Susceptibility of Pallid Sturgeon to viral hemorrhagic septicemia virus genotype IVb.

OBJECTIVE: Viral hemorrhagic septicemia virus (VHSV) is an aquatic rhabdovirus causing severe disease in freshwater and saltwater fish species. The susceptibility of endangered Pallid Sturgeon Scaphirhynchus albus to VHSV genotype IVb (VHSV-IVb) infection was investigated. METHODS: An in vitro assessment using two Pallid Sturgeon cell lines derived from skin and spleen tissue and in vivo evaluation of juvenile Pallid Sturgeon after exposure to VHSV-IVb were performed. RESULT: Plaque assay and RT-PCR results confirmed VHSV-IVb replication in Pallid Sturgeon cell lines. Sturgeon were also susceptible to VHSV-IVb infection after immersion and injection exposures during laboratory experiments. However, after widespread mortality occurred in all treatment groups, including negative control fish, it was determined that the Pallid Sturgeon stock fish were infected with Missouri River sturgeon iridovirus (MRSIV) prior to experimental challenge. Nevertheless, mortalities were equal or higher among VHSV-exposed fish than among negative controls (MRSIV infected), and histopathological assessments indicated reduced hematopoietic cells in spleen and kidney tissues and hemorrhage in the gastrointestinal organs only in fish from the VHSV treatment. CONCLUSION: These results indicate that Pallid Sturgeon is a susceptible host for VHSV-IVb, but the degree of pathogenicity was confounded by the underlying MRSIV infection. Research comparing susceptibility of specific pathogen-free and MRSIV-infected fish to VHSV-IVb is needed to accurately assess the vulnerability of Pallid Sturgeon to VHSV-IVb.

Comparative virulence of spring viremia of carp virus (SVCV) genotypes in two koi varieties.

Spring viremia of carp virus (SVCV), is a lethal freshwater pathogen of cyprinid fish, and Cyprinus carpio koi is a primary host species. The virus was initially described in the 1960s after outbreaks occurred in Europe, but a global expansion of SVCV has been ongoing since the late 1990s. Genetic typing of SVCV isolates separates them into 4 genotypes that are correlated with geographic origin: Ia (Asia), Ib and Ic (Eastern Europe), and Id (Central Europe). We compared infectivity and virulence of 8 SVCV strains, including 4 uncharacterized Chinese Ia isolates and representatives of genotypes Ia-d in 2 morphologically distinct varieties of koi: long-fin semi-scaled Beni Kikokuryu koi and short-fin fully scaled Sanke koi. Mortality ranged from 4 to 82% in the Beni Kikokuryu koi and 0 to 94% in the Sanke koi following immersion challenge. Genotype Ia isolates of Asian origin had a wide range in virulence (0-94%). Single isolates representing the European genotypes Ib and Ic were moderately virulent (38-56%). Each virus strain produced similar levels of mortality in both koi breeds, with the exception of the SVCV Id strain that appeared to have both moderate and high virulence phenotypes (60% in Beni Kikokuryu koi vs. 87% in Sanke koi). Overall SVCV strain virulence appeared to be a dominant factor in determining disease outcomes, whereas intraspecies variation, based on koi variety, had less of an impact. This study is the first side-by-side comparison of Chinese SVCV isolates and genotype Ia-d strain virulence in a highly susceptible host.

Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA.

Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

Molecular identification of erythrocytic necrosis virus (ENV) from the blood of Pacific herring (Clupea pallasii).

Viral erythrocytic necrosis (VEN) is a condition affecting the red blood cells of more than 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. Among populations of Pacific herring (Clupea pallasii) on the west coast of North America the disease causes anemia and elevated mortality in periodic epizootics. Presently, VEN is diagnosed by observation of typical cytoplasmic inclusion bodies in stained blood smears from infected fish. The causative agent, erythrocytic necrosis virus (ENV), is unculturable and a presumed iridovirus by electron microscopy. In vivo amplification of the virus in pathogen-free laboratory stocks of Pacific herring with subsequent virus concentration, purification, DNA extraction, and high-throughput sequencing were used to obtain genomic ENV sequences. Fragments with the highest sequence identity to the family Iridoviridae were used to design four sets of ENV-specific polymerase chain reaction (PCR) primers. Testing of blood and tissue samples from experimentally and wild infected Pacific herring as well as DNA extracted from other amphibian and piscine iridoviruses verified the assays were specific to ENV with a limit of detection of 0.0003 ng. Preliminary phylogenetic analyses of a 1448 bp fragment of the putative DNA polymerase gene supported inclusion of ENV in a proposed sixth genus of the family Iridoviridae that contains other erythrocytic viruses from ectothermic hosts. This study provides the first molecular evidence of ENV's inclusion within the Iridoviridae family and offers conventional PCR assays as a means of rapidly surveying the ENV-status of wild and propagated Pacific herring stocks.

Susceptibility of Koi and Yellow Perch to infectious hematopoietic necrosis virus by experimental exposure.

Infectious hematopoietic necrosis virus (IHNV) is a novirhabdoviral pathogen that originated in western North America among anadromous Pacific salmonids. Severe disease epidemics in the late 1970s resulting from IHNV's invasion into farmed Rainbow Trout Oncorhynchus mykiss in North America, Asia, and Europe emphasized IHNV's ability to adapt to new hosts under varying rearing conditions. Yellow Perch Perca flavescens and Koi Carp Cyprinus carpio (hereafter, "Koi") are aquaculture-reared fish that are highly valued in sport fisheries and the ornamental fish trade, respectively, but it is unknown whether these fish species are vulnerable to IHNV infection. In this study, we exposed Yellow Perch, Koi, and steelhead (anadromous Rainbow Trout) to IHNV by intraperitoneal injection (10(6) PFU/fish) and by immersion (5.7×10(5) PFU/mL) for 7 h, and monitored fish for 28 d. The extended immersion exposure and high virus concentrations used in the challenges were to determine if the tested fish had any level of susceptibility. After experimental exposure, Yellow Perch and Koi experienced low mortality (<6%) compared with steelhead (>35%). Virus was found in dead fish of all species tested and in surviving Yellow Perch by plaque assay and quantitative reverse transcription polymerase chain reaction (qPCR), with a higher prevalence in Yellow Perch than Koi. Infectious virus was also detected in Yellow Perch out to 5 d after bath challenge. These findings indicate that Yellow Perch and Koi are highly resistant to IHNV disease under the conditions tested, but Yellow Perch are susceptible to infection and may serve as possible virus carriers.

Virulence of viral hemorrhagic septicemia virus (VHSV) genotypes Ia, IVa, IVb, and IVc in five fish species.

The susceptibility of yellow perch Perca flavescens, rainbow trout Oncorhynchus mykiss, Chinook salmon O. tshawytscha, koi Cyprinus carpio koi, and Pacific herring Clupea pallasii to 4 strains of viral hemorrhagic septicemia virus (VHSV) was assessed. Fish were challenged via intraperitoneal injection with high (1 × 106 plaque-forming units, PFU) and low (1 × 103 PFU) doses of a European strain (genotype Ia), and North American strains from the West coast (genotype IVa), Great Lakes (genotype IVb), and the East coast (genotype IVc). Pacific herring were exposed to the same VHSV strains, but at a single dose of 5 × 103 PFU ml-1 by immersion in static seawater. Overall, yellow perch were the most susceptible, with cumulative percent mortality (CPM) ranging from 84 to 100%, and 30 to 93% in fish injected with high or low doses of virus, respectively. Rainbow trout and Chinook salmon experienced higher mortalities (47 to 98% CPM) after exposure to strain Ia than to the other virus genotypes. Pacific herring were most susceptible to strain IVa with an average CPM of 80% and moderately susceptible (42 to 52% CPM) to the other genotypes. Koi had very low susceptibility (≤5.0% CPM) to all 4 VHSV strains. Fish tested at 7 d post challenge were positive for all virus strains, with yellow perch having the highest prevalence and concentrations of virus, and koi the lowest. While genotype Ia had higher virulence in salmonid species, there was little difference in virulence or host-specificity between isolates from subtypes IVa, IVb, and IVc.

Kinetics of viral load and erythrocytic inclusion body formation in Pacific herring artificially infected with erythrocytic necrosis virus.

Viral erythrocytic necrosis (VEN) is a condition that affects marine and anadromous fish species, including herrings and salmonids, in the Atlantic and Pacific oceans. Infection is frequently associated with severe anemia and causes episodic mortality among wild and hatchery fish when accompanied by additional stressors; VEN can be presumptively diagnosed by (1) light microscopic identification of a single characteristic-a round, magenta-colored, 0.8-µm-diameter inclusion body (IB) within the cytoplasm of erythrocytes and their precursors on Giemsa-stained blood films; or (2) observation (via transmission electron microscopy [TEM]) of the causative iridovirus, erythrocytic necrosis virus (ENV), within erythrocytes or their precursors. To better understand the kinetics of VEN, specific-pathogen-free Pacific herring Clupea pallasii were infected with ENV by intraperitoneal injection. At 1, 4, 7, 10, 14, 21, and 28 d postexposure, samples of blood, spleen, and kidney were collected and assessed (1) via light microscopy for the number of intracytoplasmic IBs in blood smears and (2) via TEM for the number of virions within erythrocytes. The mean prevalence of intracytoplasmic IBs in the blood cells increased from 0% at 0-4 d postexposure to 94% at 28 d postexposure. Viral load within circulating red blood cells peaked at 7 d postexposure, fell slightly, and then reached a plateau. However, blood cells observed within the kidney and spleen tissues demonstrated high levels of ENV between 14 and 28 d postexposure. The results indicate that the viral load within erythrocytes does not correlate well with IB prevalence and that the virus can persist in infected fish for more than 28 d.

Characterization of the mutant spectra of a fish RNA virus within individual hosts during natural infections.

Infectious hematopoietic necrosis virus (IHNV) is an RNA virus that causes significant mortalities of salmonids in the Pacific Northwest of North America. RNA virus populations typically contain genetic variants that form a heterogeneous virus pool, referred to as a quasispecies or mutant spectrum. This study characterized the mutant spectra of IHNV populations within individual fish reared in different environmental settings by RT-PCR of genomic viral RNA and determination of partial glycoprotein gene sequences of molecular clones. The diversity of the mutant spectra from ten in vivo populations was low and the average mutation frequencies of duplicate populations did not significantly exceed the background mutation level expected from the methodology. In contrast, two in vitro populations contained variants with an identical mutational hot spot. These results indicated that the mutant spectra of natural IHNV populations is very homogeneous, and does not explain the different magnitudes of genetic diversity observed between the different IHNV genogroups. Overall the mutant frequency of IHNV within its host is one of the lowest reported for RNA viruses.

Phylogeography of infectious haematopoietic necrosis virus in North America.

Infectious hematopoietic necrosis virus (IHNV) is a rhabdoviral pathogen that infects wild and cultured salmonid fish throughout the Pacific Northwest of North America. IHNV causes severe epidemics in young fish and can cause disease or occur asymptomatically in adults. In a broad survey of 323 IHNV field isolates, sequence analysis of a 303 nucleotide variable region within the glycoprotein gene revealed a maximum nucleotide diversity of 8.6 %, indicating low genetic diversity overall for this virus. Phylogenetic analysis revealed three major virus genogroups, designated U, M and L, which varied in topography and geographical range. Intragenogroup genetic diversity measures indicated that the M genogroup had three- to fourfold more diversity than the other genogroups and suggested relatively rapid evolution of the M genogroup and stasis within the U genogroup. We speculate that factors influencing IHNV evolution may have included ocean migration ranges of their salmonid host populations and anthropogenic effects associated with fish culture.