Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonids in North America, Europe and Asia that is phylogenetically classified into five major virus genogroups (U, M, L, E and J). The geographic range of the U and M genogroup isolates overlap in the North American Columbia River Basin and Washington Coast region, where these genogroups pose different risks depending on the species of Pacific salmon (Oncorhynchus spp.). For certain management decisions, there is a need to both test for IHNV presence and rapidly determine the genogroup. Herein, we report the development and validation of a U/M multiplex reverse transcription, real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) protein gene. The new U/M RT-rPCR is a rapid, sensitive, and repeatable assay capable of specifically discriminating between North American U and M genogroup IHNV isolates. However, one M genogroup isolate obtained from commercially cultured Idaho rainbow trout (O. mykiss) showed reduced sensitivity with the RT-rPCR test, suggesting caution may be warranted before applying RT-rPCR as the sole surveillance test in areas associated with the Idaho trout industry. The new U/M assay had high diagnostic sensitivity (DSe > 94%) and specificity (DSp > 97%) in free-ranging adult Pacific salmon, when assessed relative to cell culture (reference standard) and the previously validated universal N RT-rPCR. The good diagnostic performance of the new U/M assay indicates the new test is suitable for surveillance, diagnosis, and confirmation of IHNV in Pacific salmon from the Pacific Northwest regions where the U and M genogroups overlap.

Infectious hematopoietic necrosis virus (IHNV) represents one of the most critical challenges for salmonids in the Pacific Northwest. There are three genogroups of IHNV, designated U, M, and L; the U is further delineated into two subgroups, UC and UP, and the M is further delineated into four subgroups (MA – MD). The UP, UC and MD subgroups co-occur in the Columbia River Basin where the host species sockeye salmon, Chinook salmon, and steelhead trout spawn and rear. Field prevalence data shows that UC viruses exhibit a generalist strategy in Chinook, and steelhead, while two other virus lineages, MD and UP, are more consistent with being specialists in steelhead or sockeye salmon, respectively. The L is found in Northern California and is considered a specialist of Chinook salmon. This study sought to understand early entry and replication of the specialist and generalist IHNV strains in three salmonid hosts. Chinook, steelhead and sockeye were exposed by immersion to their specialist viruses (L, MD and UP, respectively) and to the generalist UC virus. As controls, these hosts were also exposed to buffer (mock control) and IHNV variants that were not specialist for their species (non-specialists). Mortality was monitored throughout the experiments. Fish were sampled at early timepoints post-infection (2, 5, and 8 days post-exposure). Kidney and fin tissues were taken to represent external and internal tissues, respectively. Viral load was assessed by reverse transcriptase quantitative PCR (RT-qPCR) targeting the nucleocapsid (N) gene of IHNV. Additional fish from each experimental group were sampled for histopathology but only a subset were processed and analyzed.

Infectious hematopoietic necrosis virus (IHNV) represents one of the most critical challenges for salmonids in the Pacific Northwest. There are three genogroups of IHNV, designated U, M, and L; the U is further delineated into two subgroups, UC and UP, and the M is further delineated into four subgroups (MA – MD). The UP, UC and MD subgroups co-occur in the Columbia River Basin where the host species sockeye salmon, Chinook salmon, and steelhead trout spawn and rear. Field prevalence data shows that UC viruses exhibit a generalist strategy in Chinook, and steelhead, while two other virus lineages, MD and UP, are more consistent with being specialists in steelhead or sockeye salmon, respectively. The L is found in Northern California and is considered a specialist of Chinook salmon. This study sought to understand early entry and replication of the specialist and generalist IHNV strains in three salmonid hosts. Chinook, steelhead and sockeye were exposed by immersion to their specialist viruses (L, MD and UP, respectively) and to the generalist UC virus. As controls, these hosts were also exposed to buffer (mock control) and IHNV variants that were not specialist for their species (non-specialists). Mortality was monitored throughout the experiments. Fish were sampled at early timepoints post-infection (2, 5, and 8 days post-exposure). Kidney and fin tissues were taken to represent external and internal tissues, respectively. Viral load was assessed by reverse transcriptase quantitative PCR (RT-qPCR) targeting the nucleocapsid (N) gene of IHNV. Additional fish from each experimental group were sampled for histopathology but only a subset were processed and analyzed.

This investigation sought to characterize the shedding of infectious hematopoietic necrosis virus (IHNV) in two populations of Columbia River Basin (CRB) Chinook salmon (Oncorhynchus tshawytscha). Juvenile spring- and fall-run Chinook salmon were exposed by immersion to each of three IHN virus strains from the UC, MD, and L subgroups, and then monitored for viral shedding from individual fish for 30 days. Detectable quantities of UC, MD and L IHN virus were shed by a subset of fish from each host population (1–9 out of 10 fish total in each treatment group). Viral shedding kinetics were consistent, with a rapid onset of shedding, peak shedding by 2–3 days, and then a rapid decline to below detectable levels by 7 days’ post-exposure to IHNV. Intraspecies variation was observed as spring Chinook salmon shed more UC virus than fall fish: spring Chinook salmon shed UC virus in greater numbers of fish, with 22-fold higher mean peak shedding magnitude, 33-fold higher mean total virus shed per fish, and 900-fold higher total virus shed per treatment group. The L and MD viruses had comparable shedding at intermediate levels in each host population. All viral shedding occurred well before host mortality began, and shedding magnitude did not correlate with virulence differences. Overall, the greater shedding of UC virus from spring Chinook salmon, combined with low virulence, indicates a uniquely high transmission potential that may explain the predominance of UC viruses in CRB Chinook salmon. This also suggests that spring-run fish may contribute more to the ecology of IHNV in the CRB than fall-run Chinook salmon.

This investigation sought to characterize the shedding of infectious hematopoietic necrosis virus (IHNV) in two populations of Columbia River Basin (CRB) Chinook salmon (Oncorhynchus tshawytscha). Juvenile spring- and fall-run Chinook salmon were exposed by immersion to each of three IHN virus strains from the UC, MD, and L subgroups, and then monitored for viral shedding from individual fish for 30 days. Detectable quantities of UC, MD and L IHN virus were shed by a subset of fish from each host population (1–9 out of 10 fish total in each treatment group). Viral shedding kinetics were consistent, with a rapid onset of shedding, peak shedding by 2–3 days, and then a rapid decline to below detectable levels by 7 days’ post-exposure to IHNV. Intraspecies variation was observed as spring Chinook salmon shed more UC virus than fall fish: spring Chinook salmon shed UC virus in greater numbers of fish, with 22-fold higher mean peak shedding magnitude, 33-fold higher mean total virus shed per fish, and 900-fold higher total virus shed per treatment group. The L and MD viruses had comparable shedding at intermediate levels in each host population. All viral shedding occurred well before host mortality began, and shedding magnitude did not correlate with virulence differences. Overall, the greater shedding of UC virus from spring Chinook salmon, combined with low virulence, indicates a uniquely high transmission potential that may explain the predominance of UC viruses in CRB Chinook salmon. This also suggests that spring-run fish may contribute more to the ecology of IHNV in the CRB than fall-run Chinook salmon.

Viral hemorrhagic septicemia virus (VHSV) is an aquatic rhabdovirus causing severe disease in numerous freshwater and saltwater fish species. Initially the virus was been found to cause disease in European fish populations starting around 1938 and was first detected in North America in the late 1980s. Of the four VHSV genotypes (I, II, III, and IV), the North American subtype IVb isolates have a broad host range. To determine whether endangered pallid sturgeon Scaphirhynchus albus, are susceptible to VHSV-IVb infection, juvenile pallid sturgeon and two pallid sturgeon cell lines derived from skin and spleen tissue were tested. Detection of viable virus via a plaque assay and molecular detection of the virus results by a RT-PCR (reverse transcription-polymerase chain reaction) confirmed VHSV-IVb in vitro replication in pallid sturgeon cell lines. Pallid sturgeon were also susceptible to VHSV-IVb infection when exposed to the virus by immersion at concentration of 5 X 10e5 plaque forming units per milliliter (pfu/ml) and by injection at a dose of 1 X 10e6 plaque forming units per fish (pfu/fish) during 28-day long challenge experiments. However, after widespread mortality occurred in all treatment groups, including control fish, it was determined that the pallid sturgeon stock fish were infected with Missouri River Sturgeon Iridovirus (MRSIV) prior to experimental challenge. Nevertheless, VHSV-exposed fish suffered equal or higher mortalities (38 – 48%) than mock treated (MRSIV-infected) fish (29 – 38%) and histopathology samples showed reduced hematopoietic cells in spleen and kidney tissues and hemorrhage in the gastrointestinal organs only in VHSV-treated fish. These results suggest that pallid sturgeon are susceptible to VHSV-IVb infection, but the degree of pathogenicity was confounded by the underlying MRSIV infection. Our data also suggest that pallid sturgeon may serve as carriers of VHSV because the virus was isolated from surviving fish that showed no clinical signs, yet were positive for both VHSV and MRSIV. Research comparing susceptibility of pathogen-free and MRSIV-infected fish to VHSV-IVb is needed to accurately assess the vulnerability of pallid sturgeon to VHSV-IVb.

Viral hemorrhagic septicemia virus (VHSV) is an aquatic rhabdovirus causing severe disease in numerous freshwater and saltwater fish species. Initially the virus was been found to cause disease in European fish populations starting around 1938 and was first detected in North America in the late 1980s. Of the four VHSV genotypes (I, II, III, and IV), the North American subtype IVb isolates have a broad host range. To determine whether endangered pallid sturgeon Scaphirhynchus albus, are susceptible to VHSV-IVb infection, juvenile pallid sturgeon and two pallid sturgeon cell lines derived from skin and spleen tissue were tested. Detection of viable virus via a plaque assay and molecular detection of the virus results by a RT-PCR (reverse transcription-polymerase chain reaction) confirmed VHSV-IVb in vitro replication in pallid sturgeon cell lines. Pallid sturgeon were also susceptible to VHSV-IVb infection when exposed to the virus by immersion at concentration of 5 X 10e5 plaque forming units per milliliter (pfu/ml) and by injection at a dose of 1 X 10e6 plaque forming units per fish (pfu/fish) during 28-day long challenge experiments. However, after widespread mortality occurred in all treatment groups, including control fish, it was determined that the pallid sturgeon stock fish were infected with Missouri River Sturgeon Iridovirus (MRSIV) prior to experimental challenge. Nevertheless, VHSV-exposed fish suffered equal or higher mortalities (38 – 48%) than mock treated (MRSIV-infected) fish (29 – 38%) and histopathology samples showed reduced hematopoietic cells in spleen and kidney tissues and hemorrhage in the gastrointestinal organs only in VHSV-treated fish. These results suggest that pallid sturgeon are susceptible to VHSV-IVb infection, but the degree of pathogenicity was confounded by the underlying MRSIV infection. Our data also suggest that pallid sturgeon may serve as carriers of VHSV because the virus was isolated from surviving fish that showed no clinical signs, yet were positive for both VHSV and MRSIV. Research comparing susceptibility of pathogen-free and MRSIV-infected fish to VHSV-IVb is needed to accurately assess the vulnerability of pallid sturgeon to VHSV-IVb.

Theory of the evolution of pathogen specialization suggests that a specialist pathogen gains high fitness in one host, but this comes with fitness loss in other hosts. By contrast, a generalist pathogen does not achieve high fitness in any host, but gains ecological fitness by exploiting different hosts, and has higher fitness than specialists in non-specialized hosts. As a result, specialist pathogens are predicted to have greater variation in fitness across hosts, and generalists would have lower fitness variation across hosts. We test these hypotheses by measuring pathogen replicative fitness as within-host viral loads from the onset of infection to the beginning of virus clearance, using the rhabdovirus infectious hematopoietic necrosis virus (IHNV) in salmonid fish. Based on field prevalence and virulence studies, IHNV subgroups UP, MD, and L are specialists, causing infection and mortality in sockeye salmon, steelhead, and Chinook salmon juveniles, respectively. The UC subgroup evolved naturally from a UP ancestor and is a generalist infecting all three host species but without causing severe disease. We show that specialist subgroups had highest peak and mean viral loads in the hosts in which they are specialized, and they had low viral loads in non-specialized hosts, resulting in large variation in viral load across hosts. Viral kinetics show that the mechanisms of specialization involve both the ability to maximize early virus replication and to avoid clearance at later times, with different mechanisms of specialization evident in different host-virus combinations. Additional nuances in the data included different fitness levels for non-specialist interactions, reflecting different trade-offs for specialist viruses in other hosts. The generalist UC subgroup reached intermediate viral loads in all hosts and showed the smallest variation in fitness across hosts. Evolution of the UC generalist from an ancestral UP sockeye specialist was associated with fitness increases in steelhead and Chinook salmon, but only slight decrease in fitness in sockeye salmon, consistent with low- or no-cost generalism. Our results support major elements of specialist-generalist theory, providing evidence of a specialist-generalist continuum in a vertebrate pathogen. These results also quantify within-host replicative fitness tradeoffs resulting from the natural evolution of specialist and generalist virus lineages in multi-host ecosystems.

Environmental variation has important effects on host-pathogen interactions, affecting large-scale ecological processes such as the severity and frequency of epidemics. However, less is known about how the environment modulates viral fitness traits and within host infection processes. Viral genetic variation, fish host immune response and environmental parameters such as temperature have been reported to strongly influence the replication and clearance of infectious hematopoietic necrosis virus (IHNV), a significant pathogen of salmon and trout. Here, we quantified the effect of water temperature on probability of infection across time following exposure of steelhead (Oncorhynchus mykiss) to a field isolate of IHNV. Warm water temperatures accelerated IHNV replication compared to colder water temperatures in cell lines, which have a more limited host immune response. In vivo challenge experiments also demonstrated a higher replication rate of IHNV at warmer water temperatures, but IHNV persisted for a shorter amount of time at these warmer temperatures and led to lower overall mortality compared to colder temperatures. Furthermore, fish were found to have higher prevalence of neutralizing antibodies at warmer water temperatures compared to colder temperatures. These results support the hypothesis that IHNV clearance or persistence is modulated by temperature, and this difference was influenced by temperature effects on the host immune responses. At later time points, the viral RNA that persisted was most commonly localized in the kidney and spleen pooled tissue; these tissues are composed of hematopoietic cells that are favored targets of the virus. By partitioning the effect of environmental variation into independent and common effects of host and pathogen responses, we can better understand the environmental regulation of host-pathogen interactions within hosts. Our results therefore provide insights into how different host-pathogen systems could react to environmental change.

Environmental variation has important effects on host-pathogen interactions, affecting large-scale ecological processes such as the severity and frequency of epidemics. However, less is known about how the environment modulates viral fitness traits and within host infection processes. Viral genetic variation, fish host immune response and environmental parameters such as temperature have been reported to strongly influence the replication and clearance of infectious hematopoietic necrosis virus (IHNV), a significant pathogen of salmon and trout. Here, we quantified the effect of water temperature on probability of infection across time following exposure of steelhead (Oncorhynchus mykiss) to a field isolate of IHNV. Warm water temperatures accelerated IHNV replication compared to colder water temperatures in cell lines, which have a more limited host immune response. In vivo challenge experiments also demonstrated a higher replication rate of IHNV at warmer water temperatures, but IHNV persisted for a shorter amount of time at these warmer temperatures and led to lower overall mortality compared to colder temperatures. Furthermore, fish were found to have higher prevalence of neutralizing antibodies at warmer water temperatures compared to colder temperatures. These results support the hypothesis that IHNV clearance or persistence is modulated by temperature, and this difference was influenced by temperature effects on the host immune responses. At later time points, the viral RNA that persisted was most commonly localized in the kidney and spleen pooled tissue; these tissues are composed of hematopoietic cells that are favored targets of the virus. By partitioning the effect of environmental variation into independent and common effects of host and pathogen responses, we can better understand the environmental regulation of host-pathogen interactions within hosts. Our results therefore provide insights into how different host-pathogen systems could react to environmental change.