Global spread and evolution of viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia virus (VHSV) is a rhabdovirus that infects over 48 species of teleosts and is lethal in many. VHSV threatens marine and aquatic fisheries. VHSV was first discovered outside Europe in 1988 in fish from the Pacific coast of North America. In 1994, VHSV was discovered in Newfoundland. In 2003, VHSV was isolated from fish in Lake St. Clair (Michigan and Ontario). In this study, we used 46 nucleotide sequences for the glycoprotein gene from 12 studies and 150 nucleotide sequences for the nucleoprotein gene from nine studies. We combined phylogenetics and a geographic information system to visualize the transmission paths of VHSV lineages. We also reconstructed the spread of VHSV lineages through optimization of geographic data for viral isolates on phylogenetic trees. We demonstrate that VHSV was transmitted from the North Atlantic Ocean and/or Baltic Sea to the Atlantic coast of North America and Japan in independent events. From the Atlantic coast, the virus was transmitted independently to the Laurentian Great Lakes and the Pacific coast of Canada and the contiguous United States. From the Pacific Northwest, the virus was transmitted to Asia and Alaska in independent events. These results clarify the debate ongoing in the literature on the geographic spread of VHSV.     

Development and validation of a novel Taqman‐based real‐time RT‐PCR assay suitable for demonstrating freedom from viral haemorrhagic septicaemia virus

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.     

FishPathogens.eu/vhsv: a user-friendly viral haemorrhagic septicaemia virus isolate and sequence database

A database has been created, http://www.FishPathogens.eu , with the aim of providing a single repository for collating important information on significant pathogens of aquaculture, relevant to their control and management. This database will be developed, maintained and managed as part of the European Community Reference Laboratory for Fish Diseases function. This concept has been initially developed for viral haemorrhagic septicaemia virus and will be extended in future to include information on other significant aquaculture pathogens. Information included for each isolate comprises sequence, geographical origin, host origin and useful key literature. Various search mechanisms make it easy to find specific groups of isolates. Search results can be presented in several different ways including table-based, map-based and graph-based outputs. When retrieving sequences, the user is given freedom to obtain data from any selected part of the genome of interest. The output of the sequence search can be readily retrieved as a FASTA file ready to be imported into a sequence alignment tool of choice, facilitating further molecular epidemiological study.     

Comparative susceptibility among three stocks of yellow perch,Perca flavescens (Mitchill), to viral haemorrhagic septicaemia virus strain IVb from the Great Lakes

The Great Lakes strain of viral haemorrhagic septicaemia virus IVb (VHSV‐IVb) is capable of infecting a wide number of naive species and has been associated with large fish kills in the Midwestern United States since its discovery in 2005. The yellow perch, Perca flavescens (Mitchill), a freshwater species commonly found throughout inland waters of the United States and prized for its high value in sport and commercial fisheries, is a species documented in several fish kills affiliated with VHS. In the present study, differences in survival after infection with VHSV IVb were observed among juvenile fish from three yellow perch broodstocks that were originally derived from distinct wild populations, suggesting innate differences in susceptibility due to genetic variance. While all three stocks were susceptible upon waterborne exposure to VHS virus infection, fish derived from the Midwest (Lake Winnebago, WI) showed significantly lower cumulative % survival compared with two perch stocks derived from the East Coast (Perquimans River, NC and Choptank River, MD) of the United States. However, despite differences in apparent susceptibility, clinical signs did not vary between stocks and included moderate‐to‐severe haemorrhages at the pelvic and pectoral fin bases and exophthalmia. After the 28‐day challenge was complete, VHS virus was analysed in subsets of whole fish that had either survived or succumbed to the infection using both plaque assay and quantitative PCR methodologies. A direct correlation was identified between the two methods, suggesting the potential for both methods to be used to detect virus in a research setting.     

Development of neutralizing antibody responses in muskellunge, Esox masquinongy (Mitchill), experimentally exposed to viral haemorrhagic septicaemia virus (genotype IVb)

A complement‐dependent 50% plaque neutralization test was used to assess the neutralizing antibody response in sera of muskellunge, Esox masquinongy, experimentally infected with viral haemorrhagic septicaemia virus (VHSV, genotype IVb) by immersion. Groups of muskellunge were challenged with varying concentrations of VHSV: Group 1 with 10² plaque‐forming units (pfu) mL^?1, Group 2 with 4 × 10³ pfu mL^?1, Group 3 with 10⁵ pfu mL^?1 and Group 4 with 0 pfu mL^?1. The fish were held at a temperature of 11 ± 1 °C and were sampled over a 20‐week period. Neutralizing antibodies were not detected in sera of any of the negative control fish throughout the study. Low neutralizing titres were detected in Groups 1–3 by 6 days post‐infection (p.i.). Neutralizing titres of <80 were not detected again until 3, 4 and 7 weeks p.i. for Groups 2, 3 and 1, respectively, with peak titres for those groups occurring 16, 11 and 17 weeks p.i., respectively. VHSV was detected in serum for up to 11 weeks p.i. Results of this study show that survivors can be detected by a serological technique, despite being virus negative. This may benefit the investigation of VHSV IVb distribution in the Great Lakes and the study of host immune responses to this emerging sublineage.     

Viral haemorrhagic septicaemia virus in marine fish and its implications for fish farming--a review

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.     

Whole-genome next-generation sequencing and phylogenetic characterization of viral haemorrhagic septicaemia virus in Korea

Whole-genome next-generation sequencing was used to investigate the local evolution of viral haemorrhagic septicaemia virus, a serious pathogen affecting economically important fish such as rainbow trout and turbot in Europe and olive flounder in Asia. Sequence analysis showed that all isolates were genotype IVa, but could be classified further into four subgroups (K1–K4). In addition, genomic regions encompassing the nucleoprotein, phosphoprotein, matrix protein and non-virion protein genes, as well as the seven non-coding regions, were relatively conserved, whereas glycoprotein and RNA-dependent RNA polymerase genes were variable in the coding region. Taken together, the data demonstrate that whole-genome next-generation sequencing may be useful for future surveillance, prevention and control strategies against viral haemorrhagic septicaemia.     

Isolation of viral haemorrhagic septicaemia virus from mummichog, stickleback, striped bass and brown trout in eastern Canada

Viral haemorrhagic septicaemia virus (VHSV) was isolated from mortalities occurring in populations of mummichog, Fundulus heteroclitus, stickleback, Gasterosteus aculeatus aculeatus, brown trout, Salmo trutta, and striped bass, Morone saxatilis, in New Brunswick and Nova Scotia, Canada. The isolated viral strains produced a cytopathic effect on the epithelioma papillosum cyprini cell line. Serum neutralization indicated the virus was VHSV and sequencing identified the rhabdovirus isolates as the North American strain of VHSV. Phylogenetic analysis indicated that the isolates are closely related and form a distinguishable subgroup of North American type VHSV. To our knowledge, this is the first report of VHSV in mummichog and striped bass.     

A study of the susceptibility of Atlantic halibut,Hippoglossus hippoglossus (L.), to viral haemorrhagic septicaemia virus isolated from turbot,Scophthalmus maximus (L.)

The susceptibility of Atlantic halibut, Hippoglossus hippoglossus (L.), to viral haemorrhagic septicaemia virus (VHSV) was tested. Juvenile halibut of approximately 5 g weight were subjected to challenge by intraperitoneal injection, cohabitation and immersion to a VHSV isolate from an outbreak of the disease in turbot, Scophthalmus maximus (L.). The intraperitoneal injection gave the highest mortality rate of 28% after 50 days. The cohabitee group suffered 19% mortality rate and the immersion group only 2%. Control groups included turbot exposed either by intraperitoneal injection or immersion which suffered mortality rates of 93 and 50%, respectively. The results suggest that halibut are markedly less susceptible to VHSV than turbot.     

Isolation and identification of a viral haemorrhagic septicaemia virus (VHSV) isolate from wild largemouth bass Micropterus salmoides in China

Fish rhabdoviruses are a family of viruses responsible for large‐scale fish die‐offs worldwide. Here, we reported the isolation and identification of a member of rhabdoviruses from wild largemouth bass (Micropterus salmoides) in the coastal area of the Pearl River Estuary, China. This virus isolate was identified as viral haemorrhagic septicaemia virus (VHSV) by specific RT‐PCR. Furthermore, the virus (VHSVLB2018) was isolated by cell culture using fathead minnow cells and confirmed by RT‐PCR. Electron microscopy showed the presence of bullet‐shaped viral particles in the cytoplasm of infected cells. The complete sequencing of VHSVLB2018 confirmed that it was genome configuration typical of rhabdoviruses. Phylogenetic analysis based on whole‐genome sequences and G gene nucleotides sequences revealed that VHSVLB2018 was assigned to VHSV genogroup Ⅳa. The pathogenicity of VHSVLB2018 was determined in infection experiments using specific pathogen‐free largemouth bass juveniles. VHSVLB2018‐infected fish showed typical clinical signs of VHSV disease, including darkened skin, petechial haemorrhages and pale enlarged livers, with the cumulative mortalities reached 63.3%–93.3% by 7 days post‐infection. VHSVLB2018 was re‐isolated from dead fish and confirmed by RT‐PCR. Together, this is the first report of isolation and identification of a VHSV isolate from wild largemouth bass in China.     

Viral replication in excised fin tissues (VREFT) corresponds with prior exposure of Pacific herring, Clupea pallasii (Valenciennes), to viral haemorrhagic septicaemia virus (VHSV)

Procedures for a viral replication in excised fin tissue (VREFT) assay were adapted to Pacific herring, Clupea pallasii, and optimized both to reduce processing time and to provide the greatest resolution between naïve herring and those previously exposed to viral haemorrhagic septicaemia virus (VHSV), Genogroup IVa. The optimized procedures included removal of the left pectoral fin from a euthanized fish, inoculation of the fin with >10⁵ plaque‐forming units (PFU) mL^?1 VHSV for 1 h, rinsing the fin in fresh medium six times to remove unadsorbed virions, incubation of the fin in fresh medium for 4 days and enumeration of the viral titre in a sample of the incubation medium by plaque assay. The optimized VREFT assay was effective at identifying the prior exposure history of laboratory‐reared Pacific herring to VHSV. The geometric mean VREFT value was significantly greater (P < 0.01) among naïve herring (1.2 × 10³ PFU mL^?1) than among groups that survived exposure to VHSV (1.0–2.9 × 10² PFU mL^?1); additionally, the proportion of cultures with no detectable virus was significantly greater (P = 0.0002) among fish that survived exposure to VHSV (39–47%) than among naïve fish (3.3%). The optimized VREFT assay demonstrates promise for identifying VHSV exposure history and forecasting disease potential in populations of wild Pacific herring.     

Epidemiological aspects of viral haemorrhagic septicaemia virus genotype II isolated from Baltic herring, Clupea harengus membras L

This study was carried out to clarify the role of wild fish, especially Baltic herring, Clupea harengus membras L., in the epidemiology of viral haemorrhagic septicaemia virus (VHSV) in brackish water in Finland. Baltic herring with no visible signs of disease were collected from the Archipelago Sea, the Gulf of Bothnia and the eastern Gulf of Finland. In total, 7580 herring were examined by virus isolation as 758 pooled samples and 3029 wild salmonid broodfish as pooled samples during 2004-2006. VHSV was isolated from 51 pooled herring samples in bluegill fibroblast-2 cells, but not in epithelioma papulosum cyprini cells. The majority of isolations were from the coastal archipelago and from fish caught during the spawning season. Based on glycoprotein (G) gene sequences, the virus was classified as a member of genotype II of VHSV. Pairwise comparisons of the G gene regions of herring isolates revealed that all the isolates were closely related, with 98.8-100% nucleotide homology. Phylogenetic analyses revealed that they were closely related to the strains isolated previously from herring and sprat, Sprattus sprattus (L.), in Gotland and to the VHSV isolates from European river lamprey, Lampetra fluviatilis (L.), in the rivers that flow into the Bothnian Bay. The infection in Baltic herring is likely to be independent of the VHSV Id epidemic in farmed rainbow trout, Oncorhynchus mykiss (Walbaum).     

Experimental infection of turbot, Psetta maxima (L.), with strains of viral haemorrhagic septicaemia virus isolated from wild and farmed marine fish

The susceptibility of turbot, Psetta maxima, to infection with two strains of viral haemorrhagic septicaemia virus (VHSV) obtained from wild Greenland halibut, Reinhardtius hippoglossoides, and from farmed turbot was examined. A marine VHSV strain known to be highly pathogenic for turbot was also utilized for comparative purposes. Fish were infected by intra-peritoneal (i.p.), immersion or cohabitation, and maintained at two different temperatures (8 and 15 degrees C). Infection trials showed that the three VHSV isolates were pathogenic for turbot fingerlings by i.p. injection at both temperatures, with high levels of mortality. Virus was recovered from most pools of dead fish i.p. challenged, but not from surviving fish. Although clinical signs were not induced following waterborne exposure, viral growth was obtained from some pools of surviving fish challenged by immersion with strain GH40 from Greenland halibut, which indicates that the virus can survive in sea water and infect other fish via horizontal transmission. Furthermore, although low, the clinical signs and mortality observed in fish cohabitating with turbot challenged with strain GH40 confirms horizontal transmission and indicates that the passage through fish increases the virulence of this strain for turbot. These findings indicate that Greenland halibut, as other wild fish, may play an important role in the epizootiology of VHSV and suggest a potential risk for the turbot farming industry.     

Outbreak of viral haemorrhagic septicaemia (VHS) in lumpfish (Cyclopterus lumpus) in Iceland caused by VHS virus genotype IV

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.