Comparative Molecular Characterization of Novel and Known Piscine Toti-Like Viruses.

Totiviridae is a virus family well known to infect uni-cellular organisms like fungi and protozoa. In more recent years, viruses characterized as toti-like viruses, have been found in primarily arthropods, but also a couple in planarians and piscine species. These toti-like viruses share phylogenetic similarities to totiviruses; however, their genomes also includes additional coding sequences in either 5' or 3' ends expected to relate to more advanced infection mechanisms in more advanced hosts. Here, we applied next generation sequencing (NGS) technologies and discovered three new toti-like viruses, one in wild common carp and one in bluegill from the USA and one in farmed lumpsucker from Norway. These are named common carp toti-like virus 1 (CCTLV-1), bluegill toti-like virus 1 (BGTLV-1), and Cyclopterus lumpus toti-like virus (CLuTLV), respectively. The genomes of these viruses have been characterized and compared to the three previously known piscine toti-like viruses, piscine myocarditis virus (PMCV) found in Atlantic salmon and the two from golden shiner, now named golden shiner toti-like virus 1 and 2 (GSTLV-1 and -2), and also to totiviruses and other toti-like viruses. We found that four piscine toti-like viruses had additional gene(s) in the 3' end of the genome, and also clustered phylogenetically based on both capsid and RdRp-genes. This cluster constituted a distant branch in the Totiviridae, and we suggest this should be defined as a separate genus named Pistolvirus, to reflect this major cluster of piscine toti-like viruses. The remaining two piscine toti-like viruses differentiated from these by lacking any additional 3' end genes and also by phylogenetical relation, but were both clustering with arthropod viruses in two different clusters.

Pasteurella spp. Infections in Atlantic salmon and lumpsucker.

The use of cleaner fish as a delousing method in Norwegian salmonid aquaculture has increased tremendously over the last few years. This has led to the emergence of a new large industry of farming lumpsuckers (Cyclopterus lumpus L.). The use of lumpsuckers as cleaner fish has, however, not been problem-free. Bacterial diseases cause high mortalities with pasteurellosis as one of the major emerging diseases. During the past few years, outbreaks of pasteurellosis in farmed Atlantic salmon (Salmo salar L.) have become more frequent. This has led to an increasing concern that this disease will become common in salmon farming as well. The purpose of this study was to investigate the susceptibility of Atlantic salmon to Pasteurella spp. infection and the possibility of lumpsuckers transmitting pasteurellosis to Atlantic salmon. Atlantic salmon were experimentally challenged, either by bath or by cohabitation with challenged lumpsuckers, using two different strains of Pasteurella spp. (originating from lumpsucker and Atlantic salmon, respectively). No clinical signs of pasteurellosis were observed on any of the Atlantic salmon. The lumpsuckers were, however, equally susceptible to both isolates. In addition, clear differences in histopathological changes were observed between individuals challenged with the two isolates.

Multigene Expression Assay for Assessment of the Immune Status of Atlantic Salmon.

We report the development of a multigene gene expression assay on the BioMark HD platform for the evaluation of immune competence (ImCom) in farmed Atlantic salmon. The first version of the assay included 92 genes selected on the basis of transcriptome analyses in 54 trials that challenged the immune system; annotations were taken into account to represent the key pathways of innate and adaptive immunity. ImCom was tested on samples collected from seven independent projects. Fish were reared from the start feeding to eight months in the sea at eight units in different parts of Norway. Several tissues were analyzed. Linear discriminant analysis (LDA) showed that no more than 10 genes were required to separate groups, and a set of 46 immune genes was sufficient for any task. The second version of the assay was tested in the gills of two groups of high-performing healthy smolts and in groups with intermediate and high mortality rates (IM and HM, respectively). A set of 645 gill samples from clinically healthy Atlantic salmon was used as a reference. The IM group showed general suppression of immunity. All HM group salmon were above the threshold by the squared deviation from the reference. This group showed marked upregulation of genes involved in acute stress and inflammation: mmp-9, mmp-13, hsp70, il-1b, lect2, and cathelicidin. Further work will clarify the boundaries of the norm and explore various cases of impaired immunity.

Indications for a vertical transmission pathway of piscine myocarditis virus in Atlantic salmon (Salmo salar L.).

Losses due to cardiomyopathy syndrome (CMS) keep increasing in salmon-producing countries in the North-Atlantic. Recently, Piscine myocarditis virus (PMCV) has been detected in post-smolts shortly after sea-transfer, indicating a possible carry-over from the hatcheries. In addition, there are reports of prevalences of PMCV as high as 70%-90% in certain groups of broodfish, and a recent outbreak of CMS in the Faroe Islands has been linked to the importation of eggs from a CMS-endemic area. Thus, there is a need to investigate whether PMCV can be transmitted vertically from infected broodstock to their progeny. In the present study, samples from eggs, larvae, fingerlings and presmolt originating from PMCV-positive broodstock from two commercial Atlantic salmon producers were tested for PMCV. The prevalence of PMCV in the broodstock was 98% in the hearts, 69% in the roe and 59% in the milt. Piscine myocarditis virus was detected in all stages of the progeny until and including the 40 g stage. Piscine myocarditis virus was also detected in presmolt sampled for tissue tropism. This provides farmers with several options for minimizing the risk of transfer of PMCV from broodstock to progeny, including screening of broodstock and aiming to use only those that are negative for PMCV or have low levels of virus.

Monitoring infection with Piscine myocarditis virus and development of cardiomyopathy syndrome in farmed Atlantic salmon (Salmo salar L.) in Norway.

An epidemiological study was carried out in Norway in 2015-2018, investigating the development of infection with Piscine myocarditis virus (PMCV) and development of cardiomyopathy syndrome (CMS) in farmed Atlantic salmon. Cohorts from 12 sites were followed and sampled every month or every other month from sea transfer to slaughter. PMCV was detected at all sites and in all sampled cages, and fish in six sites developed clinical CMS. The initial infection happened between 1 and 7 months post-sea transfer, and the median time from infection with PMCV until outbreak of CMS was 6.5 months. Generally, fish from sites with CMS had higher viral titre and a higher prevalence of PMCV, compared to sites that did not develop clinical CMS. The virus persisted until the point of slaughter at most (11 out of 12) of the sites. The detection of PMCV in all sites suggests that PMCV is more widespread than previously known. Screening for PMCV as a tool to monitor impending outbreaks of CMS must be supported by observations of the health status of the fish and risk factors for development of disease.

A new aquareovirus causing high mortality in farmed Atlantic halibut fry in Norway.

A new aquareovirus was isolated from cultured Atlantic halibut (Hippoglossus hippoglossus) fry at a facility where massive mortalities had occurred during the start-feeding phase. The same virus was also detected in juveniles (about 10 grams) of the 2013 generation at two other production sites, but not in larger fish from generations 2007-2012. The virus replicated in BF-2 and CHSE-214 cell cultures and produced syncytia and plaque-like cytopathic effects. This Atlantic halibut reovirus (AHRV) was associated with necrosis of the liver and pancreas, syncytium formation in these tissues, and distinct viroplasm areas within the syncytium in halibut fry. Transmission electron microscopy revealed that the viroplasm contained virions, non-enveloped, icosahedral particles approximately 70 nm in diameter with a double capsid layer, amorphous material, and tubular structures. The RNA-dependent RNA polymerase (RdRp) gene from the AHRV isolates showed the highest amino acid sequence identity (80 %) to an isolate belonging to the species Aquareovirus A, Atlantic salmon reovirus TS (ASRV-TS). A partial sequence from the putative fusion-associated small transmembrane (FAST) protein of AHRV was obtained, and this sequence showed the highest amino acid sequence identity (46.8 %) to Green River Chinook virus which is an unassigned member of the genus Aquareovirus, while a comparison with isolates belonging to the species Aquareovirus A showed <33 % identity. A proper assessment of the relationship of AHRV to all members of the genus Aquareovirus, however, is hampered by the absence of genetic data from members of several Aquareovirus species. AHRV is the first aquareovirus isolated from a marine coldwater fish species and the second reovirus detected in farmed fish in Norway. A similar disease of halibut fry, as described in this paper, has also been described in halibut production facilities in Canada and Scotland.

Characterization of 'Candidatus Syngnamydia salmonis' (Chlamydiales, Simkaniaceae), a bacterium associated with epitheliocystis in Atlantic salmon (Salmo salar L.).

Two Chlamydiales have previously been found to infect Atlantic salmon (Salmo salar L.), Candidatus Piscichlamydia salmonis and Candidatus Clavichlamydia salmonicola. Both develop intracellularly in cyst-like inclusions in gill cells, generally referred to as epitheliocysts. Here, we present evidence for the association of a novel species of Chlamydiales with epitheliocystis in Atlantic salmon. Based on its partial 16S rRNA gene sequence, it is a new member of the family Simkaniaceae, and a 95.7 % identity to the type species Candidatus Syngnamydia venezia suggests inclusion in the candidate genus Syngnamydia. The presence of the bacterium in epitheliocysts in gills of Atlantic salmon was demonstrated by RNA-RNA hybridization. Ultrastructurally, the novel bacterium produces pleomorphic reticulate bodies and elementary bodies (EBs) with a characteristic morphology. The EBs are short rods with a terminal disc-like cap area, a sub-apical spherical vacuole-like electron-lucent structure and a post-equatorial nucleoid. We propose the name Candidatus Syngnamydia salmonis for this new agent from epitheliocysts in seawater-reared salmon .

'Cand. Actinochlamydia clariae' gen. nov., sp. nov., a unique intracellular bacterium causing epitheliocystis in catfish (Clarias gariepinus) in Uganda.

BACKGROUND AND OBJECTIVES: Epitheliocystis, caused by bacteria infecting gill epithelial cells in fish, is common among a large range of fish species in both fresh- and seawater. The aquaculture industry considers epitheliocystis an important problem. It affects the welfare of the fish and the resulting gill disease may lead to mortalities. In a culture facility in Kampala, Uganda, juveniles of the African sharptooth catfish (Clarias gariepinus) was observed swimming in the surface, sometimes belly up, showing signs of respiratory problems. Histological examination of gill tissues from this fish revealed large amounts of epitheliocysts, and also presence of a few Ichthyobodo sp. and Trichodina sp. METHODS AND RESULTS: Sequencing of the epitheliocystis bacterium 16S rRNA gene shows 86.3% similarity with Candidatus Piscichlamydia salmonis causing epitheliocystis in Atlantic salmon (Salmo salar). Transmission electron microscopy showed that the morphology of the developmental stages of the bacterium is similar to that of members of the family Chlamydiaceae. The similarity of the bacterium rRNA gene sequences compared with other chlamydia-like bacteria ranged between 80.5% and 86.3%. Inclusions containing this new bacterium have tubules/channels (termed actinae) that are radiating from the inclusion membrane and opening on the cell surface or in neighbouring cells. CONCLUSIONS: Radiation of tubules/channels (actinae) from the inclusion membrane has never been described in any of the other members of Chlamydiales. It seems to be a completely new character and an apomorphy. We propose the name Candidatus Actinochlamydia clariae gen. nov., sp. nov. (Actinochlamydiaceae fam. nov., order Chlamydiales, phylum Chlamydiae) for this new agent causing epitheliocystis in African sharptooth catfish.

Characterization of a VHS virus genotype III isolated from rainbow trout (Oncorhychus mykiss) at a marine site on the west coast of Norway.

BACKGROUND: Norwegian production of rainbow trout (Oncorhynchus mykiss) has been without any outbreaks of VHS for many years until the disease emerged in a farm in western Norway in November 2007. The fish were, in addition to VHS virus, positive for gill chlamydia-like bacteria, Flavobacterium psychrophilum, and a microsporidian. A new VHS virus genotype III was isolated from the fish in RTgill-W1 cells and the complete coding region (11,065 nucleotides) was sequenced. This virus was also used in a challenge experiment to see if it could cause any mortality in rainbow trout in sea water. RESULTS: This is the first time a nearly complete sequence of a genotype III virus isolate has been presented. The organization of the genes is the same as in the other VHS virus genotypes studied (GI and GIV). Between the ORFs are nontranslated regions that contain highly conserved sequences encompassing the polyadenylation signal for one gene, and the putative transcription initiation site of the next gene. The intergenic regions vary in length from 74 nt to 128 nt. The nucleotide sequence is more similar to genotype I isolates compared to isolates from genotype II and IV. Analyses of the sequences of the N and G protein genes show that this new isolate is distinct from other VHS virus isolates and groups closely together with isolates from genotype III. In a challenge experiment, using intraperitoneal (ip) injection of the isolate, co-habitation with infected fish, and bath challenge, mortalities slightly above 40% were obtained. There was no significant difference in mortality between the bath challenged group and the ip injected group, while the mortality in the co-habitation group was as low as 30%. CONCLUSIONS: All VHS virus isolates in genotype III are from marine fish in the North East Atlantic. Unlike the other known genotype III isolates, which are of low virulence, this new isolate is moderately virulent. It was not possible to detect any changes in the virus genome that could explain the higher virulence. A major problem for the study of virulence factors is the lack of information about other genotype III isolates.

Paranucleospora theridion n. gen., n. sp. (Microsporidia, Enterocytozoonidae) with a Life Cycle in the Salmon Louse (Lepeophtheirus salmonis, Copepoda) and Atlantic Salmon (Salmo salar).

Paranucleospora theridion n. gen, n. sp., infecting both Atlantic salmon (Salmo salar) and its copepod parasite Lepeophtheirus salmonis is described. The microsporidian exhibits nuclei in diplokaryotic arrangement during all known life-cycle stages in salmon, but only in the merogonal stages and early sporogonal stage in salmon lice. All developmental stages of P. theridion are in direct contact with the host cell cytoplasm or nucleoplasm. In salmon, two developmental cycles were observed, producing spores in the cytoplasm of phagocytes or epidermal cells (Cycle-I) and in the nuclei of epidermal cells (Cycle-II), respectively. Cycle-I spores are small and thin walled with a short polar tube, and are believed to be autoinfective. The larger oval intranuclear Cycle-II spores have a thick endospore and a longer polar tube, and are probably responsible for transmission from salmon to L. salmonis. Parasite development in the salmon louse occurs in several different cell types that may be extremely hypertrophied due to P. theridion proliferation. Diplokaryotic merogony precedes monokaryotic sporogony. The rounded spores produced are comparable to the intranuclear spores in the salmon in most aspects, and likely transmit the infection to salmon. Phylogenetic analysis of P. theridion partial rDNA sequences place the parasite in a position between Nucleospora salmonis and Enterocytozoon bieneusi. Based on characteristics of the morphology, unique development involving a vertebrate fish as well as a crustacean ectoparasite host, and the results of the phylogenetic analyses it is suggested that P. theridion should be given status as a new species in a new genus.

ISA virus in Chile: evidence of vertical transmission.

Infectious salmon anaemia virus (ISAV), genus Isavirus (family Orthomyxoviridae), is present in all large salmon (Salmo salar)-producing countries around the North Atlantic. The target species for this virus are members of the genus Salmo, but the virus may also replicate in other salmonids introduced to the North Atlantic (Oncorhychus spp.). Existing ISA virus isolates can be divided into two major genotypes, a North American (NA) and a European (EU) genotype, based on phylogenetic analysis of the genome. The EU genotype can be subdivided into several highly supported clades based on analysis of segments 5 (fusion protein gene) and 6 (hemagglutinin-esterase gene). In 1999 an ISA virus belonging to the NA genotype was isolated from Coho salmon in Chile, and in 2007 the first outbreaks of ISA in farmed Atlantic salmon was observed. Several salmon farms in Chile were affected by the disease in 2007, and even more farms in 2008. In this study, ISA virus has been isolated from salmon in a marine farm suffering an outbreak of the disease in 2008 and from smolts with no signs of ISA in a fresh water lake. Sequencing of the partial genome of these ISA viruses, followed by phylogenetic analysis including genome sequences from members of the NA and EU genotypes, showed that the Chilean ISA virus belongs to the EU genotype. The Chilean ISA virus groups in a clade with exclusively Norwegian ISA viruses, where one of these isolates was obtained from a Norwegian brood stock population. All salmonid species in the southern hemisphere have been introduced from Europe and North America. The absence of natural hosts for ISA viruses in Chile excludes the possibility of natural reservoirs in this country, and the close relationship between contemporary ISA virus strains from farmed Atlantic salmon in Chile and Norway suggest a recent transmission from Norway to Chile. Norway export large amounts of Atlantic salmon embryos every year to Chile; hence, the best explanation for the Norwegian ISA virus in Chile is transmission via these embryos, i.e. vertical or transgenerational transmission. This supports other studies showing that the ISA virus can be transmitted vertically.

The complete genome sequence of the Atlantic salmon paramyxovirus (ASPV).

The complete RNA genome of the Atlantic salmon paramyxovirus (ASPV), isolated from Atlantic salmon suffering from proliferative gill inflammation (PGI), has been determined. The genome is 16,965 nucleotides in length and consists of six nonoverlapping genes in the order 3'- N - P/C/V - M - F - HN - L -5', coding for the nucleocapsid, phospho-, matrix, fusion, hemagglutinin-neuraminidase and large polymerase proteins, respectively. The gene junctions contain highly conserved transcription start and stop signal sequences and trinucleotide intergenic regions similar to those of other Paramyxoviridae. The ASPV P-gene expression strategy is like that of the respiro- and morbilliviruses, which express the phosphoprotein from the primary transcript, and edit a portion of the mRNA to encode the accessory proteins V and W. It also encodes the C-protein by ribosomal choice of translation initiation. Pairwise comparisons of amino acid identities, and phylogenetic analysis of deduced ASPV protein sequences with homologous sequences from other Paramyxoviridae, show that ASPV has an affinity for the genus Respirovirus, but may represent a new genus within the subfamily Paramyxovirinae.

Characterization of 'Candidatus Clavochlamydia salmonicola': an intracellular bacterium infecting salmonid fish.

The phylum Chlamydiae contains obligate intracellular bacteria, several of which cause disease in their hosts. Morphological studies have suggested that this group of bacteria may be pathogens of fish, causing cysts in epithelial tissue - epitheliocystis. Recently, the first genetic evidence of a chlamydial aetiology of this disease in seawater reared Atlantic salmon from Norway and Ireland was presented, and the agent was given the name 'Candidatus Piscichlamydia salmonis'. In this article we present molecular evidence for the existence of a novel Chlamydiae that also may cause epitheliocystis in Norwegian salmonids. This novel Chlamydiae has been found in salmonid fish from freshwater, and based on its partial 16S rRNA gene, it may constitute a third genus in the family Chlamydiaceae, or a closely related sister family. By using whole-mount RNA-RNA hybridization we demonstrate how infected cells are distributed in a patchy manner on a gill arch. The morphology of the novel Chlamydiae includes the characteristic head-and-tail cells that have been described earlier from salmonid fish suffering from epitheliocystis. We propose the name 'Candidatus Clavochlamydia salmonicola' for this agent of epitheliocystis in freshwater salmonids.

RNA-dependent RNA polymerase from Atlantic halibut nodavirus contains two signals for localization to the mitochondria.

Nodaviruses encode an RNA-dependent RNA polymerase called Protein A that is responsible for replication of the viral RNA segments. The intracellular localization of Protein A from a betanodavirus isolated from Atlantic halibut (AHNV) was studied in infected fish cells and in transfected mammalian cells expressing Myc-tagged wild type Protein A and mutants. In infected cells Protein A localized to cytoplasmic structures resembling mitochondria and in transfected mammalian cells the AHNV Protein A was found to co-localize with mitochondrial proteins. Two independent mitochondrial targeting signals, one N-terminal comprising residues 1-40 and one internal consisting of residues 225-246 were sufficient to target both Protein A deletion mutants and enhanced green fluorescent protein (EGFP) to the mitochondria. The N-terminal signal corresponds to the mitochondrial targeting sequence of the Flock House Virus (FHV) Protein A while the internal signal is similar to the single targeting signal previously found in Greasy Grouper Nervous Necrosis Virus (GGNNV) Protein A.