Viral Haemorrhagic Septicemia Virus (VHSV) Isolated from Atlantic Herring, Clupea harengus, Causes Mortality in Bath Challenge on Juvenile Herring.

Viral hemorrhagic septicaemia virus (VHSV) has been demonstrated to cause high mortalities in a wide range of teleosts, farmed as well as wild. In Europe, VHSV of genotypes Ib, Id, II, and III have been detected in wild fish, including Atlantic herring Clupea harengus, but disease outbreaks have not been observed in Atlantic herring and the effects on wild stocks are not well documented. Here, we have tested two VHSV isolates from herring (genotypes Ib and III, from the western coasts of Norway and Denmark, respectively) in a challenge experiment with herring (mean weight 2.59 g, SD 0.71 g) caught on the west coast of Denmark. The Norwegian genotype Ib isolate (NO-F-CH/2009) showed an accumulated mortality of 47% compared to 6% mortality with the Danish genotype III isolate 4p168 and zero in the unchallenged control group. In both groups, we found positive rt-RT-PCR and positive immunohistochemistry of VHSV from days 6 and 8 onward. With both isolates, the organs mainly affected were the heart and kidney. The results demonstrate the susceptibility of Atlantic herring to VHSV, and both genotypes gave pathological findings in several organs. Genotype III showed a low mortality rate, and the importance of this genotype for herring is therefore not determined. Genotype Ib showed both high prevalence and mortality, and this genotype is therefore likely to have a negative effect on wild Atlantic herring stocks. Further examinations to determine how VHSV can affect wild Atlantic herring stocks are needed.

Virulence marker candidates in N-protein of viral haemorrhagic septicaemia virus (VHSV): virulence variability within VHSV Ib clones.

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.

Spatio-temporal risk factors for viral haemorrhagic septicaemia (VHS) in Danish aquaculture.

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.

Typing of viral hemorrhagic septicemia virus by monoclonal antibodies.

Seven mAbs with specific reaction patterns against each of the four genotypes and eight subtypes of viral hemorrhagic septicemia virus (VHSV) were produced, aiming to establish an immunoassay for typing VHSV isolates according to their genotype. Among the mAbs, VHS-1.24 reacted with all genotypes except genotype Ie, whilst mAb VHS-9.23 reacted with all genotypes except genotype III. mAb VHS-3.80 reacted with genotypes Ib, Ic, Id and II. mAb VHS-7.57 reacted with genotypes II and IVa, and mAb VHS-5.18 with genotype Ib only. Interestingly, mAb VHS-3.75 reacted with all of the genotype III isolates except a rainbow trout-pathogenic isolate from the west coast of Norway, and reacted in addition with the IVb isolate, CA-NB00-01, from the east coast of the USA. Finally, mAb VHS-1.88 reacted with all genotype IVb isolates from the Great Lakes, but not with CA-NB00-01. In conclusion, we can distinguish between all four genotypes and between five of eight subtypes of VHSV by testing isolates in immunoassay using a panel of nine mAbs. By Western blotting and transfection of cell cultures, it was shown that mAb VHS-1.24 recognized an epitope on the viral phosphoprotein (P), whilst all others recognized antigenic determinants on the nucleoprotein (N). From amino acid alignments of the various genotypes and subtypes of VHSV isolates, it was possible to determine the epitope specificity of mAb VHS-1.24 to be aa 32-34 in the P-protein; the specificities of mAbs VHS-3.80, VHS-7.57 and VHS-3.75 were found to be aa 43 and 45-48, aa 117 and 121, and aa 103, 118 and 121 of the N-protein, respectively.

First isolation and genotyping of viruses from recent outbreaks of viral haemorrhagic septicaemia (VHS) in Slovenia.

In November and December 2007, the virus causing viral haemorrhagic septicaemia (VHS) was detected in rainbow trout Oncorhynchus mykiss from 2 fish farms in Slovenia. During 2008 and 2009 the infection spread only among rainbow trout farms and 4 new outbreaks were confirmed. High mortality and clinical signs of VHS were observed among the diseased fish. VHSV was confirmed by virus isolation, immunoperoxidase test, reverse transcriptase polymerase chain reaction (RT-PCR) and phylogenetic analysis. Based on 1 complete (1524 nucleotides [nt]) and 9 partial (600 nt) glycoprotein gene nucleotide sequences, 9 VHSV isolates from the 6 VHS outbreaks were genetically closely related (99 to 100% identity), and were classified into the Subgroup I-a of Genotype I, most closely related to the German isolates Dstg21-07, Dstg36-06, and Dstg54-1-07 (99 to 100% identity). Phylogenetic analysis and epidemiological investigations confirmed that the VHS virus had been (re)introduced with imported live fish, and that subsequent outbreaks were linked to the initial infection. Our study shows that direct nucleotide sequencing of RT-PCR products, amplified from the tissue of VHSV-infected fish, represents a reliable tool for fast routine genotyping in diagnostic laboratories. This is the first report of a natural epidemic associated with VHSV infection in Slovenia since the eradication of the disease in 1977.

Viral haemorrhagic septicaemia (VHS) outbreaks in Finnish rainbow trout farms.

In Finland, viral haemorrhagic septicaemia virus (VHSV) was diagnosed for the first time in 2000 from 4 rainbow trout farms in brackish water. Since then the infection has spread and, by the end of 2004, VHSV had been isolated from 24 farms in 3 separate locations: 2 in the Baltic Sea and 1 in the Gulf of Finland. The pathogenicity of 3 of these isolates from 2 separate locations was analysed in infection experiments with rainbow trout fry. The cumulative mortalities induced by waterborne and intraperitoneal challenge were approximately 40 and 90 %, respectively. Pair-wise comparisons of the G and NV gene regions of Finnish VHSV isolates collected between 2000 and 2004 revealed that all isolates were closely related, with 99.3 to 100% nucleotide identity, which suggests the same origin of infection. Phylogenetic analysis revealed that they were closely related to the old freshwater isolates from rainbow trout in Denmark and to one old marine isolate from cod in the Baltic Sea, and that they were located close to the presumed ancestral source. As the Finnish isolates induce lower mortality than freshwater VHSV isolates in infection experiments, they could represent an intermediate stage of marine isolates evolving towards pathogenicity in rainbow trout.

Prevalence of viral haemorrhagic septicaemia virus in Danish marine fishes and its occurrence in new host species.

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.

Experimental infection of rainbow trout Oncorhynchus mykiss with viral haemorrhagic septicaemia virus isolates from European marine and farmed fishes.

The susceptibility of rainbow trout Oncorhynchus mykiss to infection with various isolates of viral haemorrhagic septicaemia virus (VHSV) was examined. A total of 8 experiments with rainbow trout ranging from 0.6 to 6.2 g was conducted for 139 isolates originating from wild marine fishes in European waters (115 isolates), farmed turbot from Scotland and Ireland (2 isolates), and farmed rainbow trout (22 isolates). The isolates were tested by immersion and/or intraperitoneal injection either as pooled or single isolates. The isolates from wild marine fishes did not cause mortality by immersion while some of the isolates caused mortality when injected. All VHSV isolates from farmed rainbow trout caused significant mortality by immersion. Currently, pathogenicity trials are the only way to differentiate VHSV isolates from wild marine fishes and farmed rainbow trout. The 2 farmed turbot isolates did not cause mortality by immersion, supporting the view that they originated from the marine environment.

Age- and weight-dependent susceptibility of rainbow trout Oncorhynchus mykiss to isolates of infectious haematopoietic necrosis virus (IHNV) of varying virulence.

The virulence of 5 European and 1 North American isolate of infectious haematopoietic necrosis virus (IHNV) was compared by infecting female sibling rainbow trout ('Isle of Man' strain) of different weights and ages (2, 20 and 50 g). The fish were exposed to 10(4) TCID50 IHNV per ml of water by immersion, and the mortality was recorded for 28 d. Two new IHNV isolates from Germany were included in the investigation. One was isolated from European eels kept at 23 degrees C (+/- 2 degrees C) and the other was not detectable by immunofluorescence with commercially available monoclonal antibodies recognising the viral G protein. The results showed that IHNV isolates of high or low virulence persisted in rainbow trout of all ages/weights for 28 d, with the exception of fish over 15 g in the eel IHNV (DF [diagnostic fish] 13/98)-infected groups from which the virus could not be reisolated on Day 28. The smallest fish were most susceptible to an infection with any of the IHNV isolates. The lowest cumulative mortality (18%) was observed in fingerlings infected with the North American isolate HAG (obtained from Hagerman Valley), and the highest mortality (100%) in DF 04/99 infected fish. The DF 04/99 and O-13/95 viruses caused mortality in fish independent of their weight or age. The isolates FR-32/87 and I-4008 were virulent in fish up to a weight of 20 g and caused no mortality in larger fish. In the IHNV HAG- and DF 13/98 (eel)-infected rainbow trout, no signs of disease were observed in fish weighing between 15 and 50 g. An age/weight related susceptibility of rainbow trout was demonstrated under the defined conditions for all IHNV isolates tested.