Genetic grouping and geographic distribution of Piscine orthoreovirus-1 (PRV-1) in farmed Atlantic salmon in Norway.

Piscine orthoreovirus-1 (PRV-1) is the causative agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). However, it has been shown that PRV-1 variants differ in their ability to induce HSMI. The objective of this work was to identify the PRV-1 variants in Norwegian aquaculture and their geographical distribution. Sequencing and subsequent analysis of the five genomic segments (S1, S4, M2, L1 and L2) putatively linked to virulence, made out the basis of the study. Thirty-seven Norwegian PRV-1 isolates were sequenced, and they grouped into eight genogroups based on combinations of the five analyzed genomic segments. Two groups were defined as high-virulent and two low-virulent, based on comparison with PRV-1 reference isolates with known virulence. The remaining four groups were of unknown virulence. The geographic distribution indicated a higher frequency of the high-virulent isolates in the mid- and northern regions. The present study confirms circulation of both high- and low-virulent isolates of PRV-1 in farmed Atlantic salmon in Norway. To reduce the impact of PRV-1 related disease, detection and differentiation between high- and low-virulent genogroups of PRV-1 could be a targeted approach for reduction of high-virulent variants.

Piscine Orthoreovirus-1 Isolates Differ in Their Ability to Induce Heart and Skeletal Muscle Inflammation in Atlantic Salmon (Salmo salar).

Piscine orthoreovirus 1 (PRV-1) is the causative agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). The virus is widespread in Atlantic salmon and was present in Norway long before the first description of HSMI in 1999. Furthermore, in Canada the virus is prevalent in farmed Atlantic salmon but HSMI is not and Canadian isolates have failed to reproduce HSMI experimentally. This has led to the hypothesis that there are virulence differences between PRV-1 isolates. In this study we performed a dose standardized challenge trial, comparing six PRV-1 isolates, including two Norwegian field isolates from 2018, three historical Norwegian isolates predating the first report of HSMI and one Canadian isolate. The Norwegian 2018 isolates induced lower viral protein load in blood cells but higher plasma viremia. Following peak replication in blood, the two Norwegian 2018 isolates induced histopathological lesions in the heart consistent with HSMI, whereas all three historical Norwegian and the Canadian isolates induced only mild cardiac lesions. This is the first demonstration of virulence differences between PRV-1 isolates and the phenotypic differences are linked to viral proteins encoded by segment S1, M2, L1, L2 and S4.

Molecular tracing confirms that infection with infectious pancreatic necrosis virus follows the smolt from hatchery to grow-out farm.

Infectious pancreatic necrosis (IPN) is an important restraint to production of salmonids in aquaculture globally. In order to implement efficacious mitigation strategies for control of this disease, it is important to understand infection routes under current production systems. IPN virus has been shown to be transmitted vertically in Rainbow trout, from broodstock to fingerlings in hatcheries, and there is circumstantial evidence suggesting that vertical transmission can also occur in Atlantic salmon, in addition to horizontal transmission between grow-out fish in farms. In this study, we show that the smolt carries infection with IPN from hatchery to the marine farm. We do this by comparing sequences from fish groups taken both in hatcheries and on corresponding marine grow-out farms. We use statistical analysis to prove that sequences obtained from the same fish group in both hatchery and marine farm are more similar than sequences obtained from random fish groups on hatcheries and marine farms.

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.

Viral encephalopathy and retinopathy (VER) in Atlantic salmon Salmo salar after intraperitoneal challenge with a nodavirus from Atlantic halibut Hippoglossus hippoglossus.

Homogenate of tissue from juveniles of Atlantic halibut Hippoglossus hippoglossus suffering from viral encephalopathy and retinopathy (VER) was used to challenge smolt of Atlantic salmon Salmo salar with an initial average weight of 110 g. The nodavirus was administered in the form of an intraperitoneal injection, and the fish were kept for 134 d post challenge. Genotype characterisation of the nodavirus was performed by sequencing the RNA1 and RNA2 segments, and a quantitative real-time PCR (Q-PCR) assay was developed. Tissues from different organs were stained by immunohistochemistry (IHC). Samples were collected at random on Days 7, 25, 45, 69, 125 and 134 after challenge. Mortality, clinical signs and pathology of VER were observed only in the challenged group. The Q-PCR detected positive fish only in the challenged group, all of which were positive on all days of sampling. An increase in relative virus concentrations was observed from Day 7 to Day 25 post challenge. The increased level of virus concentration was maintained in the medulla oblongata throughout the experiment, suggesting persistence or slow elimination of the virus over time. The IHC detected positive cells on Days 34, 70 and 74. These results suggest that the nodavirus is transported to the medulla oblongata from the intraperitoneal injection site and is able to replicate in salmon. When injected, this nodavirus isolate caused mortality and established a persistent infection in the challenged salmon throughout the experiment. This susceptibility suggests that co-location of salmon and marine species should be avoided until further studies of possible transmission have been carried out.

Cloning and identification of the infectious salmon anaemia virus haemagglutinin.

Infectious salmon anaemia virus (ISAV) is an orthomyxo-like virus that causes serious disease in Atlantic salmon (Salmo salar). Like the orthomyxoviruses, ISAV has been shown to possess haemagglutinin (HA) activity. This study presents the cloning, expression and identification of the ISAV HA gene, which was isolated from a cDNA library by immunoscreening. The HA gene contained an ISAV-specific conserved nucleotide motif in the 5' region and a 1167 bp open reading frame encoding a protein with a predicted molecular mass of 42.4 kDa. The HA gene was expressed in a baculovirus system. A monoclonal antibody (MAb) shown previously to be directed against the ISAV HA reacted with insect cells infected with recombinant baculovirus. Salmon erythrocytes also adsorbed to these cells and adsorption was inhibited by the addition of either the ISAV-specific MAb or a polyclonal rabbit serum prepared against purified virus, confirming the virus specificity of the reaction. Immunoblot analyses indicated that ISAV HA, in contrast to influenza virus HA, is not posttranslationally cleaved. Sequence comparisons of the HA gene from five Norwegian, one Scottish and one Canadian isolate revealed a highly polymorphic region that may be useful in epidemiological studies.