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