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Viral hemorrhagic septicemia virus (VHSV) infects over 70 fish species inhabiting marine, brackish or freshwater environments throughout the Northern Hemisphere. Over its geographic range, 4 VHSV genotypes and multiple subtypes exist. Here, we describe the development and validation of a rapid, sensitive and specific real-time reverse transcription quantitative PCR assay (RT-qPCR) that amplifies sequence from representative isolates of all VHSV genotypes (I, II, III and IV). The pan-specific VHSV RT-qPCR assay reliably detects 100 copies of VHSV nucleoprotein RNA without cross-reacting with infectious hematopoietic necrosis virus, spring viremia of carp virus or aquatic birnavirus. Test performance characteristics evaluated on experimentally infected Atlantic salmon Salmo salar L. revealed a diagnostic sensitivity (DSe) ≥93% and specificity (DSp) = 100%. The repeatability and reproducibility of the procedure was exceptionally high, with 93% agreement among test results within and between 2 laboratories. Furthermore, proficiency testing demonstrated the VHSV RT-qPCR assay to be easily transferred to and performed by a total of 9 technicians representing 4 laboratories in 2 countries. The assay performed equivalent to the traditional detection method of virus isolation via cell culture with the advantage of faster turnaround times and high throughput capacity, further suggesting the suitability of the use of this VHSV RT-qPCR in a diagnostic setting.

Summary 1.There is considerable debate over the impact of seal predation on salmonid populations in both the Atlantic and Pacific oceans. Conventional hard-part analysis of scats has suggested that salmonids represent a minor component of the diet of grey seals ( Halichoerus grypus ) and harbour seals ( Phoca vitulina ) in the UK. However, it is unclear whether this is an accurate reflection of the diet or due to methodological problems. To investigate this issue, we applied quantitative PCR (qPCR) to examine the presence of salmonids in the diet of seals in the Moray Firth, UK, during the summers of 2003 and 2005. 2. Two qPCR assays were designed to detect Atlantic salmon Salmo salar and sea trout Salmo trutta DNA in field samples and experimentally spiked scats. The proportion of scats sampled in the field that were positive for salmonid DNA was low ( ≈ 10%). However, the DNA technique consistently resulted in more positive scats than when hard-part analysis was used.3. An experimental study using spiked scat material revealed a highly significant negative relationship between Ct values obtained from the Atlantic salmon qPCR assay and the proportion of Atlantic salmon material added to scats. The Ct value denotes the cycle number at which the increasing fluorescence signal of target DNA crosses a threshold value. Ct values from field-collected seal scats suggested they contained a very low concentration of salmonid remains (1-5%) based on an approximate calibration curve constructed from the experimental data. 4. Synthesis and applications . The qPCR assay approach was shown to be highly efficient and consistent in detection of salmonids from seal scats, and to be more sensitive than conventional hard-parts analysis. Nevertheless, our results confirm previous studies indicating that salmonids are not common prey for seals in these Scottish estuaries. These studies support current management practice, which focuses on control of the small number of seals that move into key salmonid rivers, rather than targeting the larger groups of animals that haul-out in nearby estuaries.