Identification of the 3' and 5' terminal sequences of the 8 rna genome segments of european and north american genotypes of infectious salmon anemia virus (an orthomyxovirus) and evidence for quasispecies based on the non-coding sequences of transcripts

• Published in: Virology Journal
• Main Authors: Riveroll Angela, Simard Nathalie, Salonius Kira, Kibenge Molly, Kulshreshtha Vikas, Kibenge Frederick
• Format: Article in Journal/Newspaper
• Language: English
• Published: BMC 2010
• Subjects: Infectious and parasitic diseases; RC109-216; Norway; Atlantic salmon; Salmo salar
• Online Access: https://doi.org/10.1186/1743-422X-7-338; https://doaj.org/article/611c7f1b117f464f84afc620acc32401

Abstract Background Infectious salmon anemia (ISA) virus (ISAV) is a pathogen of marine-farmed Atlantic salmon ( Salmo salar ); a disease first diagnosed in Norway in 1984. This virus, which was first characterized following its isolation in cell culture in 1995, belongs to the family Orthomyxoviridae , genus, Isavirus . The Isavirus genome consists of eight single-stranded RNA segments of negative sense, each with one to three open reading frames flanked by 3' and 5' non-coding regions (NCRs). Although the terminal sequences of other members of the family Orthomyxoviridae such as Influenzavirus A have been extensively analyzed, those of Isavirus remain largely unknown, and the few reported are from different ISAV strains and on different ends of the different RNA segments. This paper describes a comprehensive analysis of the 3' and 5' end sequences of the eight RNA segments of ISAV of both European and North American genotypes, and evidence of quasispecies of ISAV based on sequence variation in the untranslated regions (UTRs) of transcripts. Results Two different ISAV strains and two different RNA preparations were used in this study. ISAV strain ADL-PM 3205 ISAV-07 (ADL-ISAV-07) of European genotype was the source of total RNA extracted from ISAV-infected TO cells, which contained both viral mRNA and cRNA. ISAV strain NBISA01 of North American genotype was the source of vRNA extracted from purified virus. The NCRs of each segment were identified by sequencing cDNA prepared by three different methods, 5' RACE (Rapid amplification of cDNA ends), 3' RACE, and RNA ligation mediated PCR. Sequence analysis of five clones each derived from one RT-PCR product from each NCR of ISAV transcripts of segments 1 to 8 revealed significant heterogeneity among the clones of the same segment end, providing unequivocal evidence for presence of intra-segment ISAV quasispecies. Both RNA preparations (mRNA/cRNA and vRNA) yielded complementary sequence information, allowing the simultaneous identification and confirmation of the 3' ...