Aase B. Mikalsen

Cardiomyopathy Syndrome of Atlantic Salmon (Salmo salar L.) Is Caused by a Double-Stranded RNA Virus of the Totiviridae Family

ABSTRACT 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.

Characterization of the Infectious Salmon Anemia Virus Fusion Protein

ABSTRACT Infectious salmon anemia virus (ISAV) is an orthomyxovirus causing serious disease in Atlantic salmon (Salmo salar L.). This study presents the characterization of the ISAV 50-kDa glycoprotein encoded by segment 5, here termed the viral membrane fusion protein (F). This is the first description of a separate orthomyxovirus F protein, and to our knowledge, the first pH-dependent separate viral F protein described. The ISAV F protein is synthesized as a precursor protein, F0, that is proteolytically cleaved to F1 and F2, which are held together by disulfide bridges. The cleaved protein is in a metastable, fusion-activated state that can be triggered by low pH, high temperature, or a high concentration of urea. Cell-cell fusion can be initiated by treatment with trypsin and low pH of ISAV-infected cells and of transfected cells expressing F, although the coexpression of ISAV HE significantly improves fusion. Fusion is initiated at pH 5.4 to 5.6, and the fusion process is coincident with the trimerization of the F protein, or most likely a stabilization of the trimer, suggesting that it represents the formation of the fusogenic structure. Exposure to trypsin and a low pH prior to infection inactivated the virus, demonstrating the nonreversibility of this conformational change. Sequence analyses identified a potential coiled coil and a fusion peptide. Size estimates of F1 and F2 and the localization of the putative fusion peptide and theoretical trypsin cleavage sites suggest that the proteolytic cleavage site is after residue K276 in the protein sequence.

Characterization of the Infectious Salmon Anemia Virus Genomic Segment That Encodes the Putative Hemagglutinin

ABSTRACT The genomic segment encoding the putative hemagglutinin of infectious salmon anemia virus (ISAV) is described. Expression of the putative hemagglutinin in a salmon cell line demonstrated hemadsorptive properties of the protein for salmon erythrocytes. The polypeptide was recognized by an ISAV-specific monoclonal antibody. Nucleotide sequencing indicated the occurrence of a variable region in the hemagglutinin gene.

In situ localisation of major histocompatibility complex class I and class II and CD8 positive cells in infectious salmon anaemia virus (ISAV)-infected Atlantic salmon.

It is assumed that the mobilisation of a strong cellular immune response is important for the survival of Atlantic salmon infected with infectious salmon anaemia virus (ISAV). In this study, the characterisation of immune cell populations in tissues of non-ISAV infected Atlantic salmon and during the early viraemia of ISAV was undertaken. Immunohistochemical investigations of spleen, head kidney and gills using monoclonal antibodies against recombinant proteins from MHC I, II and CD8 were performed on tissues from Atlantic salmon collected day 17 post-challenge in a cohabitant infection model. The localisations of MHC I and II in control salmon were consistent with previous reports but this study presents novel observations on the distribution of CD8 labelled cell populations in Atlantic salmon including the description of significant mucosal populations in the gills. The distribution of MHC I, MHC II and CD8 positive cell populations differed between control salmon and cohabitant salmon in the early stages of ISAV infection. The changes in MHC I labelled cells differed between organs in ISAV cohabitants but all investigated organs showed a decreased presence of MHC II labelled cells. Together with a clustering of CD8 labelled cells in the head kidney and a reduced presence of CD8 labelled cells in the gills, these observations support the early mobilisation of cellular immunity in the response of Atlantic salmon to ISAV infection. However, differences between the present study and the findings from studies investigating immune gene mRNA expression during ISAV infection suggest that viral strategies to interfere with protein expression and circumvent the host immune response could be operative in the early response to ISAV infection.

Atlantic Salmon Reovirus Infection Causes a CD8 T Cell Myocarditis in Atlantic Salmon (Salmo salar L.)

Heart and skeletal inflammation (HSMI) of farmed Atlantic salmon (Salmo salar L.) is a disease characterized by a chronic myocarditis involving the epicardium and the compact and spongious part of the heart ventricle. Chronic myositis of the red skeletal muscle is also a typical finding of HSMI. Piscine reovirus (PRV) has been detected by real-time PCR from farmed and wild salmon with and without typical changes of HSMI and thus the causal relationship between presence of virus and the disease has not been fully determined [1]. In this study we show that the Atlantic salmon reovirus (ASRV), identical to PRV, can be passaged in GF-1 cells and experimental challenge of naïve Atlantic salmon with cell culture passaged reovirus results in cardiac and skeletal muscle pathology typical of HSMI with onset of pathology from 6 weeks, peaking by 9 weeks post challenge. ASRV replicates in heart tissue and the peak level of virus replication coincides with peak of heart lesions. We further demonstrate mRNA transcript assessment and in situ characterization that challenged fish develop a CD8+ T cell myocarditis.

Detection of Infectious pancreatic necrosis virus in subclinically infected Atlantic salmon by virus isolation in cell culture or real-time reverse transcription polymerase chain reaction: influence of sample preservation and storage

Infectious pancreatic necrosis, an important problem of the salmon industry worldwide, is caused by Infectious pancreatic necrosis virus (IPNV). Fish surviving an IPNV infection become virus carriers, and the identification of infected fish is highly relevant to disease control. The target organ for IPNV diagnosis is the kidney, where the virus persists, usually with low virus loads. The current study documents a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay that proved 100 times more sensitive than a conventional RT-PCR. Cell culture and real-time RT-PCR were compared for their ability to detect IPNV in carrier Atlantic salmon kidney samples after different preservation and storage procedures. Storage of whole tissue at −80°C for 1 month and storage of tissue homogenized in transport medium (TM) at +4°C for 1 week before investigation in cell cultures resulted in a marked reduction of virus infectivity. For detection by real-time RT-PCR, storage of whole tissue was suboptimal, whereas storage of tissue homogenized in TM did not affect virus detection. The results of the present study demonstrate that both cell culture and real-time RT-PCR are reliable tests for the detection of low amounts of IPNV in kidneys of carrier Atlantic salmon, and both methods are relatively robust against minor preservation and storage deviations, or both. Preservation of tissues in RNA stabilization solution seems only necessary when samples are to be shipped at ambient temperatures or when laboratory testing might be delayed. Independent of detection method, these results indicate that for long-term storage, samples are best kept at −80°C after homogenization in TM.

Characterization of a novel calicivirus causing systemic infection in atlantic salmon (Salmo salar L.): proposal for a new genus of caliciviridae.

The Caliciviridae is a family of viruses infecting humans, a wide range of animals, birds and marine fish and mammals, resulting in a wide spectrum of diseases. We describe the identification and genetic characterization of a novel calicivirus replicating in Atlantic salmon. The virus has a high prevalence in farmed salmon and is found in fish suffering from several diseases and conditions and also in presumable healthy fish. A challenge and vaccination trial shows that the calicivirus replicates in Atlantic salmon and establishes a systemic infection, which can be reduced by vaccination with formalin-inactivated virus preparation. The virus, named Atlantic salmon calicivirus (ASCV), is found in two genetically distinct variants, a cell culture isolated and a variant sequenced directly from field material. The genomes are 7,4 kb and contain two open reading frames where typical conserved amino acid motifs and domains predict a gene order reminiscent of calicivirus genomes. Phylogenetic analysis performed on extracted capsid amino acid sequences segregated the two ASCV variants in a unique cluster sharing root with the branch of noroviruses infecting humans and the unassigned Tulane virus and St-Valérien like viruses, infecting rhesus monkey and pig, respectively, with relatively large distance to the marine calicivirus subgroup of vesiviruses. Based on the analyses presented, the ASCV is predicted to represent a new genus of Caliciviridae for which we propose the name Salovirus.

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

Salmon pancreas disease virus (SPDV) also referred to as salmonid alphavirus (SAV) is a virus causing pancreas disease in Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss). Although the virus causes an economically important disease, relatively few full-length genome sequences of SAV strains are currently available. Here, we report full-length genome sequences of nine SAV3 strains from sites farming Atlantic salmon geographically spread along the Norwegian coastline. The virus genomes were sequenced directly from infected heart tissue, to avoid culture selection bias. Sequence analysis confirmed a high level of sequence identity within SAV3 strains, with a mean nucleotide diversity of 0.11 %. Sequence divergence was highest in 6K and E2, while lowest in the capsid protein and the non-structural proteins (nsP4 and nsP2). This study reports for the first time that numerous defective viruses containing genome deletions are generated during natural infection with SAV. Deletions occurred in all virus strains and were not distributed randomly throughout the genome but instead tended to aggregate in certain areas. We suggest imprecise homologous recombination as an explanation for generation of defective viruses with genome deletions. The presence of such viruses, provides a possible explanation for the difficulties in isolating SAV in cell culture. Primary virus isolation was successfully achieved for only two of eight strains, despite extensive attempts using three different cell lines. Both SAV isolates were easily propagated further and concomitant viral deletion mutants present in clinically infected heart tissue were maintained following serial passage in CHH-1 cells.

Expression, antigenicity and studies on cell receptor binding of the hemagglutinin of infectious salmon anemia virus

Summary.Infectious salmon anemia (ISA) virus belongs to the proposed genus Isavirus of Orthomyxoviridae and is an emerging pathogen in Atlantic salmon (Salmo salar) farming. The hemagglutinin-esterase (HE) of ISA virus share several characteristics with the influenza virus hemagglutinin. This study reports recombinant expression of different ISA virus HE mutants in fish cell lines. Some introduced mutations, representing minimal differences in single amino acid residues, resulted in remarkable effects on expression efficiency in general and on surface expression specifically. Receptor binding assays showed that amino acid residues in the N-terminal half part are important in receptor binding, either being directly involved in the binding, or by influencing the structure of the binding site. Deletion of the putative N-glycosylation sites of the ISA virus HE, located near the transmembrane region, did not influence expression, receptor binding properties or staining by either a neutralising MAb, or salmon convalescent sera. The humoral immune response of Atlantic salmon after ISA virus infection showed weak neutralising activity and the results indicated that it was directed against HE.

Evaluation of histopathology, real-time PCR and virus isolation for diagnosis of infectious salmon anaemia in Norwegian salmon using latent class analysis.

Infectious salmon anaemia (ISA) is a disease of saltwater farmed Atlantic salmon, Salmo salar L., caused by the orthomyxovirus, ISA virus (Mjaaland, Rimstad, Falk & Dannevig 1997), that in recent years has caused considerable economic losses to the Atlantic salmon farming industry worldwide. There is no cure for ISA, hence most efforts are directed towards monitoring and control programmes. For such programmes to be effective, quick, reliable tests are needed. Virus isolation (VI) (Dannevig, Falk & Namork 1995), reverse transcriptase-polymerase chain reaction (RT-PCR) (Mjaaland et al. 1997) and indirect fluorescent antibody testing (IFAT) (Falk & Dannevig 1995; Falk, Namork & Dannevig 1998) as well as histopathology (HP) (Evensen et al. 1991; Speilberg, Evensen & Dannevig 1995; Thorud & Djupvik 1988; Byrne, MacPhee, Ostland, Johnson & Ferguson 1998) have been commonly used in the diagnosis of ISA. Recently, real time RT-PCR (RRT-PCR) has been introduced as a more rapid, sensitive and probably relatively more specific test (Mackay, Arden & Nitsche 2002; Munir & Kibenge 2004; OIE 2009). Validation and evaluation studies of commonly used ISA diagnostic tests including VI, RT-PCR and IFAT, as well as HP, have been carried out with complementary results (Nerette, Dohoo & Hammel 2005; Snow, McKay, McBeath, Black, Doig, Kerr, Cunningham, Nylund & Devold 2006; Raynard et al. 2003). So far, however, there is no information available regarding the comparative performance of the RRT-PCR with respect to the commonly used tests in the detection of ISA virus from naturally infected Atlantic salmon. RRT-PCR is a test with higher analytic sensitivity than RTPCR (for ISAV, a sensitivity of 1000 times higher than RT-PCR; Plarre, Devold, Snow & Nylund 2005) and with a low detection limit, warranting investigation regarding its agreement with the other tests that detect the presence of replicating virus or an obvious pathology inflicted by the virus. Thus, the objective of this study was to evaluate and compare the diagnostic performance of RRTPCR, VI and HP in a population of naturally infected salt-water farmed Atlantic salmon. As none of these tests could be considered as a perfect reference test, a latent class analysis allowing the estimation of test characteristics in the absence of a gold standard was applied. The study was carried out in two populations of Atlantic salmon in a fish farm located in northern Norway (random samples from two cages, Journal of Fish Diseases 2010, 33, 529–532 doi:10.1111/j.1365-2761.2010.01139.x