Marius Karlsen

Transmission of infectious salmon anaemia virus (ISAV) in farmed populations of Atlantic salmon (Salmo salar)

Summary.In the present study, 24 smolt production sites were screened for the presence of infectious salmon anaemia virus (ISAV) with the help of a specific real-time RT PCR assay, and 22 of these sites had smolts that were positive. If these smolt production sites are representative for the prevalence of ISAV in Norwegian smolts, then most marine production sites must be considered to be positive for ISAV. In addition, 92 European ISAV isolates have been genotyped based on the hemagglutinin-esterase gene (HE), and their distribution pattern was analysed. This pattern has been coupled to information about the origin of smolt, eggs, and broodfish in those cases where it has been possible to obtain such information, and with information about ISAV in neighbouring farms. The pattern suggests that an important transmission route for the ISAV could be that the salmon farming industry in Norway is circulating some of the isolates in the production cycle, i.e. some sort of vertical or transgenerational transmission may occur. It has also been shown that avirluent ISAV isolates are fairly common in Norwegian farmed salmon. Based on this, it is hypothesized that the change from avirulent to virulent ISAV isolates is a stochastic event that is dependent on the replication frequency of the virus and the time available for changes in a highly polymorphic region (HPR) of the HE gene to occur. This, and the possibility that only avirluent ISAV isolates are vertically transmitted, may explain why ISA most often occurs at marine sites and why no more than about 15 farms get ISA every year in Norway.

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

Summary.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) × 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.

New clade of betanodaviruses detected in wild and farmed cod ( Gadus morhua ) in Norway

Betanodaviruses have been isolated and detected in both farmed and wild fish species worldwide. They are classified in five clusters, and all are connected to mortalities in farmed fish. The clusters do not represent specific geographical areas or host species, but one cluster, barfin flounder nervous necrosis virus (BFNNV), is mainly associated with cold water fish species. This study presents the first species-specific clade within the BFNNV cluster. This clade consists of six isolates from wild and farmed Atlantic cod in Norway and is genetically distinct from other betanodaviruses in the North Atlantic. Screening of farmed and wild cod in Norway shows that betanodaviruses are present in wild fish on the west coast of Norway, including migratory cod, but so far we have not detected any betanodavirus-positive wild cod in northern Norway. The presence of significant amounts of betanodaviruses in wild cod represents a serious challenge for the management of viral nervous necrosis in farmed cod in Norway. Betanodavirus-positive farmed cod were present both in western and northern Norway. Mortalities in three cod farms were suspected to be caused by betanodaviruses; however, in two of these, other pathogens may have been responsible for or strongly contributed to the mortalities.

Evolution of infectious salmon anaemia virus (ISA virus)

Infectious salmon anaemia virus, ISA virus (genus Isavirus, family Orthomyxoviridae), emerged in Norwegian salmon culture in the mid-80s. The genome consists of eight segments coding for at least 10 proteins. ISA viruses show many of similarities to influenza A viruses but differ in many important aspects such as the number of hosts, the host population structure and the route of transmission. The only known hosts and reservoirs for ISA viruses are salmonids found in countries surrounding the North Atlantic. In this study, four different segments of the genome of about 100 ISA viruses have been sequenced in an attempt to understand the evolution of ISA viruses and how these viruses are maintained in and transmitted between populations of farmed Atlantic salmon. The four gene segments code for the nucleoprotein (NP), the putative acid polymerase (PA), the fusion protein (F) and the haemagglutinin-esterase (HE). Analysis of these four genes showed that the substitution rates of the internal proteins (NP and PA) are lower than those of the two surface proteins (F and HE). All four segments are evolving at a lower rate than similar genes in influenza A viruses. The ISA virus populations consist of avirulent viruses and pathogenic strains with variable virulence in Atlantic salmon. Recombination resulting in inserts close to the proteolytic-cleavage site of the precursor F0 protein and deletions in the stalk region of the HE protein seem to be responsible for the transition from avirulent ISA viruses to pathogenic strains. It is also shown that reassortment is a frequent event among the dominating ISA viruses in farmed Atlantic salmon. The pattern that is obtained after phylogenetic analysis of the four gene segments from ISA viruses suggests that the variation is limited to a few distinct clades and that no major changes have occurred in the ISA virus population in Norway since the first viruses were isolated. Calculation of the time of most recent common ancestor (TMRCA) suggests that the Norwegian ISA viruses separated from the European subtype found in North America between 1932 and 1959. The TMRCA data also suggest that the ISA viruses in Chile were transmitted from Norway in the period from 1995 to 2007, depending on which of the four genes were used in the analysis.

The amino terminus of the salmonid alphavirus capsid protein determines subcellular localization and inhibits cellular proliferation

Salmonid alphavirus (SAV) is the most divergent member of the family Togaviridae and constitutes a threat to farming of salmonid fish in Europe. Here, we report cloning, expression and preliminary functional analysis of the capsid protein of SAV, confirming it to be expressed as an approximately 31-kDa protein in infected cells. The protein localizes strictly to the cytoplasm in Chinook salmon embryo cells, and either to the nucleus or cytoplasm in bluegill fry cells. An expression study of full-length and different truncated versions of the SAV capsid fused to the enhanced green fluorescent protein demonstrated that the localization is independent of other viral components in both cell lines, and controlled by the N-terminal 82 aa, which include a conserved, predicted helix and a downstream positively charged region. Thus, the results suggest that the SAV capsid possesses a cell-type-dependent potential for nuclear import and export. Moreover, the SAV capsid and its N-terminal 82 aa were shown to be associated with inhibition of cellular proliferation, a hallmark of the cytopathic effect caused by SAV. These results highlight that the SAV capsid is a multifunctional protein with possible importance for pathogenesis.

Development of infectious cDNA clones of Salmonid alphavirus subtype 3

BackgroundSalmonid alphavirus (SAV) is a widespread pathogen in European aquaculture of salmonid fish. Distinct viral subtypes have been suggested based on sequence comparisons and some of these have different geographical distributions. In Norway, only SAV subtype 3 have so far been identified. Little is known about viral mechanisms important for pathogenesis and transmission. Tools for detailed exploration of SAV genomes are therefore needed.ResultsInfectious cDNA clones in which a genome of subtype 3 SAV is under the control of a CMV promoter were constructed. The clones were designed to express proteins that are putatively identical to those previously reported for the SAVH20/03 strain. A polyclonal antiserum was raised against a part of the E2 glycoprotein in order to detect expression of the subgenomic open reading frame (ORF) encoding structural viral proteins. Transfection of the cDNA clone revealed the expression of the E2 protein by IFAT, and in serial passages of the supernatant the presence of infectious recombinant virus was confirmed through RT-PCR, IFAT and the development of a cytopathic effect similar to that seen during infection with wild type SAV. Confirmation that the recovered virus originated from the infectious plasmid was done by sequence identification of an introduced genetic tag. The recombinant virus was infectious also when an additional ORF encoding an EGFP reporter gene under the control of a second subgenomic alphavirus promoter was added. Finally, we used the system to study the effect of selected point mutations on infectivity in Chinook salmon embryo cells. While introduced mutations in nsP2197, nsP3263 and nsP3323 severely reduced infectivity, a serine to proline mutation in E2206 appeared to enhance the virus titer production.ConclusionWe have constructed infectious clones for SAV based on a subtype 3 genome. The clones may serve as a platform for further functional studies.

A naturally occurring substitution in the E2 protein of Salmonid alphavirus subtype 3 changes viral fitness.

Phylogenetic analyses of the Salmonid alphavirus subtype 3 (SAV3) epizootic have suggested that a substitution from proline to serine in the receptor binding protein E2 position 206 has occurred after the introduction of virus from a wild reservoir to farmed salmonid fish in Norway. We modelled the 3D structure of P62, the uncleaved E3-E2 precursor, of SAVH20/03 based on its sequence homology to the Chikungunya virus (CHIKV), and studied in vitro and in vivo effects of the mutation using reverse genetics. E2(206) is located on the surface of the B-domain of E2, which is associated with receptor attachment in alphaviruses. Recombinant virus expressing the E2(206S) codon replicated slower and produced significantly less genomic copies than virus expressing the ancestral E2(206P) codon in vitro in Bluegill Fry (BF2) cells. The E2(206S) mutant was out-competed by the E2(206P) mutant after 5 passages in an in vitro competition assay, confirming that the substitution negatively affects the efficacy of virus multiplication in cell culture. Both mutants were highly infectious to Atlantic salmon (Salmo salar), produced similar viral RNA loads in gills, heart, kidney and brain, and induced similar histopathologic changes in these organs. The E2(206S) mutant produced a less persistent infection in salmon and was shed more rapidly to water than the E2(206P) mutant. Reduced generation time through more rapid shedding could therefore explain why a serine in this position became dominant in the viral population after SAV3 was introduced to farmed salmon from the wild reservoir.