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In British Columbia, Canada (BC), aquaculture of finfish in ocean netpens has the potential for pathogen transmission between wild and farmed species due to the sharing of an aquatic environment. Viral hemorrhagic septicemia virus (VHSV) is enzootic in BC and causes serious disease in wild Pacific herring, Clupea pallasii, which often enter and remain in Atlantic salmon, Salmo salar, netpens. Isolation of VHSV from farmed Atlantic salmon has been previously documented, but the effects on the health of farmed salmon and the wild fish sharing the environment are unknown. To determine their susceptibility, Atlantic salmon were exposed to a pool of 9 isolates of VHSV obtained from farmed Atlantic salmon in BC by IP-injection or by waterborne exposure and cohabitation with diseased Pacific herring. Disease intensity was quantified by recording mortality, clinical signs, histopathological changes, cellular sites of viral replication, expression of interferon-related genes, and viral tissue titers. Disease ensued in Atlantic salmon after both VHSV exposure methods. Fish demonstrated gross disease signs including darkening of the dorsal skin, bilateral exophthalmia, light cutaneous hemorrhage, and lethargy. The virus replicated within endothelial cells causing endothelial cell necrosis and extensive hemorrhage in anterior kidney. Infected fish demonstrated a type I interferon response as seen by up-regulation of genes for IFNα, Mx, and ISG15. In a separate trial infected salmon transmitted the virus to sympatric Pacific herring. The results demonstrate that farmed Atlantic salmon can develop clinical VHS and virus can persist in the tissues for at least 10 weeks. Avoiding VHS epizootics in Atlantic salmon farms would limit the potential of VHS in farmed Atlantic salmon, the possibility for further host adaptation in this species, and virus spillback to sympatric wild fishes., (Published by Elsevier B.V.)

Viral haemorrhagic septicaemia virus (VHSV) is a fish pathogen found throughout the Northern Hemisphere and is capable of infecting and causing mortality in numerous marine and freshwater hosts. In the coastal waters of British Columbia, Canada, the virus has been detected for 20 yr with many occurrences of mass mortalities among populations of Pacific herring Clupea pallasii (Valenciennes) and sardine Sardinops sagax as well as detections among cultured Atlantic Salmo salar and Chinook Oncorhynchus tshawytscha salmon. We compared nucleotide sequence of the full glycoprotein (G) gene coding region (1524 nt) of 63 VHSV isolates sampled during its recorded presence from 1993 to 2011 from 6 species and a total of 29 sites. Phylogenetic analysis showed that all isolates fell into sub-lineage IVa within the major VHSV genetic group IV. Of the 63 virus isolates, there were 42 unique sequences, each of which was ephemeral, being repeatedly detected at most only 1 yr after its initial detection. Multiple sequence types were revealed during single viral outbreak events, and genetic heterogeneity was observed within isolates from individual fish. Moreover, phylogenetic analysis revealed a close genetic linkage between VHSV isolates obtained from pelagic finfish species and farmed salmonids, providing evidence for virus transmission from wild to farmed fish.

Viral haemorrhagic septicaemia virus (VHSV) is the agent of a disease that causes mortality events in marine and freshwater fish. It is one of the most important pathogens in European rainbow trout (Oncorhynchus mykiss) aquaculture. Four major genotypes of the virus are recognised reflecting different geographic and host ranges. Genotyping of VHS isolates is important for disease management enabling monitoring of disease spread into new geographical regions or susceptible species. This study sought to develop molecular tools for rapid and efficient classification of European VHSV genotypes. Specificity of genotype-specific real-time reverse transcription polymerase chain reaction (RT-qPCR) assays targeting the viral nucleoprotein (N) gene was tested using 66 viral isolates. All designed Taqman(®) RT-qPCR assays were genotype specific, displayed a high sensitivity and together constituted a diagnostic method for the rapid discrimination of European VHSV genotypes. Practical diagnostic applications of such assays demonstrated in this study include: (1) rapid genotype determination of isolates; and (2) identification of mixed-genotype isolates originating from pooled samples in areas where genotype distribution is known to overlap. However, the most important application will be supporting international VHSV surveillance programmes through the provision of a rapid specific and sensitive isolate characterisation method., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Viral hemorrhagic septicemia virus (VHSV) genotype IVa causes mass mortality in wild Pacific herring, a species of economic value, in the Northeast Pacific Ocean. Young of the year herring are particularly susceptible and can be carriers of the virus. To understand its pathogenesis, tissue and cellular tropisms of VHSV in larval and juvenile Pacific herring were investigated with immunohistochemistry, transmission electron microscopy, and viral tissue titer. In larval herring, early viral tropism for epithelial tissues (6d post-exposure) was indicated by foci of epidermal thickening that contained heavy concentrations of virus. This was followed by a cellular tropism for fibroblasts within the fin bases and the dermis, but expanded to cells of the kidney, liver, pancreas, gastrointestinal tract and meninges in the brain. Among wild juvenile herring that underwent a VHS epizootic in the laboratory, the disease was characterized by acute and chronic phases of death. Fish that died during the acute phase had systemic infections in tissues including the submucosa of the gastrointestinal tract, spleen, kidney, liver, and meninges. The disease then transitioned into a chronic phase that was characterized by the appearance of neurological signs including erratic and corkscrew swimming and darkening of the dorsal skin. During the chronic phase viral persistence occurred in nervous tissues including meninges and brain parenchymal cells and in one case in peripheral nerves, while virus was mostly cleared from the other tissues. The results demonstrate the varying VHSV tropisms dependent on the timing of infection and the importance of neural tissues for the persistence and perpetuation of chronic infections in Pacific herring., (Published by Elsevier B.V.)

Viral hemorrhagic septicemia virus (VHSV) is a fish rhabdovirus that causes disease in a broad range of marine and freshwater hosts. The known geographic range includes the Northern Atlantic and Pacific Oceans, and recently it has invaded the Great Lakes region of North America. The goal of this work was to characterize genetic diversity of Great Lakes VHSV isolates at the early stage of this viral emergence by comparing a partial glycoprotein (G) gene sequence (669 nt) of 108 isolates collected from 2003 to 2009 from 31 species and at 37 sites. Phylogenetic analysis showed that all isolates fell into sub-lineage IVb within the major VHSV genetic group IV. Among these 108 isolates, genetic diversity was low, with a maximum of 1.05% within the 669 nt region. There were 11 unique sequences, designated vcG001 to vcG011. Two dominant sequence types, vcG001 and vcG002, accounted for 90% (97 of 108) of the isolates. The vcG001 isolates were most widespread. We saw no apparent association of sequence type with host or year of isolation, but we did note a spatial pattern, in which vcG002 isolates were more prevalent in the easternmost sub-regions, including inland New York state and the St. Lawrence Seaway. Different sequence types were found among isolates from single disease outbreaks, and mixtures of types were evident within 2 isolates from individual fish. Overall, the genetic diversity of VHSV in the Great Lakes region was found to be extremely low, consistent with an introduction of a new virus into a geographic region with previously naive host populations.

Viral hemorrhagic septicemia virus (VHSV) infects over 70 fish species inhabiting marine, brackish or freshwater environments throughout the Northern Hemisphere. Over its geographic range, 4 VHSV genotypes and multiple subtypes exist. Here, we describe the development and validation of a rapid, sensitive and specific real-time reverse transcription quantitative PCR assay (RT-qPCR) that amplifies sequence from representative isolates of all VHSV genotypes (I, II, III and IV). The pan-specific VHSV RT-qPCR assay reliably detects 100 copies of VHSV nucleoprotein RNA without cross-reacting with infectious hematopoietic necrosis virus, spring viremia of carp virus or aquatic birnavirus. Test performance characteristics evaluated on experimentally infected Atlantic salmon Salmo salar L. revealed a diagnostic sensitivity (DSe) > or = 93% and specificity (DSp) = 100%. The repeatability and reproducibility of the procedure was exceptionally high, with 93% agreement among test results within and between 2 laboratories. Furthermore, proficiency testing demonstrated the VHSV RT-qPCR assay to be easily transferred to and performed by a total of 9 technicians representing 4 laboratories in 2 countries. The assay performed equivalent to the traditional detection method of virus isolation via cell culture with the advantage of faster turnaround times and high throughput capacity, further suggesting the suitability of the use of this VHSV RT-qPCR in a diagnostic setting.

Three North American and 1 European viral hemorrhagic septicemia virus (VHSV) isolates taken from either a marine, freshwater, or estuarine host were assessed for survivability in raw and filtered freshwater and seawater at temperatures ranging from 4 to 30 degrees C. All 4 isolates were substantially more stable in freshwater than in seawater, and higher survival was observed at lower water temperatures. The average time required for 99.9% inactivation of VHSV in raw freshwater at 15 degrees C was 13 d, while in raw seawater VHSV was inactivated within an average of 4 d. No consistent correlation was observed between the origin and the stability of the virus isolates. Freshwater isolates were not always the most stable in freshwater; similarly, seawater isolates were not consistently more stable in seawater. Virus survival was greatly enhanced in filtered freshwater with some virus strains remaining infective after 1 yr at 4 degrees C.