Elucidation of mechanism for host response to VHSV infection at varying temperatures in vitro and in vivo through proteomic analysis.

Seasonal temperature has a major influence on the infectivity of pathogens and the host immune system. Viral hemorrhagic septicemia virus (VHSV) is one such pathogen that only causes the mortality of fish at low temperatures. This study aims to discover the host defense mechanism and pathway for resistance to VHSV at higher temperatures. We first observed the VHSV infection patterns at low and higher temperatures in fathead minnow (FHM) cells (20 °C and 28 °C) and zebrafish (15 °C and 25 °C). In comparison to the 20 °C infection, FHM cells infected at 28 °C showed decreased apoptosis, increased cell viability, and reduced VHSV N gene expression. In zebrafish, infection at 25 °C caused no mortality and significantly reduced the N gene copy number in comparison to infection at 15 °C. To explore the antiviral infection mechanisms induced by high temperature in vitro and in vivo, the changes in the proteomic profile were measured through UPLC-MSE analysis. ACADL, PTPN6, TLR1, F7, A2M, and GLI2 were selected as high temperature-specific biomarkers in the FHM cell proteome; and MYH9, HPX, ANTXR1, APOA1, HBZ, and MYH7 were selected in zebrafish. Increased immune response, anticoagulation effects, and the formation of lymphocytes from hematopoietic stem cells were analyzed as functions that were commonly induced by high temperature in vitro and in vivo. Among these biomarkers, GLI2 was predicted as an upstream regulator. When treated with GANT58, a GLI-specific inhibitor, cell viability was further reduced due to GLI2 inhibition during VHSV infection at varying temperatures in FHM cells, and the mortality in zebrafish was induced earlier at the low temperature. Overall, this study discovered a new mechanism for VHSV infection in vitro and in vivo that is regulated by GLI2 protein.

In vivo study of viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus coexistence in Senegalese sole (Solea senegalensis).

The effect of IPNV-VHSV coinfection and superinfection on the mortality caused by both viruses in Senegalese sole has been analysed. No effect was observed after coinfection. However, a clear viral interference was recorded between a primary IPNV and a subsequent VHSV infection, which led to a survival increase in the infected sole of 50% points when compared with fish infected only with VHSV. The significantly higher Mx transcriptional values in the fish pre-exposed to IPNV (at least at first days after superinfection) and the increased daily mortality when low Mx transcriptional levels were recorded suggest that Mx may be involved in the protective effect against VHSV infection. However, in fish subjected to VHSV primary/IPNV secondary infection, no interference was observed.

Differentially expressed genes after viral haemorrhagic septicaemia virus infection in olive flounder (Paralichthys olivaceus).

A strain of viral haemorrhagic septicaemia virus (VHSV) was isolated from cultured olive flounder (Paralichthys olivaceus) during epizootics in South Korean. This strain showed high mortality to olive flounder in in vivo challenge experiment. The complete genomic RNA sequences were determined and phylogenetic analysis of the amino acid sequences of glycoprotein revealed that this isolate was grouped into genotype IVa of genus Novirhabdovirus. Expression profile of genes in olive flounder was analyzed at day 1 and day3 after infection with this VHSV isolate by using cDNA microarray containing olive flounder 13K cDNA clones. Microarray analysis revealed 785 up-regulated genes and 641 down-regulated genes by at least two-fold in virus-infected fish compared to healthy control groups. Among 785 up-regulated genes, we identified seven immune response-associated genes, including the interferon (IFN)-induced 56-kDa protein (IFI56), suppressor of cytokine signaling 1 (SOCS1), interleukin 8 (IL-8), cluster of differentiation 83 (CD83), α-globin (HBA), VHSV-induced protein-6 (VHSV6), and cluster of differentiation antigen 9 (CD9). Our results confirm previous reports that even virulent strain of VHSV induces expression of genes involved in protective immunity against VHSV.

Phylogenetic analysis of the glycoprotein gene of viral hemorrhagic septicemia virus from Iranian trout farms points towards a common European origin.

Viral haemorrhagic septicaemia virus (VHSV), a member of family Rhabdoviridae and genus Novirhabdoviridae, causes mortality in numerous marine and freshwater hosts located in northern hemisphere. To evaluate the genetic diversity of VHSV from the North and South West of Iran, the sequences of a 1483bp nt region of the glycoprotein gene were determined for four Iranian isolates. These sequences were analysed to evaluate their genetic relatedness with 86 worldwide isolates representing the four known genogroups of VHSV. Phylogenetic analysis by nucleotide sequences showed that all the VHSV isolates studied were closest related to the 19 fresh water strains from Germany grouped within the European genogroup Ia-2. This finding indicates that Iranian VHSV most likely was introduced to Iran by the movement of contaminated fish fry from a source in Europe.

Susceptibility of goldsinny wrasse, Ctenolabrus rupestris L. (Labridae), to viral haemorrhagic septicaemia virus (VHSV) genotype III: Experimental challenge and pathology.

Cleaner fish, such as wrasse, are being increasingly used to combat the sea lice infestation of Atlantic salmon (Salmo salar L.) in many European countries. To determine susceptibility of the goldsinny wrasse (Ctenolabrus rupestris L.) and pathogenesis of the viral haemorrhagic septicaemia virus (VHSV) genotype III isolate 12-654, previously associated with VHSV infection in the Shetland Islands in 2012, fish were experimentally challenged by intraperitoneal injection (IP), bath immersion and cohabitation routes. Cumulative proportion of moribund wrasse reached 17% following the virus immersion challenge while by the IP-route moribunds exceeded 50% within 14days post-challenge. Typical signs of VHS as reported in rainbow trout (Oncorhynchus mykiss), were not observed in moribund goldsinny wrasse. The most pronounced histopathological changes, consistent regardless of the route of infection, were observed within the heart and included atrium myofibril degeneration, focal infiltration and multifocal necrosis, with prominent swelling of the endocardium and occasional detachment. Pathological changes in the atrium were associated with presence of the viral antigen as confirmed by a positive immunohistochemical staining. Virus clearance and heart tissue recovery were noted although further experiments are required to confirm these observations. The results of a cohabitation experiment confirmed that goldsinny wrasse shed viable virus and therefore represent a risk of virus transmission to other VHSV susceptible species. Similarities between the pathology in goldsinny wrasse induced through the controlled experimental challenges and that of wrasse spp. from an infection occurrence in Shetland are discussed.

A mortality event in wrasse species (Labridae) associated with the presence of viral haemorrhagic septicaemia virus.

Viral haemorrhagic septicaemia (VHS) is an infectious disease of farmed and wild fish and has an extensive host range in both freshwater and marine environments. In December 2012, a wrasse population consisting of ballan, Labrus bergylta (Ascanius), corkwing, Symphodus melops (L.), cuckoo, Labrus mixtus L., goldsinny, Ctenolabrus rupestris (L.), and rock cook, Centrolabrus exoletus (L.), held at a marine hatchery in the Shetland Isles, Scotland, experienced a mortality event. Approximately 10 000 wrasse were being held at the facility on behalf of an Atlantic salmon, Salmo salar L., aquaculture company prior to being deployed for the biological control of parasites on marine pen Atlantic salmon, aquaculture sites. Fish Health Inspectors from Marine Scotland Science initiated a diagnostic investigation, and subsequent diagnostic testing confirmed the site to be VHSV positive by qRT-PCR and virus isolation followed by ELISA. A VHSV genotype-specific qRT-PCR assay revealed that the isolates belonged to genotype III, the European marine strain of the virus. The virus genotype was further confirmed by nucleic acid sequencing of the partial nucleoprotein (N) and glycoprotein (G) genes followed by BLAST nucleotide searches. This study reports for the first time the detection of VHSV within multiple wrasse species and highlights the need for a comprehensive risk-based approach to the use of wrasse and other finfish species as biological controls within the aquaculture industry.

High virulence differences among phylogenetically distinct isolates of the fish rhabdovirus viral hemorrhagic septicaemia virus are not explained by variability of the surface glycoprotein G or the non-virion protein Nv.

Viral hemorrhagic septicaemia virus (VHSV) is an important viral pathogen in European rainbow trout farming. Isolates from wild marine fish and freshwater trout farms show highly different virulence profiles: isolates from marine fish species cause little or no mortality in rainbow trout following experimental waterborne challenge, whilst challenge with rainbow trout isolates results in high levels of mortality. Phylogenetic analyses have revealed that the highly virulent trout-derived isolates from freshwater farms have evolved from VHSV isolates from marine fish host species over the past 60 years. Recent isolates from rainbow trout reared in marine zones show intermediate virulence. The present study aimed to identify molecular virulence markers that could be used to classify VHSV isolates according to their ability to cause disease in rainbow trout. By a reverse genetics approach using a VHSV-related novirhabdovirus [infectious hematopoietic necrosis virus (IHNV)], four chimaeric IHNV-VHSV recombinant viruses were generated. These chimaeric viruses included substitution of the IHNV glyco- (G) or non-structural (Nv) protein with their counterparts from either a trout-derived or a marine VHSV strain. Comparative challenge experiments in rainbow trout fingerlings revealed similar levels of survival induced by the recombinant (r)IHNV-VHSV chimaeric viruses regardless of whether the G or Nv genes originated from VHSV isolated from a marine fish species or from rainbow trout. Interestingly, recombinant IHNV gained higher virulence following substitution of the G gene with those of the VHSV strains, whilst the opposite was the case following substitution of the Nv genes.

Expression kinetics of key genes in the early innate immune response to Great Lakes viral hemorrhagic septicemia virus IVb infection in yellow perch (Perca flavescens).

The recently discovered strain of viral hemorrhagic septicemia virus, VHSV-IVb, represents an example of the introduction of an extremely pathogenic rhabdovirus capable of infecting a wide variety of new fish species in a new host-environment. The goal of the present study was to delineate the expression kinetics of key genes in the innate immune response relative to the very early stages of VHSV-IVb infection using the yellow perch (Perca flavescens) as a model. Administration of VHSV-IVb by IP-injection into juvenile yellow perch resulted in 84% cumulative mortality, indicating their high susceptibility to this disease. In fish sampled in the very early stages of infection, a significant up-regulation of Mx gene expression in the liver, as well as IL-1ß and SAA activation in the head kidney, spleen, and liver was directly correlated to viral load. The potential down-regulation of Mx in the hematopoietic tissues, head kidney and spleen, may represent a strategy utilized by the virus to increase replication.

Resistance to a rhabdovirus (VHSV) in rainbow trout: identification of a major QTL related to innate mechanisms.

Health control is a major issue in animal breeding and a better knowledge of the genetic bases of resistance to diseases is needed in farm animals including fish. The detection of quantitative trait loci (QTL) will help uncovering the genetic architecture of important traits and understanding the mechanisms involved in resistance to pathogens. We report here the detection of QTL for resistance to Viral Haemorrhagic Septicaemia Virus (VHSV), a major threat for European aquaculture industry. Two induced mitogynogenetic doubled haploid F2 rainbow trout (Oncorhynchus mykiss) families were used. These families combined the genome of susceptible and resistant F0 breeders and contained only fully homozygous individuals. For phenotyping, fish survival after an immersion challenge with the virus was recorded, as well as in vitro virus replication on fin explants. A bidirectional selective genotyping strategy identified seven QTL associated to survival. One of those QTL was significant at the genome-wide level and largely explained both survival and viral replication in fin explants in the different families of the design (up to 65% and 49% of phenotypic variance explained respectively). These results evidence the key role of innate defence in resistance to the virus and pave the way for the identification of the gene(s) responsible for resistance. The identification of a major QTL also opens appealing perspectives for selective breeding of fish with improved resistance.

Comparative susceptibility among three stocks of yellow perch, Perca flavescens (Mitchill), to viral haemorrhagic septicaemia virus strain IVb from the Great Lakes.

The Great Lakes strain of viral haemorrhagic septicaemia virus IVb (VHSV-IVb) is capable of infecting a wide number of naive species and has been associated with large fish kills in the Midwestern United States since its discovery in 2005. The yellow perch, Perca flavescens (Mitchill), a freshwater species commonly found throughout inland waters of the United States and prized for its high value in sport and commercial fisheries, is a species documented in several fish kills affiliated with VHS. In the present study, differences in survival after infection with VHSV IVb were observed among juvenile fish from three yellow perch broodstocks that were originally derived from distinct wild populations, suggesting innate differences in susceptibility due to genetic variance. While all three stocks were susceptible upon waterborne exposure to VHS virus infection, fish derived from the Midwest (Lake Winnebago, WI) showed significantly lower cumulative % survival compared with two perch stocks derived from the East Coast (Perquimans River, NC and Choptank River, MD) of the United States. However, despite differences in apparent susceptibility, clinical signs did not vary between stocks and included moderate-to-severe haemorrhages at the pelvic and pectoral fin bases and exophthalmia. After the 28-day challenge was complete, VHS virus was analysed in subsets of whole fish that had either survived or succumbed to the infection using both plaque assay and quantitative PCR methodologies. A direct correlation was identified between the two methods, suggesting the potential for both methods to be used to detect virus in a research setting.

Genetically similar VHSV isolates are differentially virulent in olive flounder Paralichthys olivaceus.

Two viral hemorrhagic septicemia virus (VHSV) isolates, VHSV-KR-CJA and VHSV-KR-YGH, were isolated from viral hemorrhagic septicemia disease outbreaks in flounder farms in South Korea. The VHSV-KR-CJA isolate was isolated from a flounder farm with high mortality (80%), while the VHSV-KR-YGH isolate was isolated from a flounder farm with low mortality (15%), suggesting that these isolates differ in virulence. The virulence of these isolates was evaluated in juvenile flounder via intraperitoneal injection. Consistent with their virulence in the field, mortality data revealed that the VHSV-KR-CJA isolate was highly pathogenic (cumulative mortality of 80%), while the VHSV-KR-YGH isolate was less pathogenic in flounder (cumulative mortality of 20%). To characterize the genotypes of these viruses, the full open reading frames (ORFs) encoding nucleoprotein N, phosphoprotein P, matrix protein M, glycoprotein G, nonstructural viral protein NV, and polymerase L of these viruses were sequenced and analyzed. Sequence analysis revealed that both isolates are genetically very similar (identical amino acid sequences for P, M, NV, and L and >99.7 and 99.8% amino acid sequence identity for N and G, respectively). Phylogenetic analysis indicated that both of these viruses belong to the Genotype IVa group, suggesting that they originated from a common ancestral virus. The low pathogenicity VHSV strain may potentially evolve to become a more pathogenic strain through only a few nucleotide substitutions. Further functional analyses of mutations in VHSV genes are necessary to identify factors that determine VHSV pathogenicity in flounder.

Viral tropism and pathology associated with viral hemorrhagic septicemia in larval and juvenile Pacific herring.

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.

Mortality and carrier status of bluegills exposed to viral hemorrhagic septicemia virus genotype IVb at different temperatures.

The emergence of the viral hemorrhagic septicemia virus (VHSV) genotype IVb (VHSV-IVb) in the Great Lakes of North America has led to concern that the virus might spread to natural fisheries and aquaculture in the southern USA. We exposed bluegills Lepomis macrochirus to VHSV-IVb by intraperitoneal injection at six temperatures from 10 degrees C to 30 degrees C and followed the disease course by quantitative real-time reverse transcriptase polymerase chain reaction (qrt-RT-PCR). Mortality of injected fish was 90% at 10 degrees C, 35% at 14 degrees C, and 10% at 18 degrees C; no mortality attributable to VHSV was observed at temperatures of 22-30 degrees C. In survivors tested at 21 d postchallenge, viral copies and prevalence determined by qrt-RT-PCR were inversely related to temperature, and VHSV-IVb could not be detected in fish held at temperatures above 22 degrees C. Similar results were obtained for bluegills that were exposed by cohabitation with the intraperitoneally injected fish. Acclimation of the fish to 12 degrees C after 21 d at higher temperatures did not appear to cause a re-emergence of the virus. Based on our findings, the temperature range of VHSV-IVb appears to be the same as published values for VHSV genotype I, which has an optimum of 9-12 degrees C and an upper limit of 18-20 degrees C.

Toll-like receptors and interferon associated immune factors in viral haemorrhagic septicaemia virus-infected olive flounder (Paralichthys olivaceus).

Pattern recognition receptor (PRR) toll-like receptors (TLRs), antiviral agent interferon (IFN) and the effector IFN stimulated genes (ISGs) play pivotal role in antiviral innate immunity of a host. The present in-vivo experiment was conducted to investigate the role of these innate immune factors in early phase as well as during recovery of viral haemorrhagic septicaemia virus (VHSV) infection by quantitative real-time reverse transcriptase polymerase chain reaction. A less lethal VHSV infection was generated in olive flounder (Paralichthys olivaceus) and was sampled at 3, 6, and 12h post infection (hpi), and 1, 2, 4, and 7 days post infection (dpi). At 3 hpi, the VHSV N gene was detected in three out of five fish and all five fish showed a relative fold increase of TLR 2, TLR 7, interleukin 8 (IL 8), IFN regulatory factor 3 (IRF 3), IRF 7, and ISG 15. Viral copies rapidly increased at 12 hpi then remained high until 2 dpi. When viral copy numbers were high, a higher expression of immune genes IL 1ß, IRF 3, IRF 7, Type I IFN, ISG 15 and Mx was observed. Viral copies were drastically reduced in 4 and 7 dpi fish, and also the immune response was considerably reduced but remained elevated, except for ISG 15 which found equal to control in 7 dpi fish. A high degree of correlation was observed between immune genes and viral copy number in each of the sampled fish at 12 hpi. A fish with ascites sampled at 7 dpi displayed high viral copy but under-expressed immune genes except for Mx. When viral copies were high at 1 and 2 dpi, both TLR 2 and TLR 7 were down-regulated, perhaps indicating immune suppression by the virus. The quick and prolonged elevated expression of the immune genes indicates their crucial role in survival of host against VHSV.

Heavy oil exposure induces high moralities in virus carrier Japanese flounder Paralichthys olivaceus.

The relationship between chemical exposure and disease outbreak in fish has not been fully defined due to the limitations of experimental systems (model fish and pathogens). Therefore, we constructed a system using the Japanese flounder, Paralichthys olivaceus, and viral haemorrhagic septicemia virus (VHSV), and evaluated it by heavy oil (HO) exposure. The fish were exposed to HO at 0.3, 0.03, 0.003, and 0 g/L following VHSV infection at doses of 10(2.5) or 10(3.5) tissue culture infectious dose (TCID)50/fish. As a result, groups given the dual stressors showed more than 90% mortality. Although VHSV infection at 10(2.5) and 10(3.5) TCID50/fish without HO exposure also induced high mortality, at 68.8% and 81.3%, respectively, HO exposure induced faster and higher mortality in the virus carrier fish, indicating that chemical stressors raise the risk of disease outbreak in fish. The experimental system established in this study could be useful for chemical risk assessment.

Zebrafish fin immune responses during high mortality infections with viral haemorrhagic septicemia rhabdovirus. A proteomic and transcriptomic approach.

BACKGROUND: Despite rhabdoviral infections being one of the best known fish diseases, the gene expression changes induced at the surface tissues after the natural route of infection (infection-by-immersion) have not been described yet. This work describes the differential infected versus non-infected expression of proteins and immune-related transcripts in fins and organs of zebrafish Danio rerio shortly after infection-by-immersion with viral haemorrhagic septicemia virus (VHSV). RESULTS: Two-dimensional differential gel electrophoresis detected variations on the protein levels of the enzymes of the glycolytic pathway and cytoskeleton components but it detected very few immune-related proteins. Differential expression of immune-related gene transcripts estimated by quantitative polymerase chain reaction arrays and hybridization to oligo microarrays showed that while more transcripts increased in fins than in organs (spleen, head kidney and liver), more transcripts decreased in organs than in fins. Increased differential transcript levels in fins detected by both arrays corresponded to previously described infection-related genes such as complement components (c3b, c8 and c9) or class I histocompatibility antigens (mhc1) and to newly described genes such as secreted immunoglobulin domain (sid4), macrophage stimulating factor (mst1) and a cluster differentiation antigen (cd36). CONCLUSIONS: The genes described would contribute to the knowledge of the earliest molecular events occurring in the fish surfaces at the beginning of natural rhabdoviral infections and/or might be new candidates to be tested as adjuvants for fish vaccines.

Failure of population recovery in relation to disease in Pacific herring.

Following an estimated 60% decline in population abundance in early 1993, recovery of the Pacific herring Clupea pallasii population of Prince William Sound, Alaska, USA, has been impaired by disease. Comprehensive epidemiological study from 1994 through 2002 validated an age-structured assessment (ASA) model of disease and population abundance; from 2003 to 2006, the impact of disease was modeled by analyzing only 2 lesions: ulcers and white foci in the heart. The ASA model identified increased natural mortality since 1993 that can be explained by (1) epidemics associated with ulcers (prevalence about 3%) and the North American strain of viral hemorrhagic septicemia virus (VHSV Type IVa; prevalence up to 14%) in 1994 and 1998 and (2) relatively high prevalence of the mesomycetozoean Ichthyophonus hoferi from 1994 through 2006, including epidemics with the greatest sample prevalence in 2001 (38%, by histopathology) and 2005 (51%, estimated histopathology prevalence). Fourteen other parasites occurred at prevalence > 10%, but none were considered significant contributors to fish mortality. We predict that if natural mortality after 1994 had returned to background levels that best fit the model from 1980 to 1992 (0.25 yr(-1)), population biomass in 2006 would have been 3 times the best estimate, despite relatively poor recruitment since 1994. In conclusion, disease information can be used to explain and predict changes in populations that have confounded traditional fisheries assessment.

The Laurentian Great Lakes strain (MI03) of the viral haemorrhagic septicaemia virus is highly pathogenic for juvenile muskellunge, Esox masquinongy (Mitchill).

The Great Lakes strain of viral haemorrhagic septicaemia virus (VHSV) isolated from adult subclinical muskellunge, Esox masquinongy (Mitchill), in Lake St. Clair, MI, USA was shown to be highly pathogenic in juvenile muskellunge through intraperitoneal (i.p.) injection and waterborne challenge. Mortality began as early as 3 days after exposure in waterborne challenged fish, whereas fish infected by the i.p. route experienced the first mortality by 5 days post-infection (p.i.). The median lethal intraperitoneal injection dose (IP-LD(50)) was approximately 2.21 plaque forming units (PFU) as opposed to the median lethal immersion challenge dose (IM-LD(50)) of 1.7 x 10(4) PFU mL(-1). A high, medium and low dose of infection caused acute, subacute and chronic progression of the disease, respectively, as was evident by the cumulative mortality data. Clinical signs of disease observed in dead and moribund fish were very pale gills, dermal petechial haemorrhages along the flanks, severe nuchal haemorrhages, intramuscular haemorrhages at the fin-muscle junction and focal haemorrhaging on the caudal peduncle. Internal lesions included livers that were pale, discoloured and friable, and kidneys that were either congested or degenerative in appearance, and petechial to ecchymotic haemorrhages on the swim bladder wall. Histopathologic examination demonstrated massive haemorrhages in the swimbladder wall and muscle, severe vacuolation and multifocal necrosis of the liver, multifocal necrosis of the gills and depletion of lymphoid tissues within the spleen. Kidney tissues also exhibited a mixed pattern of degeneration that included tubular necrosis, interstitial oedema and congestion. Virus was recovered from kidney and spleen tissues through tissue culture and reverse transcriptase-polymerase chain reaction (RT-PCR).

Characterization of a VHS virus genotype III isolated from rainbow trout (Oncorhychus mykiss) at a marine site on the west coast of Norway.

BACKGROUND: Norwegian production of rainbow trout (Oncorhynchus mykiss) has been without any outbreaks of VHS for many years until the disease emerged in a farm in western Norway in November 2007. The fish were, in addition to VHS virus, positive for gill chlamydia-like bacteria, Flavobacterium psychrophilum, and a microsporidian. A new VHS virus genotype III was isolated from the fish in RTgill-W1 cells and the complete coding region (11,065 nucleotides) was sequenced. This virus was also used in a challenge experiment to see if it could cause any mortality in rainbow trout in sea water. RESULTS: This is the first time a nearly complete sequence of a genotype III virus isolate has been presented. The organization of the genes is the same as in the other VHS virus genotypes studied (GI and GIV). Between the ORFs are nontranslated regions that contain highly conserved sequences encompassing the polyadenylation signal for one gene, and the putative transcription initiation site of the next gene. The intergenic regions vary in length from 74 nt to 128 nt. The nucleotide sequence is more similar to genotype I isolates compared to isolates from genotype II and IV. Analyses of the sequences of the N and G protein genes show that this new isolate is distinct from other VHS virus isolates and groups closely together with isolates from genotype III. In a challenge experiment, using intraperitoneal (ip) injection of the isolate, co-habitation with infected fish, and bath challenge, mortalities slightly above 40% were obtained. There was no significant difference in mortality between the bath challenged group and the ip injected group, while the mortality in the co-habitation group was as low as 30%. CONCLUSIONS: All VHS virus isolates in genotype III are from marine fish in the North East Atlantic. Unlike the other known genotype III isolates, which are of low virulence, this new isolate is moderately virulent. It was not possible to detect any changes in the virus genome that could explain the higher virulence. A major problem for the study of virulence factors is the lack of information about other genotype III isolates.