title: Discovery of variant infectious salmon anaemia virus (ISAV) of European genotype in British Columbia, Canada.
abstract: BACKGROUND
  Infectious salmon anaemia (ISA) virus (ISAV) belongs to the genus Isavirus, family Orthomyxoviridae. ISAV occurs in two basic genotypes, North American and European. The European genotype is more widespread and shows greater genetic variation and greater virulence variation than the North American genotype. To date, all of the ISAV isolates from the clinical disease, ISA, have had deletions in the highly polymorphic region (HPR) on ISAV segment 6 (ISAV-HPRΔ) relative to ISAV-HPR0, named numerically from ISAV-HPR1 to over ISAV-HPR30. ISA outbreaks have only been reported in farmed Atlantic salmon, although ISAV has been detected by RT-PCR in wild fish. It is recognized that asymptomatically ISAV-infected fish exist. There is no universally accepted ISAV RT-qPCR TaqMan® assay. Most diagnostic laboratories use the primer-probe set targeting a 104 bp-fragment on ISAV segment 8. Some laboratories and researchers have found a primer-probe set targeting ISAV segment 7 to be more sensitive. Other researchers have published different ISAV segment 8 primer-probe sets that are highly sensitive.
  
  
  METHODS
  In this study, we tested 1,106 fish tissue samples collected from (i) market-bought farmed salmonids and (ii) wild salmon from throughout British Columbia (BC), Canada, for ISAV using real time RT-qPCR targeting segment 8 and/or conventional RT-PCR with segment 8 primers and segment 6 HPR primers, and by virus isolation attempts using Salmon head kidney (SHK-1 and ASK-2) cell line monolayers. The sequences from the conventional PCR products were compared by multiple alignment and phylogenetic analyses.
  
  
  RESULTS
  Seventy-nine samples were "non-negative" with at least one of these tests in one or more replicates. The ISAV segment 6 HPR sequences from the PCR products matched ISAV variants, HPR5 on 29 samples, one sample had both HPR5 and HPR7b and one matched HPR0. All sequences were of European genotype. In addition, alignment of sequences of the conventional PCR product segment 8 showed they had a single nucleotide mutation in the region of the probe sequence and a 9-nucleotide overlap with the reverse primer sequence of the real time RT-qPCR assay. None of the classical ISAV segment 8 sequences in the GenBank have this mutation in the probe-binding site of the assay, suggesting the presence of a novel ISAV variant in BC. A phylogenetic tree of these sequences showed that some ISAV sequences diverted early from the classical European genotype sequences, while others have evolved separately. All virus isolation attempts on the samples were negative, and thus the samples were considered "negative" in terms of the threshold trigger set for Canadian federal regulatory action; i.e., successful virus isolation in cell culture.
  
  
  CONCLUSIONS
  This is the first published report of the detection of ISAV sequences in fish from British Columbia, Canada. The sequences detected, both of ISAV-HPRΔ and ISAV-HPR0 are of European genotype. These sequences are different from the classical ISAV segment 8 sequences, and this difference suggests the presence of a new ISAV variant of European genotype in BC. Our results further suggest that ISAV-HPRΔ strains can be present without clinical disease in farmed fish and without being detected by virus isolation using fish cell lines.
authors.affiliation: Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Ave., Charlottetown, P.E.I., C1A 4P3, Canada. mkibenge@upei.ca.
authors.forename: Molly Jt
authors.initials: MJ
authors.lastname: Kibenge
authors.affiliation: Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Ave., Charlottetown, P.E.I., C1A 4P3, Canada. tiwamoto@upei.ca.|Current address: Diagnostic Services Unit, Atlantic Veterinary College, University of Prince Edward Island, 550 University Ave., Charlottetown, P.E.I., C1A 4P3, Canada. tiwamoto@upei.ca.
authors.forename: Tokinori
authors.initials: T
authors.lastname: Iwamoto
authors.affiliation: Department of Computer Science, University of Prince Edward Island, 550 University Ave., Charlottetown, P.E.I., C1A 4P3, Canada. ywang@upei.ca.
authors.forename: Yingwei
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Raincoast Research Society, Box 399, 390 1st Street, Sointula, BC, V0N 3E0, Canada. alexandramorton5@gmail.com.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Morton
authors.affiliation: Department of Statistics and Actuarial Science, Simon Fraser University, 8888 University Drive, Burnaby, B.C., V5A 1S6, Canada. routledg@sfu.ca.
authors.forename: Richard
authors.initials: R
authors.lastname: Routledge
authors.affiliation: Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Ave., Charlottetown, P.E.I., C1A 4P3, Canada. kibenge@upei.ca.
authors.forename: Frederick Sb
authors.initials: FS
authors.lastname: Kibenge
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001955:British Columbia; D005393:Fish Diseases / Q000821:virology; D014644:Genetic Variation; D005838:Genotype; D046829:Isavirus / Q000145:classification* / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D012367:RNA, Viral; D060888:Real-Time Polymerase Chain Reaction; D020317:Salmo salar / Q000821:virology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D012367:RNA, Viral