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Infectious pancreatic necrosis viruses (IPNVs) belonging to the family Birnaviridae display a high degree of antigenic variability, pathogenicity, and differences in outbreak mortality in salmonid species. To determine if virus isolates of Sp serotype differ in virulence, fry of Atlantic salmon (Salmo salar L.) were challenged with nine different field strains. These viruses caused either high mortality and severe pathological changes or low mortality and no lesions. To study the molecular basis for the variation in virulence of IPNV, complete nucleotide sequences of segment A of all these strains as well as segment B of three selected strains were determined. All viruses tested had a unique genome sequence. Only minor differences were noted in the genes encoding VP1, VP3, and VP4 proteins, whereas most changes were observed in the gene encoding the VP2 protein. A high level of variation was found in the small open reading frame (ORF), which encodes a 15-kDa nonstructural (NS) polypeptide also known as VP5. One of the strains lacked the initiation codon for this protein, whereas the other four could encode a truncated version of the NS protein. Additional data obtained by sequencing of the NS and VP2 genes directly from diseased fish demonstrated changes in the VP2 gene after two passages in cell culture, which could possibly be associated with attenuation. Comparison of the deduced amino acid sequences of the NS and VP2 genes reveals that the virulent strains possess a 12-kDa coding NS gene and have residues Thr, Ala, Thr/Ala, and Tyr/His at positions 217, 221, 247, and 500 of the VP2 gene, respectively-the motifs identified in this study to be involved in the virulence of IPNV.

Cardiomyopathy syndrome (CMS) of farmed and wild Atlantic salmon (Salmo salar L.) is a disease of yet unknown etiology characterized by a necrotizing myocarditis involving the atrium and the spongious part of the heart ventricle. Here, we report the identification of a double-stranded RNA virus likely belonging to the family Totiviridae as the causative agent of the disease. The proposed name of the virus is piscine myocarditis virus (PMCV). On the basis of the RNA-dependent RNA polymerase (RdRp) sequence, PMCV grouped with Giardia lamblia virus and infectious myonecrosis virus of penaeid shrimp. The genome size of PMCV is 6,688 bp, with three open reading frames (ORFs). ORF1 likely encodes the major capsid protein, while ORF2 encodes the RdRp, possibly expressed as a fusion protein with the ORF1 product. ORF3 seems to be translated as a separate protein not described for any previous members of the family Totiviridae. Following experimental challenge with cell culture-grown virus, histopathological changes are observed in heart tissue by 6 weeks postchallenge (p.c.), with peak severity by 9 weeks p.c. Viral genome levels detected by real-time reverse transcription (RT)-PCR peak earlier at 6 to 7 weeks p.c. The virus genome is detected by in situ hybridization in degenerate cardiomyocytes from clinical cases of CMS. Virus genome levels in the hearts from clinical field cases correlate well with the severity of histopathological changes in heart tissue. The identification of the causative agent for CMS is important for improved disease surveillance and disease control and will serve as a basis for vaccine development against the disease.

Infectious pancreatic necrosis viruses (IPNVs) exhibit a wide range of virulence in salmonid species. In previous studies, we have shown that the amino acid residues at positions 217 and 221 in VP2 are implicated in virulence. To pinpoint the molecular determinants of virulence in IPNV, we generated recombinant IPNV strains using the cRNA-based reverse-genetics system. In two virulent strains, residues at positions 217 and 247 were replaced by the corresponding amino acids of a low-virulence strain. The growth characteristics of the recovered chimeric strains in cell culture were similar to the low-virulence strains, and these viruses induced significantly lower mortality in Atlantic salmon fry than the parent strains did in in vivo challenge studies. Furthermore, the virulent strain was serially passaged in CHSE-214 cells 10 times and was completely characterized by nucleotide sequencing. Deduced amino acid sequence analyses revealed a single amino acid substitution of Ala to Thr at position 221 in VP2 of this virus, which became highly attenuated and induced 15% cumulative mortality in Atlantic salmon fry, compared to 68% mortality induced by the virulent parent strain. The attenuated strain grows to higher titers in CHSE cells and can be distinguished antigenically from the wild-type virus by use of a monoclonal antibody. However, the virulent strain passaged 10 times in RTG-2 cells was stable, and it retained its antigenicity and virulence. Our results indicate that residues Thr at position 217 (Thr217) and Ala221 of VP2 are the major determinants of virulence in IPNV of the Sp serotype. Highly virulent isolates possess residues Thr217 and Ala221; moderate-to low-virulence strains have Pro217 and Ala221; and strains containing Thr221 are almost avirulent, irrespective of the residue at position 217.

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ABSTRACT. The present study describes pathological changes and bacteriological findings in 'winter ulcer' in Atlantic salmon Salmo salar L. The transmissibility of the disease was also evaluated under experimental conditions. Skin changes were characterized by ulcers of varying size, and were categorized as acute to subacute, chronic, and regenerative/reparative. In the acute stages, lesions were superficial with scale loss and mild inflammation, while in the subacute stages ulcers were present that extended down to the underlying n~uscle. Histologically, the chronic stages were characterized by a severe inflammation of the dermis and of the interstitial muscle tissue. In the regeneration/reparation stages, a hyperplastic epidermis covered granulation tissue. Bacteriological investigations carried out in salmon sampled from 8 different farms with winter ulcer identified 2 groups of bacteria that were common in affected fish. The examinations performed in the present study indicate that both these groups belonged to the genus Vibrio, termed sp. 1 and sp. 2, respectively. Immunohistochemically, Vibrio sp. 1 and 2 were identified in situ associated with muscle tissue degeneration. Experimental infection with Vibno sp. 1 induced a disease similar to winter ulcer in Atlantic salmon, while inoculation with Vibrio sp. 2 had no effect. Cohabitation experiments showed that winter ulcer can be transmitted from diseased to healthy individuals, and that injection was not required to induce the process. However, mechanical skin lesions were a predisposing factor for ulcer formation. The present study provides evidence that winter ulcer is caused by an infection with a Vibrio-like bacterium, and that the disease can be transmitted through cohabitation and injection Pathological changes were not pathognomonic, and the severity of changes varied.

Lysozyrne activity was studied in vanous species of fish (13 mild species and cultured rainbow trout Salmo gairdnen and Atlantic salmon Salmo salar). Marked interspecies variation prevailed, most strikingly between rainbow trout and Atlantic salmon; lysozyme activity in the former was at least 20 times greater than in the latter. Tissue distribution was studied in more depth in rainbow trout. Kidneys appeared to have the highest lysozyme levels, followed in descending order by alimentary tract, spleen, slan mucus, serum, gills, liver and muscle. This pattern is compatible with the assumption that lysozymes play an important role in body defence. Using gel filtration chromatography, 2 lysozyrne-like variants with molecular weights (MW) of ca 14 500 (high activity) and 23 000 daltons (low activity) were found in rainbow trout kidn.ey. Only low MW activities (between 13 000 and 12 000 daltons) were detected in Atlanlc salmon. Relevant future research approaches, as well as possible or speculative applications of lysozymes in disease control, are discussed.