Development of a sensitive and controlled real-time RT-PCR assay for viral haemorrhagic septicaemia virus (VHSV) in marine salmonid aquaculture

A survey was undertaken to determine the potential distribution of viral haemorrhagic septicaemia virus (VHSV) in marine cage-based salmonid farms in Scotland. A rapid, accurate and sensitive quantitative real-time RT-PCR (qRT-PCR) assay was developed, targeting a conserved region of the nucleoprotein (N) gene of the virus. The qRT-PCR assay was shown to be more sensitive than the conventional VHSV RT-PCR. A validation protocol included several different virus isolates as the target and confirmed that the assay could detect all European VHSV genotypes (I, II and III). Both endogenous and exogenous controls were designed to control for integrity of template and distinguish between true VHSV positives and contamination with the positive control material. In total, the universal European VHSV qRT-PCR assay with exogenous positive control was applied to screen 2040 individual Atlantic salmon Salmo salar and 150 individual rainbow trout Oncorhynchus mykiss. No evidence of the presence of VHSV in association with either salmonid species in Scottish marine farms was detected. However, both marine Atlantic salmon and rainbow trout are still considered possible carriers of VHSV, which remains a potential threat to freshwater farming. Therefore, a continued surveillance of these species in marine environment is recommended.     

Genetic relatedness among Japanese,American and European isolates of viral hemorrhagic septicemia virus (VHSV) based on partial G and P genes

Molecular virological analyses of 8 Japanese VHSV (viral hemorrhagic septicemia virus) isolates from wild and farmed Japanese flounder Paralichthys olivaceus were performed to investigate their genetic relatedness to American and European isolates of VHSV. Phylogenetic analyses based on the partial nucleotide sequences of G and P genes revealed that there are 2 genogroups of VHSV in Japan. The first one represented by the Obama25 isolate is closely related to the American isolates (Genogroup I) while the other, the KRRV9601 isolate, is closely related to the traditional European isolates (Genogroup III). The 2 types of Japanese VHSV showed differences in the relative mobility of the G protein and intensity of the antibody reaction on the P and M proteins. The Obama25 type of VHSV is widely distributed as a native virus in the coastal areas of western Japan and has been responsible for the occurrence of VHSV infection in farmed Japanese flounder while the KRRV9601 isolate is considered to have been introduced from a foreign country.     

Experimental horizontal transmission of viral hemorrhagic septicemia virus (VHSV) in Japanese flounder Paralichthys olivaceus

Infection by viral hemorrhagic septicemia virus (VHSV) has recently occurred among wild and farmed Japanese flounder Paralichthys olivaceus in Japan. In the present study, horizontal transmission of VHSV among Japanese flounder was experimentally demonstrated by immersion challenge. Exposure to a flounder isolate (Obama25) of VHSV revealed a dose-response, with higher mortality (81 and 70%) at the 2 higher exposure levels (6.0 and 4.0 log10 TCID50 ml(-1)). In a second experiment, high titers of VHSV were expressed from moribund and dead flounder based on virus detection in holding-tank waters 2 to 3 d prior to death of the fish and 1 d after death. The virus could not be detected in tank waters 2 d after death. Finally, a third cohabitation experiment in small tanks demonstrated horizontal transmission of VHSV from experimentally infected to uninfected fish.     

Effects of temperature on infectivity and of commercial freezing on survival of the North American strain of viral hemorrhagic septicemia virus (VHSV)

Temperature affected the growth of the North American strain of viral hemorrhagic septicemia virus (VHSV) in experimentally infected cell cultures and in Pacific sardine Sardinops sagax. In addition, commercial freezing significantly reduced the infectivity of VHSV in tissues of experimentally infected sardine. Isolates of VHSV representing the geographic range of North American VHSV replicated in the EPC (Epithelioma papulosum cyprini) cell line at 10, 15 and 20 degrees C, but the more northern isolates from British Columbia, Canada, demonstrated significantly reduced growth at 20 degrees C compared to VHSV from more southern locations (p <0.001). An injection challenge of Pacific sardine with VHSV from California resulted in 66.7% mortality at a seawater temperature of 13 degrees C compared to 6.7% at 20 degrees C. Commercial blast-freezing of sardine experimentally infected with VHSV reduced median concentrations of virus in the kidney and spleen from 5.25 x 10(6) to 5.5 x 10(3) pfu (plaque-forming units) g(-1). Decreased growth of the California isolate of VHSV at higher temperatures following experimental infection of the sardine and reduced virus survival following commercial freezing of infected sardine are factors that would lessen the risk of transmission of VHSV through frozen baitfishes.     

Shedding of viral hemorrhagic septicemia virus (Genotype IVb) by experimentally infected muskellunge (<Emphasis Type="Italic">Esox masquinongy</Emphasis>)

Previous experimental infection demonstrated that juvenile muskellunge (Esox masquinongy) can survive experimental infection of viral hemorrhagic septicemia virus, Genotype IVb (VHSV IVb) at a low concentration of exposure. Herein we report that survivors of experimental infection with VHSV IVb shed the virus into the surrounding environment for an extended period of time. When muskellunge were exposed to VHSV IVb by immersion at a concentration of 1,400 plaque forming units (PFU)/ml, VHSV IVb was detected in the water of surviving fish for up to 15 weeks postexposure (p.e.) with the highest levels of shedding occurring between weeks 1 and 5 p.e. We estimated that each juvenile muskellunge can shed upwards of 1.36×10(5) PFU/fish/h after initial exposure signifying the uptake and amplification of VHSV to several orders of magnitude above the original exposure concentration. Muskellunge surviving low concentration exposure were re-infected with VHSV IVb by immersion at week 22 p.e. at concentrations ranging from 0 to 10(6) PFU/ml. Viral shedding was detected in all re-exposed fish, including mock rechallenged controls up to 15 consecutive weeks. Rates of viral shedding were substantially higher following rechallenge in the first 5 weeks. The highest rate of viral shedding was approximately 4.6×10(6) PFU/fish/h and shedding did not necessarily correspond to the re-exposure VHSV concentration. The results of this study shed new light into the dynamics of VHSV IVb shedding in a highly susceptible host and provide useful insights to fishery managers to design effective control strategies to this deadly virus.     

Occurrence of viral haemorrhagic septicaemia virus (VHSV) in wild marine fish species in the coastal regions of Norway

This study was aimed at determining the occurrence of viral haemorrhagic septicaemia virus (VHSV) in selected stocks of wild fish species in the coastal regions of Norway. Six cruises were undertaken covering areas which included the coastal regions of northern Norway, the Norwegian Sea, the Barents Sea, and the Skagerrak area down to the Danish border. Collected, pooled samples of internal organs (kidney, spleen and heart) from 8395 fish were examined for the presence of VHSV by inoculation on BF-2 cells. Identification of virus was performed by a standard ELISA procedure with monoclonal antibody IP5B11, which is specific for the VHSV nucleocapsid protein (N-protein). VHSV was isolated from blue whiting and Norway pout in Skagerrak. No positive samples were detected in the northern coastal regions of Norway, the Norwegian Sea, or the Barents Sea. The findings indicate a very low occurrence of VHSV in the coastal regions of Norway. Geographically, the only positive samples were obtained from fish collected in areas where VHSV has previously been found in different species of fish.     

Screening for Viral Hemorrhagic Septicemia Virus in Marine Fish along the Norwegian Coastal Line

Viral hemorrhagic septicemia virus (VHSV) infects a wide range of marine fish species. To study the occurrence of VHSV in wild marine fish populations in Norwegian coastal waters and fjord systems a total of 1927 fish from 39 different species were sampled through 5 research cruises conducted in 2009 to 2011. In total, VHSV was detected by rRT-PCR in twelve samples originating from Atlantic herring (Clupea harengus), haddock (Melanogrammus aeglefinus), whiting (Merlangius merlangus) and silvery pout (Gadiculus argenteus). All fish tested positive in gills while four herring and one silvery pout also tested positive in internal organs. Successful virus isolation in cell culture was only obtained from one pooled Atlantic herring sample which shows that today''s PCR methodology have a much higher sensitivity than cell culture for detection of VHSV. Sequencing revealed that the positive samples belonged to VHSV genotype Ib and phylogenetic analysis shows that the isolate from Atlantic herring and silvery pout are closely related. All positive fish were sampled in the same area in the northern county of Finnmark. This is the first detection of VHSV in Atlantic herring this far north, and to our knowledge the first detection of VHSV in silvery pout. However, low prevalence of VHSV genotype Ib in Atlantic herring and other wild marine fish are well known in other parts of Europe. Earlier there have been a few reports of disease outbreaks in farmed rainbow trout with VHSV of genotype Ib, and our results show that there is a possibility of transfer of VHSV from wild to farmed fish along the Norwegian coast line. The impact of VHSV on wild fish is not well documented.     

Detection of rainbow trout antibodies against viral haemorrhagic septicaemia virus (VHSV) by neutralisation test is highly dependent on the virus isolate used

Three serological tests, enzyme linked immunosorbent assay (ELISA), 50% plaque neutralisation test (50%PNT) and Western blotting (WB), were used to detect antibodies against viral haemorrhagic septicaemia virus (VHSV) in 50 rainbow trout broodstock from a rainbow trout farm endemically infected with VHS but with no clinical signs of infection. When the sera were examined by 50%PNT using the VHSV reference isolate DK-F1 or the heat attenuated DK-F25 mutant strain, no neutralizing antibodies were found. In contrast, when one of the virus isolates from the farm (homologous virus) was used in the 50%PNT, 90% of the fish were found to be positive. By examining a panel of different VHSV isolates in 50%PNT, it was demonstrated that the virus isolate used as test antigen could significantly affect the sensitivity and titre determination in 50%PNT for detection of rainbow trout antibodies against VHSV. When the sera were examined for the presence of VHSV antibodies by ELISA or WB, 61% were found to be positive. When conducting WB analysis, the viral glycoprotein was the protein most frequently recognized, followed by the viral nucleoprotein.     

Production of the 57 kDa major surface antigen by a non-agglutinating strain of the fish pathogen Renibacterium salmoninarum

Both the prevalence and tissue titer of viral hemorrhagic septicemia virus (VHSV) increased in Pacific herring Clupea pallasi following their introduction into net pens (pounds) used in the closed pound spawn-on-kelp (SOK) fishery in Prince William Sound, Alaska. VHSV was also found in water samples from inside and outside the SOK pounds after herring had been confined for several days; however, water samples taken near wild free-ranging, spawning herring either failed to test positive or tested weakly positive for virus. Little or no virus was found in tissue samples from free-ranging, spawning herring captured from the vicinity of the pounds, nor did the prevalence of VHSV increase following spawning as it did in impounded herring. The data indicated that increased prevalences of VHSV were correlated with confinement of herring for the closed pound SOK fishery and that infection was spread within the pounds through waterborne exposure to virus particles originating from impounded fish. In addition, pounds containing predominantly young fish had higher prevalences of VHSV, suggesting that older fish may be partially immune, perhaps as a result of previous infection with the virus. Operation of SOK pounds during spawning seasons in which young herring predominate may amplify the disease and possibly exacerbate the population fluctuations observed in wild herring stocks.     

Comparison of two birnavirus-rhabdovirus coinfections in fish cell lines

Aquabirnaviruses, such as the infectious pancreatic necrosis virus (IPNV), Novirhabdoviruses, such as the infectious hematopoiteic necrosis virus (IHNV) and the viral hemorrhagic septicemia virus (VHSV), cause considerable losses to the salmonid industry worldwide. Coinfections of 2 viruses have been described, but the interactions between rhabdoviruses and birnaviruses have not been examined closely. Using virus titration, flow cytometry and RT-PCR assays, we compared the effect of IPNV on the replication of IHNV and VHSV in tissue culture cells. RT-PCR assays indicated that simultaneous infection of IPNV with VHSV does not affect the replication of the rhabdovirus either in the first or successive passages; the infective titers were similar in single and double infections. In contrast, coinfection of IPNV with IHNV induced a fall in infectivity, with reduced expression of IHNV viral antigens in BF-2 cells from Lepomis macrochirus and a loss of 4.5 log10 units of the infective titer after 3 successive passages. It was possible to stimulate BF-2 cells to produce significant interferon-like activity against IHNV but not against VHSV.     

Using 96-well tissue culture polystyrene plates and a fluorescence plate reader as tools to study the survival and inactivation of viruses on surfaces

A method for studying the behavior of viruses on surfaces has been developed and is illustrated by determining the temperatures that inactivate adsorbed viral hemorrhagic septicemia virus (VHSV) and the concentration of 1-propanol that disinfected surfaces with adsorbed VHSV and chum salmon virus (CSV). VHSV is a rhabdovirus; CSV, a reovirus, and they were detected with two fish cell lines, EPC and CHSE-214, respectively. When polystyrene tissue culture surfaces were incubated with virus, rinsed, and left to dry, they still supported the attachment and spreading of cell lines and after 7 days these cells showed the characteristic CPE of the viruses. Thus cells appeared to be infected directly from surfaces on which viruses had been adsorbed. Applying this property to 96-well plates allowed duplicate surfaces to be examined for their infectiousness or support of CPE. For each treatment 80 replicate surfaces in a 96-well plate were tested at one time and the results expressed as the number of wells showing CPE. VHSV adsorbed to polystyrene was inactivated by drying in the dark at temperatures above 14 °C, but remained infectious for at least 15 days of drying at 4 °C. For chemical sterilization of polystyrene surfaces with adsorbed virus, disinfection was achieved with 1-propanol at 40% for VHSV and at 60% for CSV. As CPE can be conveniently monitored in 96-well plates with a fluorescence plate reader, this method can be used to rapidly evaluate a variety of treatments for their ability to inactivate surface-bound viruses.     

Susceptibility of juvenile sole Solea senegalensis to marine isolates of viral haemorrhagic septicaemia virus from wild and farmed fish

The susceptibility of sole Solea senegalensis to infection with 3 viral haemorrhagic septicaemia virus (VHSV) strains obtained from wild Greenland halibut Reinhardtius hippoglossoides and farmed turbot Psetta maxima was demonstrated. Fish were infected by an intraperitoneal (i.p.), immersion or cohabitational route, and maintained at 16 degrees C. Infection trials showed that VHSV isolates were pathogenic for sole fingerlings by i.p. injection and waterborne exposure causing moderate levels of mortality (10 to 55%). In addition, the mortality observed in fish cohabitating with i.p.-infected sole confirms horizontal transmission of the virus. However, the low rates of mortality registered in this challenge suggest that there is a low dissemination of virus by the i.p.-infected sole, which results in lower secondary challenge of the cohabitating fish. External signs of disease included haemorrhaging of the ventral area and ascitic fluid in the body cavity. Dead fish were tested for VHSV by both cell culture and RT-PCR assay, using pools of kidney and spleen from 10 individuals. Virus was recovered from most of the pools composed of dead fish. The results obtained in this study not only demonstrate the susceptibility of sole to the VHSV strains employed but also indicate that wild VHSV marine isolates represent a potential risk for sole aquaculture.     

Nonvirion protein of novirhabdovirus suppresses apoptosis at the early stage of virus infection

Viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) are members of the genus Novirhabdovirus within the Rhabdoviridae family, which can cause severe hemorrhagic disease in fresh- and saltwater fish worldwide. These viruses carry an additional nonvirion (NV) gene, which codes for the nonstructural NV protein that has been implicated to play a role in viral pathogenesis. To determine the precise biological function of this NV gene and its gene product, we generated NV-deficient and NV knockout recombinant VHSVs, using reverse genetics. Comparisons of the replication kinetics and markers for virus-induced apoptosis indicated that the NV-deficient and NV knockout mutant viruses induce apoptosis earlier in cell culture than the wild-type recombinant VHSV. These results suggest that the NV protein has an antiapoptotic function at the early stage of virus infection. Furthermore, we created a chimeric VHSV, in which the NV gene of VHSV was replaced by the IHNV NV gene, which was capable of suppressing apoptosis in cell culture. These results show that the NV protein of other members of Novirhabdovirus can restore the NV protein function. In this study, we also investigated the kinetics of VHSV replication during a single round of viral replication and examined the mechanism of VHSV-induced apoptosis. Our results show that VHSV infection induced caspases 3, 8 and 9 in cell culture.     

The role of viral hemorrhagic septicemia virus (VHSV) NV gene in TNF-α- and VHSV infection-mediated NF-κB activation

The role of viral hemorrhagic septicemia virus (VHSV) NV gene in nuclear factor-κB (NF-κB) activation was investigated. Epithelioma papulosum cyprini (EPC) cells pre-treated with tumor necrosis factor (TNF)-α showed a strong resistance against VHSV infection, but cells treated with TNF-α after VHSV infection showed no resistance, suggesting that immediate early TNF-α-mediated responses inhibit VHSV replication. Activation of NF-κB is a key step in TNF-α-mediated immunomodulatory pathways. In this study, activation of NF-κB by TNF-α exposure was inhibited in EPC cells harboring NV gene expressing vectors, indicating that the NV gene of VHSV can suppress TNF-α-mediated NF-κB activation. Furthermore, the NV gene knock-out recombinant VHSV (rVHSV-ΔNV-EGFP) induced significantly higher NF-κB activity in EPC cells than wild-type VHSV, suggesting that VHSV adopted a strategy to suppress early activation of NF-κB in host cells through and NV gene.