Genetic stability within the Norwegian subtype of salmonid alphavirus (family Togaviridae)

Abstract

Salmonid alphavirus (SAV) (family Togaviridae) causes mortality in Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss W.) in Norway, France, UK, and Ireland. At least three subtypes of SAV exist: SPDV in UK/Ireland, SDV in France/UK, and the recently reported Norwegian salmonid alphavirus (NSAV) in western Norway. During 2003 and 2004, disease caused by NSAV was reported for the first time in northern Norway, more than 800 km away from the enzootic area in western Norway. The present study has investigated the phylogenetic relationships among 20 NSAV isolates, based on a 1221-nt-long segment covering part of the capsid gene, E3, and part of the E2 gene, collected over a period of eight years. The results revealed genetic homogeneity among NSAV isolates, including those from northern Norway. The SDV or SPDV subtypes were not found in diseased Norwegian fish. A substitution rate of 1.70 (+/-1.03) x 10(-4) nt subst/site/year was obtained for the NSAV subtype by maximum likelihood analysis. The second aim of this study was to clarify whether NSAV changes genotypically in cell culture by culturing a NSAV isolate through 20 passages in CHSE-214 cells. Sequencing of almost the entire genome (11530 nt) after 20 passages revealed four nucleotide substitutions, all resulting in amino acid substitutions. One of these substitutions, serine to proline in E2 position 206, was also found to have occurred in field isolates.

Identification of a wild reservoir of salmonid alphavirus in common dab Limanda limanda, with emphasis on virus culture and sequencing

“…Salmon lice have been suggested as a potential vector for SAV. Although SAV may be detected in salmon lice by PCR (Karlsen et al 2006, Petterson et al 2009), viral replication in lice and the consequent transfer of the virus to a new host has not been proven. The discovery of viral RNA in areas remote from fish farming would suggest that common dab are natural carriers of the virus and that the viral RNA is not the result of fish farming activity.…”

Natural infection of Atlantic salmon (Salmo salar L.) with salmonid alphavirus 3 generates numerous viral deletion mutants

“…no). The virus genomes were sequenced directly from heart tissues, as it is well known that during passage in cell culture, adaptation can occur and genetic changes can be introduced into the virus genome (Karlsen et al, 2006). Due to technical difficulties with direct sequencing, all PCR products were cloned prior to sequencing.…”

“…It has been speculated that only minor populations of viral quasispecies present in clinical SAV samples have possibilities of growth in cell cultures (Weston et al, 2002), and a serine to proline substitution at position 206 in the E2 protein has been associated with the appearance of CPE in CHSE-214 cells (Karlsen et al, 2006). It was also suggested to affect in vivo virulence although this has not been documented experimentally.…”

“…As primary virus isolation from field outbreaks has been difficult and yielded relatively few numbers of isolates (McLoughlin & Graham, 2007), the understanding has been that there is need for some degree of selection or adaptation before virus can be isolated in cell culture (Castric et al, 1997;Nelson et al, 1995). A previous study has shown that mutations occur following serial passage in CHSE-214 cells, and four amino acid substitutions were identified in the genome between passage 3 and 20 of one defined isolate of SAV3 (Karlsen et al, 2006). This paper describes sequence analysis of full-length viral genomes of SAV3 from nine sites geographically spread along the Norwegian coastline.…”

“…However, following the first detection of a SAV2-related virus in 2011, this subtype is now frequently found in PD outbreaks in mid-Norway (Hjortaas et al, 2013). The common understanding has been that there is little genetic variation within subtypes (Fringuelli et al, 2008;Karlsen et al, 2006;Weston et al, 2005), and previously published sequence data from the Norwegian strains show only minor amino acid differences between the strains analysed (Jansen et al, 2010;Karlsen et al, 2006). However, very few full-length SAV sequences have been published, and the SAV3 fulllength sequences previously reported come from the same geographical area, and might therefore not be representative of the entire Norwegian coast.…”