Cooperative Fish and Wildlife Research Units Program: Washington
Education, Research and Technical Assistance for Managing Our Natural Resources

Many of the most important and destructive pathogens of finfish are viruses in the family Rhabdoviridae. The rhabdovirus Infectious hematopoietic necrosis virus (IHNV) is the most significant viral pathogen of salmonid fish hosts and it is endemic to nearly all Pacific watersheds of western North America. Numerous field isolates of IHNV have been characterized and found to fall into three major genetic groups, called genogroups U, M, and L. Geographic ranges of U and M genogroup IHNV overlap in much of the Columbia River basin. Individual strains are known to differ in virulence and show host-specific virulence, e.g. U genogroup IHNV has high virulence in sockeye salmon (Oncorhynchus nerka), but low virulence in rainbow trout (O. mykiss). Conversely, M genogroup IHNV has low virulence in sockeye salmon but high virulence in rainbow trout. The mechanistic basis of these virulence differences is unknown. This project will use various experimental approaches to explore and elucidate the basis of virulence of IHNV. This will include investigations of viral features and also host immune responses to both viral infection and vaccination against IHNV. In addition to well established endemic viruses such as IHNV, there are recently emerging viruses of great importance, such as the rhabdovirus Viral hemorrhagic septicemia virus (VHSV) in the Great Lakes. As time and funding permit researchers will explore viral and host factors associated with virulence of multiple strains of VHSV and possibly other important viruses that cause mortality in wild or cultured fish in North America. Objectives Objective 1. Investigate virulence and pathogenicity of important established and emerging fish viral pathogens Objective 2. Investigate viral fitness and mechanisms of viral virulence Objective 3. Characterize host immune response to viral infection Objective 4. Characterize vaccine efficacy and immune response to vaccination Methods Objective 1. Investigate virulence and pathogenicity of important established and emerging fish viral pathogens Virulence of different field strains of IHNV will be characterized in controlled wet-laboratory challenges. Variables will include different host fish species and/or stocks, different fish ages, different viral challenge routes (immersion or intraperitoneal injection), or different doses of challenge virus. Challenges will typically be done in duplicate groups of 20 fish, with mock-challenged groups to control for non-viral associated mortality. Virulence will be defined in terms of final cumulative percent mortality (CPM) after a 28 day observation period, and kinetics of mortality curves over time. As funding permits, virulence of different strains of important emerging fish viruses such as VHSV may also be examined, using strict containment in our Aquatic Biosafety level 3 wetlab when appropriate. The goal of this objective is to characterize variation in viral virulence and identify strains that reproducibly demonstrate high and low virulence under specific controlled conditions. Objective 2. Investigate viral fitness and mechanisms of viral virulence For viruses fitness is defined as the ability to replicate and produce infectious progeny in a given environment. Replication of different virus strains will be assessed and compared in in vivo growth curve studies in which fish are infected with individual strains in batch and then separated into individual beakers to prevent cross-contamination. At specified time points 1-10 days post-infection 5-15 fish will be euthanized and viral progeny will be quantified in each fish using a quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) assay for viral RNA. Rate of replication and maximum yield attained will be measures of viral fitness: our hypothesis will be that more virulent viruses will have higher fitness, i.e. they will replicate faster and/or to higher yield. Competitive fitness of selected pairs of virus strains will also be tested in co-infections to see if higher virulence viruses out-compete lower virulence viruses in the same host. These assays will require development of genotype-specific qRT-PCR assays to quantify each viral strain within mixed strain progeny populations. Objective 3. Characterize host immune response to viral infection Selected fish host species will be infected with high and low virulence IHNV strains and at specified time points subgroups of fish will be euthanized to collect samples of various tissues (e.g. kidney, spleen). Innate immune responses of these fish will be characterized by extracting RNA from these tissue samples and conducting qRT-PCR assays for up-regulation (or down-regulation) of innate immune genes such as interferons, interferon-stimulated genes (ISGs, such as Mx), and virally-induced genes (VIGs) such as Vig-1 and Vig-8. Adaptive immunity will be similarly profiled at later time points with assays for immune-related genes such as IgM, T-cell receptor, Il-10 and Il-12. The goal of this objective is to profile the immune responses qualitatively (which genes are turned on) and quantitatively (magnitude of increase in gene expression). Adaptive immunity will also be assessed by quantifying IHNV-specific neutralizing antibody (NAb) titers and NAb specificity in sera collected from fish 6-12 weeks post-infection. We hypothesize that the immune responses to high and low virulence virus strains will differ in ways that are informative for understanding the basis of virulence. As funding permits, immune responses to other viral species may be characterized for comparison with findings from IHNV studies. Objective 4. Characterize vaccine efficacy and immune response to vaccination Although vaccines for fish rhabdoviruses have been explored since the 1970s there are no commercially available vaccines for IHNV or VHSV in widespread use. In the last ten years novel vaccines comprised of plasmid DNA carrying the viral surface glycoprotein (G protein) have been shown to be exceptionally successful in eliciting protective immunity against IHNV or VHSV. We have constructed DNA vaccines carrying G protein genes from both U and M genogroup IHNV strains. Efficacy of these vaccines will be compared in different fish hosts, and the innate and adaptive immune responses stimulated by the vaccines will be characterized and compared as described in objective 3. The results of these experiments will allow us to compare the immune responses to vaccination with the responses to viral infection, and will contribute to continued development of these novel vaccines for eventual use in cultured fish.