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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.

Piscine orthoreovirus infects various salmonid fish species, and the infection is associated with diseases such as heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). There are no vaccines available or genetically selected resistant hosts that can efficiently control piscine orthoreovirus (PRV) infection. Currently, the only prophylactic measure against PRV is general biosecurity measures aiming to break the transmission cycle. Methods to eradicate infectious virus from contaminated facilities are desirable, but the knowledge on how to inactivate PRV is lacking. A major bottleneck for inactivation studies is the lack of ability to propagate PRV in cell culture. Therefore, in this study we developed an in vivo model for detection of infectious PRV particles after treatment of the virus with inactivation tools such as heat, pH, iodine, UV and commercially available disinfectants. The results show that standard iodine treatment is efficient in inactivation of the virus, and similarly are high and low pH extremes and treatment with Virocid, a commercially available disinfectant. A UV dose of at least 50 mJ/cm2 is required for inactivation, and the virus has high resistance against heat treatment.

Infectious pancreatic necrosis virus (IPNV) is a common pathogen of rainbow trout (Oncorhynchus mykiss) in Turkey. We found that 455 of 1,676 sample pools tested were IPNV positive. Positive samples were found in all geographical regions where sampling was conducted. Sequence and phylogenetic analyses of VP2 from 30 isolates representing all regions showed that the viruses were highly similar in sequence and grouped within Genogroup 5 (serotype Sp-A2). No correlations between sequences, sampling sites or geographical origins were identified. Although clinical disease was evident in several farms, analyses of the amino acid sequence of VP2 showed that all virus strains harboured the P217 T221 motif, assumed to be associated with low virulence. We conclude that IPNV is prevalent in Turkish rainbow trout farms and that the viruses are very homogenous and likely to be of European origin. Frequent exchange of eggs and live fish within the farming industry may explain the homogeneity of the IPNV.

Piscine orthoreovirus (PRV-1) can cause heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). The virus targets erythrocytes in the acute peak phase, followed by cardiomyocytes, before the infection subsides into persistence. The persistent phase is characterized by high level of viral RNA, but low level of viral protein. The origin and nature of persistent PRV-1 are not clear. Here, we analyzed for viral persistence and activity in various tissues and cell types in experimentally infected Atlantic salmon. Plasma contained PRV-1 genomic dsRNA throughout an 18-week long infection trial, indicating that viral particles are continuously produced and released. The highest level of PRV-1 RNA in the persistent phase was found in kidney. The level of PRV-1 ssRNA transcripts in kidney was significantly higher than that of blood cells in the persistent phase. In-situ hybridization assays confirmed that PRV-1 RNA was present in erythroid progenitor cells, erythrocytes, macrophages, melano-macrophages and in some additional un-characterized cells in kidney. These results show that PRV-1 establishes a productive, persistent infection in Atlantic salmon and that erythrocyte progenitor cells are PRV target cells. Erythroid Progenitor Cells in Atlantic Salmon (Salmo salar) May Be Persistently and Productively Infected with Piscine Orthoreovirus (PRV)

Heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar) was first diagnosed in Norway in 1999. The disease is caused by Piscine orthoreovirus-1 (PRV-1). The virus is prevalent in farmed Atlantic salmon, but not always associated with disease. Phylogeny and sequence analyses of 31 PRV-1 genomes collected over a 30-year period from fish with or without HSMI, grouped the viral sequences into two main monophylogenetic clusters, one associated with HSMI and the other with low virulent PRV-1 isolates. A PRV-1 strain from Norway sampled in 1988, a decade before the emergence of HSMI, grouped with the low virulent HSMI cluster. The two distinct monophylogenetic clusters were particularly evident for segments S1 and M2. Only a limited number of amino acids were unique to the association with HSMI, and they all located to S1 and M2 encoded proteins. The observed co-evolution of the S1-M2 pair coincided in time with the emergence of HSMI in Norway, and may have evolved through accumulation of mutations and/or segment reassortment. Sequences of S1-M2 suggest selection of the HSMI associated pair, and that this segment pair has remained almost unchanged in Norwegian salmon aquaculture since 1997. PRV-1 strains from the North American Pacific Coast and Faroe Islands have not undergone this evolution, and are more closely related to the PRV-1 precursor strains not associated with clinical HSMI.

Piscine orthoreovirus (PRV) causes heart- and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). Erythrocytes are the main target cells for PRV. HSMI causes significant economic losses to the salmon aquaculture industry, and there is currently no vaccine available. PRV replicates and assembles within cytoplasmic structures called viral factories, mainly organized by the non-structural viral protein µNS. In two experimental vaccination trials in Atlantic salmon, using DNA vaccines expressing different combinations of PRV proteins, we found that expression of the non-structural proteins µNS combined with the cell attachment protein σ1 was associated with an increasing trend in lymphocyte marker gene expression in spleen, and induced moderate protective effect against HSMI.

Over a 5 day period in October 2007 a boil-water notice was served on the majority of Oslo, capital city of Norway, as a result of a combination of bacteriological findings (coliforms, intestinal enterococci, and E. coli), and very low numbers of Cryptosporidium oocysts and Giardia cysts in 10 L water samples taken from the water distribution network. The water source had been regularly monitored for these parasites and generally found to be negative. Over 460,000 residents were affected by the boil-water notice, as were many thousands of businesses. Despite an extensive outbreak of waterborne giardiasis in Bergen, Norway during 2004/2005, occurrence of parasites in Norwegian drinking water supplies has apparently continued to be considered to be of minimal relevance by Norwegian health authorities. Here we describe the background and occurrence of the episode in Oslo, including the species of Cryptosporidium detected, and use this event, in conjunction with incidents from other countries, as a basis to discuss the following issues: 1) under which circumstances should the occurrence of Cryptosporidium oocysts and Giardia cysts in water supplies trigger the issue of a boil-water notice, and 2) the possibilities and probabilities of post-treatment contamination events in the water distribution network.

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.