating in the cells that was not causing cytopathic effect and sure enough in the liver cell line LMH we did have the chicken astra virus replicating in these cells the bottom panel is infected self-control we did use a nuclear stain that's what these red stains are and then the cytoplasm of the LMH cells were staining positive for the astra virus these viruses do replicate in the cytoplasm so we were able to confirm that we had isolated an Astra buyers the last thing on our list was to try to reproduce the disease now that we had an Astra virus isolates we wanted to put this back into chickens and see if we could reproduce the disease we were pretty excited at this point in time it had taken us several years to get to this point and we're pretty certain that this was it we were going to inoculate these chicks they were going to show body weight suppression and they were going to have cystic a neuropathy and we did that and there was no cystic Interop a--they we had some body weight suppression but our hallmark lesion was not present in these infected chickens so our thought was maybe we needed to back passage this and see if by pathogen this isolated virus back through Birds maybe somehow we would restore the phenotype this was our thinking restore the phenotype of what we were seeing in the field so we took Dale boilers we inoculated them with the Ostrava ROS isolates seven days later we harvested the intestines at the same time we had negative or uninfected chickens and we harvested those intestines as well because we didn't want to we wanted to be able to see if there was if there were any other components and the GI tract of these boilers that might be contributing to body weights depression and those intestines were processed and put into a second group of day old boilers and we repeated this process for six passages what I'm showing here is the percent body weight suppression compared to the uninfected controls and you can see that starting really by passage for we started seeing significant body weight suppression by the fifth passage we had a 17% body weight suppression and then also we were looking at the duodenal loops from these bursts this is from an RSS positive control and then past one past three and past six from our astra virus passages and when we looked at the the crypts from these passages we saw an increase in the number of cystic crypts by passage as well as the size of these cystic crypts with each passage so this was really the first time that we or anyone was able to reproduce the clinical disease of renting and stenting syndrome the last part of this quest for our identifying the etiology of renting and stenting that I want to share with you is the development of a capsid protein vaccine because we had cloned the capsid protein we wanted to utilize that protein to vaccinate boiler' breeders and then take the progeny from those broiler breeders and put them through our RSS challenge model so we vaccinated reuler breeders three times in during the the course of the study we brought them into production and we collected the eggs we set the eggs hatch the eggs and put the progeny through our RSS challenge model the birds or the the progeny was not completely protected but the capsid protein vaccine did mitigate weight suppression and it did result in a reduction of cystic lesions in the progeny that were challenged so it's difficult to tell how the progeny would have fared out under field or commercial conditions because there are so many other contributing factors that the mitigation that we saw under experimental conditions may have been maybe lost under field conditions but this this was promising for us unfortunately we don't have any commercial vaccines for enteric viruses there are some companies that during this time elected to put a variety of viruses that were identified into inactivated oil adjuvanted vaccines and use those in their breeders when we tested progeny from those vaccinated breeders and this challenge model they weren't protected so other the wrong viruses were included in this vaccine or the wrong viruses were included in the vaccine we have we have several other experimental vaccines that have been reported but so far nothing that that's commercially available it just in wrapping up there's been a great deal of advancement in understanding the biome of diseased broilers over the course of the past couple of years as a biology community we aren't as advanced as what I've heard over the past day and as far as understanding the interactions of these viruses and the hosts guts but certainly we're making some strides NGS sequencing and metagenomic analysis has been performed and reported several times using different target populations of chickens and turkeys these studies have been used to identify very complex viral community and the gut novel viruses have been identified unknown viral sequences much like we found in our earlier study and then also bacteria phages have been identified in the gut contents from diseased Birds we don't really know what the role of some of these viruses are in in disease if any certainly we continue to see a high representation of our rio verde astro verde and pokorny verde what I'm showing here is just a metagenomic analysis of some commercial flocks that were having enteric issues this was done by Michael day at the US Department of Agriculture and he identified a astro Rio and pakora viruses new viruses such as pico burner virus the H E virus and turdy virus have also been identified in some of these metagenomic studies but we're still in the very early stages of understanding how these viruses might be playing a role in enteric disease another interesting finding from dr. days study was the association of some of these enteric viruses with gut bacteria so in this chart the larger the circle the more positively or negatively associated the virus is with the bacteria the blue circles and and the intensity of the blue circles indicate a very strong association between the viruses with the gut bacteria and the red circles indicate a low degree of association so just as an example we have here that chicken Astro virus had a fairly high association with Clostridium and rumen okok AC in this study and so this was a really interesting outcome and I think this is more of the type of work that we'll be seeing in the very near future as we try to get a better understanding of these viruses we need to study more the mucosal immunity and the gut associated lymphoid tissue Galt is the key amia logic tissue and the mucosal immune system and recent a study that was published in the frontiers of immunology in May actually identified it as a command center for the humoral immune response of course this work was that they reported on was actual vaccination of hens with an e coli bactrim so it wasn't associated with viruses but the methods that they used to evaluate the immune response to that doctrine were perfect for what we need to do for viruses so challenges that we have are looking at the the changes in inter peril disease especially in the no antibiotics ever or antibody antibiotic free production systems likely these changes will also change the landscape for the viral pathogens and certainly time of discovery seems like we're able to get so much more done now with all the high-throughput sequencing this will will certainly generate data more rapidly than we've been able to generate in the past so the team at Georgia that helped with the renting and stunting dr. Egbert Muntz he's now with Bohringer cameo King BJ dua Raj airflow diamond Vanessa and then also a great things to our funding agencies [Music] you