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.     

Investigation of round goby viral haemorrhagic septicaemia outbreak in New York

Eleven viral haemorrhagic septicaemia virus (VHSV) genotype IVb isolates were sequenced, and their genetic variation explored to determine the source of a VHS outbreak on the eastern shore of Cayuga Lake. An active fish kill of round gobies (Neogobius melanostomus, Pallas) was intensively sampled at King Ferry, NY and nearby Long Point State Park in May 2017. Gross lesions observed on 67 moribund round gobies and two rock bass (Ambloplites rupestris, Rafinesque) included moderately haemorrhagic internal organs and erythematous areas on the head, flank, and fins. RT‐qPCR tests for VHSV were positive for all 69 fish. Viral isolation on epithelioma papulosum cyprinid cells showed cytopathic effect characteristic of VHSV for six round goby samples from King Ferry. The complete nucleotide sequence of the VHSV IVb genomes of five Cayuga Lake round goby isolates were derived on an Illumina platform along with 2017 VHSV IVb isolates from round gobies collected from the following: Lake Erie near Dunkirk, NY; the St. Lawrence River near Clayton and Cape Vincent, NY; and Lake St. Lawrence near Massena, NY. The phylogenetic tree created from these aligned sequences and four other complete VHSV IVb genomes shows Cayuga Lake isolates are closely related to the Lake Erie isolates.     

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.     

Factors controlling the early stages of viral haemorrhagic septicaemia epizootics: low exposure levels, virus amplification and fish-to-fish transmission

Viral haemorrhagic septicaemia virus, Genogroup IVa (VHSV), was highly infectious to Pacific herring, Clupea pallasii (Valenciennes), even at exposure doses occurring below the threshold of sensitivity for a standard viral plaque assay; however, further progression of the disease to a population‐level epizootic required viral amplification and effective fish‐to‐fish transmission. Among groups of herring injected with VHSV, the prevalence of infection was dose‐dependent, ranging from 100%, 75% and 38% after exposure to 19, 0.7 and 0.07 plaque‐forming units (PFU)/fish, respectively. Among Pacific herring exposed to waterborne VHSV (140 PFU mL^?1), the prevalence of infection, geometric mean viral tissue titre and cumulative mortality were greater among cohabitated herring than among cohorts that were held in individual aquaria, where fish‐to‐fish transmission was prevented. Fish‐to‐fish transmission among cohabitated herring probably occurred via exposure to shed virus which peaked at 680 PFU mL^?1; shed virus was not detected in the tank water from any isolated individuals. The results provide insights into mechanisms that initiate epizootic cascades in populations of wild herring and have implications for the design of VHSV surveys in wild fish populations.     

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.     

A mortality event in wrasse species (Labridae) associated with the presence of viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia (VHS) is an infectious disease of farmed and wild fish and has an extensive host range in both freshwater and marine environments. In December 2012, a wrasse population consisting of ballan, Labrus bergylta (Ascanius), corkwing, Symphodus melops (L.), cuckoo, Labrus mixtus L., goldsinny, Ctenolabrus rupestris (L.), and rock cook, Centrolabrus exoletus (L.), held at a marine hatchery in the Shetland Isles, Scotland, experienced a mortality event. Approximately 10 000 wrasse were being held at the facility on behalf of an Atlantic salmon, Salmo salar L., aquaculture company prior to being deployed for the biological control of parasites on marine pen Atlantic salmon, aquaculture sites. Fish Health Inspectors from Marine Scotland Science initiated a diagnostic investigation, and subsequent diagnostic testing confirmed the site to be VHSV positive by qRT-PCR and virus isolation followed by ELISA. A VHSV genotype-specific qRT-PCR assay revealed that the isolates belonged to genotype III, the European marine strain of the virus. The virus genotype was further confirmed by nucleic acid sequencing of the partial nucleoprotein (N) and glycoprotein (G) genes followed by BLAST nucleotide searches. This study reports for the first time the detection of VHSV within multiple wrasse species and highlights the need for a comprehensive risk-based approach to the use of wrasse and other finfish species as biological controls within the aquaculture industry.     

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.     

The first report of viral haemorrhagic septicaemia in farmed rainbow trout, Oncorhynchus mykiss (Walbaum), in the United Kingdom

Viral haemorrhagic septicaemia (VHS) was diagnosed in rainbow trout in the UK in May 2006. VHS virus (VHSV) was isolated from fingerlings showing typical histopathological lesions at a single rainbow trout farm site experiencing high mortality. The virus was confirmed as VHSV by serological and molecular biological tests. Phylogenetic analysis based on the complete glycoprotein gene sequence revealed that the isolate was closely related (99% nucleotide identity) to several Danish isolates from 1991 to 2000 and was assigned to VHSV genogroup Ia. The pathogenicity of the isolate was determined in infection experiments using rainbow trout fry. Following waterborne challenge, cumulative mortalities reached 96.67-100% by 12 days post-infection. This represents the first isolation of a pathogenic freshwater VHSV in the UK.     

Potential distribution of the viral haemorrhagic septicaemia virus in the Great Lakes region

Viral haemorrhagic septicaemia virus (VHSV) genotype IVb has been responsible for large‐scale fish mortality events in the Great Lakes of North America. Anticipating the areas of potential VHSV occurrence is key to designing epidemiological surveillance and disease prevention strategies in the Great Lakes basin. We explored the environmental features that could shape the distribution of VHSV, based on remote sensing and climate data via ecological niche modelling. Variables included temperature measured during the day and night, precipitation, vegetation, bathymetry, solar radiation and topographic wetness. VHSV occurrences were obtained from available reports of virus confirmation in laboratory facilities. We fit a Maxent model using VHSV‐IVb reports and environmental variables under different parameterizations to identify the best model to determine potential VHSV occurrence based on environmental suitability. VHSV reports were generated from both passive and active surveillance. VHSV occurrences were most abundant near shore sites. We were, however, able to capture the environmental signature of VHSV based on the environmental variables employed in our model, allowing us to identify patterns of VHSV potential occurrence. Our findings suggest that VHSV is not at an ecological equilibrium and more areas could be affected, including areas not in close geographic proximity to past VHSV reports.     

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.     

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 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.     

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.     

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).