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The monoclonal antibody (mAb) IP5B11, which is used worldwide for the diagnosis of viral haemorrhagic septicaemia (VHS) in fish, reacts with all genotypes of VHS virus (VHSV). The mAb exceptionally also reacts with the carpione rhabdovirus (CarRV). Following next generation genome sequencing of CarRV and N protein sequence alignment including five kinds of fish novirhabdoviruses, the epitope recognized by mAb IP5B11 was identified. Dot blot analysis confirmed the epitope of mAb IP5B11 to be associated with the region N219 to N233 of the N protein of VHSV. Phylogenetic analysis identified CarRV as a new member of the fish novirhabdoviruses.

In November 2016, infectious pancreatic necrosis virus (IPNV) was isolated from a broodstock female of landlocked sea trout (Salmo trutta) in Lake Vänern in Sweden. VP2 gene sequencing placed the IPNV isolate in genogroup 6, for which pathogenicity is largely unknown. Lake Vänern hosts landlocked sea trout and salmon populations that are endangered, and thus the introduction of new pathogens poses a major threat. In this study we characterized the novel isolate by conducting an infection trial on three salmonid species present in Lake Vänern, whole genome sequencing of the isolate, and prevalence studies in the wild sea trout and salmon in Lake Vänern. During the infection trial, the pathogenicity of the Swedish isolate was compared to that of a pathogenic genogroup 5 isolate. Dead or moribund fish were collected, pooled, and analyzed by cell culture to identify infected individuals. In the trial, the Swedish isolate was detected in fewer sample pools in all three species compared to the genogroup 5 isolate. In addition, the prevalence studies showed a low prevalence (0.2–0.5%) of the virus in the feral salmonids in Lake Vänern. Together the data suggest that the novel Swedish IPNV genogroup 6 isolate is only mildly pathogenic to salmonids.

Viral Nervous Necrosis (VNN) causes high mortality and reduced growth in farmed European sea bass (Dicentrarchus labrax) in the Mediterranean. In the current studies, we tested a novelPichia-produced virus-like particle (VLP) vaccine against VNN in European sea bass, caused by the betanodavirus “Red-Spotted Grouper Nervous Necrosis Virus” (RGNNV). European sea bass were immunized with a VLP-based vaccine formulated with different concentrations of antigen and with or without adjuvant. Antibody response was evaluated by ELISA and serum neutralization. The efficacy of these VLP-vaccine formulations was evaluated by an intramuscular challenge with RGNNV at different time points (1, 2 and 10 months post-vaccination) and both dead and surviving fish were sampled to evaluate the level of viable virus in the brain. The VLP-based vaccines induced an effective protective immunity against experimental infection at 2 months post-vaccination, and even to some degree at 10 months post-vaccination. Furthermore, the vaccine formulations triggered a dose-dependent response in neutralizing antibodies. Serologic response and clinical efficacy, measured as relative percent survival (RPS), seem to be correlated with the administered dose, although for the individual fish, a high titer of neutralizing antibodies prior to challenge was not always enough to protect against disease. The efficacy of the VLP vaccine could not be improved by formulation with a water-in-oil (W/O) adjuvant. The developed RGNNV-VLPs show a promising effect as a vaccine candidate, even without adjuvant, to protect sea bass against disease caused by RGNNV. However, detection of virus in vaccinated survivors means that it cannot be ruled out that survivors can transmit the virus.

Koi herpesvirus (KHV) infections cause high mortality in carp (Cyprinus carpio). This study compared the susceptibility of silver crucian carp (Carassius auratus langsdorfii), also called ginbuna, and koi carp to KHV infection. Silver crucian carp and koi carp were challenged with KHV by both intraperitoneal injection and immersion, respectively, and kept in tanks at 22°C. All KHV-exposed koi carp died within 14 days post-infection (dpi), whereas no clinics nor mortality was observed in the KHV-exposed silver crucian carp. KHV DNA was detected in both koi and silver crucian carp shortly after infection. At 7 dpi, the copy numbers of KHV genome were increased in koi carp but decreased in silver crucian carp. Using reverse transcriptase PCR, KHV mRNA was detected in koi carp but not in silver crucian carp. Cell cultivation on common carp brain (CCB) cell samples from koi carp caused KHV-associated cytopathic effects in CCB cells. Therefore, we concluded that KHV replicated in koi carp but not in silver crucian carp and that silver crucian carp is not susceptible to infection with KHV.

Several reverse genetics systems for viral haemorrhagic septicaemia virus (VHSV) have been developed over the last decade. These systems have been based on genotype Ia, IVa and IVb isolates and have used the fish cell line EPC, which is less susceptible to some VHSV isolates belonging to genotype I and genotypes II and III. While developing a reverse genetics system in our laboratories for VHSV genotype Ib, we realized that the isolate in interest (SE SVA 1033 9C) did not grow in EPC cells and it was necessary to adapt the reverse genetics protocols to the BF-2 fish cell line. This cell line is very sensitive to high temperatures and is therefore not compatible with the original protocols based on the use of recombinant vaccinia virus (vTF7-3) as a provider of the T7 RNA polymerase (T7-RNAP) to the system, which includes incubation periods at 37 °C. Transfection efficiency was assessed in BF-2 cells using a reporter plasmid and it showed to be highest when using Lipofectamine™ 3000 compared to other transfection reagents. A luciferase assay was performed to determine the optimal activity of T7-RNAP in BF-2 cells with different amounts of vTF7-3. We successfully recovered recombinant VHSV (rVHSV) in BF-2 cells by reducing the incubation time at 37 °C after transfection to both 3 and 6 h. Another strategy we attempted successfully was to transfect mammalian BHK-21 cells, which are routinely used to propagate vTF7-3, and after the 37 °C incubation period, a BF-2 cell suspension was added hypothesizing that the virions formed in the transfected mammalian cells would infect the subsequently added fish cells at 15 °C incubation over the following days. We have successfully recovered rVHSV from both BHK-21 with a BF-2 cells suspension as well as a new protocol for VHSV reverse genetics in BF-2 cells has been established.

International audience; Viral hemorrhagic septicemia virus (VHSV) is a highly contagious virus leading to high mortality in a large panel of freshwater and marine fish species. VHSV isolates originating from marine fish show low pathogenicity in rainbow trout. The analysis of several nearly complete genome sequences from marine and freshwater isolates displaying varying levels of virulence in rainbow trout suggested that only a limited number of amino acid residues might be involved in regulating the level of virulence. Based on a recent analysis of 55 VHSV strains, which were entirely sequenced and phenotyped in vivo in rainbow trout, several amino acid changes putatively involved in virulence were identified. In the present study, these amino acid changes were introduced, alone or in combination, in a highly-virulent VHSV 23-75 genome backbone by reverse genetics. A total of 35 recombinant VHSV variants were recovered and characterized for virulence in trout by bath immersion. Results confirmed the important role of the NV protein (R116S) and highlighted a major contribution of the nucleoprotein N (K46G and A241E) in regulating virulence. Single amino acid changes in these two proteins drastically affect virus pathogenicity in rainbow trout. This is particularly intriguing for the N variant (K46G) which is unable to establish an active infection in the fins of infected trout, the main portal of entry of VHSV in this species, allowing further spread in its host. In addition, salmonid cell lines were selected to assess the kinetics of replication and cytopathic effect of recombinant VHSV and discriminate virulent and avirulent variants. In conclusion, three major virulence markers were identified in the NV and N proteins. These markers explain almost all phenotypes (92.7%) observed in trout for the 55 VHSV strains analyzed in the present study and herein used for the backward validation of virulence markers. The identification of VHSV specific virulence markers in this species is of importance both to predict the in vivo phenotype of viral isolates with targeted diagnostic tests and to improve prophylactic methods such as the development of safer live-attenuated vaccines.

Infectious haematopoietic necrosis virus (IHNV) is the causative agent of infectious haematopoietic necrosis (IHN), a disease of salmonids responsible for great economic losses. The disease occurs in most parts of the world where rainbow trout is reared but has not been previously reported in North Macedonia. In this study, 150 pooled samples in total, each consisting of organ mix of 10 freshly killed rainbow trout Oncorhynchus mykiss, were collected from 50 trout farms by the Food and Veterinary Agency of North Macedonia as part of the annual surveillance plan for IHN and viral haemorrhagic septicaemia (VHS) control. Screening of samples was done by cell culture and real-time RT-PCR (qRT-PCR). All 150 tested samples were VHS virus (VHSV) qRT-PCR negative. Two samples from different trout farms were IHNV qRT-PCR positive. On cell culture, 1 IHNV qRT-PCR positive sample caused cytopathic effect after 2 passages on EPC cells. The virus, isolated from an asymptomatic rainbow trout fry, was identified by qRT-PCR and designated as MAKIHNV1. The phylogenetic reconstruction indicates that the isolated virus belongs to the European E genogroup, more specifically within the E-1 clade, and is similar to the German, Italian and Iranian isolates. This study has revealed for the first time the presence of IHNV in rainbow trout in North Macedonia. However, it is not possible to make interpretations about the source of infection from the phylogenetic analysis, and the origin of MAKIHNV1 remains unclear.

Viral hemorrhagic septicemia virus (VHSV) is a highly contagious virus leading to high mortality in a large panel of freshwater and marine fish species. VHSV isolates originating from marine fish show low pathogenicity in rainbow trout. The analysis of several nearly complete genome sequences from marine and freshwater isolates displaying varying levels of virulence in rainbow trout suggested that only a limited number of amino acid residues might be involved in regulating the level of virulence. Based on a recent analysis of 55 VHSV strains, which were entirely sequenced and phenotyped in vivo in rainbow trout, several amino acid changes putatively involved in virulence were identified. In the present study, these amino acid changes were introduced, alone or in combination, in a highly-virulent VHSV 23–75 genome backbone by reverse genetics. A total of 35 recombinant VHSV variants were recovered and characterized for virulence in trout by bath immersion. Results confirmed the important role of the NV protein (R116S) and highlighted a major contribution of the nucleoprotein N (K46G and A241E) in regulating virulence. Single amino acid changes in these two proteins drastically affect virus pathogenicity in rainbow trout. This is particularly intriguing for the N variant (K46G) which is unable to establish an active infection in the fins of infected trout, the main portal of entry of VHSV in this species, allowing further spread in its host. In addition, salmonid cell lines were selected to assess the kinetics of replication and cytopathic effect of recombinant VHSV and discriminate virulent and avirulent variants. In conclusion, three major virulence markers were identified in the NV and N proteins. These markers explain almost all phenotypes (92.7%) observed in trout for the 55 VHSV strains analyzed in the present study and herein used for the backward validation of virulence markers. The identification of VHSV specific virulence markers in this species is of importance both to predict the in vivo phenotype of viral isolates with targeted diagnostic tests and to improve prophylactic methods such as the development of safer live-attenuated vaccines.

The commercial production of lumpfish

Simple Summary Replication studies are frequently based on viral production, which provides limited information to understand certain processes. Therefore, to discover which failures in the viral haemorrhagic septicaemia virus (VHSV) replication cycle might be involved in the differences in its virulence on fish, a different approach has been taken. Our results have demonstrated that adsorption and morphogenesis are the steps most involved in the modulation of virulence, although failures in the synthesis step were also observed. As a potential application of our results, we believe that this kind of knowledge relating in vivo virulence to in vitro markers could help reduce the need for experimental infections in animals, representing a step forward in ethical issues. Abstract The viral haemorrhagic septicaemia virus (VHSV), a single-stranded negative-sense RNA novirhabdovirus affecting a wide range of marine and freshwater fish species, is a main concern for European rainbow trout (Oncorhynchus mykiss) fish farmers. Its genome is constituted by six genes, codifying five structural and one nonstructural proteins. Many studies have been carried out to determine the participation of each gene in the VHSV virulence, most of them based on genome sequence analysis and/or reverse genetics to construct specific mutants and to evaluate their virulence phenotype. In the present study, we have used a different approach with a similar aim: hypothesizing that a failure in any step of the replication cycle can reduce the virulence in vivo, we studied in depth the in vitro replication of VHSV in different cell lines, using sets of strains from different origins, with high, low and moderate levels of virulence for fish. The results demonstrated that several steps in the viral replication cycle could affect VHSV virulence in fish, including adsorption, RNA synthesis and morphogenesis (including viral release). Notably, differences among strains in any step of the replication cycle were mostly strain-specific and reflected only in part the in vivo phenotype (high and low virulent). Our data, therefore, support the need for further studies aimed to construct completely avirulent VHSV recombinants targeting a combination of genes rather than a single one in order to study the mechanisms of genes interplay and their effect on viral phenotype in vitro and in vivo.

Viral haemorrhagic septicaemia (VHS) is one of the most serious viral diseases in salmonid and olive flounder farms. Various diagnostic methods for detecting VHS virus (VHSV) are described in the VHS chapter of the World Organization for Animal Health (OIE) Aquatic Diagnostic Manual. A conventional reverse transcription-PCR (cRT-PCR) targeting the viral nucleocapsid gene is recommended for the detection of VHSV and, to some extent, for genotypic classification. However, the recommended assay exhibits low sensitivity for the detection of VHSV genotype IVa isolates and often shows non-specific amplicons when the RNA template is extracted from non-infected fish cell lines. For these reasons, it is necessary to develop a new RT-PCR method for the foolproof detection of all VHSV genotypes and elimination of non-specific results. In this study, we selected five candidate primer sets that target the VHSV nucleoprotein (N) gene, and selected the most sensitive among them (3F/2R). We then established the optimal reaction conditions for these primers, and ensured that no non-specific amplification had occurred in the fish tissues, fish cell lines, or heterologous viruses. The analytical sensitivity of the novel cRT-PCR was compared to that of cell culture assays, real-time RT-PCR, and other cRT-PCR methods and was found to be as sensitive as or superior to the other methods for detecting all VHSV genotypes. Our newly developed cRT-PCR assay was tested with 80 isolates, representing a collection of all known VHSV genotypes worldwide. Clear and unique amplicons were amplified from all 80 VHSV isolates. The reproducibility, and partly the robustness, of the assay were confirmed by an inter-laboratory proficiency tests including nine laboratories. A high diagnostic sensitivity and specificity was confirmed on tissue material from affected fish. In conclusion a highly robust, sensitive and specific cRT-PCR for detection of VHSV was developed and validated.

Ranaviruses are globally emerging pathogens negatively impacting wild and cultured fish, amphibians, and reptiles. Although conventional and diagnostic real-time PCR (qPCR) assays have been developed to detect ranaviruses, these assays often have not been tested against the known diversity of ranaviruses. Here we report the development and partial validation of a TaqMan real-time qPCR assay. The primers and TaqMan probe targeted a conserved region of the major capsid protein (MCP) gene. A series of experiments using a 10-fold dilution series of Frog virus 3 (FV3) MCP plasmid DNA revealed linearity over a range of 7 orders of magnitude (107-101), a mean correlation coefficient (R2) of >0.99, and a mean efficiency of 96%. The coefficient of variation of intra- and inter-assay variability ranged from Cyprinivirus. DNA from fish tissue homogenates previously determined to be positive or negative for the ranavirus Epizootic hematopoietic necrosis virus by virus isolation demonstrated a diagnostic sensitivity of 95% and a diagnostic specificity of 100%. The reported qPCR assay provides an improved expedient diagnostic tool and can be used to elucidate important aspects of ranaviral pathogenesis and epidemiology in clinically and sublinically affected fish, amphibians, and reptiles.

Viral haemorrhagic septicaemia virus (VHSV) Genotype IVb has been isolated from amphipods belonging to the genus Diporeia, but it has yet to be established whether crustacean zooplankton act as vectors of this virus for fish species. Therefore, we evaluated the viability of infectious VHSV in the water flea Moina macrocopa. VHSV was re-isolated from replicate groups of M. macrocopa that had been immersed with 108.0, 107.0, and 105.0 TCID50 ml-1 of VHSV (DK-3592B, Genotype Ia). Furthermore, 40 M. macrocopa that had been immersed with 108.0 TCID50 ml-1 of VHSV for 72 h had VHSV titers of 102.7-104.3 TCID50. Thus, VHSV was clearly taken up by M. macrocopa and remained viable in this crustacean for several days. However, no mortality was observed over a 28 d period in rainbow trout Oncorhynchus mykiss that were fed VHSV-contaminated M. macrocopa for 14 d, and we found that the virus titer significantly decreased after a 4 h incubation with pyloric caecal extracts from rainbow trout, indicating that passage through the gut is likely to result in a significant decrease in viral titer. This may explain why consumption of prey containing low levels of VHSV did not result in clinical VHS.

Piscine orthoreovirus genotype 1 (PRV-1) is widespread in farmed Atlantic salmon (Salmo salar L.) populations in northern Europe, Canada and Chile. PRV-1 occurs in wild fish in Norway and Canada; however, little information of its geographical distribution in wild populations is currently available, and the effect of PRV-1 infection in wild populations is currently unknown. In this study, we present the findings of a survey conducted on 1,130 wild salmonids sampled in Denmark, Sweden, Ireland, Faroe Islands, France, Belgium and Greenland between 2008 and 2017. PRV-1 is reported for the first time in wild salmonids in Denmark, Sweden, Faroe Island and Ireland. The annual PRV-1 prevalence ranged from 0% in France, Belgium and Greenland to 43% in Faroe Islands. In total, 66 samples tested positive for PRV-1, including Atlantic salmon broodfish returning to spawn and Atlantic salmon collected at the feeding ground north of Faroe Islands. The phylogenetic analysis of S1 sequences of the PRV-1 isolates obtained in this survey did not show systematic geographical distribution. This study sheds light on the spread and genetic diversity of the virus identified in populations of free-living fish and provides rationale for screening wild broodfish used in restocking programmes.

Publisher's version (útgefin grein), A novel viral haemorrhagic septicaemia virus (VHSV) of genotype IV was isolated from wild lumpfish (Cyclopterus lumpus), brought to a land-based farm in Iceland, to serve as broodfish. Two groups of lumpfish juveniles, kept in tanks in the same facility, got infected. The virus isolated was identified as VHSV by ELISA and real-time RT-PCR. Phylogenetic analysis, based on the glycoprotein (G) gene sequences, may indicate a novel subgroup of VHSV genotype IV. In controlled laboratory exposure studies with this new isolate, there was 3% survival in the I.P. injection challenged group while there was 90% survival in the immersion group. VHSV was not re-isolated from fish challenged by immersion. In a cohabitation trial, lumpfish infected I.P. (shedders) were placed in tanks with naïve lumpfish as well as naïve Atlantic salmon (Salmo salar L.). 10% of the lumpfish shedders and 43%–50% of the cohabiting lumpfish survived after 4 weeks. 80%–92% of the Atlantic salmon survived, but no viral RNA was detected by real-time RT-PCR nor VHSV was isolated from Atlantic salmon. This is the first isolation of a notifiable virus in Iceland and the first report of VHSV of genotype IV in European waters., H2020 SFS-2014-2 ParaFishControl, Grant/ Award Number: 634429; European Union Reference Laboratory for Fish Diseases Grant Decision SI2.725290

Red mark syndrome (RMS) is a skin disease of rainbow trout, the prevalence of which has increased in Europe over the last two decades. Hallmark symptoms are large, haemorrhagic skin lesions. It is believed that the disease is bacterial and caused by a Midichloria-like organism (MLO). However, the bacterium has never been isolated or cultured in vitro, and is only known from its 16S rDNA sequence. Thus there is no vaccine for the disease, and no other officially recognized way of ameliorating RMS symptoms. Here we investigate for the first time the effect on RMS of in-feed treatment with three types of antibiotics: Florfenicol, oxolinic acid and oxytetracycline under controlled experimental conditions using a cohabitation model of disease transfer. In short, 160 rainbow trout were cohabited with seeder fish, which showed the classical skin pathology of RMS and tested positive for MLO. After 55 days at 12 °C the cohabitants (now weighing 223 ± 57 g) started showing very early signs of RMS-related skin pathology and were randomly divided into 8 tanks (4 treatment groups in duplicate). The fish were fed medicated (or control) feed for 10 days. The fish were evaluated visually after 7 and 14 days and finally terminated after 20 days where skin samples were taken for testing for MLO by qPCR. All three types of antibiotics significantly affected the monitored disease parameters: Macroscopic skin lesions were less severe and less MLO 16S rDNA could be detected from skin samples by qPCR in antibiotics-fed fish compared to fish that had not received antibiotics.

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a serious infective, notifiable disease affecting common carp and varieties. In survivors, infection is generally characterized by a subclinical latency phase with restricted viral replication. The CyHV-3 genome is difficult to detect in such carrier fish that represent a potential source of dissemination if viral reactivation occurs. In this study, the analytical and diagnostic performance of an alternative serum neutralization (SN) method based on the detection of CyHV-3-specific antibodies was assessed using 151 serum or plasma samples from healthy and naturally or experimentally CyHV-3-infected carp. French CyHV-3 isolate 07/108b was neutralized efficiently by sera from carp infected with European, American and Taiwanese CyHV-3 isolates, but no neutralization was observed using sera specific to other aquatic herpesviruses. Diagnostic sensitivity, diagnostic specificity and repeatability of 95.9%, 99.0% and 99.3%, respectively, were obtained, as well as a compliance rate of 89.9% in reproducibility testing. Neutralizing antibodies were steadily detected in infected carp subjected to restrictive or permissive temperature variations over more than 25 months post-infection. The results suggest that this non-lethal diagnostic test could be used in the future to improve the epidemiological surveillance and control of CyHV-3 disease.

We report the first description of a new Rhabdoviridae tentatively named eelpout rhabdovirus (EpRV genus Perhabdovirus). This virus was associated with mass mortalities in eelpout (Zoarces viviparous, Linnaeus) along the Swedish Baltic Sea coast line in 2014. Diseased fish showed signs of central nervous system infection, and brain lesions were confirmed by histology. A cytopathogenic effect was observed in cell culture, but ELISAs for the epizootic piscine viral haemorrhagic septicaemia virus (VHSV), infectious pancreas necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and spring viraemia of carp virus (SVCV) were negative. Further investigations by chloroform inactivation, indirect fluorescence antibody test and electron microscopy indicated the presence of a rhabdovirus. By deep sequencing of original tissue suspension and infected cell culture supernatant, the full viral genome was assembled and we confirmed the presence of a rhabdovirus with 59.5% nucleotide similarity to the closest relative Siniperca chuatsi rhabdovirus. The full-genome sequence of this new virus, eelpout rhabdovirus (EpRV), has been deposited in GenBank under accession number KR612230. An RT-PCR based on the L-gene sequence confirmed the presence of EpRV in sick/dead eelpout, but the virus was not found in control fish. Additional investigations to characterize the pathogenicity of EpRV are planned.

Four major genotypes of viral haemorrhagic septicaemia virus (VHSV), which have been isolated from many marine and freshwater fish species, are known to differ in virulence. While fast and low-cost genotyping systems based on monoclonal antibodies (MAbs) have been developed for typing of VHSV virulence, there is a need for supplementing the knowledge. In particular, 2 field isolates from viral haemorrhagic septicaemia (VHS) outbreaks in sea-reared rainbow trout Oncorhynchus mykiss in Sweden, SE-SVA-14 and SE-SVA-1033 (both genotype Ib), have yielded contradictory reactions. In the present study, upon cloning by limited dilution, both isolates appeared to be heterogeneous in terms of reactivity with nucleo (N)-protein-specific MAbs as well their gene sequences. Infection trials in rainbow trout further revealed differences in the virulence of these virus clones derived from the same primary isolate. Based on a comparative analysis of the entire genome of the clones tested, we suggest that the differences in virulence are tentatively linked to substitutions of amino acids (aa) in the N-protein region covered by aa 43-46 and aa position 168, or a combination of the two. The fact that such minor naturally occurring genetic differences affect the virulence implies that even low-virulent VHSV isolates in the marine environment should be considered as a potential threat for the trout farming industry. The described MAbs can represent useful tools for initial risk assessment of disease outbreaks in farmed trout by marine VHSV isolates.

Infectious hematopoietic necrosis virus (IHNV) is endemic in farmed rainbow trout in continental Europe and in various salmonid fish species at the Pacific coast of North America. IHN has never occurred in European Atlantic salmon (Salmo salar) farms, but is considered as a major threat for the European salmon industry. Another virus, Piscine orthoreovirus (PRV), is widespread in the sea phase of Atlantic salmon, and is identified as the causative agent of heart and skeletal muscle inflammation. The aim of this study was to investigate the interactions between a primary PRV infection and a secondary IHNV infection under experimental conditions. A PRV cohabitation challenge was performed with Atlantic salmon. At peak of PRV viremia the fish were challenged by immersion with an IHNV genogroup E isolate. Clinical signs and morbidity were monitored. Target organs were sampled at selected time points to assess viral loads of both pathogens. Antiviral immune response and presence of histopathological findings were also investigated. Whereas the PRV-negative/IHNV positive group suffered significant decrease in survival caused by IHNV, the PRV infected groups did not suffer any morbidity and showed negligible levels of IHNV infection. Antiviral response genes were induced, as measured in spleen samples, from PRV infected fish prior to IHNV challenge. In conclusion, PRV-infection protects Atlantic salmon against IHNV infection and morbidity, most likely by inducing a protective innate antiviral response. Electronic supplementary material The online version of this article (10.1186/s13567-018-0524-z) contains supplementary material, which is available to authorized users.

Genotype IVb of viral haemorrhagic septicaemia virus (VHSV) was isolated for the first time in the Great Lakes basin in 2003, where it spread and caused mass mortalities in several wild fish species throughout the basin. In order to prevent further spreading of the disease and to assess risks of new genotypes invading new watersheds, basic microbiological information such as pathogenicity studies are essential. In this study, experimental infections were conducted on 7 indigenous freshwater fish species from Japan by immersion with a VHSV genotype IVb isolate. In Expt 1, cumulative mortalities in bluegill Lepomis macrochirus used as positive controls, Japanese fluvial sculpin Cottus pollux, and iwana Salvelinus leucomaenis pluvius were 50, 80 and 0%, respectively. In Expt 2, cumulative mortalities of 100, 100 and 10% were observed in Japanese fluvial sculpin C. pollux, Japanese rice fish Oryzias latipes and yoshinobori Rhinogobius sp., respectively. No mortality was observed in honmoroko Gnathopogon caerulescens, akaza Liobagrus reini or Japanese striped loach Cobitis biwae. VHSV was detected by RT-PCR from samples of kidney, spleen, and brain from all dead fish, and virus re-isolation by cell culture was successful from all dead fish. We detected the virus in the brain from a few surviving bluegill 50 d post exposure by both cell culture and RT-PCR. These results revealed that VHSV IVb could become a serious threat to wild freshwater fish species in Japan, and that some surviving fish might become healthy carriers of the virus.

A rhabdovirus was isolated in cell culture inoculated with tissue material from diseased grayling, Thymallus thymallus (L.), originating from a fish farm affected by a mortality episode in Poland. Diagnostics tests showed that the virus was not related to novirhabdoviruses known in Europe, nor to vesiculovirus-like species, except perch rhabdovirus (PRhV) with which it shared moderate serological relations. However, RT-PCR with PRhV probes gave negative results. To identify the virus, a random-priming sequence-independent single primer amplification was adopted. Surprisingly, two of the obtained sequences exhibited a high identity (>99%) with hirame rhabdovirus (HIRRV), a novirhabdovirus usually found in fish in marine Asiatic countries, for instance Japan, China and Korea. The full-length sequence of the phosphoprotein gene (P) demonstrated a higher identity of the present isolate with HIRRV from China compared with the Korean isolate. An identical viral sequence was also found in brown trout, Salmo trutta trutta L., affected by mortalities in a second farm in the same region, after a likely contamination from the grayling farm. To our knowledge, this is the first report of HIRRV in Europe, and in two hosts from fresh water that have not been described before as susceptible species.

Complete and transparent reporting of key elements of diagnostic accuracy studies for infectious diseases in cultured and wild aquatic animals benefits end-users of these tests, enabling the rational design of surveillance programs, the assessment of test results from clinical cases and comparisons of diagnostic test performance. Based on deficiencies in the Standards for Reporting of Diagnostic Accuracy (STARD) guidelines identified in a prior finfish study (Gardner et al. 2014), we adapted the Standards for Reporting of Animal Diagnostic Accuracy Studies-paratuberculosis (STRADAS-paraTB) checklist of 25 reporting items to increase their relevance to finfish, amphibians, molluscs, and crustaceans and provided examples and explanations for each item. The checklist, known as STRADAS-aquatic, was developed and refined by an expert group of 14 transdisciplinary scientists with experience in test evaluation studies using field and experimental samples, in operation of reference laboratories for aquatic animal pathogens, and in development of international aquatic animal health policy. The main changes to the STRADAS-paraTB checklist were to nomenclature related to the species, the addition of guidelines for experimental challenge studies, and the designation of some items as relevant only to experimental studies and ante-mortem tests. We believe that adoption of these guidelines will improve reporting of primary studies of test accuracy for aquatic animal diseases and facilitate assessment of their fitness-for-purpose. Given the importance of diagnostic tests to underpin the Sanitary and Phytosanitary agreement of the World Trade Organization, the principles outlined in this paper should be applied to other World Organisation for Animal Health (OIE)-relevant species.

Viral haemorrhagic septicaemia (VHS) is a serious disease in several fish species. VHS is caused by the rhabdovirus viral haemorrhagic septicaemia virus (VHSV). To prevent spreading of the pathogen, it is important to use a fast, robust, sensitive and specific diagnostic tool to identify the infected fish. Traditional diagnosis based on isolation in cell culture followed by identification using, for example, ELISA is sensitive and specific but slow. By switching to RT-PCR for surveillance and diagnosis of VHS the time needed before a correct diagnosis can be given will be considerably shortened and the need for maintaining expensive cell culture facilities reduced. Here we present the validation, according to OIE guidelines, of a sensitive and specific Taqman-based real-time RT-PCR. The assay detects all isolates in a panel of 79 VHSV isolates covering all known genotypes and subtypes, with amplification efficiencies of approximately 100%. The analytical and diagnostic specificity of the real-time RT-PCR is close to 1, and the analytical and diagnostic sensitivity is comparable with traditional cell-based methods. In conclusion, the presented real-time RT-PCR assay has the necessary qualities to be used as a VHSV surveillance tool on par with cell culture assays.

Viral haemorrhagic septicaemia (VHS), caused by the novirhabdovirus VHSV, often leads to significant economic losses to European rainbow trout production. The virus isolates are divided into 4 distinct genotypes with additional subgroups including sublineage Ia, isolates of which are the main source of outbreaks in European rainbow trout farming. A significant portion of Danish rainbow trout farms have been considered endemically infected with VHSV since the first disease outbreak was observed in the 1950s. However, following a series of sanitary programs starting in 1965, VHSV has not been detected in Denmark since January 2009. Full-length G- genes of all Danish VHSV isolates that were submitted for diagnostic analyses in the period 2004�2009 were sequenced and analysed. All 58 Danish isolates from rainbow trout grouped with sublineage Ia isolates. Furthermore, VHSV isolates from infected Danish freshwater catchments appear to have evolved into a distinct clade within sublineage Ia, herein designated clade Ia-1, whereas trout isolates originating from other continental European countries cluster in another distinct clade, designated clade Ia-2. In addition, phylogenetic analyses indicate that VHSV Ia-1 strains have caused a few outbreaks in Germany and the UK. It is likely that viruses have been transmitted from infected site(s) out of the Danish environment, although a direct transmission pathway has not been identified. Furthermore, VHSV Ia-2 isolates seem to have been transmitted to Denmark at least once. Interestingly, one viral isolate possibly persisted in a Danish watershed for nearly 4 yr without detection whereas other subclades of VHSV isolates appear to have been eliminated, probably because of implemented eradication procedures.

Passage of cell cultures may adversely influence cell susceptibility to virus infection through selection of cell clones that thrive in vitro but may not necessarily display high sensitivity to virus infection. Susceptibility to a given virus can therefore vary not only between cell lines and laboratories, but also between lineages of the same cell line. To minimise the occurrence of false negatives in a cell culture based surveillance system, we have investigated methods, to select cell lineages that are relatively superior in their susceptibility to a panel of virus isolates. The procedures compare susceptibility between cell lines and between lineages within a laboratory and between laboratories (Inter-laboratory Proficiency Test). The objective being that the most sensitive cell line and lineages are routinely selected for diagnostic purposes. In comparing cell lines, we simulated “non-cell-culture-adapted” virus by propagating the virus in heterologous cell lines to the one tested. A stock of test virus was produced and stored at − 80 °C and tests were conducted biannually. This procedure becomes complicated when several cell lines are in use and does not account for variation among lineages. In comparing cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test (Ariel et al., in press), which is organised by the European Community Reference Laboratory for Fish Diseases (CRL) in Denmark. In the year 2000, infected organ material rather than cell-culture-adapted virus was included in the test, to approach a realistic assessment of the variability in cell sensitivity for surveillance purposes within a cell line and between laboratories. In terms of economic and practical considerations as well as attempting to approach a realistic test system, we suggest the optimal procedure for susceptibility testing of fish cell lines for virus isolation to be a combination of biannual tests within the laboratory to compare cell lineages combined with the Inter-laboratory Proficiency Test.

Part of the functions and duties of the European Community Reference Laboratory for Fish Diseases (CRL) is to organise periodic comparative tests of diagnostic procedures at Community level as described in Council Directive 2006/88/EC (Anonymous, 2006), previously outlined in Council Directive 93/53/EEC (Anonymous, 1993). A qualitative and quantitative proficiency test was carried out annually since 1996. These tests were primarily designed to assess the ability of participating laboratories to identify the listed viruses: viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV) on list II in Council Directive 91/67/EEC, annex A (Anonymous, 1991). The tests have typically consisted of five coded ampoules of lyophilised supernatant from infected cell cultures. Participants were asked to identify and quantify the contents within a deadline of 8 weeks. The CRL collated the answers and processed them statistically and graphically to provide the individual laboratory with a unique picture of its performance in relation to the other participants. Each participant was assigned a code number to ensure discretion. Occasionally the test was designed with additional purposes; other common fish viruses were included for differential diagnosis, cell line susceptibility within and between laboratories were tested, and the ability of laboratories to detect double infections and differentiate between genotypes was also part of the test. This paper describes the different approaches in designing the test over a 10-year period and comments on the overall performance of participants during that period.

We describe the finding of a novel viral haemorrhagic septicaemia virus (VHSV) Genotype III strain that caused disease of both a neurological and septicaemic nature in seawater-farmed rainbow trout Oncorhynchus mykiss in Storfjorden, Norway. In November 2007, an outbreak of VHS associated with slightly elevated mortality was confirmed at a seawater site rearing rainbow trout (90 to 440 g). Within 3 to 4 mo, the disease was recognised in 3 neighbouring sea sites with ongrowing rainbow trout. The clinical, gross pathological and histopathological findings were in accordance with VHS, and the diagnosis was confirmed by the detection of VHSV in brain and internal tissues by immunohistochemistry, cell culture and reverse transcriptase PCR (RT-PCR). Sequence analysis of the G-gene revealed that the isolated virus clustered with VHSV Genotype III and that the Norwegian isolate represents a unique strain of VHSV. The pathogenicity of the virus strain to rainbow trout and Atlantic salmon Salmo salar was examined using infection experiments. In immersion trials, the Norwegian isolate produced a cumulative mortality of 70% in rainbow trout, while nearly 100% mortality was obtained after intraperitoneal injection of the virus. For Atlantic salmon, no mortality was observed in immersion trials, whereas 52% mortality was observed after intraperitoneal injection. The Norwegian isolate thus represents the first VHSV of Genotype III pathogenic to rainbow trout.

A selection of 16 field isolates of Photobacterium damselae from marine rainbow trout farms in Denmark was subjected to phenotypic and genotypic characterization and pathogenicity to fish. All isolates belonged to the subspecies damselae, being positive for haemolysis, motility and urease. There were considerable differences in haemolytic properties, some isolates presenting a broad zone of haemolysis and others only a narrow zone. Pulsed-field gel electrophoresis revealed a high diversity indicating that P. damselae subsp. damselae is an opportunistic, not clonal pathogen in Danish marine rainbow trout. Virulence of the strains to rainbow trout was highly variable with LD(50) values ranging from 3.9 x 10(3) to 1.5 x 10(8) cfu at 20 degrees C. The virulence was significantly higher at 20 degrees C than at 13 degrees C. The strains with the strongest haemolytic properties were the most virulent suggesting a strong involvement of haemolysin in the pathogenesis. The pathological changes were consistent with a bacterial septicaemia and the haemorrhages were more pronounced than for most other bacterial infections.

Juvenile rainbow trout Oncorhynchus mykiss were experimentally infected by immer- sion with viral haemorrhagic septicaemia virus (VHSV), infectious haematopoietic necrosis virus (IHNV) or with both viruses. The presence of neutralizing antibodies in the sera of infected fish were analysed by 50% plaque neutralization tests (50%PNT). In Group 1 (infected with VHSV) and Group 2 (infected with IHNV) neutralizing antibodies were found in 41% and 21% of the serum samples, respectively. No cross-reacting antibodies were found in these 2 groups. In Group 3 (infected with both viruses) 30% of the samples showed neutralizing antibodies against VHSV, 21% against IHNV and 12% against both viruses. Fish in Group 3 developed a double specific antibody reaction whose kinetics and intensity (mean of log10 titres) were similar to the antibody response of the single infected groups.

Viral haemorrhagic septicaemia (VHS) is a notifiable fish disease, whose causative agent is a rhabdovirus isolated from a wide range of fish species, not only in fresh but also in marine and brackish waters. Phylogenetic studies have identified four major genotypes, with a strong geographical relationship. In this study, we have designed and validated a new procedure – named binary multiplex RT-qPCR (bmRT-qPCR) – for simultaneous detection and typing of all four genotypes of VHSV by real-time RT-PCR based on dual-labelled probes and composed by two multiplex systems designed for European and American/Asiatic isolates, respectively, using a combination of three different fluorophores. The specificity of the procedure was assessed by including a panel of 81 VHSV isolates covering all known genotypes and subtypes of the virus, and tissue material from experimentally infected rainbow trout, resulting in a correct detection and typing of all strains. The analytical sensitivity was evaluated in a comparative assay with titration in cell culture, observing that both methods provided similar limits of detection. The proposed method can be a powerful tool for epidemiological analysis of VHSV by genotyping unknown samples within a few hours.

Viral haemorrhagic septicaemia virus (VHSV) has, in recent decades, been isolated from an increasing number of free-living marine fish species. So far, it has been isolated from at least 48 fish species from the northern hemisphere, including North America, Asia and Europe, and fifteen different species including herring, sprat, cod, Norway pout and flatfish from northern European waters. The high number of VHSV isolations from the Baltic Sea, Kattegat, Skagerrak, the North Sea and waters around Scotland indicate that the virus is endemic in these waters. The VHSV isolates originating from wild marine fish show no to low pathogenicity to rainbow trout and Atlantic salmon, although several are pathogenic for turbot. Marine VHSV isolates are so far serologically indistinguishable from freshwater isolates. Genotyping based on VHSV G- and N-genes reveals four groups indicating the geographical origin of the isolates, with one group representing traditional European freshwater isolates and isolates of north European marine origin, a second group of marine isolates from the Baltic Sea, a third group of isolates from the North Sea, and a group representing North American isolates. Examples of possible transfer of virus from free-living marine fish to farmed fish are discussed, as are measures to prevent introduction of VHSV from the marine environment to aquaculture.

In order to analyse the occurrence of viral haemorrhagic septicaemia virus (VHSV) in the marine waters around Denmark, staff from the Danish Institute for Food and Veterinary Research participated in 5 research cruises during 1998 to 2002 as a follow-up to 4 research cruises performed in 1996 to 1997. In total, 16,655 fish were examined virologically as 3569 samples. Forty fish species and 3 invertebrate species were represented. VHSV was isolated from 133 samples representing 8 species: herring Clupea harengus, sprat Sprattus sprattus, dab Limanda limanda, flounder Platichthys flesus, plaice Pleuronectes platessa, cod Gadus morhua, sand eel Ammodytes sp. and sand goby Pomatochistus minutus. Calculations showed that VHSV was more prevalent in the Baltic Sea in an area between Zealand and the island of Bornholm and the waters surrounding Bornholm than in the Kattegat, Skagerrak and along the North Sea coast of Denmark. This is the first report on the isolation of VHSV from dab, flounder and plaice and the first publication on VHSV from sand eel from Europe and sand goby.

One hundred and forty-eight wild whitefish, Coregonus lavaretus (L.), were caught by electrofishing and sampled for virological examination in December 1999 and 2000, during migration from the brackish water feeding grounds to the freshwater spawning grounds, where the whitefish may come into contact with farmed rainbow trout. All samples were examined on cell cultures. No viruses were isolated. Three viral haemorrhagic septicaemia virus (VHSV) isolates of different origin were tested in infection trials by immersion and intraperitoneal (IP) injection, using 1.5 g farmed whitefish: an isolate from wild caught marine fish, a farmed rainbow trout isolate with a suspected marine origin and a classical freshwater isolate. The isolates were highly pathogenic by IP injection where 99-100% of the whitefish died. Using an immersion challenge the rainbow trout isolates were moderately pathogenic with approximately 20% mortality, whereas the marine isolate was virtually non-pathogenic. At the end of the experiment it was possible to isolate VHSV from survivors infected with the marine and suspected marine isolates. Because of the low infection rate in wild whitefish in Denmark, the role of whitefish in the spread of VHSV in Denmark is probably not significant. The experimental studies, however, showed that whitefish are potential carriers of VHSV as they suffer only low mortality after infection but continue to carry virus.

A novel rhabdovirus, preliminary designated as the Sea trout rhabdovirus 28/97 (STRV 28/97), was isolated from sea trout (Salmo trutta trutta) in Sweden in 1996. The fish showed central nervous symptoms, and at the autopsy petechial bleedings in the mesenteric fat were visible. STRV 28/97 was shown to be serologically related to the vesiculotype rhabdovirus 903/87 isolated from brown trout (Salmo trutta lacustris) in Finland [1,3]. The sequences for the nucleocapsid protein, phosphoprotein, matrix protein, glycoprotein and beginning of the polymerase protein of STRV 28/97 were determined. At the amino acid level the genes were over 97% similar to virus 903/87. The nucleocapsid proteins, glycoproteins and beginning of the polymerase protein of STRV 28/97 and virus 903/87 were clustered with the vesiculoviruses and the phosphoproteins close to the vesiculoviruses in protein parsimony analysis. The matrix proteins formed a distinct clade in protein parsimony analysis.

Wild freshwater eel populations have dramatically declined in recent past decades in Europe and America, partially through the impact of several factors including the wide spread of infectious diseases. The anguillid rhabdoviruses eel virus European X (EVEX) and eel virus American (EVA) potentially play a role in this decline, even if their real contribution is still unclear. In this study, we investigate the evolutionary dynamics and genetic diversity of anguiillid rhabdoviruses by analysing sequences from the glycoprotein, nucleoprotein and phosphoprotein (P) genes of 57 viral strains collected from seven countries over 40 years using maximum-likelihood and Bayesian approaches. Phylogenetic trees from the three genes are congruent and allow two monophyletic groups, European and American, to be clearly distinguished. Results of nucleotide substitution rates per site per year indicate that the P gene is expected to evolve most rapidly. The nucleotide diversity observed is low (2–3 %) for the three genes, with a significantly higher variability within the P gene, which encodes multiple proteins from a single genomic RNA sequence, particularly a small C protein. This putative C protein is a potential molecular marker suitable for characterization of distinct genotypes within anguillid rhabdoviruses. This study provides, to our knowledge, the first molecular characterization of EVA, brings new insights to the evolutionary dynamics of two genotypes ofAnguillid rhabdovirus, and is a baseline for further investigations on the tracking of its spread.

Viral haemorrhagic septicaemia (VHS) is an economically very important fish disease in the Northern hemisphere. When VHS virus was first isolated in Denmark 50 yr ago, more than 80% of the 800 Danish fish farms were considered to be infected, but vigilant surveillance and eradication programmes have led to a drastic reduction in prevalence, and finally, to complete eradication of VHS. Denmark is expected to obtain official status as an approved VHS free member state within the EU in November 2013. Data on outbreaks within the country have been collected throughout the years, and in this study these data are combined with the geographical coordinates of fish farms to identify clusters of high disease prevalence and other risk factors. These analyses revealed a statistically significant cluster in the south-western part of the country, which persisted throughout the study period. Being situated within such a cluster was a significant risk factor for VHS (p < 0.001). For freshwater rainbow trout farms situated inland, the number of upstream farms was a determining risk factor for VHS, as was distance to nearest VHS infected farm and year. Whether the farm used fresh or marine water in their production did not have any influence on the risk of VHS, when accounting for whether the farm was situated inside a cluster of high risk. This information can be used when deciding risk-based surveillance programs. Viral haemorrhagic septicaemia (VHS) is an economically very important fish disease in the northern hemisphere. When the VHS virus was first isolated in Denmark 50 yr ago, more than 80% of the 800 Danish fish farms were considered to be infected, but vigilant surveillance and eradication programmes led to a drastic reduction in prevalence, and finally, to complete eradication of VHS. Denmark thus obtained official status as an approved VHS-free member state within the European Union in November 2013. Data on outbreaks within the country have been collected since 1970, and here we combined these data with the geographical coordinates of fish farms to identify clusters of high disease prevalence and other risk factors. Our analyses revealed a statistically significant cluster in the southwestern part of the country, which persisted throughout the study period. Being situated within such a cluster was a significant risk factor for VHS. For freshwater rainbow trout farms situated inland, the number of upstream farms was a determining risk factor for VHS, as was distance to the nearest VHS-infected farm and year. Whether the farm used fresh or marine water in production did not have any influence on the risk of VHS, when accounting for whether the farm was situated inside a cluster of high risk. This information can be used when implementing risk-based surveillance programmes.

The distribution of different phenotypes of the complement component C3 and a possible correlation with body weight were investigated in five Danish strains (A, C, D, E, J) of farmed trout (Oncorhynchus mykiss Walbaum). All strains showed the presence of the f1 and f2 alleles; two of these strains also expressed the s allele. Most common was the f1 allele (> 68%); rarest was the s allele (

To study immunological and immunogenetical parameters related to resistance against viral haemorrhagic septicaemia (VHS), attempts to make gynogenetic strains of rainbow trout selected for high and low resistance to VHS were initiated in 1988. The first gynogenetic generation of inbreeding resulted in the more resistant offspring E8 and the low resistance offspring K3; the K3 offspring having the same high mortality as the susceptible reference strain of outbred trout in infection trials. A second gynogenetic generation derived from the E8 strain resulted in some low resistance offspring, and two gynogenetic families in which all, or nearly all, fish survived challenge with VHS virus. In this study, an attempt to associate the distribution of different MHC class II genotypes with low and high resistance gynogenetic offspring was performed. Two different MHC haplotypes could be distinguished, and in both low and high resistance families all three genotypes were found, which could be explained by the fact that the mother fish carried the heterozygous genotype. Although no significant differences in MHC II genotypes were found between the high and low resistance offspring, a significantly different distribution of haplotypes in the low resistance offspring was observed, that could not be explained by a one- or two-locus model.

In the field of fish diseases, the amount of relevant information available is enormous. Internet-based databases are an excellent tool for keeping track of the available knowledge in the field. Fishpathogens.eu was launched in June 2009 with the aim of collecting, storing and sorting data on fish pathogens. The first pathogen to be included was the rhabdovirus, viral haemorrhagic septicaemia virus (VHSV). Here, we present an extension of the database to also include infectious haematopoietic necrosis virus (IHNV). The database is developed, maintained and managed by the European Community Reference Laboratory for Fish Diseases and collaborators. It is available at http://www.fishpathogens.eu/ihnv.

In order to analyse the occurrence of viral haemorrhagic septicaemia virus (VHSV) in the marine environment surrounding Denmark, fish tissue samples were collected on four cruises with the research vessel H/S Dana in 1996 and 1997. The sampling comprised 923 samples totalling 7344 fish representing 29 different species. VHSV was isolated from 24 fish samples from the Baltic Sea, four samples from Skagerrak and three samples from the North Sea. The virus-positive host species included herring Clupea harengus (11 isolates), sprat Sprattus sprattus (eight isolates), cod Gadus morhua (six isolates), rockling Rhinonemus cimbrius (one isolate), Norway pout Trisopterus esmarkii (one isolate), blue whiting Micromesistius poutassou (one isolate), whiting Merlangius merlangus (two isolates) and lesser argentine Argentina sphyraena (one isolate). VHSV has previously been reported from cod and herring, but not from the other five species. A virus belonging to serogroup II of the aquatic birnaviruses was isolated from three samples of flounder Platichthys flesus and three samples of dab Limanda limanda and a virus preliminary identified as iridovirus (lymphocystis virus) was isolated from seven samples of long rough dab Hippoglossoides platessoides.

Viral hemorrhagic septicemia virus (VHSV) is the rhabdovirus that causes most disease problems in farmed rainbow trout in Europe. Survivors of infection are usually immune to reinfection but as with other fish viruses, development of a modern recombinant vaccine has been complicated by the limited knowledge of the immune mechanisms and antigens involved in induction of immunity. Neutralizing and protective monoclonal antibodies recognize the envelope glycoprotein (G protein) which is the only viral protein known to be present on the surface of the virus particle. Immunoblotting analyses with monoclonal antibodies as well as with sera from immunized trout have indicated that protein conformation plays an important role in neutralization epitopes. The virus neutralizing activity often found in sera from convalescent trout is highly dependent on a poorly defined complementing activity in normal trout serum. Attempts to demonstrate involvement of the complement component C3 were not successful, but inhibition experiments indicated that the classical pathway for complement activation was needed. Being the target of neutralizing antibodies, the G protein is an obvious candidate for a recombinant vaccine. However, recombinant forms of the G protein expressed in Escherichia coli have been poorly immunogenic in fish, presumably due to incorrect protein conformation. Expression in insect cells has resulted in more potent products but, more recently, considerably higher levels of protection were found following vaccination with naked DNA encoding the G protein under the control of a CMV promotor. Genetic resistance to VHS would be a desirable alternative to vaccination but the time required to obtain this makes it a long-time goal. Results from breeding programs in France and Denmark nevertheless indicate that such a strategy may provide considerable improvement in resistance.

Rabbit antisera against viral hemorrhagic septicemia virus (VHSV) produced by two immunization procedures were compared for neutralization and immunochemical properties against homologous and heterologous strains. The VHSV isolate used as the immunogen was a member of a serogroup not neutralized by previously available antisera. The results from this study suggested that frequent intravenous (IV) injections of rabbits with viral antigens were superior to adjuvant-mediated, combined subcutaneous and intraperitoneal (SC/IP) injections for the production of neutralizing antisera. All IV injected rabbits produced high neutralization titers against the homologous VHSV isolate but not against an isolate from a different serogroup. The SC/IP injected rabbits had no significant neutralization titers against either the homologous VHSV strain or two isolates of a heterologous VHSV strain. Sera from all injected rabbits reacted in indirect immunofluorescence (IF) assays with either strain; however, the SC/I...

Eleven European National Reference Laboratories participated in an inter-laboratory comparison of the susceptibility of 5 selected cell lines to 3 fish pathogenic viruses. The test included viral hemorrhagic septicaemia virus (VHSV); infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV), and the cell lines derived from bluegill fry (BF-2), chinook salmon embryo (CHSE-214), epithelioma papulosum cyprini (EPC), fathead minnow (FHM) and rainbow trout gonad (RTG-2). The results showed that for isolation of VHSV, BF-2 and RTG-2 cells performed equally well and had higher sensitivity compared to the other cell lines. For IHNV, EPC and FHM cells gave the best results, and for IPNV it was BF-2 and CHSE-214 cells. FHM cells showed the largest variability among laboratories, whereas EPC was the cell line showing the smallest variability.

Summary A sanitation programme for stamping-out viral haemorrhagic septicaemia (VHS) was implemented in Denmark in 1965. The programme has resulted in a dramatic reduction in the number of infected rainbow trout farms, from 5400 to 26. The programme is carried out on a voluntary basis at the expense of the involved fish farm owners. The fact that financial support for the eradication of VHS and IHN may be made available from the European Union may enhance the efforts towards further eradication of the disease in Europe. The finding of a VHS virus (VHSV)-like virus in the marine environment is a matter of major concern, but improved diagnostic procedures may enable discrimination between the highly pathogenic freshwater strains and the apparently less virulent marine strains.

A new research programme involving eight Danish institutions is described. The programme started in 1993 and is expected to run for 5 years. The primary objective of the research initiative is to exploit and integrate the knowledge of several institutions and disciplines for the benefit of the production of rainbow trout. The programme includes several projects with aspects of disease prevention, genetics, and nutrition. In most of the projects, the work has been divided into stages of 2 and 3 years, respectively. During a 2 year period, production, management, and health status are recorded at the participating fish farms, and all data are organized in a database. Diseases cause major problems in rainbow trout production, therefore a great deal of the effort in this programme deals with diseases caused by vira, bacteria and parasites. On the basis of the database, epidemiological examinations are carried out as well as investigations of the possibilities of preventive measures and cost-benefit analyses. In the genetic studies, polymorphic genetic markers will be developed and used for analysis of the genetic structure of selected fish stocks. Microsatellites will be developed and introduced in the study. Primarily genetic differences between lines/strains and their crossings will be estimated with the purpose of describing the genetic level and the importance of additive and non-additive genetic effects. In the nutritional area the product quality and pollution questions will be in focus.