Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonid fishes in North America, Europe and Asia and is reportable to the World Organization for Animal Health (OIE). Phylogenetic analysis has identified 5 major virus genogroups of IHNV worldwide, designated U, M, L, E and J; multiple subtypes also exist within those genogroups. Here, we report the development and validation of a universal IHNV reverse-transcriptase real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) gene. Properties of diagnostic sensitivity (DSe) and specificity (DSp) were defined using laboratory-challenged steelhead trout Oncorhynchus mykiss, and the new assay was compared to the OIE-accepted conventional PCR test and virus isolation in cell culture. The IHNV N gene RT-rPCR had 100% DSp and DSe and a higher estimated diagnostic odds ratio (DOR) than virus culture or conventional PCR. The RT-rPCR assay was highly repeatable within a laboratory and highly reproducible between laboratories. Field testing of the assay was conducted on a random sample of juvenile steelhead collected from a hatchery raceway experiencing an IHN epizootic. The RT-rPCR detected a greater number of positive samples than cell culture and there was 40% agreement between the 2 tests. Overall, the RT-rPCR assay was highly sensitive, specific, repeatable and reproducible and is suitable for use 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°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°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°C. KEY WORDS: Viral hemorrhagic septicemia virus · Virus inactivation · Great Lakes VHSV · Genotype IVb · Virus survival · Environmental factors/effects Resale or republication not permitted without written consent of the publisherDis Aquat Org 82: [171][172][173][174][175][176][177][178] 2008 (Meyers & Winton 1995, Stone et al. 1997, Dixon 1999, Einer-Jensen et al. 2004, Snow et al. 2004.Historically, VHSV in North America had only been recovered from marine, estuarine and anadromous fish populations occurring in coastal waters of the Pacific Ocean from Alaska to California and the Atlantic Ocean waters of Maine, New Brunswick and Nova Scotia (Meyers & Winton 1995, Olivier 2002, Hedrick et al. 2003. However, in the spring of 2005, VHSV was isolated for the first time from a mass mortality event in a freshwater environment in North America. The virus was isolated from dead or dying freshwater drum Aplodinotus grunniens in Lake Ontario (Lumsden et al. 2007). Following this initial discovery, an archived viral isolate obtained from diseased muskellunge Esox masquinongy in Lake St. Clair in 2003 was identified as VHSV, indicating the earliest detection of the virus in the Great Lakes (Elsayed et al. 2006). The virus has also been associated with large mortalities of round gobies Neogobius melanostomus in Lake Ontario and the Saint Lawrence River (Groocock et al. 2007). To date, this freshwater strain of VHSV has been isolated from 28 species of fish in the Great Lakes regions of Canada and the USA including Lake Michigan, Lake Huron, Lake St. Clair, Lake Erie, Lake Ontario, the Saint Lawrence River and from inland lakes in New York, Michigan, Wisconsin and Ohio (USDA & APHIS 2007).Phylogenetic analyses based on nucleotide sequence of nucleo-and glycoprotein genes illustrated that VHSV isolates group into 4 major genotypes that generally correlate with geographic location (Benmansour et al. 1997, Einer-Jensen et al. 2004Snow et al. 2004. Genotype I encompasses European freshwater and north European marine isolates; Genotype II represents marine isolates originating from the Baltic Sea; Genot...

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) ≥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.

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.

A Jaundice Syndrome occurs sporadically among sea-pen-farmed Chinook Salmon in British