Genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV)

The nucleotide sequences of a specific region of the nucleoprotein gene were compared in order to investigate the genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV). Analysis of the sequence from 128 isolates of diverse geographic and host origin renders this the most comprehensive molecular epidemiological study of marine VHSV conducted to date. Phylogenetic analysis of nucleoprotein gene sequences confirmed the existence of the 4 major genotypes previously identified based on N- and subsequent G-gene based analyses. The range of Genotype I included subgroups of isolates associated with rainbow trout aquaculture (Genotype Ia) and those from the Baltic marine environment (Genotype Ib) to emphasise the relatively close genetic relationship between these isolates. The existence of an additional genotype circulating within the Baltic Sea (Genotype II) was also confirmed. Genotype III included marine isolates from around the British Isles in addition to those associated with turbot mariculture, highlighting a continued risk to the development of this industry. Genotype IV consisted of isolates from the marine environment in North America. Taken together, these findings suggest a marine origin of VHSV in rainbow trout aquaculture. The implications of these findings with respect to the future control of VHSV are discussed. The capacity for molecular phylogenetic analysis to resolve complex epidemiological problems is also demonstrated and its likely future importance to disease management issues highlighted.     

Rainbow trout surviving infections of viral haemorrhagic septicemia virus (VHSV) show lasting antibodies to recombinant G protein fragments

Rainbow trout antibodies (Abs) binding to recombinant fragments (frgs) derived from the protein G of the viral haemorrhagic septicemia virus (VHSV)-07.71 strain, could be detected by ELISA (frg-ELISA) in sera from trout surviving laboratory-controlled infections. Abs were detected not only by using sera from trout infected with the homologous VHSV isolate but also with the VHSV-DK-201433 heterologous isolate, which had 13 amino acid changes. Sera from healthy trout and/or from trout surviving infectious haematopoietic necrosis virus (IHNV) infection, were used to calculate cut-off absorbances to differentiate negative from positive sera. Specific anti-VHSV Abs could then be detected by using any of the following frgs: frg11 (56-110), frg15 (65-250), frg16 (252-450) or G21-465. While high correlations were found among the ELISA values obtained with the different frgs, no correlations between any frg-ELISA and complement-dependent 50% plaque neutralization test (PNT) titres could be demonstrated. Between 4 and 10 weeks after VHSV infection, more trout sera were detected as positives by using heterologous frg-ELISA rather than homologous PNT. Furthermore, the percentage of positive sera detected by frg11-ELISA increased with time after infection to reach 100%, while those detected by complement-dependent PNT decreased to 29.4%, thus confirming that the lack of neutralizing Abs does not mean the lack of any anti-VHSV Abs in survivor trout sera. Preliminary results with sera from field samples suggest that further refinements of the frg-ELISA could allow detection of anti-VHSV trout Abs in natural outbreaks caused by different heterologous VHSV isolates. The homologous frg-ELISA method could be useful to follow G immunization attempts during vaccine development and/or to best understand the fish Ab response during VHSV infections. The viral frgs approach might also be used with other fish species and/or viruses.     

Phylogenetic analysis of viral haemorrhagic septicaemia virus (VHSV) isolates from France (1971-1999)

The nucleotide sequences of a specific region of the glycoprotein gene were compared among 63 strains of viral haemorrhagic septicaemia virus (VHSV) isolated from fish in France between 1971 and 1999. The analysis was performed on a region corresponding to amino acids 238 to 331 of the glycoprotein gene, also designated the V2 region and previously shown to accumulate most of the mutations. The sequences of many VHSV isolates were found to be identical or very conserved. An isolate, designated L59X, obtained from elver in the Loire estuary, depicted a higher degree of divergence compared to the other French isolates. The deduced amino-acid sequences were analysed together with the results of neutralisation tests performed using monoclonal antibody 168m4 specific to serotype 1. Non-neutralised VHSV strains had mutations in the region corresponding to the previously described 168m4 epitope. Phylogenetic analysis showed that all the VHSV isolates studied, except L59X, belong to genotype I, previously described as containing VHSV strains isolated from continental Europe. Most of the VHSV isolates studied were found to be genetically related to one of the previously described VHSV strains representative of the major serotypes. Isolate L59X, which was the only French marine strain studied, was found to belong to genotype II, previously shown to encompass the VHSV strains isolated from the British Isles coastal waters. Overall there was a good correlation between the geographical origin of the studied isolates and their genetic characteristics.     

Generation and characterization of NV gene-knockout recombinant viral hemorrhagic septicemia virus (VHSV) genotype IVa

A recombinant viral hemorrhagic septicemia virus (rVHSV-deltaNV-EGFP) containing the enhanced green fluorescent protein (EGFP) gene instead of the NV gene was produced using the reverse-genetics method. For use as a positive control, another recombinant virus (rVHSV-wild) was also generated, which had an identical nucleotide sequence to the wild-type VHSV genome except for a few artificially replaced nucleotides. The rVHSVs were rescued using a system controlled by T7 RNA polymerase supplied by a retroviral vector. Generation of rVHSV-deltaNV-EGFP and rVHSV-wild was confirmed by sequencing of RT-PCR products, and rescue of infectious rVHSVs was confirmed by observation of plaque formation. Replication efficiency of rVHSV-wild was distinctly lower than that of wild-type VHSV, suggesting that the artificially replaced nucleotides, especially when immediately preceding the G or NV gene start codons, might affect the replication of the virus. Replication of rVHSV-deltaNV-EGFP was slightly lower than that of rVHSV-wild when epithelioma papulosum cyprini cells were infected with multiplicity of infection (MOI) 1.0, but much lower when cells were infected with MOI 0.00001. These results suggest that the NV gene plays an important role in VHSV replication through interactions with host-cell responses, and the lower replication ability of rVHSV-wild compared to wild-type VHSV might be caused by replaced nucleotides just before the NV gene open reading frame (ORF) rather than the G gene ORF. In olive flounder Paralichthys olivaceus, rVHSV-wild produced slower-progressing mortalities than wild-type VHSV, whereas rVHSV-deltaNV-EGFP pathogenesis was highly attenuated. These results suggest that the NV protein of VHSV may play an important role not only in viral replication but also in viral pathogenesis.     

Oral immunization of olive flounder (Paralichthys olivaceus) with recombinant live viral hemorrhagic septicemia virus (VHSV) induces protection against VHSV infection

A recombinant viral hemorrhagic septicemia virus (rVHSV-ΔNV-EGFP) that has enhanced green fluorescent protein (EGFP) gene instead of NV gene was previously generated using reverse genetics technology. In this study, potential of the rVHSV-ΔNV-EGFP to be used as a live oral vaccine candidate was assessed. The presence of the recombinant virus in internal organs of orally administered olive flounder (Paralichthys olivaceus) was analyzed by semi-quantitative RT-PCR. Although the recombinant VHSV-specific band was detected only when the number of PCR cycle was increased to 35, the band was detected from internal organs, such as kidney, spleen, and liver of fish that were reared at either 15 °C or 20 °C till even 20 days, suggesting that a few orally administered rVHSV-ΔNV-EGFP might be transported to internal organs, and might keep weak replication ability in the organs. VHSV-neutralizing activity was induced by oral immunization of olive flounder with the NV gene knock-out recombinant VHSV not only in skin and intestinal mucus but also in serum, suggesting that mucosal and systemic adaptive immune responses were elicited by oral immunization. In challenge experiment, groups of fish immunized with 10?, 10?, and 2 × 10? PFU of rVHSV-ΔNV-EGFP/fish showed 25%, 50%, and 70% of relative percent survival (RPS), respectively. The RPSs were elevated to 60%, 75%, and 90% by a boost immunization in fish boost immunized with 10?, 10?, and 2 × 10? PFU of rVHSV-ΔNV-EGFP, respectively. The cumulative mortality of fish in the control groups was 100%. Conclusionly, the present results demonstrate that the NV gene knock-out recombinant VHSV administered orally to olive flounder can induce dose- and boosting-dependent VHSV-neutralizing antibody in mucus and serum, and can provide a high protection in olive flounder against a virulent VHSV challenge.     

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.     

Immune response of heterologous recombinant antigenic protein of viral hemorrhagic septicemia virus (VHSV) in mice

Viral hemorrhagic septicemia (VHS) is an important infectious disease in fish worldwide caused by viral hemorrhagic septicemia virus (VHSV). VHSV is the causative agent of serious systemic diseases in fish, affecting a number of teleost fish species. In this study, VHSV glycoprotein (G), including its epitope, as a subunit vaccine candidate, was expressed in tobacco plant (Nicotiana tabacum). The recombinant gene, VHSVG, was fused to the immunoglobulin Fc fragment and extended with the endoplasmic reticulum (ER) retention signal (KDEL) to generate VHSVG-FcK. The recombinant expression vector for VHSVG-FcK was transferred into Agrobacterium tumefaciens (LBA4404), and plant transformation was conducted N. tabacum. Polymerase chain reaction (PCR) was performed to confirm gene insertion and VHSVG-FcK protein expression was confirmed by immunoblot analysis. VHSVG-FcK protein was successfully purified from tobacco plant leaves. Furthermore, ELISA analysis showed that mice serum immunized with the plant-derived VHSVG-FcK (VHSVGP-FcK) had a high absorbance against VHSVG-FcK, indicating that the plant-derived recombinant subunit vaccine protein VHSVG-FcK can induce immune response. Taken together, this recombinant vaccine protein can be expressed in plant expression systems and can be appropriately assembled to be functional in immunogenicity.     

Variable domain antibodies specific for viral hemorrhagic septicemia virus (VHSV) selected from a randomized IgNAR phage display library

Phage display libraries are used to screen for nucleotide sequences that encode immunoglobulin variable (V) regions that are specific for a target antigen. We previously constructed an immunoglobulin new antigen receptor (IgNAR) phage display library. Here we used this library to obtain an IgNAR V region that is specific for viral hemorrhagic septicemia virus (VHSV). A phage clone (clone 653) was found to be specific for VHSV by the biopanning method. The V region of clone 653 was used to construct a 6 × His tagged recombinant IgNAR-653 V protein (rIgNAR-653) using the Escherichia coli pET system. The rIgNAR-653 protein bound specifically to VHSV, confirming its activity.     

Risk of introducing viral hemorrhagic septicemia virus (VHSV) to the Chilean South Pacific via sardine imports from Europe

Chile imports from Spain 100s of metric tons of frozen sardine Sardina pilchardus fished in European oceans, which, with several other clupeids, are presumed susceptible to infection with viral hemorrhagic septicemia virus (VHSV). The frozen sardines are directly introduced into the sea as bait to catch southern hake Merluccius australis in the same areas where wild and pen-raised salmonids are present. A simulation model was therefore developed to evaluate the potential risk of infection of wild Chilean southern hake with VHSV from imported bait. The model indicated that VHSV-susceptible fish species present in Chilean waters, like southern hake, are not at immediate risk of infection. However, sensitivity analyses showed that infectious doses at lower concentrations of VHSV combined with higher VHSV-prevalence import scenarios could likely result in VHSV infections of a moderate number of indigenous southern hake (> or =54 fish yr(-1)).     

Modeling the secondary spread of viral hemorrhagic septicemia virus (VHSV) by commercial shipping in the Laurentian Great Lakes

Researchers have only begun to study the role of shipping in the spread of invasive species in the Laurentian Great Lakes despite a well-documented history of introductions in these lakes due to ballast water release. Here, we determine whether ballast water discharge was a likely vector of spread of the fish disease, viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb), throughout the Great Lakes and St. Lawrence Seaway. Three models were developed to assess whether the spread of VHSV was due to (1) chance (random model), or (2) ballast water discharge (location model), and whether (3) increased propagule pressure, as measured by the number of visitations by ships carrying ballast water from VHSV infected areas, increased the likelihood of a discharge location becoming infected with VHSV (propagule pressure model). The third model was also used to assess the probable point of initial introduction of VHSV. Presence and absence accuracies and weighted Cohen’s kappa were calculated to determine which models best predicted observed presences and absences of VHSV. Location models explain the patterns of VHSV detections better than random models, and inclusion of “propagule pressure” often improved model fit; however, the relationship is weak likely because of a long lag time between introduction and detection, a high rate of false negatives in reporting, and the possible contribution of other vectors of spread. Montreal was also identified as the more likely introduction site of VHSV, rather than Lake St. Clair, the site where the virus was first detected.     

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.     

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.     

An outbreak of VHSV (viral hemorrhagic septicemia virus) infection in farmed Japanese flounder Paralichthys olivaceus in Japan.

A rhabdoviral disease occurred in farmed populations of market sized Japanese flounder (hirame) Paralichthys olivaceus in the Seto Inland Sea of Japan in 1996. The causative agent was identified as viral hemorrhagic septicemia virus (VHSV) based on morphological, immunological, and genetic analyses. Diseased fish that were artificially injected with a representative virus isolate showed the same pathological signs and high mortality as observed in the natural outbreak. This is the first report of an outbreak of VHSV infection in cultured fish in Japan. Clinical signs of diseased fish included dark body coloration, an expanded abdomen due to ascites, congested liver, splenomegaly, and a swollen kidney. Myocardial necrosis was most prominent and accompanied by inflammatory reactions. Necrotic lesions also occurred in the liver, spleen and hematopoietic tissue, and were accompanied by circulatory disturbances due to cardiac failure. Hemorrhagic lesions did not always appear in the lateral musculature. Transmission electron microscopy revealed many rhabdovirus particles and associated inclusion bodies containing nucleocapsids in the necrotized myocardium. The histopathological findings indicated that the necrotizing myocarditis could be considered a pathognomonic sign of VHSV infection in Japanese flounder.     

Diagnostic capacity for viral haemorrhagic septicaemia virus (VHSV) infection in rainbow trout (Oncorhynchus mykiss) is greatly increased by combining viral isolation with specific antibody detection

Detection of disease specific antibodies in farmed rainbow trout (Oncorhynchus mykiss) has been proposed as an alternative or supplement to the currently approved procedures for diagnosis and surveillance in this species. In samples from natural outbreaks of the disease viral haemorrhagic septicaemia (VHS) at two freshwater farms in southern Denmark serologic testing was used to broaden the diagnostic window from outbreak to diagnosis in the laboratory as compared to traditional procedures of isolation and identification of the virus. The serologic assay clearly increased the chance of detecting present or previous infections where the pathogen could not be isolated by standard methods (indicating older infections where the virus had been cleared). Our data allowed us to monitor the levels of neutralising antibodies in relation to the presence of the virus in fish experiencing two different types of outbreaks at two different farms. By sequence analysis of the viral glycoprotein from selected isolates we found no evidence for escape mutants having developed in the fish showing high titres of neutralising antibodies.     

Protection and antibody response induced by intramuscular DNA vaccine encoding for viral haemorrhagic septicaemia virus (VHSV) G glycoprotein in turbot (Scophthalmus maximus)

Turbot (Scophthalmus maximus) is a high-value farmed marine flatfish with growing demand and production levels in Europe susceptible to turbot-specific viral haemorrhagic septicaemia virus (VHSV) strains. To evaluate the possibility of controlling the outbreaks of this infectious disease by means of DNA vaccination, the gpG of a VHSV isolated from farmed turbot (VHSV(860)) was cloned into an expression plasmid containing the human cytomegalovirus (CMV) promoter (pMCV1.4-G(860)). In our experimental conditions, DNA immunised turbots were more than 85% protected against VHSV(860) lethal challenge and showed both VHSV-gpG specific and neutralizing antibodies. To our knowledge this is the first report showing the efficacy of turbot genetic immunisation against a VHSV. Work is in progress to determine the contribution of innate and adaptive immunity to the protective response elicited by the immunization.