Source: UNIVERSITY OF WASHINGTON submitted to
ECOLOGICAL DRIVERS OF TRANSMISSIONS, EMERGENCE, AND DISPLACEMENT OF AN AQUATIC VIRUS IN FISH HOSTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
0229951
Grant No.
2012-67015-19960
Project No.
WNW-2012-03366
Proposal No.
2012-03366
Multistate No.
(N/A)
Program Code
A1222
Project Start Date
Aug 15, 2012
Project End Date
Aug 14, 2015
Grant Year
2012
Project Director
Naish, K.
Recipient Organization
UNIVERSITY OF WASHINGTON
4333 BROOKLYN AVE NE
SEATTLE,WA 98195
Performing Department
School of Aquatic and Fisheries Sciences
Non Technical Summary
The emergence of pathogens into new hosts and geographic regions is a widespread problem for a variety of wildlife, human, and agricultural diseases. Although pathogen emergence has been broadly studied in many terrestrial systems, there is relatively little knowledge of the transmission processes that drive aquatic pathogen emergence. Due to expansions of, aquaculture, aquatic habitat loss, fisheries harvest, and aquatic species conservation, the emergence of pathogens in aquatic ecosystems has become a critical issue. As such, there is increasing demand from aquatic animal health managers for tools to predict pathogen disease outbreaks and emergence. Aquatic pathogens have unique transmission properties and understanding emergence events in aquatic hosts requires information on a variety of ecological, environmental, host, and viral factors. Here we propose to develop informative models of transmission dynamics for the salmon pathogen infectious hematopoietic necrosis (IHN) virus. We will focus on documented viral emergence and displacement events that have occurred in Pacific Northwest river systems by testing viral genetic types associated with these events in relevant fish host populations that vary in IHN virus susceptibility. This will involve utilizing a novel integrative approach that combines process, data, and transmission network models, informed by epidemiological and ecological data, in vivo viral fitness experiments, and genetic and immunological markers of host susceptibility and phenotypic plasticity. Our goal is to elucidate general principles and ecological processes that drive IHN virus transmission, emergence, and displacement. Results from this project will have impacts on the economic health of rural and tribal communities, as well as State- and region-wide fish hatchery programs, by improving their return on salmon rearing investment and overall fish health. Longer term impacts will be felt by conservation programs, with the reduction in risks to threatened and endangered fish populations through better disease control. Finally, managers of a variety of global aquatic systems, like those concerned with recent fish virus disease outbreaks in the US Great Lakes and the international Atlantic salmon industry, will benefit from what we learn about the drivers of aquatic pathogen emergence.
Animal Health Component
20%
Research Effort Categories
Basic
75%
Applied
20%
Developmental
5%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3110810107025%
3110810108025%
3110810109025%
3110810110125%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
0810 - Finfish;

Field Of Science
1070 - Ecology; 1080 - Genetics; 1090 - Immunology; 1101 - Virology;
Goals / Objectives
We propose to develop informative models of transmission dynamics for infectious hematopoietic necrosis (IHN) virus, a globally important pathogen of salmon and trout. This study will focus on documented viral emergence and displacement events that have occurred in Pacific Northwest river systems by testing IHN viral genotypes associated with these events in relevant fish host populations that vary in IHN virus susceptibility. Aim 1 will quantify the relative importance of spatially- and temporally-associated variables in IHN viral epidemic events and genotype divergence in Pacific Northwest river systems. Aim 2 will define viral factors associated with transmission and fitness of four IHN viral variants involved in emergence and displacement events in the field. Aim 3 will quantify the interaction between genetic and environmental factors that affect host susceptibility to viral disease and emergence. Aim4 will integrate experimental and field data in a Bayesian hierarchical model to generate inference about viral transmission, prediction of emergence, and testing of virus control strategies. We will rapidly disseminate the result of our interdisciplinary research through established ties with specific groups that can utilize the data to inform policy decisions about fish resource management. Dissemination of results will be achieved by direct interaction with these groups, and by continued updating of our existing online database of IHN virus genetic typing. A new synthetic database will also be a product of Aim 1 and will also be provided freely online on the Western Fisheries Research Center (U.S. Geological Survey) website. As part of our outreach plan we will develop a curriculum entitled Fundamental Drivers of Aquatic Pathogen Transmission that will be offered at the end of the project as a continuing education course at the annual AFS Fish Health meeting, and as an online version available through the Western Fisheries Research Center websites
Project Methods
In Aim 1, we will create a synthetic database that includes all available diagnostic and genetic typing data for field-collected infectious hematopoietic necrosis (IHN) virus samples and the associated measures of environmental and ecological conditions. This will be done by careful reconciliation of reports from various agencies and integration of geospatial data at varying scales. This database will allow us to evaluate ecological determinants of IHN viral transmission and interactions between environmental and/or population characteristics that may influence transmission and viral genotype emergence. In Aim 2, the goal is to define virulence, infectious dose and virus shedding of four IHN viral types under laboratory conditions. These four IHN virus types sequentially emerged in the steelhead trout in the Columbia River basis with each field emergence displacing the former type. This analysis will determine the statistical differences in virulence between types and how it correlates with viral displacement and emergence. We will assess the transmission potential of the four viral types in terms of the kinetics and quantity of virus shed from infected fish. Viral fitness in co-infection and super-infection for pairs of IHN viral types associated with field displacement events will also be evaluated. Finally, we will address the probabilities of long-term infection outcomes and determine the frequencies and duration of viral persistence compared to the frequency of viral clearance over a 1 year period. In Aim 3, the focus shifts to the interaction between genetic and environmental factors that affect host susceptibility to viral disease and emergence. We will compare the reactions of host populations that differ in IHNV susceptibility, under two different temperature regimes, to determine whether host susceptibility a stable trait when environmental conditions shift and if the kinetics of the immune response and infection dynamics differ depending on temperature. Markers associated with IHNV resistance will be identified using genome wide association studies and these markers will be evaluated in other steelhead populations. In Aim 4, we will integrate experimental and field data in a Bayesian hierarchical model to generate inference about viral transmission, prediction of emergence, and testing of control strategies. This will include defining landscape-level dynamics of IHNV and assessing the relative importance of different IHNV transmission routes. This will allow us to test hypotheses regarding IHNV persistence in populations, the influence of host susceptibility and environment on IHNV expansion, and how management actions influences IHNV transmission. Furthermore, we will address the hypothesis that areas where multiple viral types have emerged in the past will share predictive traits with future sites of emergence and displacement. Finally, the effects of management options for dealing with epidemics and their effects on potential emergence events will be evaluated within the model.

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.

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