Genomic characterization, phylogenetic position and in situ localization of a novel putative mononegavirus in Lepeophtheirus salmonis.

The complete genome sequence of a novel mononegavirus, Lepeophtheirus salmonis negative-stranded RNA virus 1 (LsNSRV-1), obtained from a salmonid ectoparasite, Lepeophtheirus salmonis was determined. The viral genome contains five open reading frames encoding three unknown proteins (ORF I, II and III), a putative glycoprotein (G), and a large (L) protein. Phylogenetic analysis placed LsNSRV-1 in the recently established mononegaviral family Artoviridae. LsNSRV-1 showed a prevalence of around 97% and was detected in all L. salmonis developmental stages. Viral genomic and antigenomic RNA was localized to nerve tissue, connective tissue, epithelial cells of the gut, subepidermal tissue, exocrine and cement glands, as well as the testis, vas deferens and spermatophore sac of male L. salmonis and the ovaries and oocytes of females. Viral RNA was detected in both the cytoplasm and the nucleoli of infected cells, and putative nuclear export and localization signals were found within the ORF I, III and L proteins, suggesting nuclear replication of LsNSRV-1. RNA interference (RNAi) was induced twice during development by the introduction of a double-stranded RNA fragment of ORF I, resulting in a transient knockdown of viral RNA. A large variation in the knockdown level was seen in adult males and off springs of knockdown animals, whereas the RNA level was more stable in adult females. Together with the localization of viral RNA within the male spermatophore and female oocytes and the amplification of viral RNA in developing embryos, this suggests that LsNSRV-1 is transmitted both maternally and paternally. Small amounts of viral RNA were detected at the site where chalimi were attached to the skin of Atlantic salmon (Salmo salar). However, as the RNAi-mediated treatment did not result in LsNSRV-1-negative offspring and the virus failed to replicate in the tested fish cell cultures, it is difficult to investigate the influence of secreted LsNSRV-1 on the salmon immune response.

High-Resolution Screening of Viral Communities and Identification of New Pathogens in Fish Using Next-Generation Sequencing.

Discovery of viral genomes in fish has historically been based on viral enrichment, random priming, cloning, and Sanger sequencing. However, the development of next-generation sequencing has enabled the possibility to sequence the entire virome of a tissue sample. This has led to an enormous increase in discovery of new viruses. In this chapter, we describe a simple and rapid method for viral discovery in fish. The method is based on Illumina sequencing of total RNA from diseased tissue or cell culture and in silico removal of host RNA.

The complete genome sequence of CrRV-Ch01, a new member of the family Rhabdoviridae in the parasitic copepod Caligus rogercresseyi present on farmed Atlantic salmon (Salmo salar) in Chile.

We have determined the complete genome sequence of a new rhabdovirus, tentatively named Caligus rogercresseyi rhabdovirus Ch01 (CrRV-Ch01), which was found in the parasite Caligus rogercresseyi, present on farmed Atlantic salmon (Salmo salar) in Chile. The genome encodes the five canonical rhabdovirus proteins in addition to an unknown protein, in the order N-P-M-U (unknown)-G-L. Phylogenetic analysis showed that the virus clusters with two rhabdoviruses (Lepeophtheirus salmonis rhabdovirus No9 and Lepeophtheirus salmonis rhabdovirus No127) obtained from another parasitic caligid, Lepeophtheirus salmonis, present on farmed Atlantic salmon on the west coast of Norway.

New virus of the family Flaviviridae detected in lumpfish (Cyclopterus lumpus).

In this study, we determined the complete coding sequence of a putative new member of the family Flaviviridae, named "Cyclopterus lumpus virus" (CLuV), which is associated with a serious disease in lumpfish (Cyclopterus lumpus). The virus was present in all tissues tested, but pathology was primarily observed in the liver and kidneys. CLuV shows low but distinct similarity to the unassigned Tamana bat virus (TABV). Unlike other known members of the family Flaviviridae, translation of the entire CLuV polyprotein is dependent on a - 1 ribosomal frameshift in the NS2A region.

Genomic characterization and phylogenetic position of two new species in Rhabdoviridae infecting the parasitic copepod, salmon louse (Lepeophtheirus salmonis).

Several new viruses have emerged during farming of salmonids in the North Atlantic causing large losses to the industry. Still the blood feeding copepod parasite, Lepeophtheirus salmonis, remains the major challenge for the industry. Histological examinations of this parasite have revealed the presence of several virus-like particles including some with morphologies similar to rhabdoviruses. This study is the first description of the genome and target tissues of two new species of rhabdoviruses associated with pathology in the salmon louse. Salmon lice were collected at different Atlantic salmon (Salmo salar) farming sites on the west coast of Norway and prepared for histology, transmission electron microscopy and Illumina sequencing of the complete RNA extracted from these lice. The nearly complete genomes, around 11,600 nucleotides encoding the five typical rhabdovirus genes N, P, M, G and L, of two new species were obtained. The genome sequences, the putative protein sequences, and predicted transcription strategies for the two viruses are presented. Phylogenetic analyses of the putative N and L proteins indicated closest similarity to the Sigmavirus/Dimarhabdoviruses cluster, however, the genomes of both new viruses are significantly diverged with no close affinity to any of the existing rhabdovirus genera. In situ hybridization, targeting the N protein genes, showed that the viruses were present in the same glandular tissues as the observed rhabdovirus-like particles. Both viruses were present in all developmental stages of the salmon louse, and associated with necrosis of glandular tissues in adult lice. As the two viruses were present in eggs and free-living planktonic stages of the salmon louse vertical, transmission of the viruses are suggested. The tissues of the lice host, Atlantic salmon, with the exception of skin at the attachment site for the salmon louse chalimi stages, were negative for these two viruses.