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.

Introduction of Viral Hemorrhagic Septicemia Virus into Freshwater Cultured Rainbow Trout Is Followed by Bursts of Adaptive Evolution.

Viral hemorrhagic septicemia virus (VHSV), a rhabdovirus infecting teleost fish, has repeatedly crossed the boundary from marine fish species to freshwater cultured rainbow trout. These naturally replicated cross-species transmission events permit the study of general and repeatable evolutionary events occurring in connection with viral emergence in a novel host species. The purpose of the present study was to investigate the adaptive molecular evolution of the VHSV glycoprotein, one of the key virus proteins involved in viral emergence, following emergence from marine species into freshwater cultured rainbow trout. A comprehensive phylogenetic reconstruction of the complete coding region of the VHSV glycoprotein was conducted, and adaptive molecular evolution was investigated using a maximum likelihood approach to compare different codon substitution models allowing for heterogeneous substitution rate ratios among amino acid sites. Evidence of positive selection was detected at six amino acid sites of the VHSV glycoprotein, within the signal peptide, the confirmation-dependent major neutralizing epitope, and the intracellular tail. Evidence of positive selection was found exclusively in rainbow trout-adapted virus isolates, and amino acid combinations found at the six sites under positive selection pressure differentiated rainbow trout- from non-rainbow trout-adapted isolates. Furthermore, four adaptive sites revealed signs of recurring identical changes across phylogenetic groups of rainbow trout-adapted isolates, suggesting that repeated VHSV emergence in freshwater cultured rainbow trout was established through convergent routes of evolution that are associated with immune escape.IMPORTANCE This study is the first to demonstrate that VHSV emergence from marine species into freshwater cultured rainbow trout has been accompanied by bursts of adaptive evolution in the VHSV glycoprotein. Furthermore, repeated detection of the same adaptive amino acid sites across phylogenetic groups of rainbow trout-adapted isolates indicates that adaptation to rainbow trout was established through parallel evolution. In addition, signals of convergent evolution toward the maintenance of genetic variation were detected in the conformation-dependent neutralizing epitope or in close proximity to disulfide bonds involved in the structural conformation of the neutralizing epitope, indicating adaptation to immune response-related genetic variation across freshwater cultured rainbow trout.

Viral haemorrhagic septicaemia virus (VHSV) remains viable for several days but at low levels in the water flea Moina macrocopa.

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.

Evaluation of blue mussel Mytilus edulis as vector for viral hemorrhagic septicemia virus (VHSV).

When viral diseases occur in aquaculture farms, the virus released into the seawater from infected animals can re-infect other susceptible species or accumulate in filter-feeding organisms. We conducted a viral hemorrhagic septicemia virus (VHSV) survivability analysis of blue mussel Mytilus edulis digestive enzymes, viral depuration, and infectivity tests via in vitro and in vivo inoculation to evaluate the infectious state. VHSV particles were not completely digested within 24 h in vitro and were maintained for 7 d in the mussel digestive gland. Mussels cohabitating with naturally VHSV-infected olive flounder Paralichthys olivaceus could accumulate the viral particles. Although the viral particles in the gill as the entrance of filter-feeding organisms are infectious, the presence of these particles in the digestive gland were not able to induce cytopathic effects in vitro. Viral particles detected by RT-PCR from bivalve mollusks (Pacific oyster Crassostrea gigas and mussel) from the field did not produce cytopathic effects in cell culture and did not replicate after intraperitoneal injection into olive flounder. Therefore, VHSV particles in blue mussel might be in a non-infectious stage and the possibilities of VHSV transmission to fish under field conditions are scarce.

Susceptibility of goldsinny wrasse, Ctenolabrus rupestris L. (Labridae), to viral haemorrhagic septicaemia virus (VHSV) genotype III: Experimental challenge and pathology.

Cleaner fish, such as wrasse, are being increasingly used to combat the sea lice infestation of Atlantic salmon (Salmo salar L.) in many European countries. To determine susceptibility of the goldsinny wrasse (Ctenolabrus rupestris L.) and pathogenesis of the viral haemorrhagic septicaemia virus (VHSV) genotype III isolate 12-654, previously associated with VHSV infection in the Shetland Islands in 2012, fish were experimentally challenged by intraperitoneal injection (IP), bath immersion and cohabitation routes. Cumulative proportion of moribund wrasse reached 17% following the virus immersion challenge while by the IP-route moribunds exceeded 50% within 14days post-challenge. Typical signs of VHS as reported in rainbow trout (Oncorhynchus mykiss), were not observed in moribund goldsinny wrasse. The most pronounced histopathological changes, consistent regardless of the route of infection, were observed within the heart and included atrium myofibril degeneration, focal infiltration and multifocal necrosis, with prominent swelling of the endocardium and occasional detachment. Pathological changes in the atrium were associated with presence of the viral antigen as confirmed by a positive immunohistochemical staining. Virus clearance and heart tissue recovery were noted although further experiments are required to confirm these observations. The results of a cohabitation experiment confirmed that goldsinny wrasse shed viable virus and therefore represent a risk of virus transmission to other VHSV susceptible species. Similarities between the pathology in goldsinny wrasse induced through the controlled experimental challenges and that of wrasse spp. from an infection occurrence in Shetland are discussed.

A survey of wild marine fish identifies a potential origin of an outbreak of viral haemorrhagic septicaemia in wrasse, Labridae, used as cleaner fish on marine Atlantic salmon, Salmo salar L., farms.

Viral haemorrhagic septicaemia virus (VHSV) was isolated from five species of wrasse (Labridae) used as biological controls for parasitic sea lice predominantly, Lepeophtheirus salmonis (Krøyer, 1837), on marine Atlantic salmon, Salmo salar L., farms in Shetland. As part of the epidemiological investigation, 1400 wild marine fish were caught and screened in pools of 10 for VHSV using virus isolation. Eleven pools (8%) were confirmed VHSV positive from: grey gurnard, Eutrigla gurnardus L.; Atlantic herring, Clupea harengus L.; Norway pout, Trisopterus esmarkii (Nilsson); plaice, Pleuronectes platessa L.; sprat, Sprattus sprattus L. and whiting, Merlangius merlangus L. The isolation of VHSV from grey gurnard is the first documented report in this species. Nucleic acid sequencing of the partial nucleocapsid (N) and glycoprotein (G) genes was carried out for viral characterization. Sequence analysis confirmed that all wild isolates were genotype III the same as the wrasse and there was a close genetic similarity between the isolates from wild fish and wrasse on the farms. Infection from these local wild marine fish is the most likely source of VHSV isolated from wrasse on the fish farms.

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.

Model for ranking freshwater fish farms according to their risk of infection and illustration for viral haemorrhagic septicaemia.

We developed a model to calculate a quantitative risk score for individual aquaculture sites. The score indicates the risk of the site being infected with a specific fish pathogen (viral haemorrhagic septicaemia virus (VHSV); infectious haematopoietic necrosis virus, Koi herpes virus), and is intended to be used for risk ranking sites to support surveillance for demonstration of zone or member state freedom from these pathogens. The inputs to the model include a range of quantitative and qualitative estimates of risk factors organised into five risk themes (1) Live fish and egg movements; (2) Exposure via water; (3) On-site processing; (4) Short-distance mechanical transmission; (5) Distance-independent mechanical transmission. The calculated risk score for an individual aquaculture site is a value between zero and one and is intended to indicate the risk of a site relative to the risk of other sites (thereby allowing ranking). The model was applied to evaluate 76 rainbow trout farms in 3 countries (42 from England, 32 from Italy and 2 from Switzerland) with the aim to establish their risk of being infected with VHSV. Risk scores for farms in England and Italy showed great variation, clearly enabling ranking. Scores ranged from 0.002 to 0.254 (mean score 0.080) in England and 0.011 to 0.778 (mean of 0.130) for Italy, reflecting the diversity of infection status of farms in these countries. Requirements for broader application of the model are discussed. Cost efficient farm data collection is important to realise the benefits from a risk-based approach.

Do imports of rainbow trout carcasses risk introducing viral haemorrhagic septicaemia virus into England and Wales?

A qualitative import risk assessment was undertaken to assess the likelihood of introduction and establishment of viral haemorrhagic septicaemia virus (VHSV) genotype 1a in England and Wales (E&W), via the processing of imported rainbow trout (Oncorhynchus mykiss) carcasses from continental Europe. The likelihood was estimated for one import from an infected farm. Four main routes by which susceptible populations could be exposed to VHSV via processing waste were considered: (i) run-off from solid waste to watercourses, (ii) contamination of birds or rodents with VHSV by scavenging solid waste, (iii) discharge of liquid waste to mains drainage, and (iv) discharge of liquid waste directly to watercourses. Data on the biophysical characteristics of VHSV, its epidemiology, fish processing practices and waste management were collected. Likelihoods for each step of the four pathways were estimated. Pathway 4 (discharge of liquid waste to a watercourse) was judged as the most likely to result in infection of susceptible individuals. Levels of virus entering the aquatic environment via pathways 1-3 were judged to be many times lower than pathway 4 due mainly to the treatment of solid waste (pathways 1 and 2) and high levels of dilution (pathways 1, 2 and 3). Thirty-four trout farms process fish, of which seven have imported carcasses for processing. Compared with other processing facilities, on-farm processing results in a higher likelihood of VHSV exposure and establishment via all four pathways. Data availability was an issue; the analysis was particularly constrained by a lack of data on the prevalence of VHSV in Europe, volume of trade of carcasses into the UK and processing practices in E&W. It was concluded that the threat of VHSV introduction into E&W could be reduced by treatment of liquid effluent from processing plants and by sourcing carcasses for on-farm processing only from approved VHSV free areas.

Experimental transmission of VHSV genotype IVb by predation.

Preliminary surveillance of wild baitfish during the 2006 viral hemorrhagic septicemia virus genotype IVb (VHSV IVb) outbreaks indicated Emerald Shiners Notropis atherinoides and Bluntnose Minnow Pimephales notatus were infected with high levels of VHSV without showing clinical signs of disease. The movement and use of baitfish was recognized as the most probable vector for the introduction of VHSV to inland waters, such as Conesus Lake and Skaneateles Lake in New York, Budd Lake in Michigan, and Little Lake Butte des Morts and Lake Winnebago in Wisconsin. While numerous government agencies implemented restrictions to stop the movement of potentially infected baitfish into new waters and prevent the spread of VHSV IVb, until now, studies to investigate whether these initial introductions were by an oral route of infection have not occurred. Our studies identified infected Fathead Minnow Pimephales promelas as suitable vectors for transmitting VHSV IVb when fed to Tiger Muskellunge ( ♂ Northern Pike Esox lucius × â™€ Muskellunge Esox masquinongy) during laboratory trials. Six of 16 Tiger Muskellunge were infected with VHSV IVb after consumption of infected Fathead Minnows when assayed with quantitative reverse transcriptase polymerase chain reaction and viral isolation in cell culture. Weekly sampling of water and feces from these Tiger Muskellunge individually reared showed intermittent shedding of VHSV IVb. Those exposed to similarly VHSV IVb-inoculated fathead minnows by cohabitation only became infected in 1 case out of 16. A similar trial of 12 Tiger Muskellunge fed Round Goby Neogobius melanostomus that survived a VHSV IVb immersion challenge did not result in infection. Overall, our findings imply that consumption of infected wild baitfish may be a risk factor for introduction of VHSV.

Trade practices are main factors involved in the transmission of viral haemorrhagic septicaemia.

Viral haemorrhagic septicaemia (VHS), caused by the novirhabdovirus viral haemorrhagic septicaemia virus (VHSV), causes significant economic problems to European rainbow trout, Oncorhynchus mykiss (Walbaum), production. The virus isolates can be divided into four distinct genotypes with additional subgroups. The main source of outbreaks in European rainbow trout farming is sublineage Ia isolates. Recently, this group of isolates has been further subdivided in to two subclades of which the Ia-2 consists of isolates occurring mainly in Continental Europe outside of Denmark. In this study, we sequenced the full-length G-gene sequences of 24 VHSV isolates that caused VHS outbreaks in Polish trout farms between 2005 and 2009. All these isolates were identified as genotype Ia-2; they divided however into two genetically distinct subgroups, that we name Pol I and Pol II. The Pol I isolates mainly caused outbreaks in the southern part of Poland, while Pol II isolates predominantly were sampled in the north of Poland, although it seems that they have been transmitted to other parts of the country. Molecular epidemiology was used for characterization of transmission pathways. This study shows that a main cause of virus transmission appears to be movement of fish. At least in Polish circumstances trading practices appear to have significant impact on spreading of VHSV infection.

Risk assessment for the design of a risk-based surveillance programme for fish farms in Switzerland (in accordance with Council Directive 2006/88/EC of the European Union).

Swiss aquaculture farms were assessed according to their risk of acquiring or spreading viral haemorrhagic septicaemia (VHS) and infectious haematopoietic necrosis (IHN). Risk factors for the introduction and spread of VHS and IHN were defined and assessed using published data and expert opinions. Among the 357 aquaculture farms identified in Switzerland, 49.3% were categorised as high risk, 49.0% as medium risk and 1.7% as low risk. According to the new Directive 2006/88/EC for aquaculture of the European Union, the frequency of farm inspections must be derived from their risk levels. A sensitivity analysis showed that water supply and fish movements were highly influential on the output of the risk assessment regarding the introduction of VHS and IHN. Fish movements were also highly influential on the risk assessment output regarding the spread of these diseases.

Oral transmission as a route of infection for viral haemorrhagic septicaemia virus in rainbow trout, Oncorhynchus mykiss (Walbaum).

Surveys among wild marine fish have revealed occurrence of viral haemorrhagic septicaemia virus (VHSV) infections in a high number of diverse fish species. In marine aquaculture of rainbow trout, preying on invading wild fish might thus be a risk factor for introduction and adaptation of VHSV and subsequent disease outbreaks. Our objective was to determine whether an oral transmission route for VHSV in rainbow trout exists. Juvenile trout were infected through oral, waterborne and cohabitation transmission routes, using a recombinant virus strain harbouring Renilla luciferase as reporter gene. Viral replication in stomach and kidney tissue was detected through bioluminescence activity of luciferase and qRT-PCR. Replication was detected in both tissues, irrespective of transmission route. Replication patterns, however, differed among transmission routes. In trout infected through oral transmission, replication was detected in the stomach prior to kidney tissue. In trout infected through waterborne or cohabitation transmission, replication was detected in kidney prior to stomach or in both tissues simultaneously. We demonstrate the existence of an oral transmission route for VHSV in rainbow trout. This implies that preying on invading infected wild fish is a risk factor for introduction of VHSV into marine cultures of rainbow trout.

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.

Ranking freshwater fish farms for the risk of pathogen introduction and spread.

A semi-quantitative model is presented to rank freshwater rainbow trout farms within a country or region with regards to the risk of becoming infected and spreading a specified pathogen. The model was developed to support a risk-based surveillance scheme for notifiable salmonid pathogens. Routes of pathogen introduction and spread were identified through a process of expert consultation in a series of workshops. The routes were combined into themes (e.g. exposure via water, mechanical transmission). Themes were weighted based on expert opinion. Risk factors for each route were scored and combined into a theme score which was adjusted by the weight. The number of sources and consignments were used to assess introduction via live fish movements onto the farm. Biosecurity measures were scored to assess introduction on fomites. Upstream farms, wild fish and processing plants were included in assessing the likelihood of introduction by water. The scores for each theme were combined to give separate risk scores for introduction and spread. A matrix was used to combine these to give an overall risk score. A case study for viral haemorrhagic septicaemia is presented. Nine farms that represent a range of farming practices of rainbow trout farms in England and Wales are used as worked examples of the model. The model is suited to risk rank freshwater salmonid farms which are declared free of the pathogen(s) under consideration. The score allocated to a farm does not equate to a quantitative probability estimate of the farm to become infected or spread infection. Nevertheless, the method provides a transparent approach to ranking farms with regards to pathogen transmission risks. The output of the model at a regional or national level allows the allocation of surveillance effort to be risk based. It also provides fish farms with information on how they can reduce their risk score by improving biosecurity. The framework of the model can be applied to different production systems which may have other routes of disease spread. Further work is recommended to validate the allocated scores. Expert opinion was obtained through workshops, where the outputs from groups were single point estimates for relative weights of risks. More formal expert opinion elicitation methods could be used to capture variation in the experts' estimates and uncertainty and would provide data on which to simulate the model stochastically. The model can be downloaded (in Microsoft(®)-Excel format) from the Internet at: http://www.cefas.defra.gov.uk/6701.aspx.

Assessment of exotic fish disease introduction and establishment in the United Kingdom via live fish transporters.

Live fish transporters returning empty from mainland Europe may mechanically introduce exotic pathogens and parasites to the UK. A qualitative risk assessment approach was adopted to investigate the likelihood of introduction and establishment in rainbow trout farms of viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN) and Gyrodactylus salaris via this route. A scenario tree was developed and estimates made for the likelihood of each step based on a review of the available information. The level of uncertainty associated with each step was qualitatively assessed. The likelihood of a lorry becoming contaminated with any of the 3 hazards was only greater than negligible if the lorry made movements between farms in mainland Europe. In these circumstances, the overall likelihood of introduction and establishment was extremely low (would occur very rarely), extremely low to negligible and very low (would occur rarely) for VHS, IHN and G. salaris, respectively. A high level of uncertainty existed due to the lack of data on farm-level prevalence, minimum infectious dose (of the viral hazards) and the large variability in duration and conditions of transport. A telephone survey of live fish transporters found that cleaning and disinfection practices after return to the UK were implemented. Currently, no UK-based transporters make movements between farms in mainland Europe. Thus, the likelihood that UK-owned transporters may become infected is negligible. Changes in the way in which UK-based live fish transporters operate in mainland Europe need to be monitored and development of a code of practice to minimise the risk of disease introduction considered.

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.

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.