Journal of Fish DiseasesVolume 33, Issue 8 p. 701-704 Current lineages of the epithelioma papulosum cyprini (EPC) cell line are contaminated with fathead minnow, Pimephales promelas, cells J Winton, J Winton US Geological Survey, Western Fisheries Research Center, Seattle, WA, USASearch for more papers by this authorW Batts, W Batts US Geological Survey, Western Fisheries Research Center, Seattle, WA, USASearch for more papers by this authorP DeKinkelin, P DeKinkelin INRA-Centre de Recherches, Jouy-en-Josas, FranceSearch for more papers by this authorM LeBerre, M LeBerre INRA-Centre de Recherches, Jouy-en-Josas, FranceSearch for more papers by this authorM Bremont, M Bremont INRA-Centre de Recherches, Jouy-en-Josas, FranceSearch for more papers by this authorN Fijan, N Fijan Department of Biology and Pathology of Fish and Bees, University of Zagreb, Zagreb, Croatia*Deceased Deceased.Search for more papers by this author J Winton, J Winton US Geological Survey, Western Fisheries Research Center, Seattle, WA, USASearch for more papers by this authorW Batts, W Batts US Geological Survey, Western Fisheries Research Center, Seattle, WA, USASearch for more papers by this authorP DeKinkelin, P DeKinkelin INRA-Centre de Recherches, Jouy-en-Josas, FranceSearch for more papers by this authorM LeBerre, M LeBerre INRA-Centre de Recherches, Jouy-en-Josas, FranceSearch for more papers by this authorM Bremont, M Bremont INRA-Centre de Recherches, Jouy-en-Josas, FranceSearch for more papers by this authorN Fijan, N Fijan Department of Biology and Pathology of Fish and Bees, University of Zagreb, Zagreb, Croatia*Deceased Deceased.Search for more papers by this author First published: 06 July 2010 https://doi.org/10.1111/j.1365-2761.2010.01165.xCitations: 153 J Winton, US Geological Survey, Western Fisheries Research Center, USGS, 6505 NE 65th Street, Seattle, WA 98115, USA (e-mail:[email protected]) Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. 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Viral haemorrhagic septicaemia virus (VHSV) was isolated from muskellunge, Esox masquinongy (Mitchill), caught from the NW portion of Lake St Clair, Michigan, USA in 2003. Affected fish exhibited congestion of internal organs; the inner wall of the swim bladder was thickened and contained numerous budding, fluid-filled vesicles. A virus was isolated using fish cell lines inoculated with a homogenate of kidney and spleen tissues from affected fish. Focal areas of cell rounding and granulation appeared as early as 24 h post-inoculation and expanded rapidly to destroy the entire cell sheet by 96 h. Electron microscopy revealed virions that were 170-180 nm in length by 60-70 nm in width having a bullet-shaped morphology typical of rhabdoviruses. The virus was confirmed as VHSV by reverse transcriptase-polymerase chain reaction. Sequence analysis of the entire nucleoprotein and glycoprotein genes revealed the virus was a member of the North American genotype of VHSV; however, the isolate was sufficiently distinct to be considered a separate sublineage, suggesting its origin may have been from marine species inhabiting the eastern coastal areas of the USA or Canada.

The initial characterization of a rhabdovirus isolated from a single, asymptomatic starry flounder (Platichthys stellatus) collected during a viral survey of marine fishes from the northern portion of Puget Sound, Washington, USA, is reported. Virions were bullet-shaped and approximately 100 nm long and 50 nm wide, contained a lipid envelope, remained stable for at least 14 days at temperatures ranging from -80 to 5degreesC and grew optimally at 15degreesC in cultures of epithelioma papulosum cyprini (EPC) cells. The cytopathic effect on EPC cell monolayers was characterized by raised foci containing rounded masses of cells. Pyknotic and dark-staining nuclei that also showed signs of karyorrhexis were observed following haematoxylin and eosin, May-Grunwald Giemsa and acridine orange staining. PAGE of the structural proteins and PCR assays using primers specific for other known fish rhabdoviruses, including Infectious hematopoietic necrosis virus, Viral hemorrhagic septicemia virus, Spring viremia of carp virus, and Hirame rhabdovirus, indicated that the new virus, tentatively termed starry flounder rhabdovirus (SFRV), was previously undescribed in marine fishes from this region. In addition, sequence analysis of 2678 nt of the amino portion of the viral polymerase gene indicated that SFRV was genetically distinct from other members of the family Rhabdoviridae for which sequence data are available. Detection of this virus during a limited viral survey of wild fishes emphasizes the void of knowledge regarding the diversity of viruses that naturally infect marine fish species in the North Pacific Ocean.

To assess the risk associated with processed rainbow trout (Oncorhynchus mykiss) produced in an area where infectious haematopoietic necrosis (IHN) virus is endemic, 240 fish weighing between 225 grams (g) and 500 g which exhibited spiral curvature or spinal compression deformity types were tested by virus isolation and polymerase chain reaction (PCR) techniques. Rainbow trout aged approximately one year which exhibited spiral deformities were considered to have a high likelihood of previously being infected with IHN virus (IHNV). The fish were gutted and degilled by automated machines and kept cool while specimens were collected for testing. Since internal organs and gills are removed during fish processing for food products, portions of the skin and muscle were collected from the area of the deformity, as well as brain tissue from each fish. Tissue homogenates were tested on epithelioma papulosum cyprini and chinook salmon embryo (CHSE)-214 cell lines which had been pre-treated with polyethylene glycol, using standard methods. Samples on CHSE-214 cells were incubated for 21 days at 15degreesC, then blind passaged for another incubation period of 21 days. Nested reverse transcription PCR (RT-PCR) used the central 1231 base pair portion of the glycoprotein gene to detect IHNV. All brain and skin-muscle homogenates gave negative results for the presence of IHNV by virus isolation and nested RT-PCR. To assess virus clearance experimentally, groups of 100 specific-pathogen-free rainbow trout (mean weight, 100 g) were either intraperitoneally injected with IHNV or sham infected and held separately in 385-litre aquaria in virus-free water at 15degreesC. In all, 33% of the infected fish died. Ten days after the last mortality or 34 days post infection, 6 fish from each treatment were sacrificed at weekly intervals for 6 weeks. Kidney and brain homogenates from individual fish were tested by virus isolation and nested RT-PCR using the nucleoprotein gene. All tissue homogenates gave negative results for IHNV. These results provide scientific information which can be used to assess the risk associated with the movement of processed rainbow trout from an area where IHNV is endemic.

Specific-pathogen-free Pacific herring Clupea pallasi were reared in the laboratory from eggs and then challenged at 5, 9, and 13 months of age by waterborne exposure to low (10(1.5-2.5) plaque-forming units [PFU] per milliliter), medium (10(3.5-4.5) PFU/mL), or high (10(5.5-6.5) PFU/mL) levels of a North American isolate of viral hemorrhagic septicemia virus (VHSV). The fish were extremely susceptible to the virus, showing clinical disease, mortality approaching 100%,, and only a limited increase in resistance with age. Mortality began 4-6 d after exposure and peaked at approximately day 7 in fish exposed to high levels of virus. Whereas the mean time to death showed a significant dose response (P < 0.001), the percent mortality and virus titers in dead fish were generally high in all groups regardless of initial challenge dose. External signs of disease were usually limited to 1-2-mm hemorrhagic areas on the lower jaw and isthmus and around the eye, but 2 of 130 infected fish exhibited extensive cutaneous hemorrhaging. Histopathologic examination of tissues from moribund fish sampled at 2-8 d after exposure revealed multifocal coagulative necrosis of hepatocytes, diffuse necrosis of interstitial hematopoietic tissues in the kidney, diffuse necrosis of the spleen, epidermis, and subcutis, and occasional necrosis of pancreatic acinar cells. Virus titers in tissues of experimentally infected herring were first detected 48 h after exposure and peaked 6-8 d after exposure at 10(7.7) PFU/g. Fish began shedding virus at 48 h after exposure with titers in the Row-through aquaria reaching 10(2.5) PFU/mL at 4-5 d after exposure, just before peak mortality. When the water flow was turned off for 3 h, titers in the water rose to 10(3.5) PFU/mL, and the amount of virus shed by infected fish (on average, greater than 10(6.5) PFU/h per fish) appeared sufficient to sustain a natural epizootic among schooling herring. Taken together, these data suggest that VHSV could be a significant limiting factor for populations of Pacific herring.

Twelve neutralizing monoclonal antibodies (MAbs) against the fish rhabdovirus, infectious haematopoietic necrosis virus (IHNV), were used to select 20 MAb escape mutants. The nucleotide sequence of the entire glycoprotein (G) gene was determined for six mutants representing differing cross-neutralization patterns and each had a single nucleotide change leading to a single amino acid substitution within one of three regions of the protein. These data were used to design nested PCR primers to amplify portions of the G gene of the 14 remaining mutants. When the PCR products from these mutants were sequenced, they also had single nucleotide substitutions coding for amino acid substitutions at the same, or nearby, locations. Of the 20 mutants for which all or part of the glycoprotein gene was sequenced, two MAbs selected mutants with substitutions at amino acids 230-231 (antigenic site I) and the remaining MAbs selected mutants with substitutions at amino acids 272-276 (antigenic site II). Two MAbs that selected mutants mapping to amino acids 272-276, selected other mutants that mapped to amino acids 78-81, raising the possibility that this portion of the N terminus of the protein was part of a discontinuous epitope defining antigenic site II. CLUSTAL alignment of the glycoproteins of rabies virus, vesicular stomatitis virus and IHNV revealed similarities in the location of the neutralizing epitopes and a high degree of conservation among cysteine residues, indicating that the glycoproteins of three different genera of animal rhabdoviruses may share a similar three-dimensional structure in spite of extensive sequence divergence.