Progress 08/15/12 to 08/14/17

Outputs
Target Audience: Target Audience: State, federal, tribal, and private fish health professionals. Effort: Rachel Breyta presented a talk entitled IHNV detection and typing in the Northwest, at the 67th meeting of Pacific Northwest Fish Health Protection Committee, Manchester, Washington. Date: September 13, 2016 Target Audience: USDA Animal Health and Well-being researchers, program staff, and policy makers Effort: Andrew Wargo presented a poster entitled Drivers of transmission, emergence, and displacement of an aquatic virus in fish hosts, with all project personnel as co-authors. Date: December 2, 2016 Target Audience: State, federal, tribal, and private fish health professions. Effort: Gael Kurath presented a talk entitled Update on IHN virus typing and research, at the 68th meeting of Pacific Northwest Fish Health Protection Committee, semi-annual meeting, Sacramento, California. Date: February 14, 2017 Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training activities Kurath and McKenney mentored a first year University of Washington Pathobiology graduate student, Andrew Gustin, in a 3 month rotation research project that investigated persistence of virus isolates in the final virulence experiment. Mentoring included training in RNA extraction and qPCR methods, data analysis, and synthesis of results. Breyta hosted fish health colleagues Sharon Clouthier and Eric Anderson from Canada for a week of training in coalescent phylogenetic analyses of large DNA viruses in sturgeon. Breyta worked with Kurath to update her capabilities in coalescent phylogenetic analyses of RNA viral sequence data. Breyta trained McKenney in sequencing for genotyping of virus field isolates and data analysis. Kurath mentored Wargo during a one month summer visit to the Western Fisheries Research Center for writing of papers, and data and concepts from the project were discussed regularly. Naish and Purcell mentored Brieuc in the development of manuscripts for publication. Professional Development Kurath mentored Fisheries graduate student Daniel Hernandez as supervisor for his PhD research on Ecology of IHNV in Chinook salmon, and assisted with preparation for written and oral exams. These were successfully completed and he is now a PhD candidate. How have the results been disseminated to communities of interest? Aim 1. Virus genotyping has continued and is communicated with fish health partners directly by email, and in regional fish health forums such as the semi-annual Pacific Northwest Fish Health Protection meetings. Virus genotyping data and associated metadata is publically available in the MEAP-IHNV database at <gis.nacse.org/ihnv>, and the new IHNV-VGS surveillance database is available as supporting information in the Data Papers section of the Ecological Society of America journal Ecology (Breyta 2017). Aim 2. Results of virus characterization studies have been presented at several stakeho;lder meetings and scientific conferences. IHNV shedding work have been published as Wargo et al., 2017, in the journal VIrus Research, reaching a general virology audience beyond fish health. Aim 3. Naish and Brieuc have assisted broader community efforts in the development of a reference genome for steelhead / rainbow trout (Oncorhyncus mykiss). Whole genome sequencing data from this project has been made avaiable publically, and an advanced genome assembly has been released on Genbank by our collaborators. Results of the genome wide association analysis have been developed into two manuscripts, which will be submitted for review shorlt. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1) Major activities completed / experiments conducted; Aim 1:Completed analyses and developed manuscripts Aim 2. Surviving fish from the final virulence experiment were processed to investigate persistence of 12 virus isolates (3 in each of 4 displacement genotypes) at 28 days after infection. Aim 3: Final anayses on the evoluton of disease resistance in five salmon populations was completed. Manuscripts were developed. Aim 4:Completed analyses and developed manuscripts 2) Data collected; Aim 2. Numbers of fish from with detectable viral RNA persisting at 28 days after infection, and viral RNA copy number in individual fish 3) Summary statistics and discussion of results Aim 2. For each of 12 virus isolates virus persisted in 70-90% of the fish at 28 days after infection, confirming the longer persistence observed in a 9 month study with one isolate. There were no significant differences between genotypes. Aim 3: A large number of markers associated with disease resistance were detected in five steelhed populations.Three chromosomes in steelhead comprised regions that had large effect sizes, and one of these regions displayed evidence for divergence between a resistant and non-resistant population. 4) Key outcomes or other accomplishments realized. Aim 1. A key outcome from the new IHNV-VGS database is recognition of both specialist and generalist IHNV lineages within the study region, which comprises a major change in knowledge regarding our understanding of IHNV ecology in multiple sympatric hosts. Aim 2. Results of final virulence experiment confirmed evolution of increasing virulence as a driver of viral displacement, and also confirmed that virus persists in steelhead trout much longer than previously recognized. Both of these factors are fundamental new knowledge relevant to virus transmission dynamics. Aim 3. There is strong evidence that steelhead populatons recently derived from a common ancestor evolve resistance rapidly, and this resistance can influence the host-pathogen dynamics and spread of IHNV in natural and hatchery systems. Aim 4: Two papers were completed and published

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Breyta R, McKenney D, Tesfaye T, Ono K, Kurath G. Increasing virulence associated with serially emergent genotypes of a fish rhabdovirus. Oral presentation by R. Breyta. American Fisheries Society National Fish Health Section Meeting, Lansing Michigan, March 1, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Kurath G, Breyta R, Brieuc M, Batts B, Naish K, La Deau S, Purcell M, Winton J. Genomic analysis of a global reference set of 24 IHN virus isolates. Poster presentation by G Kurath. American Fisheries Society National Fish Health Section Meeting, Lansing Michigan, March 2, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Citation: Kurath G, Breyta R, Black A, Hernandez D. Evolution of specialist and generalist lineages of a salmon virus in a complex multi-host ecosystem. Oral presentation by G Kurath, 9th Virus Evolution Workshop, State College PA, March 10, 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Citation: Kurath G, Breyta R, Black A, Hernandez D. Evolution of a generalist lineage of infectious hematopoietic necrosis virus in Pacific salmon. 10th International Symposium on Viruses of Lower Vertebrates, Budapest, Hungary, June 6, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Kurath G, Breyta R, Black A, Hernandez D. Generalist and specialist IHN virus in Pacific Northwest salmonids. Western Fish Disease Workshop, Suquamish, WA. June 21, 2017.
• Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Ferguson, PFB, Breyta, R, Brito, I, Kurath, G, and S.L. LaDeau. An epidemiological model of virus transmission in salmonid fish of the Columbia River Basin. Ecological Modelling. In Review
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Breyta R. Insights and mysteries: how infectious diseases impact their host population genetics. Oral presentation by R. Breyta, invited keynote speaker, Annual meeting of Southeastern Society of Parasitologists.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Black A, Breyta RB, Bedford T, Kurath G. Geography and host species shape the evolutionary dynamics of U genogroup infectious hematopoietic necrosis virus. Virus Evolution 2(2):vew034.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Wargo A, Scott RJ, Kerr B, Kurath G. Replication and shedding kinetics of infectious hematopoietic necrosis virus in rainbow trout. Virus Research 227:200-211.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Breyta, R., Brito, I., Ferguson, P., Kurath, G., Naish, K., Purcell, M.K., Wargo, A. and S.L. LaDeau*. 2017. Transmission routes maintaining a viral pathogen of steelhead trout within a complex multi-host assemblage. Ecology and Evolution. DOI: 10.1002/ece3.3276
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Breyta, R., Brito, I., Kurath, G. and S.L. LaDeau*. 2017. Infectious Hematopoietic necrosis virus virological and genetic surveillance 2000-2012. Data Papers Ecology doi:10.1002/ecy.1634

Progress 08/15/15 to 08/14/16

Outputs
Target Audience:Target Audience: Scientists and policy makers working on the science and implantation of hatchery reform Effort: Kerry Naish co-organized a special session at the American Fisheries Society "Interactions between Hatchery and Wild Salmonids: International Understanding of the Risks, Benefits, and Options for Management" Date: August 16-20, 2015 Target Audience: Pacific Northwest Fish Health Protection Committee, representing state, federal, tribal, and private fish health professionals. Semi-annual meeting. Effort: Gael Kurath presented a talk entitled Genetics and landscape analysis of IHN virus, Gleneden Beach, Oregon Date: September 15, 2015 Target Audience: Federal scientists at the Western Fisheries Research Center Principle Investigator meeting. Effort: Gael Kurath presented a talk entitled Scientific data communication and sharing: IHNV Molecular Epidemiology Database, Union, Washington Date: October 27, 2015 Target Audience: General Public Effort: Kerry Naish participated at the Discover Weekend at the Seattle Aquarium. This event was aimed at introducing ocean and marine research through interactive displays and lab-based activities. Date: November 15, 2015 Target Audience: Chinese research scientists at the Heilongjiang River Fishery Institute. Effort: Gael Kurath presented a talk entitled IHN virus: North American and Global. Chinese Academy of Fishery Science, Harbin, China Date: November 17, 2015 Target audience: SUNY Albany faculty, staff, and students. Effort: Andrew Wargo presented a seminar entitled "Virulence Epidemiology: Harnessing Evolution to Manage Disease". Date: January 1, 2016 Target Audience: Pacific Northwest Fish Health Protection Committee, representing state, federal, tribal, and private fish health professionals. Semi-annual meeting. Effort: Rachel Breyta presented a talk entitled What exactly is IHNV doing in chinook salmon?, Santa Rosa, California. Date: January 27, 2016 Target audience: Virginia Institute of Marine Science faculty, staff, and students. Effort: Andrew Wargo presented an Aquatic Health Sciences departmental seminar entitled Ecological Drivers of Transmission, Emergence, and Displacement of an Aquatic Virus in Fish Hosts. Date: March 1, 2016. Target Audience: USGS infectious disease research scientists and policy makers. Effort: Gael Kurath presented a talk in the USGS monthly wildlife disease series entitled Infectious dose and lethal dose of a fish virus in rainbow trout. By webex. Date: March 9, 2016 Target Audience: NOAA physiology, conservation genetics, and hatchery science research scientists Effort: Kerry Naish presented a talk in the NOAA monthly seminar series on the implementation of genomic tools in disease management and hatchery reform Date: March 17, 2016 Target Audience: High School Girls Effort: Maureen Purcell co-taught a workshop called "Fish Get Sick Too!" for the Expand Your Horizons (EYH) network. Three workshop sessions presented with hands-on activities. Bellevue, WA. Date: March 25, 2016 Target Audience: US Fish and Wildlife Service Fish Health Biologists meeting. Effort: Maureen Purcell presented a talk on best practices for detecting fish pathogens including IHNV, Seattle, Washington. Date: March 30, 2016 Target Audience: US Fish and Wildlife Service Fish Health Biologists meeting. Effort: Gael Kurath presented a talk entitled Current research directions for IHNV in Pacific salmon, Seattle, Washington. Date: March 31, 2016 Target Audience: Oregon State University, undergraduate and graduate students in Fish Disease class. Effort: Gael Kurath presented a classroom lecture entitled Epidemiology and evolution of IHN virus in the Columbia River Basin, Corvallis, Oregon. Date: April 7, 2016 Target Audience: Oregon State University Microbiology graduate students and faculty at the OSU Microbiology Student Association Research Symposium. Effort: Gael Kurath presented a talk entitled Regional and global tracking of a virus in salmonid fish, Corvallis, Oregon. Date: April 8, 2016 Target Audience: International scientists, Aquaculture representatives and policy makers Effort: Kerry Naish gave an invited talk on the genetic basis of IHNV resistance at the International conference of Salmon Biology in Puerto Varas, Chile. Date: April 24 - 28, 2016 Target Audience: Fish and wildlife research scientists and policy makers. Effort: Gael Kurath presented a talk in the USGS American Fish and Wildlife Association Webinar series entitled Genetic tracking of fish viruses to control disease: examples in steelhead trout. Webinar. Date: April 28, 2016 Target Audience: University of Washington, undergraduate and graduate students in Fish Disease class. Effort: Gael Kurath presented a classroom lecture and lab entitled IHN virus in the Pacific Northwest, Seattle, Washington. Date: May 5, 2016 Target Audience: International Scientists and policy specialists in conservation Effort: Kerry Naish gave an invited talk on the application of genomic approaches to conservation and management of Pacific salmon, Congenomics conference, Porto, Portugal. Date: May 3-6, 2016 Target audience: Cornell University, School of Veterinary Medicine, faculty, staff, and students. Effort: Andrew Wargo presented a seminar entitled Enhancing Epidemiological Inference Through an Integration of Ecology and Evolution. Date: May 18, 2016 Target Audience: Fish Health community Effort: Rachel Breyta, Marine Brieuc, Paige Ferguson, Gael Kurath and Maureen Purcell co-taught a continuing education course entitled "Landscape ecology and modeling in fish health" at the 57th Western Fish Disease Workshop. Date: June 7, 2016 Target Audience: Middle school and high school students Effort: Marine Brieuc was a volunteer during the Northwest Earth and Space Sciences Pipeline Summer camp organized by WA NASA Space Grant Consortium. The aim of the camp was to teach the students about climate change through hands-on activities and games. Date: August 12, 2016 Changes/Problems:As part of the original Aim 1 concept one aspect of the database development was to incorporate environmental data such as water temperatures and water flow rates, in order to test whether environmental conditions were important covariates in the incidence of infection. In the process of collecting environmental data, it became clear that this was a more difficult process than had been anticipated. Water temperature and flow monitors are operated by numerous agencies (State Ecology Departments, USGS, hatchery programs, and others), and the location and duration of the data were incongruent with the virus diagnostic data. Significant effort was expended in 2016 to determine if thermal modeling was advanced enough to extrapolate from water temperature monitoring stations to relatively distal fish locations. Although this is an active research field for the Columbia River basin, the current capabilities were found to be insufficient. Therefore project team focused on developing collaborations in the field of freshwater ecology and thermal biology to identify ways to move forward on how water temperature impacts virus transmission and fish health (see Breyta symposium on "Fish Health in the Context of Water Temperature" in events above). This stimulated ongoing communication that will facilitate incorporation of environmental data as a future direction as we continue to develop the transmission model. What opportunities for training and professional development has the project provided? Response: Brieuc worked with Naish to deepen their knowledge of the random forest algorithm and other genome wide association studies. They co-wrote a paper, along with two other co-authors, on the best practices for association studies in fishes with random forest. Brieuc and Naish worked with Yniv Palti and Sibjorn Lien to learn about full genome sequencing. Brieuc and Naish supervised an undergraduate student, Trevor Eakes, for the development and completion of his senior thesis entitled "Influence of host sex on disease resistance to the infectious hematopoietic necrosis virus in Oncorhynchus mykiss". Brieuc and Naish participated at the 2016 Annual Symposium of the Fisheries Society of the British Isles, Bangor, Wales. The theme of the international conference was "Fish, genes & genomes: contributions to ecology, evolution & management". Naish was an invited speaker at the 3rd International Conference on Integrative Salmonid Biology in Puerto Varas, Chile. LaDeau worked with Ferguson to develop model goals, evaluate simulation code and outcomes LaDeau mentored Breyta in development of knowledge regarding infectious disease modeling and applications of genotype data during years 1-3 of the project. LaDeau worked with Brito and Breyta in development and quality checking of the IHNV diagnostics database in Aim 1, and appropriate univariate analysis of the resulting final database. This activity involved regular webex communications and has now moved forward to manuscript revision. LaDeau became primary mentor for Breyta in 2016 to provide training in large database analysis, addition of environmental variables to landscape modeling, and infectious disease modeling (Breyta transferred to Cary Institute sub-budget Dec. 1, 2015). This resulted in a career enhancement for Breyta by expanding into new research fields. Kurath mentored Wargo during three one-month summer visits to the Western Fisheries Research Center, during which analysis and interpretation of novel long-term virus shedding data and broader interpretations of all project data were regularly discussed. Wargo and Kurath worked with Breyta to enhance her fluency in virulence evolution theory, with particular focus on the project virulence data now published in Breyta et al. 2016. Kurath mentored Fisheries graduate student Daniel Hernandez as supervisor for his PhD research on Ecology of IHNV in Chinook salmon, using methods and principles developed in this project. Purcell mentored Kurath, Breyta, Thompson, McKenney, and Hernandez in biostatistical analysis of experimental data from laboratory infections studies. Breyta and Wargo mentored technician McKenney in proper estimation and statistical analyses of ID50 values from infectivity data in sub-aim 2b. Wargo and Kurath mentored technician McKenney in writing of his first manuscript for publication as first author (McKenney et al., 2016). McKenney was supported to attend a national conference and present a talk on his work at the American Fisheries Society meeting in Portland, OR in 2015. Wargo mentored technician McKenney in robotic RNA extraction techniques during two 2-week visits to the Virignia Institute of Marine Science in June and October of 2016. Breyta and Kurath mentored recent University of Washington graduate Matthew Seymour, who volunteered for 9 months and participated in virus genotype data assembly and analysis. How have the results been disseminated to communities of interest? Aim 1. Database characteristics and summary statistics were presented to agency and hatchery stakeholders at the Continuing Education course in June 2016. Updates are presented regularly at regional fish health agency meetings, including the Pacific Northwest Fish Health Protection Committee. Virus genetic typing data is available to all interested parties online in the searchable and georeferenced MEAP-IHNV database at <gis.nacse.org/ihnv. The new IHNV-VGS database and accompanying metadata will be available publically as links to the data publication abstract in Ecological Archives (Breyta et al, in press). Aim 2. Viral virulence, infectivity, shedding, fitness, and persistence of IHNV field isolates associated with major displacement events in the field were presented the Continuing Education course in June 2016. It has also been presented to the virus research community as several conferences noted earlier. Aim 3: We have presented our results at several international conferences: the American Fisheries Society conference in Portland (Aug 2015), the 3rd International Conference on Integrative Salmonid Biology in Chile (Apr 2016), the 2016 Annual Symposium of the Fisheries Society of the British Isles in Wales (July 2016). We have participated in the Continuing Education course in June 2016. We have participated in public outreach events at the Seattle Aquarium and during the NASA Summer camp. Aim 4: Model development and performance results were presented to Agency stakeholders at the Continuing Education course in June 2016. These events provided exchange of knowledge with field scientists that was essential for relevant model parameterization. A powerpoint file titled "Ecological drivers of transmission of IHNV in steelhead trout of the Columbia River basin and coastal Washington" was prepared from the talks at the stakeholders meeting in July 2015 and distributed to all attendees for sharing with their lab staffs and other colleagues. As part of the Continuing Education course in June 2016 participants each received notebooks with print-outs for each oral presentation and learning exercise. Copies of this material have been provided by course organizers to interested parties who were not able to attend the course. Project data has been communicated in numerous presentations at various venues as listed earlier. What do you plan to do during the next reporting period to accomplish the goals? Aim 1: We will publish a manuscript that is currently in revisions that details the IHNV-VGS database scope and presents summary analyses and evidence for relative roles of spawning adults, out-migrating juveniles and hatchery location 'hot spots' for facilitating IHNV transmission. A review manuscript summarizing univariate analysis of database is also in preparation targeting fish health managers and researchers (journal "Fisheries"). Genetic typing will be continued as virus isolates are received. The resulting genotypes will be analyzed for phylogenetic and landscape pattern inferences, and reported to fish health agencies for use in disease management. Aim 2. Data analysis for virus shedding studies will be completed and prepared for publication. Results of the final virulence experiment confirming increased virulence as a phenotype associated with the displacement genotypes will be presented in a synthesis review article detailed below. Analysis of data on long-term persistence of IHNV will be completed and prepared for publication. Aim 3: We will publish the results of the family-based study and the population-based study that aim at identifying regions of the genome linked to IHNV resistance. We will analyze the whole-genome sequencing data to further characterize the candidate regions of interest. Complete analysis of neutralizing antibody response in steelhead trout exposed to IHNV at three different temperatures and prepare a manuscript reporting the study. Aim 4: The validated model will be fit to the full regional and temporal scope of our project. We further intend to assess more detailed distance metrics for incorporating viral genotyping data in order to provide more precise support for specific transmission pathways. A review paper synthesizing results from the four aims of this project has been outlined and will be prepared for publication.

Impacts
What was accomplished under these goals? Management of diseases in fishes is challenging when cultured and wild fish interact. Both hatchery and wild anadromous steelhead salmon return together to reproduce in the Columbia River, and all are susceptible to a viral pathogen, IHNV, which emerges episodically, but the routes of transmission are not understood. Here, we provide the science relevant to supporting fish health management. Our database that revealed the prevalence of IHNV as 8-30% in different fish populations, similar to human immunodeficiency virus levels in the southern African countries, and confirms IHNV as a major pathogen impacting conservation of Pacific salmon and trout. We found that IHNV is evolving toward higher virulence in the field, and host susceptibility varies among different fish populations. These data have been integrated into a landscape model of viral transmission that determines how virus infection spreads, aimed at predicting disease emergence and improving virus control strategies. These results have been disseminated to federal, state, and tribal fish health management agencies, and the public. 1) Major activities completed Aim 1: The completed IHNV-VGS database contains virological and genetic surveillance data for 2000-2012 in the Columbia River Basin and Coastal Washington State. Univariate analysis is complete and support for three possible routes of transmission has been evaluated. Additional IHNV genetic typing has been conducted. Aim 2: A final virulence experiment was conducted using three IHNV isolates from each of the four genotypes rather than single isolates. Results for sub-aims 2a, 2b, and 2d have been published. Aim 3: Genome-wide association studies with fish of known phenotypes identified markers linked to IHNV resistance in a family-based study. Increased sample sizes with 5 additional populations were examined to identify conserved pathways to resistance across populations. Aim 4: A landscape IHNV transmission model has been developed and used to evaluate the relative importance of three possible transmission sources: within hatchery transmission; juvenile out-migration; and adult fish return migration. Simulations were conducted and compared with actual field data to assess model performance and impact of missing data. 2) Data collected; Aim 1: The normalized IHNV-VGS database contains 6766 records with epidemiological information for surveillance of wild and cultured fish. Genotype data was available for 66% of virus positive records. Genetic typing was conducted for 60 new virus isolates from the study region. Aim 2. Virulence of three independent virus isolates from each of the 4 viral displacement genotypes was quantified using expanded virulence measures including lethal dose estimates, survival kinetics, and proportional hazards analysis. Virus shedding samples have now all been processed. Aim 3: Over 27,000 and 38,000 polymorphic loci were identified in the family-based study and the population-based study respectively. Random forest was conducted on 350 fish from the family based study and 379 fish from the population study. Genome-wide divergence between trait-linked markers was examined between two related hatchery populations. Aim 4. A test of the IHNV Bayesian transmission model was conducted using a spatial subset of the database. The model outputs the most likely transmission route(s) and probabilities of infection for each cohort-site. Genotype data informed the relative likelihood of possible transmission. Simulations were conducted testing various levels of data completeness. 3) Summary statistics and discussion of results Aim 1: The prevalence of IHNV was 12.5% among all fish cohorts, and ranged from 8-32% in different species or age-classes. Steelhead trout had the highest prevalence, with 25-31% in juvenile or adult fish. There was spatial and temporal heterogeneity of virus, with specific focus sites of high frequency infection. Genetic data revealed 90 virus genotypes, with M types 2-fold more common than U in steelhead, and U types 4-fold more common than M in Chinook. Seven dominant genotypes collectively accounted for 77% of all records. Aim 2: The previously identified pattern of significantly increasing virulence for genotypes 007, 111, and 110 was evident in the new experiment using three virus isolates of each genotype. This confirms evolution of increasing virulence as a phenotype associated with displacement genotypes. Analysis of viral shedding studies found consistent kinetics between virus genotypes and no correlation with displacement. Aim 3: 126 loci and 227 loci associated with resistance to IHNV were identified in the family-based and population-based studies respectively. Many of the markers co-localized, highlighting genomic regions likely containing one or more genes linked to resistance to IHNV. There was significant overlap of regions linked to resistance to IHNV, as well as regions unique to each study, suggesting that some pathways to resistance might be shared whereas some might be population-specific. Some loci linked to IHNV were located in regions potentially under directional selection between QNFH and LQ. Our results support previous hypothesis that resistance to IHNV is highly polygenic. Aim 4: Model simulations of complete testing accurately estimated all parameters, and simulations with incomplete testing were largely unbiased. The model was successful at identifying the transmission route that exposed cohort-sites, with low frequency of misidentifying transmission route. The model fit to real data from 336 cohort-sites in the lower Columbia River inferred that exposure by infectious adult fish occurred frequently (31% of cohort-sites) and that the probability of infection following exposure was greatest when exposure occurred between juvenile cohorts at a site (0.22, 95% BCI = 0.14-0.31) or by infectious adult fish (0.18, 95% BCI = 0.12-0.25). 4) Key outcomes or other accomplishments realized. Aim 1. Univariate statistical analysis of the IHNV diagnostics database provided the first rigorous estimates of overall IHNV prevalence in Pacific salmonids, analysis of spatiotemporal distributions, patterns of viral occurrence in different host species and ages. The database revealed emergence and submergence of dominant M and U genotypes over time, cross-species transmission, and evolution of the UC subgroup. General host specificity patterns were confirmed, with M virus predominant in steelhead and U virus predominant in Chinook and sockeye. Aim 2. Evolution of increasing viral virulence was identified as a potential driver of emergence and displacement in the Lower Columbia River source region where these IHNV genotypes originated. A virus source and sink perspective has potential for explaining some of the complexity observed in field events. This is a substantial advance for the field of virulence evolution which to date has been informed almost exclusively by the evolution of myxomavirus virulence and host resistance in Australian rabbits. Aim 3: Various lines of evidence have identified several genomic regions linked to IHNV resistance in 5 populations of steelhead trout. 20 individuals were fully sequenced and will be analyzed in an attempt to identify the exact genes associated with IHNV resistance in the regions of interest identified. Aim 4: The positive performance of the new Bayesian IHNV transmission model is a major advance for modeling of infectious disease in aquatic environments. Novel aspects include the spatially explicit output and use of virus genotype data. The finding that virus transmission from returning adult fish is the most common source of infection for juveniles provides scientifically sound data for fish health management decisions like efforts to enhance security of hatchery water supplies as a way to interrupt virus transmission from adult fish to hatchery juveniles.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Brieuc MSO, Purcell MK, Palmer A, Naish KA (2015) Genetic variation underlying resistance to infectious hematopoietic necrosis virus in a steelhead trout (Oncorhynchus mykiss) population. Diseases of Aquatic Organisms 117:77-83.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Breyta RB, McKenney DG, Thompson TM, Kurath G. Increasing virulence, but not infectivity, associated with serially emergent virus strains of a fish rhabdovirus. Virus Evolution, 2(1):vev018. doi:10.1093/ve/vev018.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: McKenney DG, Kurath G, Wargo AR. Characterization of infectious dose and lethal dose of two strains of infectious hematopoietic necrosis virus (IHNV). Virus Research, 214:80-89. doi: 10.1016/j.virusres.2015.12.020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Brieuc MSO, Purcell MK, Kurath G, Thompson R, Naish KA: Using Random Forest to Identify Loci Linked to Resistance to Infectious Hematopoietic Necrosis Virus in Two Populations of Steelhead. Oral presentation by Naish. American Fisheries Society 2015 Annual meeting, Portland, OR, Aug 16-20, 2015
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Brieuc MSO, Purcell MK, Kurath G, Thompson R, Naish KA: The genetic basis of pathogen resistance across outbred salmon populations: A comparative analysis using Random Forest. Oral presentation by KA Naish  Invited speaker. 3rd International Conference on Integrative Salmonid Biology, Puerto Varas, Chile, April 24  27, 2016
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Naish KA, Waters CD, Brieuc MSO: Conservation genomics in Pacific salmon: from Patterns to Solutions. Oral presentation by KA Naish  Invited speaker. Congenomics 2016, Campus de Vairao, Portugal
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: McKenney DG, Kurath G, Wargo AR (2015) The Effects of lethality and infectivity on emergence and displacement events of a salmonid virus in the Pacific Northwest. American Fisheries Society meeting, Portland, OR, August 19, 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kurath G (2015) Viruses in fish: epidemiology, evolution, and the impact of disease in aquaculture. Invited Keynote talk, Virology Africa 2015 Conference, Cape Town, South Africa, December 2, 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Breyta R, Ferguson P, LaDeau S, Brito I, Kurath G, McKenney DG, Wargo A, Tesfaye T, Purcell M, Brieuc M, Naish K (2016) Modeling transmission dynamics of an acute viral pathogen of Pacific salmon in a complex aquatic ecosystem, Ecology and Evolution of Infectious Disease meeting, Ithaca, NY, June 2-4, 2016. Poster presentation
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Kurath G, Breyta R, McKenney DG, Kell A, Wargo A, Tesfaye T, Purcell M, Brieuc M, Ferguson P, LaDeau S, Naish K (2016) Virulence evolution as a driver of viral emergence and displacement in salmonid fish, Ecology and Evolution of Infectious Disease meeting, Ithaca NY, June 2-4, 2016. Poster presentation
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Kurath G, Black A, Breyta R, Bedford T (2016) Evolution of a newly recognized IHN virus subgroup, UC, in Columbia River Basin Chinook salmon and steelhead trout. 57th Western Fish Disease Workshop, Jackson Hole, Wyoming, June 9, 2016.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Breyta R, Black A, Kaufman J, Kurath G. 2016. Spatial and temporal heterogeneity of infectious hematopoietic necrosis virus in Pacific Northwest salmonids. Infection, Genetics, and Evolution 45:347-358.
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Black A, Breyta R, Bedford T, Kurath G. Geography and host species shape the evolutionary dynamics of U genogroup infectious hematopoietic necrosis virus. Virus Evolution, in press.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Breyta R, Kurath G, McKenney DG, Black A, Ferguson PFB, LaDeau S, Brito I, Wargo A, Tesfaye T, Purcell MK, Brieuc M, Naish K (2016) Landscape heterogeneity and transmission patterns of IHNV in salmon and trout of the Pacific Northwest, 57th Western Fish Disease Workshop, Jackson Hole, Wyoming, June 9, 2016.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Purcell MK, Powers, R.L., and Ballesteros, N. (2016) Persistence of infectious hematopoietic necrosis virus (IHNV) in steelhead trout. International Symposium for Fish and Shellfish Immunology. Portland ME, June 26 2016. Conference poster
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Brieuc MSO, Purcell MK, Kurath G, Thompson R, Naish KA : Evolution of resistance to infectious hematopoietic necrosis virus across populations of steelhead trout: Evidence for shared pathways? Oral presentation. 2016 Annual Symposium of the Fisheries Society of the British Isles, Bangor, Wales, July 18-22 2016
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Purcell MK, Kurath G, Winton JR. Managing infectious disease risks in salmon and steelhead hatcheries and natural population. Invited presented at symposium entitled Interactions between Hatchery and Wild Salmonids  International Understanding of the Risks, Benefits, and Options for Management. American Fisheries Society 2015 Annual meeting, Portland, OR, Aug 16-20, 2015

Progress 08/15/14 to 08/14/15

Outputs
Target Audience: Target Audience: Academic students and faculty at Cornell University Effort: Andrew Wargo presented a seminar entitled Harnessing evolution to manage disease, Ithaca, New York Date: September 15, 2014 Target Audience: General Public Effort: Marine Brieuc and Kerry Naish participated at the Discover Weekend at the Seattle Aquarium. This event was aimed at introducing ocean and marine research through interactive displays and lab-based activities. Date: November 8, 2014 Target Audience: University of Washington students and faculty in School of Aquatic and Fishery Sciences Effort: Gael Kurath presented a lunchtime talk in the "EcoLunch Seminar Series", entitled Landscape ecology of IHN virus in steelhead trout of the Columbia River Basin and coastal Washington, Seattle, WA Date: November 10, 2014 Target Audience: NIFA and fellow awardees Effort: All project scientists collaborated to present a talk to program managers and fellow awardees at the annual PI meeting held virtually. Date: November 19-21, 2014 Target Audience: Academic students and faculty at Washington State University Effort: Andrew Wargo presented a seminar entitled Virulence epidemiology, Pullman, Washington Date: February 9, 2015 Target Audience: Project collaborators Effort: All project scientists participated and presented talks at an annual project meeting in Seattle, WA Date: July 20-21 2015 Target Audience: State, Federal and tribal fish health managers from natural resource agency fish health laboratories in Washington, Oregon, and Idaho. Effort: All project scientists presented project research findings and discussed pathogen control strategies at a workshop organized in Seattle, WA Date: July 22 2015 Changes/Problems:One goal for this reporting period (2014 - 2015) was to expand our original findings on viral persistence from Aims 2e / 3b and compare persistence in both the resistant (Quinault National) and susceptible (Lake Quinault) steelhead stocks. Additionally, we sought to further extend the results of Aim 3a and evaluate the stability of IHNV-linked loci at two different temperatures (e.g. genotype by environment interaction). Neither of these studies were described in the original proposal but they were logical extensions from those studies. We obtained brood year 2015 steelhead trout from both populations to conduct these experiments, but after hatch both populations experienced unusually high, unexplained levels of mortality, despite good health of other salmonid fish in the wetlab at the time. Subsequentlypilot challenge dose studies indicated that both steelhead populations of this brood year had an unusually high IHNV resistance phenotype that differed from the previously consistent results of our studies conducted over the past six years. We suspect this variation in phenotype might be linked to the unusually high mortality that occurred at the early life stage. Due to the unusual performance of these fish the additional studies were not conducted, and as they were expansions beyond the original proposal we do not propose to conduct them in the coming reporting period. All of the animal experiments that were originally proposed in the grant have been accomplished. An additional change was that the application of expanded statistical analyses to the virulence data from sub-aim 2a was successful demonstrating statistical differences between groups, so it was not necessary to test the alternative in vivo design for virulence assays. This avoided the use of additional animals for experiments that were not planned in the original proposal. What opportunities for training and professional development has the project provided? Naish and Purcell worked with postdoctoral associate, Brieuc, on host response to viral challenges, host genome analysis and databases of host genotypes and phenotypes Naish, Purcell and Brieuc mentored an undergraduate student, Trevor Eakes, on the development on an independent research project on host genomics that will be used as a senior thesis Naish, Purcell, and Brieuc worked with post-doctoral associate Breyta to define and optimize expanded statistical analyses of virulence data, resulting in greater ability to discern statistical significance between groups. Purcell and Brieuc worked with Technician Thompson to learn new immunological and genomic techniques. Ms. Thompson then competed and was offered a new position as a federal scientist at the US Geological Survey. LaDeau worked with Postdoc Ferguson to develop model goals, evaluate simulation code and outcomes. LaDeau mentored Breyta in development of knowledge regarding infectious disease modeling and applications of genotype data. LaDeau worked with Brito and Breyta in development and quality checking of the IHNV diagnostics database in Aim 1, and appropriate univariate analysis of the resulting final database. This activity involved regular webex communications and has now moved forward to preparation of a manuscript. Ferguson was selected to attend the NIMBioSResearch Collaboration Workshop for Women in Mathematical Biology (http://www.nimbios.org/education/WS_wwmb) sponsored by the National Institute for Mathematical and Biological Synthesis (NIMBioS). This externally funded workshop was held June 22-25, 2015 in Knoxville, TN. Kurath mentored Wargo during a one month visit to the Western Fisheries Research Center in July of 2015, during which the preliminary virus shedding data and broader interpretations of all project data were regularly discussed. Wargo and Kurath worked with Breyta to enhance her fluency in virulence evolution theory, with particular focus on the project virulence data now published in Breyta et al. (in press). Breyta and Wargo mentored technician McKenney in proper estimation and statistical analyses of ID50 values from infectivity data in sub-aim 2b. McKenney attended a Summer Institutes for Statistics and Modeling of Infectious Disease course at the University of Washington, Seattle WA, from July 6-8, 2015. The course was "Probability and Statistical Inference". How have the results been disseminated to communities of interest? Aim 1. Database characteristics and summary statistics were presented to agency and hatchery stakeholders at a joint meeting in Seattle, WA in July 2015. Virus genetic typing data is available to all interested parties online in the searchable and georeferenced MEAP-IHNV database at <gis.nacse.org/ihnv. Aim 2. Virulence, infectivity, shedding, fitness, and persistence of IHNV field isolates associated with major displacement events in the field were presented to Agency and hatchery stakeholders at a joint meeting in Seattle, WA. Aim 3: We have presented results to American Fisheries Society conferences in 2014 and 2015 (outside reporting period), and have participated in our joint stakeholders meeting in July 2015. We have participated in public outreach events at the Seattle aquarium. We have also produced one publication. Details are provided in Section 2 "Target Audience" and Section 3 "Products". We have also shared our genotyping results with the developers of the SNP Chip, based at the USDA National cool and coldwater aquaculture center in Virginia. Aim 4: Model goals and summary structures were presented to Agency and hatchery stakeholders at a joint meeting in Seattle, WA. All aims: A powerpoint file titled "Ecological drivers of transmission of IHNV in steelhead trout of the Columbia River basin and coastal Washington" was prepared from the talks at the meeting and distributed to all attendees for sharing with their lab staffs and other colleagues. Project data has been communicated in numerous presentations at various venues as listed earlier. What do you plan to do during the next reporting period to accomplish the goals? Aim 1: We will publish a manuscript that is currently in revisions that details the database scope and presents summary analyses and evidence for relative roles of spawning adults, out-migrating juveniles and hatchery location 'hot spots' for facilitating IHNV transmission. Additionally, we intend to gather relevant temperature and flow data to evaluate associations with temporal variability in IHNV prevalence in our focal region. Genetic typing will be continued as virus isolates are received. The resulting genotypes will be analyzed for phylogenetic and landscape pattern inferences, and reported to fish health agencies for use in disease management. The online IHNV-MEAP database will be updated with records for over 500 additional virus isolates typed during this project. Aim 2. Processing will be completed for the remaining samples from the viral shedding studies and the shedding profiles for all 4 displacement genotypes in single and mixed infections will be compared as a measure of transmission fitness. Shedding profiles will also be compared between infections in two different steelhead populations that differ in IHNV resistance. Aim 3: Genome-wide association analysis: We will publish the results of our initial trait analysis from the two populations we surveyed. We will extend our work to surveying DNA from five populations, all with disease phenotypes known, to determine genetic variation in host resistance across the Columbia basin. Complete analysis of neutralizing antibody response in steelhead trout exposed to IHNV at three different temperatures and prepare a manuscript reporting the study. Aim 4: The model will be fit to the full regional and temporal scope of our project. We further intend to assess more detailed distance metrics for incorporating viral genotyping data in order to provide more precise support for specific transmission pathways. A continuing Education Class based on project results will be developed and presented on June 7th, 2016 at the combined meeting of the American Fisheries Society Fish Health Section and the Western Fish Disease Workshop, in Jackson Hole, Wyoming.

Impacts
What was accomplished under these goals? Impact Management of diseases in fishes is challenging when cultured & wild conspecifics interact. Significant numbers of steelhead salmon in the Columbia River return to reproduce in the wild, but these populations are also supplemented by hatchery releases. All populations are susceptible to a viral pathogen, IHNV, which emerges in different populations episodically, but the causes & routes of transmission are not well characterized. Here, we will inform policy decisions in fish resource management by providing the science relevant to developing appropriate disease management protocols. We will build a database that will permit testing of the effects of spatial & temporal environmental variables that influence viral transmission. We are experimentally testing how the virus might be transmitted from fish to fish, & how fish populations might respond to virus exposure. These data will be integrated into a model that will be used to study viral transmission, predict emergence & examine the efficacy of different virus control strategies. Results will be disseminated to management agencies & to the public. Major activities 1: A full IHNV diagnostic database has been constructed for the years 2000-current. Genetic typing data has been aligned with testing results from 169 hatcheries & 163 non-hatchery locations. 2: An LD50 virulence experiment was completed for a fifth fish host population. The in vivo portion is complete. Processing from all infectivity experiments & a study of ID50 & LD50 values in rainbow trout was completed. Results for sub-aims 2a & 2b were published. 3: Molecular markers linked to IHNV resistance were identified using a genome-wide association analysis across fish with known disease phenotypes. An assay to evaluate anti-IHNV antibodies in steelhead trout exposed to IHNV was optimized & applied to our previous persistence study. 4: A landscape transmission model was developed to evaluate the relative importance of adult fish, juvenile out-migration & hatchery characteristics in supporting IHNV transmission & persistence in the region. The model incorporates available viral genetic typing data to inform transmission connectivity. Data collected 1: The database contains 1458 records on steelhead trout from 126 unique locations & includes complimentary records in other host species. Temporal data on hatchery fish abundance were collated. Genotyping was conducted for 134 new virus isolates representing 29 viral events. 2: Virulence of the 4 viral displacement genotypes in 5 different host populations was compared using expanded virulence measures including lethal dose estimates, survival kinetics & proportional hazards analysis. Infectivity of the four genotypes was assessed as ID50 estimates in two steelhead trout populations. Sample processing & qRt-PCR have been completed for 50% of the virus shedding samples collected in 30-day shedding kinetics experiments for the 4 viral genotypes in single & mixed infections in two host populations. 3: 284 individuals were genotyped using the 50,000 SNP array for rainbow trout, where marker position has been mapped to the rainbow trout genome. Random Forest was used to detect markers linked to disease phenotypes. We tested serum from trout exposed to IHNV at 15°C to evaluate antibody response. 4: The model was tested using a subset of the data from 24 sites in the lower Columbia River. The basic outputs included likely transmission route(s) & probabilities of infection for each cohort-site given the transmission route. The genotype model used genotype data to infer the relative likelihood of possible transmission routes & probabilities of infection for each cohort site, given the transmission route. Statistics & discussion 1: Prevalence of IHNV in steelhead (25.9 % to 42.5% of adults & 16% - 28.6% of juveniles) was higher than for other host species in the database. Adult (juvenile) testing was reported at least once by 136 (142) hatcheries, & 75 (54) reported positives at least once. Ongoing viral genotyping of 134 new virus isolates found 12 different genotypes, 7 novel. The majority were U genogroup isolates from the Snake River, with an apparent waning of the dominant M genogroup viruses that emerged since 2002 2: A pattern of increasing virulence for isolates 007, 111, & 110 was consistent in all five host populations tested & correlated with serial emergence & displacements in the lower Columbia River source region during 1980-2013. Genotype 139 represented a conditional displacement that was not associated with an increase in virulence. Viral infectivity (ID50), did not correlate consistently with virulence or with viral emergence, & showed a narrow range of variation relative to that observed in virulence. Variation in resistance was confirmed among the five steelhead trout populations. Kinetics between virus genotypes are very similar in the processed viral shedding samples, with a peak in viral shedding around day 2-3 post infection & tapering rapidly by day 10. So far, no correlates with field displacement & viral shedding have been observed. 3: Over 35,500 polymorphic markers were genotyped across 284 individuals. Random Forest detected 164 loci that predicted disease phenotype. Mapping of these markers resulted in "clusters" of predictor loci that identified a few key genomic regions that likely identify candidate loci involved in resistance.Preliminary results do not support the hypothesis that fish with persistent IHNV infections may have failed to mount a sufficient adaptive immune response to clear the virus. Neutralizing antibodies were detected in all fish that tested positive for persistent IHNV RNA while fish that tested negative for IHNV RNA lacked neutralizing antibodies. 4: Model output from the lower Columbia River Basin during 2000-2012 suggests that juveniles often exposed to INHV by infected adults returning to the site or passing the site as they swam upstream. However, probabilities of infection associated with this transmission route were relatively low (mean = 0.22). Exposure within a hatchery ('self') did not occur often but associated probabilities of infection were high (mean = 0.44). Transmission by all possible routes in concert (mean = 0.54) had the highest probabilities of exposure & infection. Key outcomes 1: The first rigorous estimates of overall IHNV prevalence in Pacific salmonids on a landscape-scale have facilitated accurate analysis of virus spatial & temporal distributions, & occurrence in different host species (Chinook & steelhead) & different host life stages. Integration of the virus genotype data has revealed evidence of emergence & submergence of dominant M & U genotypes over time, cross-species transmission of virus of M viruses from steelhead to Chinook, & evolution of a previously unrecognized subgroup of U viruses that express a generalist phenotype, infecting both Chinook & steelhead. 2: Analysis of variation in viral virulence & host resistance indicated increasing viral virulence over time as a potential driver for emergence & displacement events in the endemic Lower Columbia River source region, but not in adjacent sink regions. Source & sink perspective may explain some of the complexity observed in field events. 3: We have identified key genomic regions associated with IHNV resistance in Columbia River Steelhead, providing the most comprehensive analysis in Pacific salmonids to date. 4: A simulation model that includes viral genotyping data as one component of 'distance' between IHNV positive samples successfully recovers parameters. Model fit to observed infection data from a subset of hatcheries in the mid-Columbia suggests an important role for spawning adult fish & hatchery practices in maintaining IHNV in this landscape.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Kell AM, Wargo AR, Kurath G (2014) Viral fitness does not correlate with three genotype displacement events involving infectious hematopoietic necrosis virus. Virology 464-465:146-155.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Brieuc MSO, Purcell MK, Palmer A, Naish KA (2015) Genetic variation in resistance to infectious hematopoietic necrosis virus (IHNV) in a population of steelhead trout (Oncorhynchus mykiss). Diseases of Aquatic Organisms. In press
• Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Breyta RB, McKenney DG, Thompson TM, Kurath G. Increasing virulence, but not infectivity, associated with serially emergent virus strains of a fish rhabdovirus. Virus Evolution, in press.
• Type: Journal Articles Status: Other Year Published: 2015 Citation: McKenney DG, Kurath G, Wargo AR. Characterization of infectious dose and lethal dose of two strains of infectious hematopoietic necrosis virus (IHNV). Virus Research, in revision after journal review.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Brieuc MSO, Purcell MK, Kurath G, Naish KA (2014) Heritability Estimates of Resistance to Infectious Hematopoietic Necrosis Virus (IHNV) in Two Populations of Steelhead (Oncorhynchus mykiss). American Fisheries Society Annual meeting Quebec, Canada, August 17-21.Conference poster.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Kurath G, Breyta R, Kell AM, Wargo AR (2014) Host susceptibility and viral fitness as potential drivers of IHN virus emergence and displacement events in steelhead trout. 7th International Symposium on Aquatic Animal Health, Portland, OR, September 2, 2014
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Kurath G, Wargo AR, Penaranda MMD, Kell AM (2015) Fish rhabdovirus model system for studies of viral fitness. 9th International Symposium on Viruses of Lower Vertebrates, Malaga, Spain, October 2, 2014
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Kurath G, Naish KA, Purcell MK, LaDeau S, Brieuc MS, Breyta R, Wargo AR, Brito IL, Ferguson P, Thompson R, McKenney DG, Kell AM (2014) Ecological drivers of Transmission, Emergence, and Displacement of an aquatic virus in fish hosts. 2014 USDA-NIFA AFRI Animal Health, Animal Well-Being & Food Security Awardee Workshop, Virtual conference. November 19-21, 2014.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kurath G, Breyta R, McKenney DG, Kell AM (2015) Evolution of the fish virus IHNV: Drivers of viral emergence and displacement events in the field. 8th Virus Evolution Workshop, Penn State University, State College, PA, March 3, 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Ferguson P, Brito, Breyta R, Kurath G, and LaDeau SL (2015) Modeling infectious hematopoietic necrosis virus (IHNV) transmission pathways in Pacific salmonids. Combined Ecology and Evolution of Infectious Disease and NSF EEID program meeting, Athens, GA, May 27-30th, 2015. Conference poster
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Breyta R, Brito I, Brieuc M, Kurath G, LaDeau S, Purcell MP, Naish K (2015) Capturing a complex system: building blocks for a hierarchical model of IHNV transmission in Pacific salmon. Combined Ecology and Evolution of Infectious Disease and NSF EEID program meeting, Athens, GA, May 27-30th, 2015. Conference Poster
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Breyta R, McKenney DG, Kurath G (2015) Capturing the dynamic patterns of transmission, emergence, and adaptation of IHNV in steelhead trout in the PNW. Western Fish Disease Workshop, American Fisheries Society. Steamboat Springs, CO, June 3, 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kurath G, Breyta R, McKenney DG, Kell AM (2015) Correlates of IHN virus emergence and displacement events in the field. Western Fish Disease Workshop, American Fisheries Society. Steamboat Springs, CO, June 3, 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Naish KA, Brieuc MS, Thompson R, Purcell MK (2015) Genetic basis of IHNV resistance in steelhead. Ecological Drivers of IHNV resistance. IHNV Stakeholders meeting, Seattle, WA, July 22, 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kurath G, Breyta R, McKenney DG (2015) IHN virus landscape ecology: emergence and displacement events in the field. IHNV Stakeholders meeting, Seattle, WA, July 22, 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Breyta R, Brito I, LaDeau S, Kurath G (2015) Building the IHNV landscape. IHNV Stakeholders meeting, Seattle, WA, July 22, 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Breyta R, McKenney DG, Kurath G (2015) Virulence, but not infectivity, associated with serially emergent MD types of IHNV. IHNV Stakeholders meeting, Seattle, WA, July 22, 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Purcell MK, Thompson RL (2015) Effects of temperature on IHNV persistence and immunity in steelhead trout. IHNV Stakeholders meeting, Seattle, WA, July 22, 2015.

Progress 08/15/13 to 08/14/14

Outputs
Target Audience: Target Audience: McNair Scholars program, Internship for a first generation college student with financial need and from a traditionally underrepresented group Effort: Kerry Naish, Marine Brieuc and Maureen Purcell mentored Bryn Perales, an undergraduate student, in research related to the disease interactions between hatchery and wild salmon Dates: June 2013-September 2013 Target Audience: General Public Effort: Marine Brieuc and Kerry Naish participated at the Discover Science Weekend at the Seattle Aquarium. This event aimed at introducing ocean and marine research through interactive displays and activities with scientists. Date: November 9, 2013 Target Audience: State, federal, and tribal fish health managers in Washington state Effort: Rachel Breyta and Gael Kurath presented talks on current IHNV typing and database progress and research results at the Washington State Interagency Fish Health Meeting (Olympia, WA) Date: May 13, 2014 Target Audience: Canadian fish health specialists and Atlantic salmon industry representatives Effort: Maureen Purcell gave an oral presentation and participated in a round-table discussion on improved standardization of molecular diagnostic tests for infectious hematopoietic necrosis virus (IHNV) and other aquatic viruses Date: June 17, 2014 Target Audience: Aquaculture managers Effort: Gael Kurath, Rachel Breyta, and Andrew Wargo participated in a USDA-RAPIDD sponsored field visit to aquaculture sites and meeting with managers to communicate about IHNV virulence in aquaculture (Twin Falls, ID) Date: July 15-16, 2014 Target Audience: Project collaborators Effort: All project scientists participated and presented talks at an annual project meeting in Seattle, WA Date: July 28-30, 2014 Target Audience: Peer Scientists Effort: Breyta presented a speed talk on project concepts at Research Coordination Network on Disease in Marine Ecosystems (RCN) meeting (Friday Harbor, WA) Date: August 17-20, 2014 Changes/Problems: The postdoctoral researcher involved in Aim 1 transitioned to a new job during this past year and there was a 6-month delay in hiring Paige Barlow, who will work on Aim 4 modeling goals. During this time, we discovered some important discrepancies between our GIS layer and the PTAGIS (agency) data for hatchery location that required us to revise the database and earlier analyses. Although our LD50 virulence studies in Aim 2 have revealed a consistent pattern of increasing virulence for the first three viral genotypes involved in field displacements, the differences are not statistically significant in many comparisons. Therefore we would like to refine the assay to improve its ability to detect subtle virulence differences, since small differences would be expected to evolve in the field, but they could have large biological consequences. Based on the results we have obtained thus far, we anticipate that we could increase our number of replicate tanks at each dose from 3 to 5, and compensate by decreasing the number of doses tested from 4 to 3, to get more replication into the experiment. It is our hope that this will increase our ability to detect statistically significant small variations in virulence. Recent advances in sequencing techniques and two major resources for Oncorhynchus mykiss in the past year (a draft genome and a 50K SNP chip) have enabled us to expand our original aims for genotyping in Aim 3. The creation of the SNP chip in particular will allow more reliable genotyping with low rate of missing values compared to other sequencing techniques. We are currently testing the SNP chip with a subset of the samples and will use this approach instead of RAD sequencing if the results are satisfactory. Exploratory work has slightly delayed the genotyping for Aim3a, but we believe that this new approach will greatly improve the power of this study. What opportunities for training and professional development has the project provided? LaDeau worked with Brito and Barlow to organize postdoctoral efforts around data base evaluation, code-sharing, and model structure. Kurath worked with Breyta to organize postdoctoral efforts around database collection and viral challenge experiments Naish and Purcell worked with Brieuc to organize efforts around host response to viral challenges and host genome analyses Technician Thompson worked with new technician McKenney to train him in viral stock culture and virus quantification by plaque assay, and Kurath worked with him on experimental design and data presentation. Postdoctoral researcher Breyta trained McKenney in statistics associated with viral challenge data and ID50 and LD50 values. McKenney attended a Summer Institute of Statistics and Modeling of Infectious Disease course in "R" (University of Washington, Seattle, WA) July 2014. Purcell, Naish and Brieuc hosted Ph.D. student Natalia Ballesteros for 3 months (Summer 2014). Ms. Ballesteros is originally from Columbia and is currently a student at the Centro de Investigaciones Biológicas CSIC in Madrid Spain studying the immune response to IHNV. She spent 3 months gaining experience conducting bioinformatic analysis, working closely with post-doctoral fellow Dr. Marine Brieuc. Naish, Purcell and Brieuc hosted undergraduate student Bryn Perales for 3 months (Summer 2014). Ms. Perales was an undergraduate McNair fellow, enrolled in University of California Montery Bay, preparing for entry into graduate school. She spent 3 months gaining independent research experience in genotyping, conducting bioinformatic analysis, and fish culture. How have the results been disseminated to communities of interest? Target Audience: General Public Effort: Marine Brieuc and Kerry Naish participated at the Discover Science Weekend at the Seattle Aquarium. This event aimed at introducing ocean and marine research through interactive displays and activities with scientists. Date: November 9, 2013 Target Audience: State, federal, and tribal fish health managers in Washington state Effort: Rachel Breyta and Gael Kurath presented talks on current IHNV typing and database progress and research results at the Washington State Interagency Fish Health Meeting (Olympia, WA) Date: May 13, 2014 Target Audience: Canadian fish health specialists and Atlantic salmon industry representatives Effort: Maureen Purcell gave an oral presentation and participated in a round-table discussion on improved standardization of molecular diagnostic tests for infectious hematopoietic necrosis virus (IHNV) and other aquatic viruses Date: June 17, 2014 Target Audience: Aquaculture managers Effort: Gael Kurath, Rachel Breyta, and Andrew Wargo participated in a USDA-RAPIDD sponsored field visit to aquaculture sites and meeting with managers to communicate about IHNV virulence in aquaculture (Twin Falls, ID) Date: July 15-16, 2014 Target Audience: Project collaborators Effort: All project scientists participated and presented talks at an annual project meeting in Seattle, WA Date: July 28-30, 2014 Target Audience: Peer Scientists Effort: Breyta presented a speed talk on project concepts at Research Coordination Network on Disease in Marine Ecosystems (RCN) meeting (Friday Harbor, WA) Date: August 17-20, 2014 What do you plan to do during the next reporting period to accomplish the goals? Aim 1: We will complete a manuscript that will summarize Aim 1 efforts to evaluate specific transmission scenarios. We will continue to use a simulation approach to evaluate assumptions and scenarios, especially where data are coarse. This will directly inform the Aim 4 goal of developing a spatially-explicit model to evaluate relative support for possible viral transmission pathways. Aim 2: We will complete the 5th LD50 study that is currently ongoing and compare results with all other LD50 data. We will complete the processing and qPCR for the second ID50 experiment of 2014 and analyze the data in comparison with available ID50 data in other years and other host populations. We plan to conduct one or two final LD50 studies using a modified experimental design intended to improve our ability to detect statistically significant differences in virulence between virus genotypes (see below). For viral shedding studies the processing of thousands of water samples will be conducted using robotic RNA extraction and qPCR, and results will be compared for single and mixed viral infections. Finally, we will conduct a long-term viral persistence study in LQ steelhead trout, to quantify viral persistence in this more susceptible host population. All results will be analyzed for parameterization of the modeling Aim 4 to determine if additional experiments will be valuable. Aim 3: a. We will complete the genotyping and conduct the genome-wide association study to identify loci linked to resistance to IHNV. We will also conduct a Genotype by Environment study where we will challenge fish from both populations (QNFH and LQ) at two different temperatures (6C and 10C). b. We will complete our viral persistence study. We also anticipate repeating aspects of this study to confirm results. c. Once all populations have been challenged, we will genotype the individuals to determine if resistance-associated loci are conserved across generations and host populations.

Impacts
What was accomplished under these goals? Impact Management of diseases in fishes is challenging when cultured and wild conspecifics interact. Significant numbers of steelhead salmon in the Columbia River return to reproduce in the wild, but these populations are also supplemented by hatchery releases. All populations are susceptible to a viral pathogen, IHNV, which emerges in different populations episodically, but the causes and routes of transmission are not well characterized. Here, we will inform policy decisions in fish resource management by providing the science relevant to developing appropriate disease management protocols. We are building a database that will allow us to test the effects of spatial and temporal environmental variables that influence viral transmission. We are experimentally testing how the virus might be transmitted from fish to fish, and how fish populations might respond to virus exposure. These data will be integrated into a model that will be used to study viral transmission, predict emergence and examine the efficacy of different virus control strategies. These results will be rapidly disseminated to management agencies and to the public. Major activities completed Aim 1: Assembly and quality assessment of the diagnostic IHNV database has been completed and a focused subset of the data has been selected for further analysis, representing virus detections in the Columbia River basin and Coastal Washington during the years 2000-2012. Univariate analyses of transmission factors are now in progress. Aim 2: Wetlab experiments have been conducted in support of all five sub-aims, and data analysis is complete for most experiments. Sub-aim 2d has been completed. Aim 3: Heritability estimates for IHNV resistance in two host populations were computed and genotyping analyses to detect the loci linked to resistance to IHNV were initiated. Host response and viral persistence was studied at three different temperatures. A study on variation of host susceptibility over time and across host populations was initiated. Aim 4: An initial simulation model, focused on a small portion of the Columbia basin and informed by field data and experimental results has been developed to evaluate alternative model structures and assumptions. Data collected Aim 1: The large georeferenced dataset encompassing diagnostic surveillance and viral genotyping data of IHNV for all salmonids in our study region has been revised. Aim 2: a: Virulence of four IHNV genotypes has been determined as LD50s in four different host populations that vary in historical exposure to IHNV. b: Infectivity of the four viral types has been determined as ID50s, tested three times in two host populations. c: Dynamics of viral shedding has been tested twice for the four viral types, in two fish populations. d: Studies of in vivo viral fitness components for four virus genotypes have been completed and published. e: IHNV persistence was assessed in Aim 3b. Aim 3: a. Population measures of genetic and phenotypic correlation in resistance to IHNV were estimated. Informative individuals were retained to determine the genes or regions of the genome involved in IHNV resistance. Genotyping has been initiated. 3b. The prediction that water temperature (6, 10 or 15°C) influences IHNV persistence and host immunity over a 9 month period was tested. 3c. Three populations of steelhead were challenged with IHNV at two doses to determine if resistance-associated loci are conserved across generations and host populations. Summary statistics and discussion Aim 1: Of the 170 unique hatchery sites in our database, 44% have documented IHNV incidence in adult (spawning) fish and 32% documented juvenile incidence in at least one year between 2000-2013 , with 27.33+/- 3.395 testing in both adult and juvenile fish in the same year. Most hatcheries (83.5%) tested juveniles for IHNV in at least one year: mean reporting frequency was 5.21 +/- 4.89 years, with an average of 0.89 +/- 1.87 positive IHNV years. Mean prevalence across all sites was 19.57 ± 32.56% for adults and 11.55 +/- 25.22% for juveniles. Viral genotyping was performed on samples in 458 instances (65.4%) of hatchery or wild populations of fish out of 701 positive tests. A subset of hatcheries for which we had release data (73 out of 170) revealed a positive correlation between the total number of years of IHNV detected and both the number of juvenile fish released and the number of species reared (R=0.2784, , p=.00784 and R=.331,p<0.01, respectively). Aim 2: a&b. Infectivity ID50 values from three experiments were all within one order of magnitude, ranging from 79-632 pfu/ml for all four viral types in both fish populations tested. LD50 values in 4 experiments varied more widely, by up to 2.5 orders of magnitude for the 4 viral types. Relative ranking of virulence was consistent in three fish populations, with 007M < 111M < 110M, and 139M intermediate. LD50 values were 10 to 1000-fold higher than ID50 values, and there was not a consistent relationship between virus infectivity and lethality. d. Results of a suite of viral fitness assays found no significant increase in fitness for any virus displacement pairs, conclusively demonstrating that displacements in the field were not driven by the viral fitness measures defined here. e. The viral persistence study found higher IHNV prevalence and duration than anticipated. Aim 3. a. Host heritability for total mortality was significant, (resistant population = 0.26, 95% CL 0.16-0.41; susceptible population = 0.32, 95% CL 0.12-0.61). Days to death had low heritability (resistant 0.07, 95% CL 0.02 to 0.14; non-significant for suspectible). Both genetic and phenotypic correlation with length suggested smaller susceptible fish die earlier than larger susceptible fish. b. IHNV mortality in all three temperature treatments were significantly different from each other, with the highest mortality at 6°C. Infection prevalence was 100% early in all groups but declined later. All temperature groups had individuals testing positive for IHNV RNA until 3 months post-infection indicating persistence of virus after mortality had ceased. The 15°C group cleared the virus more rapidly. Viral RNA was still detected in the 6 and 10°C groups at 6 months post-infection. Accelerated viral clearance in the 15°C group was likely due to host immunity rather than an impairment of viral replication, as cell culture studies indicated that the virus replicates efficiently at 15°C. Viral RNA persistence was most commonly detected in the kidney and less commonly in the brain, fin or gill. Key Outcomes Aim 1: There is evidence that larger hatchery programs (more species reared, more fish released) have a higher likelihood of IHNV incidence reported. Aim 2: Relative virulence of four virus types had a consistent pattern in different years and different fish populations, suggesting increases in viral virulence as a driving factor in two viral displacement events. Virulence and infectivity were not correlated, and viral fitness did not correlate with displacements. Among fish host populations, higher resistance generally correlated with more historical exposure to IHNV. Aim 3: a. There is significant opportunity for domestication selection for disease resistance in steelhead hatchery populations, possibly through correlated selection on size at age. b. Viral persistence in steelhead trout may occur more frequently than anticipated from previous studies in other salmonid hosts and could be a mechanism for long-term maintenance of the virus in natural populations. Immunity appears impaired at lower water temperatures. Aim 4: Juvenile IHNV positives at a hatchery are not well-predicted by standard transmission model (SIR) assumptions. The modeling approach requires attention to detection and reporting issues, as well as anthropogenic manipulation. Current work suggests a role for adult (spawning) fish in moving virus.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Kell AM, Wargo AR, Kurath G. 2014. Viral fitness does not correlate with three genotype displacement events involving infectious hematopoietic necrosis virus. Virology 464-465:146-155.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Brieuc MSO, M Purcell, G Kurath and KA Naish. Heritability Estimates of Resistance to Infectious Hematopoietic Necrosis Virus (IHNV) in Two Populations of Steelhead (Oncorhynchus mykiss). International Conference on Integrative Salmonid Biology Vancouver BC. June 10-12, 2014. Poster presented by Kerry Naish
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Breyta R, Kurath G. Capturing a complex system: building blocks for a hierarchical model of IHNV transmission in Pacific salmon. EEID meeting, Fort Collins. June 1-4, 2014. Poster presented by Rachel Breyta
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Kurath G, Breyta R, Wargo, A. Capturing a complex system: building blocks for a hierarchical model of IHNV transmission in Pacific salmon. EEID meeting, Fort Collins. June 1-4, 2014.

Progress 08/15/12 to 08/14/13

Outputs
Target Audience: Target Audience: Hatchery and agency staff Effort: Shared the goals of the project with hatchery and agency staff in order to acquire data. Postdoctoral associate Breyta and co-PI’s Kurath and Purcell have developed relationships with several state and federal fish health agencies, as well as with specific hatchery managers. Purcell visited the USFWS Olympia Fish Health Center to brief the fish health group on the specific goals of Aim 3 (host genetics) (April 8, 2013). Target Audience: Northwest Indian Fisheries Commission Effort: Kurath and Breyta presented project plans at a joint meeting in July 2012. Purcell presented a talk on IHNV and other viruses to the Northwest Indian Fish Commission staff, as well as other local fish health professionals (May 29, 2013). Target Audience: Pacific Northwest Fish Health Protection Committee Effort: Breyta presented IHNV research products to multiple partner fish health agencies at semi-annual meeting (Portland, OR) February 12-13, 2013. Purcell presented a talk on IHNV and other viruses at the semi-annual meeting (Ocean Shores, WA) August 19, 2013. Target Audience: Hatchery and fish health diagnostics staff Effort: Breyta and Brito visited one hatchery in July 2013, both to see the practice and to share information about research goals. Changes/Problems: Aim 3a. We deviated from the original experimental design by integrating a traditional quantitative genetics breeding design. Originally, we proposed to obtain a sampling of the entire Lake Quinault and Quinault National Steelhead trout populations. We proposed to IHNV challenge these fish at two temperatures and use genetic material from mortalities and survivors for genome wide association studies. However, instead we performed a nested half-sibling design (equaling > 56 full-sibling families) on the Lake Quinault population. We then made hybrid crosses between Lake Quinault and Quinault National populations to maximize the potential genetic variation (> 16 full-sibling families). This significantly increased our workload as we challenged ~4300 individual fish in the first series of IHNV challenge. Next, we challenged a large number of individuals (300 fish) from a few select hybrid families to obtain genetic material for a QTL map. We feel this change will significantly increase the power to detect genetic associations to IHNV resistance. However, we had to eliminate the temperature component of the aim (challenge at two temperatures) because it was not feasible due to facility constraints. The temperature component will still be included in Aim 3b. What opportunities for training and professional development has the project provided? All co-investigators participated in training activities with proiect staff. LaDeau worked with postdoctoral associate Ilana Brito to develop protocols for assembling the complex database and prioritize ecological data and questions to be evaluated. Naish and Purcell worked with postdoctoral researcher Marine Brieuc to develop skills in animal infection studies, with technician Rachel Thompson to develop a new ELISA protocol for immune analyses and with technician Alex palmer to develop research populations of fish. Kurath worked with postdoctoral researcher Rachel Breyta and technician Tarin Thompson to develop molecular biological methods needed for analysis of biological samples from in vivo infection experiments. Brito attended the Summer Institute in Statistics and Modeling in Infectious Diseases workshops, MCMC 1 and MCMC 2 (University of Washington, Seattle, WA). July 15-18, 2013. Brieuc attended the Summer Institute in Statistical Genetics, (University of Washington, Seattle, WA). July 10-19, 2013. Modules: Bayesian Statistics for Genetics; Population Genetics and Association Mapping; Introduction to QTL Mapping, Aim 2d has been conducted as part of the doctoral thesis research of graduate student Alison M. Kell. Ms. Kell successfully defended her PhD on Sept. 25th, 2013, with one of her major thesis chapters based on superinfection fitness assays for this project. As part of her work she developed and applied a novel system for investigating viral superinfection in living fish. Dirir Abdullahi, an undergraduate Louis Gates Minority Scholarship recipient at the University of Washington was trained for two academic quarters in laboratory techniques in virology. Our grant continued to support Dirir as an hourly student for another academic quarter. How have the results been disseminated to communities of interest? Kurath hosted the RAPIDD workshop: Evolution of virulence in aquacultural diseases (Seattle, WA) Nov 2-3, 2012 with Andrew Read, David Kennedy LaDeau, Brito, Breyta, Wargo, Purcell, Brieuc and Naish participated in the RAPIDD workshop: Evolution of virulence in aquacultural diseases (Seattle, WA) Nov 2-3, 2012. Brito presented a poster "Modeling the spread of IHN Virus in salmonids throughout the Northwestern United States" at NSF-EEID meeting (Athens, GA) March 16-17, 2013. Breyta presented a poster "The dynamic landscape of IHNV in Pacific Salmon" at RCN meeting (Seattle, WA) October 27-29, 2012. Breyta presented a poster "A heterogeneous landscape of host susceptibility and viral virulence: IHN virus in Pacific salmon" at NSF-EEID meeting (Athens, GA) March 16-17, 2013. Kurath presented project overview "Drivers of ecology and evolution of a fish thabdovirus in Pacific salmon" at NSF-EEID meeting in Athens, Georgia March 16, 2013. What do you plan to do during the next reporting period to accomplish the goals? Aim 1 and 4: Over the next year we will move through univariate, hypothesis driven analyses to approach the Aim 4 goal of developing a spatially-explicit model to evaluate relative support for possible viral transmission pathways, given data on diagnostic sampling, viral genotypes, host production and return rates, and ecological variables. Aim 2: Over the next year we will conduct additional experiments to confirm and refine our determinations of virulence (LD50) in Aim 2a, infectious dose (ID50) in 2b, and shedding dynamics in 2c. Viral transmission fitness will be examined by shedding assays from co-infections in support of aims 2c and 2d. Preliminary experiments for Aim 2e will also be conducted. All results will be analyzed for parameterization of the modeling Aim 4 to determine if additional experiments will be valuable. Aim 3: In the next reporting period, we plan to conduct the genetic analysis to identify loci linked to resistance to IHNV in steelhead. To achieve aim 3a, we decided to conduct the experiments in two steps: first, to identify the loci linked to IHNV resistance using mapping families as those will give us greater power; second, to examine the susceptibility of the populations at different temperatures. The experiments for this second step and the experiments to assess the effects of temperature on the immune response and the infection dynamics (aim 3b) will be conducted simultaneously in the next reporting period. Finally we will collect eggs from several steelhead populations, including Skamania and Elwha wild, in this upcoming spawning season to address aim 3c. Eyed eggs will be brought to WFRC and fish will be challenged when they reach the appropriate size, around 1g.

Impacts
What was accomplished under these goals? A: Major activities completed: Aim 1 and 4: Database creation and management is ongoing. Aim 2: In vivo wetlab experiments have been conducted to support of four of the five specific sub-aims. Data analyses have been conducted for the majority of those experiments. Aim 3: Three types of experimental crosses from two populations of steelhead that differ in susceptibility to INHV were created, and challenged. B: Specific Objectives met Aim 1 and 4: A large dataset encompassing diagnostic surveillance and viral genotytyping data of IHNV for all salmonids in our study region was assembled. All (26,878) records collected by 7 agencies (Federal - USF&W in ID, Lower Columbia and Olympia region, State: WA, ID and OR, and the Tribal NWIFC) were geo-referenced and each location was evaluated and grouped into unique 205 unique hatchery sites and 143 wild-caught sample sites. Aim 2: 2a: Define virulence of four IHNV types: determine LD50 values: A set of four viral challenge experiments was conducted by exposing groups of juvenile Lake Quinault (LQ) steelhead trout to immersion in four doses of each of the four viral variants. Mortality was monitored over 30 days and data was analyzed to define the LD50 for each virus type. These experiments will be repeated in year 3. 2b: Define infectious dose of four IHN viral types. Four experiments were conducted by exposing juvenile LQ steelhead to four doses of each of the four viral types as above, but holding fish individually and harvesting on 3 and 7 days post-exposure to assess infection status. The ID50 values obtained were used to refine experimental design for year 2. 2c: Determine dynamics of viral shedding for four IHN viral types. The in vivo experimental portion of 2c has been conducted by challenging LQ steelhead trout with each of the four viral types, and collecting water samples from each of 180 individual fish over the 30 days. Sample processing is underway. 2d: Define viral fitness in co-infection and super-infection for pairs of IHN viral types associated with displacement events. In vivo co-infection assays have been conducted providing a statistically rigorous comparison of replication fitness between virus types in each displacement pair. Fitness has also been quantified in super-infection, where reciprocal superinfection groups were tested with three time intervals between primary and secondary infection. Data analysis is complete, defining the relative superinfection fitness of each pair of virus types. The majority of work in this sub-aim is complete. 2e: Define probabilities of long-term infection outcomes: persistence vs. clearance. Work on this sub-aim will begin in year 2. Aim 3: Gametes from Quinault National Fish Hatchery (QNFH) and Lake Quinault Tribal Fish Hatchery (LQ) were collected in December 2013 and January 2014. Using a half-sibling design we created 56 QNFH families, 5 LQ families and 15 QNFH x LQ hybrid families. All the families were challenged by immersion at 10C with IHNV (2x105 PFU/mL) for 21 days. The LQ families were pooled prior to the challenge. The phenotypes recorded during the challenges were mortality, days to death and length. Tissue for each fish was stored in 95% ethanol for genetic analysis. The LQ, QNFH and QNFH x LQ steelhead were batch challenged. C: Specific results achieved Aim 1: The IHNV dataset represents the first time all federal, tribal and state records have been amassed into one cohesive dataset. The diagnostic data are extensive, specifying species and seasonal run, date, location, and whether the fish was of wild or hatchery origin.Several ecological datasets were linked to the diagnostic data (by year and or location): hatchery-specific salmon production; returning adult abundance (using counts at large dams); USGS water and air monitoring stations. Nearly 2000 viral genotype samples have been georeferenced and aligned with known diagnostic samples. Early analyses include examination of relationships among the ecological data (e.g., temperature, number of dam crossings, migration length, governing agency, fish population sizes), IHNV diagnostics and IHNV genotype evolution across the landscape. Evaluating these relationships will guide progress on Aim 4 goals in the next year. Aim 2a & 2b: The LD50 and ID50 studies indicate that there are significant differences between the four virus types in both LD50 and ID50 (Table 1). It also appears that there is an inverse relationship between the relative infectivity of a virus and its ability to kill infected hosts. An ID50/LD50 ratio closer to 1 suggests that more of the infecting dose is lethal. Table 1. LD50 and ID50 data for each virus genotype in LQ steelhead trout virus genotype 007M 111M 110M 139M LD50 1.5 x 105 1.2 x 104 4.7 x 103 1.8 x 10e4 ID50 1.9 x 102 3.8 x 102 6.3 x 102 1.2 x 102 ratio ID50:LD50 0.001 0.031 0.133 0.007 Aim 2d: Results of in vivo co-infection fitness assays found no significant difference in replication fitness for the virus types in displacement pairs 1 or 2, and in the 3rd pair the virus type that was displaced had higher fitness than the displacing virus type. This contradicts the hypothesis that viral displacement events might be driven by increasing viral replication fitness. Studies of viral superinfection fitness show that the frequency of superinfection decreases with increasing time between exposures to the primary and secondary virus, confirming that superinfection restriction does occur for IHNV. However there was generally no difference in superinfection fitness between virus types involved in displacement events. For all cases the replication of the primary virus was not significantly affected by superinfection, but secondary virus replication was significantly decreased in superinfection compared with single infection. This effect was consistent for all viruses, so that there were no differences in superinfection fitness in terms of viral replication. Aim 3: Mortality results from the IHNV challenges have confirmed that the LQ population was more susceptible to IHNV than the QN population. The hybrid families exhibit hybrid vigor; they are more resistant to IHNV than both parental populations. Heritability estimated for two traits in the QNFH population and the hybrid. Heritability of days to death (DTD) was not significantly different from 0 in the hybrid population and low in the QNFH population. However the heritability of mortality was significantly different from 0 in both populations, suggesting that we should be able to identify loci involved in IHNV resistance. Mortality Days to death QNFH 0.25 (0.16-0.41) 0.07 (0.02-0.14) QNFH x LQ 0.33 (0.13-0.62) <0.01 (0.00-0.4) Table 2. Heritability estimates for two traits. In parentheses: 95% highest posterior density interval D: Key Outcomes or other accomplishments met: Aim 2. Research in year 1 led to the following two changes in knowledge: a. Comparison of LD50 and ID50 data for the four virus types revealed significant variation in these viral traits, and revealed an unexpected relationship in which these traits did not correlate, suggesting that infectivity does not predict virulence for this virus. b. In vivo fitness assays did not find significant fitness differences, either in co-infection or superinfection, between the virus types in the three displacement pairs tested. This contradicts the hypothesis that displacement events are associated with increasing replication or superinfection fitness of the displacing virus type, and suggests that displacement events must be driven by other factors, including possibly transmission fitness (aim 2c), or other ecological or anthropogenic factors. Aim 3: DNA Sequencing and genome mapping analysis and immune response and infection dynamics (aim 3b) will be conducting during the next reporting period. Two thousand fish from the QNFH brood year 2013 are currently being held until they reach the appropriate size.

Publications