The nucleotide sequences of a specific region of the nucleoprotein gene were compared in order to investigate the genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV). Analysis of the sequence from 128 isolates of diverse geographic and host origin renders this the most comprehensive molecular epidemiological study of marine VHSV conducted to date. Phylogenetic analysis of nucleoprotein gene sequences confirmed the existence of the 4 major genotypes previously identified based on N-and subsequent G-gene based analyses. The range of Genotype I included subgroups of isolates associated with rainbow trout aquaculture (Genotype Ia) and those from the Baltic marine environment (Genotype Ib) to emphasise the relatively close genetic relationship between these isolates. The existence of an additional genotype circulating within the Baltic Sea (Genotype II) was also confirmed. Genotype III included marine isolates from around the British Isles in addition to those associated with turbot mariculture, highlighting a continued risk to the development of this industry. Genotype IV consisted of isolates from the marine environment in North America. Taken together, these findings suggest a marine origin of VHSV in rainbow trout aquaculture. The implications of these findings with respect to the future control of VHSV are discussed. The capacity for molecular phylogenetic analysis to resolve complex epidemiological problems is also demonstrated and its likely future importance to disease management issues highlighted.

Salmonid alphaviruses (SAVs), which include the aetiological agents of salmon pancreas disease (SPD) in farmed Atlantic salmon Salmo salar L. and sleeping disease (SD) in rainbow trout Oncorhynchus mykiss (Walbaum), are significant viral pathogens of European salmonid aquaculture. SAV is horizontally transmitted and the virus can survive for extended periods in seawater. A lack of convincing evidence for vertical transmission coupled to the fact that the SPD virus (SPDV) occurs in historically infected sites irrespective of fallow period duration suggests that a substantial reservoir of infection exists in the marine environment. We used a highly sensitive real-time PCR (qPCR) assay targeting a region of the SAV nsP1 gene to screen wild marine fish species for the presence of SAV in an attempt to identify such a potential reservoir. Screened fish species were caught in the vicinity of aquaculture activity in an area with a previous history of SAV infection (Shetland Isles, Scotland). SAV RNA was detected in internal organs (kidney and heart) from the flatfish species common dab Limanda limanda, long rough dab Hippoglossoides platessoides, and plaice Pleuronectes platessa. Based on these findings, sampling was extended to an area remote from aquaculture activity (Stonehaven Bay, NE coast of Scotland) from where heart tissues obtained from common dab also tested positive. While no virus could be cultivated from these samples, qPCR detections were shown to be SAV-specific by sequencing of an alternative gene region (E2) to that targeted by the qPCR assay. Analysis of these nucleotide sequences revealed minor differences to those previously obtained from farmed salmon, and subsequent phylogenetic analysis of an E2 dataset demonstrated a subtype V-like sequence.

Common dab Limanda limanda from Scottish and international waters were examined by quantitative real-time RT-qPCR for evidence of viral RNA consistent with salmonid alphaviruses (SAV). SAV prevalence in heart tissue varied between sampling sites and reached up to 17% in fish collected near the Shetland Islands, Scotland. Raw Ct values ranging from 22.31 to 39.45 were obtained from the SAV-positive tissue material using the nsP1 RT-qPCR assay. Bayesian-, likelihood-and distance-based phylogenetic analyses performed with the amplified partial E2 gene sequence dataset suggest that the dab-derived virus belongs to SAV Subtypes I, II and V. A single SAV subtype was identified from the majority of sampling sites, apart from Shetland, where Subtypes II and V were also identified. The presence of SAV RNA from common dab in regions detached from salmon aquaculture lends support to the hypothesis that common dab are bone fide wild reservoirs of SAV, independent of fish farming activity. There was no link between the occurrence of viral RNA, length and sex of the dab, water depth, or health status as recorded using the International Council for the Exploration of the Sea (ICES) guidelines. In addition, the histological changes recorded in dab could not, with certainty, be attributed to infection with SAV. Finally, and for the first time, this study demonstrated that the dab-derived SAV Subtype V virus could be successfully cultured in a salmonid cell line.

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