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.     

Susceptibility of juvenile Atlantic cod Gadus morhua to viral haemorrhagic septicaemia virus isolated from wild-caught Atlantic cod

A European strain of viral haemorrhagic septicaemia virus (VHSV) isolated from wild-caught cod Gadus morhua (H16/7/95) was shown to cause clinical disease and mortality in excess of 80% in juvenile Atlantic cod when administered by the intra-peritoneal (i.p.) route. No virus was recovered from cod cohabiting with experimentally infected fish at a ratio of 1:1, and no VHSV-associated mortality was demonstrated following immersion infection. External signs of disease in cod were the presence of exophthalmia and ascites. Virus was identified as VHSV by enzyme-linked immunosorbent assay (ELISA) and was recovered from both brain and organ pools (kidney, liver and spleen) of 100% of i.p. infected cod mortalities. Virus was also detected using an indirect immunofluorescence test on tissue imprints of kidney, liver, spleen and brain taken from moribund fish. The fact that cod were not susceptible to VHSV following waterborne exposure raises important questions surrounding the propagation, maintenance and impact of a naturally occurring reservoir of virus in the marine environment.     

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.     

Experimental susceptibility of turbot Scophthalmus maximus to viral haemorrhagic septicaemia virus isolated from cultivated turbot

Juvenile pathogen-free turbot were infected with a viral haemorrhagic septicaemia virus (VHSV) isolate recovered from turbot cultivated on the island of Gigha, West Scotland. Mortality of 100% was recorded in fish infected via the intra-peritoneal (i.p.) route. Horizontal transmission of VHSV in sea water was demonstrated by cohabitation of naive fish with i.p. infected fish at a ratio of 1:1. The total cumulative average mortality in cohabiting fish was 60% by 60 d post-infection. Turbot infected via an immersion route exhibited a cumulative average mortality of 71% by the end of the experiment. VHSV identified by enzyme-linked immunosorbent assay (ELISA) was recovered from both organ (kidney and spleen) and brain samples of individual fish that died following infection by all experimental routes. These findings pose significant implications regarding the persistence of VHSV and its role in limiting natural populations of marine fish species. In addition, the establishment of infection models for the transmission of VHSV in sea water is of fundamental importance to the development of anti-VHSV vaccines in important commercial species such as turbot.     

Distribution of viral haemorrhagic septicaemia virus in wild fish species of the North Sea, north east Atlantic Ocean and Irish Sea.

A surveillance programme was initiated on the occurrence and distribution of viral haemorrhagic septicaemia virus (VHSV) in wild marine fish. Six research cruises were undertaken in an 18 mo period during 1997 and 1998, covering the North Sea, the Atlantic waters off the north and west coasts of Scotland and the Irish Sea. A total of 19,293 fish were sampled from 23 different species including cod, haddock, Norway pout, herring and sprat. Individual fish lengths were recorded and the fish were checked for lesions, haemorrhaging and other signs of disease. Pools of organ samples were taken for virus assay. The majority of fish sampled did not display clinical signs indicative of viral haemorrhagic septicaemia. A small number of cod were found with skin lesions and haddock with skin haemorrhaging. Of the 2081 organ and skin sample pools collected, 21 tested positive for VHSV by tissue culture and enzyme-linked immunosorbent assay. Seventeen of the isolates originated from Norway pout Trisopterus esmarkii, one from cod Gadus morhua (skin lesion), one from herring Clupea harengus, one from whiting Merlangius merlangus, and one from a previously unreported host species, poor cod Trisopterus minutus.     

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.     

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

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

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.     

Virus susceptibility of the fish cell line SAF-1 derived from gilt-head seabream

The recently reported SAF-1 cell line from fins of gilt-head seabream was evaluated for susceptibility to lymphocystis disease virus (LDV) and to several salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), viral haemorrhagic septicemia virus (VHSV) and several strains of infectious pancreatic necrosis virus (IPNV). LDV, VHSV and IHNV replicated well in the cultured fin cells as demonstrated by cell lysis and increases in viral titer. The potential use of this cell line to detect viruses from fish marine species is discussed.     

Coral diversity and disease in Mexico

A total of 14 viral haemorrhagic septicaemia virus (VHSV) isolates obtained from Greenland halibut Reinhardtius hippoglossoides caught at the Flemish Cap, a fishing ground in the North Atlantic Ocean near Newfoundland, were characterised using restriction fragment length polymorphism (RFLP) and nucleotide sequence analysis. RFLP analysis was performed on a 1259 bp fragment of the glycoprotein (G) gene, and a 305 nucleotide region within the nucleoprotein (N) gene was used for sequence analysis. Representative strains of the 4 established genotypes were employed for comparative purposes. Sequencing analysis indicated that the Flemish cap isolates grouped in Genotype 3, which also includes isolates from wild fish caught in the North Sea and coastal waters of the UK and Ireland, isolates derived from outbreaks of VHS in turbot farms in the British Isles, and an isolate from European eel Anguilla anguilla caught in northern France. Characterisation using RFLPs resulted in the development of a simple and reliable method of typing VHSV at the genotype level using a 2-step restriction analysis (2-SRA) assay.     

Host and geographic range extensions of the North American strain of viral hemorrhagic septicemia virus

Viral hemorrhagic septicemia virus (VHSV) was isolated from populations of Pacific sardine Sardinops sagax from the coastal waters of Vancouver Island, British Columbia, Canada, and central and southern California, USA. The virus was also isolated from Pacific mackerel Scomber japonicus in southern California, from eulachon or smelt Thaleichthys pacificus, and surf smelt Hypomesus pretiosus pretiosus from Oregon, USA. Mortality and skin lesions typical of viral hemorrhagic septicemia in other marine fish species were observed among sardine in Canada and in a few surf smelt from Oregon, but the remaining isolates of VHSV were obtained from healthy appearing fish. The prevalence of VHSV among groups of apparently healthy sardine, mackerel and smelt ranged from 4 to 8%, in California and Oregon. A greater prevalence of infection (58%) occurred in groups of sardine sampled in Canada that sustained a naturally occurring epidemic during 1998-99. A captive group of surf smelt in Oregon exhibited an 81% prevalence of infection with clinical signs in only a few fish. The new isolates were confirmed as North American VHSV and were closely related based on comparisons of the partial nucleotide sequence of the glycoprotein (G) gene. The VHSV isolates from sardine in Canada and California were the most closely related, differing from isolates obtained from other marine fish species and salmonids in British Columbia, Canada, Alaska and Washington, USA. These new virus isolations extend both the known hosts (sardine, mackerel and 2 species of smelt) and geographic range (Oregon and California, USA) of VHSV.     

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

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

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.     

Chronic and persistent viral hemorrhagic septicemia virus infections in Pacific herring

Chronic viral hemorrhagic septicemia virus (VHSV) infections were established in a laboratory stock of Pacific herring Clupea pallasii held in a large-volume tank supplied with pathogen-free seawater at temperatures ranging from 6.8 to 11.6 degrees C. The infections were characterized by viral persistence for extended periods and near-background levels of host mortality. Infectious virus was recovered from mortalities occurring up to 167 d post-exposure and was detected in normal-appearing herring for as long as 224 d following initial challenge. Geometric mean viral titers were generally as high as or higher in brain tissues than in pools of kidney and spleen tissues, with overall prevalence of infection being higher in the brain. Upon re-exposure to VHSV in a standard laboratory challenge, negligible mortality occurred among groups of herring that were either chronically infected or fully recovered, indicating that survival from chronic manifestations conferred protection against future disease. However, some survivors of chronic VHS infections were capable of replicating virus upon re-exposure. Demonstration of a chronic manifestation of VHSV infection among Pacific herring maintained at ambient seawater temperatures provides insights into the mechanisms by which the virus is maintained among populations of endemic hosts.     

Comparative susceptibility of turbot Scophthalmus maximus to different genotypes of viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia (VHS) disease has exerted a significant impact on the development of turbot aquaculture in the British Isles. The source of such outbreaks is believed to be naturally occurring marine isolates of viral haemorrhagic septicaemia virus (VHSV), which are endemic in the marine environment of Northern Europe. Genetic studies have classified these marine VHSV isolates into genotypes based on their geographic rather than host-species origin. This study set out to explore the hypothesis that susceptibility of turbot to VHSV might be genotype specific. Immersion infection of turbot with a range of isolates, selected according to genotype, identified significant differences between susceptibility and genotype. Viruses belonging to Genotypes Ib (Baltic marine isolates) and III (North Sea/E. Atlantic marine isolates) caused significantly higher mortality than isolates from Genotypes Ia (isolates associated with rainbow trout aquaculture) and II (Baltic marine isolates). This study serves to highlight the importance of thoroughly investigating the susceptibility of any given species to the range of pathogens to which they might be exposed prior to considering them resistant to any disease. Furthermore, it highlights different risk factors that might be associated with turbot aquaculture undertaken in different environments. Finally, an increased knowledge of the relative virulence of different isolates in turbot will assist in understanding virulence determinants, which could lead to advances in disease control.     

Genotyping and pathogenicity of viral hemorrhagic septicemia virus from free-living turbot (Psetta maxima) in a Turkish coastal area of the Black Sea

Viral hemorrhagic septicemia (VHS) is one of the most serious fish viral diseases for cultured rainbow trout (Oncorhynchus mykiss), although VHS virus (VHSV) seems to be ubiquitous among marine fishes. In the present study, VHSV isolation was performed with free-living and cultured turbot (Psetta maxima) in the Trabzon coastal area of the Black Sea to evaluate participation of VHSV in mass mortalities of seed-produced turbot larvae. VHSV was detected in 14 of 66 free-living spawners (positive ratio, 21.2%), 1 of 65 free-living immature fish (1.5%) and 7 of 40 cultured brood stock (17.5%), respectively. Based on a partial glycoprotein gene nucleotide sequence, Turkish VHSV isolates were classified into the class I-e of genotype I and were the most closely related to the GE-1.2 isolate (>98% identity), which was found >20 years ago in Georgia. Thus, it was revealed that Turkish VHSV isolates were not introduced from European countries, it could be an indigenous type of VHSV distributing in the Black Sea environment. In pathogenicity tests, the Turkish isolates did not induce mortality in turbot larvae and rainbow trout fingerlings. Mass mortalities at a rate of approximately 90% occurred in turbot larvae produced by experimental seeding, although VHSV was not detected in any dead fish. Thus, it was concluded that mass mortality in the seed-produced turbot larvae was not caused by VHSV infection.     

Surveillance of Viruses in Wild Fish Populations in Areas around the Gulf of Cadiz (South Atlantic Iberian Peninsula)

This report describes a viral epidemiological study of wild fish around the Gulf of Cadiz (southwestern Iberian Peninsula) and is focused on infectious pancreatic necrosis virus (IPNV), viral hemorrhagic septicemia virus (VHSV), and viral nervous necrosis virus (VNNV). One fish species (Chelon labrosus) was sampled inside the gulf, at the mouth of the San Pedro River. Another 29 were sampled, in three oceanographic campaigns, at sites around the Bay of Cadiz. The fish were processed individually and subjected to isolation in cell culture and molecular diagnosis. VHSV was not isolated from any species. Thirteen IPNV-type isolates were obtained from barracuda (Sphyraena sphyraena), axillary seabream (Pagellus acarne), common two-banded seabream (Diplodus vulgaris), common pandora (P. erythrinus), Senegal seabream (D. bellottii), and surmullet (Mullus surmuletus). Six VNNV isolates were obtained from axillary seabream, common pandora, black seabream (Spondyliosoma cantharus), red mullet (Mullet barbatus), Lusitanian toadfish (Halobatrachus didactylus), and tub gurnard (Chelidonichtys lucerna). In the river mouth, viruses were detected only after reamplification, obtaining prevalence percentages of IPNV and VNNV (44.4 and 63.0%, respectively) much higher than those observed in the oceanographic campaigns (25.7 and 19.6%, respectively). The opposite results were obtained in the case of VHSV after reamplification: 11.1% in the river mouth and 43.6% in the oceanic locations. Analyzing the results with respect to the proximity of the sampling sites to the coast, an anthropogenic influence on wild fish is suggested and discussed. The type of viruses and the presence of natural reassortants are also discussed.     

A Reverse Genetics System for the Great Lakes Strain of Viral Hemorrhagic Septicemia Virus: the NV Gene is Required for Pathogenicity

Viral hemorrhagic septicemia virus (VHSV), belonging to the genus Novirhabdovirus in the family of Rhabdoviridae, causes a highly contagious disease of fresh and saltwater fish worldwide. Recently, a novel genotype of VHSV, designated IVb, has invaded the Great Lakes in North America, causing large-scale epidemics in wild fish. An efficient reverse genetics system was developed to generate a recombinant VHSV of genotype IVb from cloned cDNA. The recombinant VHSV (rVHSV) was comparable to the parental wild-type strain both in vitro and in vivo, causing high mortality in yellow perch (Perca flavescens). A modified recombinant VHSV was generated in which the NV gene was substituted with an enhanced green fluorescent protein gene (rVHSV-ΔNV-EGFP), and another recombinant was made by inserting the EGFP gene into the full-length viral clone between the P and M genes (rVHSV-EGFP). The in vitro replication kinetics of rVHSV-EGFP was similar to rVHSV; however, the rVHSV-ΔNV-EGFP grew 2 logs lower. In yellow perch challenges, wtVHSV and rVHSV induced 82-100% cumulative per cent mortality (CPM), respectively, whereas rVHSV-EGFP produced 62% CPM and rVHSV-ΔNV-EGFP caused only 15% CPM. No reversion of mutation was detected in the recovered viruses and the recombinant viruses stably maintained the foreign gene after several passages. These results indicate that the NV gene of VHSV is not essential for viral replication in vitro and in vivo, but it plays an important role in viral replication efficiency and pathogenicity. This system will facilitate studies of VHSV replication, virulence, and production of viral vectored vaccines.