Emergence of Salmonid Alphavirus Genotype 2 in Norway-Molecular Characterization of Viral Strains Circulating in Norway and Scotland.

Pancreas disease (PD) and sleeping disease (SD), caused by an alphavirus, are endemic in European salmonid aquaculture, causing significant mortality, reduced growth and poor flesh quality. In 2010, a new variant of salmonid alphavirus emerged in Norway, marine salmonid alphavirus genotype 2 (SAV2). As this genotype is highly prevalent in Scotland, transmission through well boat traffic was hypothesized as one possible source of infection. In this study, we performed full-length genome sequencing of SAV2 sampled between 2006 and 2012 in Norway and Scotland, and present the first comprehensive full-length characterization of Norwegian marine SAV2 strains. We analyze their relationship with selected Scottish SAV2 strains and explore the genetic diversity of SAV. Our results show that all Norwegian marine SAV2 share a recent last common ancestor with marine SAV2 circulating in Scotland and a higher level of genomic diversity among the Scottish marine SAV2 strains compared to strains from Norway. These findings support the hypothesis of a single introduction of SAV2 to Norway sometime from 2006-2010, followed by horizontal spread along the coast.

Ultra-deep pyrosequencing of partial surface protein genes from infectious Salmon Anaemia virus (ISAV) suggest novel mechanisms involved in transition to virulence.

Uncultivable HPR0 strains of infectious salmon anaemia viruses (ISAVs) infecting gills are non-virulent putative precursors of virulent ISAVs (vISAVs) causing systemic disease in farmed Atlantic salmon (Salmo salar). The transition to virulence involves two molecular events, a deletion in the highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) gene and a Q266→L266 substitution or insertion next to the putative cleavage site (R267) in the fusion protein (F). We have performed ultra-deep pyrosequencing (UDPS) of these gene regions from healthy fish positive for HPR0 virus carrying full-length HPR sampled in a screening program, and a vISAV strain from an ISA outbreak at the same farming site three weeks later, and compared the mutant spectra. As the UDPS data shows the presence of both HE genotypes at both sampling times, and the outbreak strain was unlikely to be directly related to the HPR0 strain, this is the first report of a double infection with HPR0s and vISAVs. For F amplicon reads, mutation frequencies generating L266 codons in screening samples and Q266 codons in outbreak samples were not higher than at any random site. We suggest quasispecies heterogeneity as well as RNA structural properties are linked to transition to virulence. More specifically, a mechanism where selected single point mutations in the full-length HPR alter the RNA structure facilitating single- or sequential deletions in this region is proposed. The data provides stronger support for the deletion hypothesis, as opposed to recombination, as the responsible mechanism for generating the sequence deletions in HE.

Molecular and epidemiological characterization of avian influenza viruses from gulls and dabbling ducks in Norway.

BACKGROUND: Wild aquatic birds constitute the natural reservoir for avian influenza viruses (AIVs). Separate Eurasian and American AIV gene pools exist. Here, the prevalence and diversity of AIVs in gulls and dabbling ducks in Norway were described. The influence of host species and temporal changes on AIV prevalence was examined. Five AIVs from Norway, including three from common gull (Larus canus), were analyzed along with 10 available AIV genomes from gulls in Eurasia to search for evidence of intracontinental and intercontinental reassortment of gene segments encoding the internal viral proteins. METHODS: Swabs collected from 2417 dabbling ducks and gulls in the south-west of Norway during five ordinary hunting seasons (August-December) in the period 2005-2010 were analyzed for presence of AIV. Multivariate linear regression was used to identify associations between AIV prevalence, host species and sampling time. Five AIVs from mallard (Anas platyrhynchos) (H3N8, H9N2) and common gull (H6N8, H13N2, H16N3) were full-length characterized and phylogenetically analyzed together with GenBank reference sequences. RESULTS: Low pathogenic AIVs were detected in 15.5% (CI: 14.1-17.0) of the samples. The overall AIV prevalence was lower in December compared to that found in August to November (p = 0.003). AIV was detected in 18.7% (CI: 16.8-20.6) of the dabbling ducks. A high AIV prevalence of 7.8% (CI; 5.9-10.0) was found in gulls. A similar temporal pattern in AIV prevalence was found in both bird groups. Thirteen hemagglutinin and eight neuraminidase subtypes were detected. No evidence of intercontinental reassortment was found. Eurasian avian (non H13 and H16) PB2 or PA genes were identified in five reference Eurasian gull (H13 and H16) AIV genomes from GenBank. The NA gene from the Norwegian H13N2 gull isolate was of Eurasian avian origin. CONCLUSIONS: The similar temporal pattern in AIV prevalence found in dabbling ducks and gulls, the relatively high virus prevalence detected in gulls and the evidence of intracontinental reassortment in AIVs from gulls indicate that gulls that interact with dabbling ducks are likely to be mixing vessels for AIVs from waterfowl and gulls. Our results support that intercontinental reassortment is rare in AIVs from gulls in Eurasia.

Low virulent infectious salmon anaemia virus (ISAV-HPR0) is prevalent and geographically structured in Norwegian salmon farming.

Infectious salmon anaemia (ISA) is a severe disease in farmed Atlantic salmon Salmo salar that has caused epidemic outbreaks in most salmon-producing countries worldwide. The disease is caused by virulent ISA virus (ISAV). Low virulent variants of the virus, characterised by a full-length sequence in the highly polymorphic region of segment 6 in the virus genome, have been reported with increasing frequencies. These variants of the virus, termed HPR0, have been proposed to be ancestors of virulent ISAV. We examined this idea through studies of the phylogeographic and environmental distribution of ISAV-HPR0, as well as phylogeographic associations between virulent ISAV and ISAV-HPR0. Samples from 232 fish groups were screened for ISAV. Real-time RT-PCR was used for detection of ISAV, and the ISAV haemagglutinin esterase (HE) gene was characterised for positive samples. A Mantel test was used to test phylogeographic associations between pairs of ISAV-HPR0 HE gene sequences. A rank test was used to test associations between HE gene sequences from virulent ISAV and ISAV-HPR0. ISAV-HPR0 was detected in fish groups both in freshwater and marine environments, and in juveniles, on-grown marine salmon and broodstock salmon. Genetic and geographic distances between pairs of ISAV-HPR0 HE gene sequences were positively correlated, suggesting that the population of ISAV-HPR0 is geographically structured. Finally, we found a spatial association between fish groups with virulent ISAV (n = 21) and fish groups with ISAV-HPR0 (n = 27), supporting the hypothesis that ISAV-HPR0 may undergo a transition to virulent ISAV.