title: Infectious salmon anaemia virus replication and induction of alpha interferon in Atlantic salmon erythrocytes.
abstract: BACKGROUND
  Infectious salmon anaemia (ISA) virus (ISAV), which causes ISA in marine-farmed Atlantic salmon, is an orthomyxovirus belonging to the genus Isavirus, family Orthomyxoviridae. ISAV agglutinates erythrocytes of several fish species and it is generally accepted that the ISAV receptor destroying enzyme dissolves this haemagglutination except for Atlantic salmon erythrocytes. Recent work indicates that ISAV isolates that are able to elute from Atlantic salmon erythrocytes cause low mortality in challenge experiments using Atlantic salmon. Previous work on ISAV-induced haemagglutination using the highly pathogenic ISAV strain NBISA01 and the low pathogenic ISAV strain RPC/NB-04-0851, showed endocytosis of NBISA01 but not RPC/NB-04-0851. Real-time RT-PCR was used to assess the viral RNA levels in the ISAV-induced haemagglutination reaction samples, and we observed a slight increase in viral RNA transcripts by 36 hours in the haemagglutination reaction with NBISA01 virus when the experiment was terminated. However, a longer sampling interval was considered necessary to confirm ISAV replication in fish erythrocytes and to determine if the infected cells mounted any innate immune response. This study examined the possible ISAV replication and Type I interferon (IFN) system gene induction in Atlantic salmon erythrocytes following ISAV haemagglutination.
  
  
  RESULTS
  Haemagglutination assays were performed using Atlantic salmon erythrocytes and one haemagglutination unit of the two ISAV strains, NBISA01 and RPC/NB-04-0851, of differing genotypes and pathogenicities. Haemagglutination induced by the highly pathogenic NBISA01 but not the low pathogenic RPC/NB-04-0851 resulted in productive infection as evidenced by increased ISAV segment 8 transcripts and increase in the median tissue culture infectious dose (TCID50) by 5 days of incubation. Moreover, reverse transcription (RT) quantitative PCR used to compare mRNA levels of key Type I IFN system genes in erythrocyte lysates of haemagglutination reactions with the two ISAV strains showed a higher relative fold increase of IFN-alpha in NBISA01 haemagglutinations compared to RPC/NB-04-085-1 haemagglutinations (33.0 - 44.26 relative fold increase compared to 11.29). Erythrocytes exposed to heat-inactivated virus or to polyinosinic:polycytidylic acid (polyI:C) or to L-15 medium alone (negative control assays) had minimal late induction (<3.5 relative fold increase) of STAT1 and/or ISG15 and Mx genes, whereas erythrocytes exposed to UV-inactivated virus lacked any cytokine induction.
  
  
  CONCLUSIONS
  ISAV-induced haemagglutination by a highly pathogenic virus strain results in virus uptake and productive infection of Atlantic salmon erythrocytes accompanied by significant induction of IFN-alpha. This study also highlights the critical role of ISAV strain variation in the initial stages of the virus-cell interaction during haemagglutination, and possibly in the pathogenesis of ISA. Moreover, the study shows for the first time that fish erythrocytes immunologically respond to ISAV infection.
authors.affiliation: Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, C1A 4P3, Canada. sworkenhe@upei.ca
authors.forename: Samuel T
authors.initials: ST
authors.lastname: Workenhe
authors.forename: Molly J T
authors.initials: MJ
authors.lastname: Kibenge
authors.forename: Glenda M
authors.initials: GM
authors.lastname: Wright
authors.forename: Dorota W
authors.initials: DW
authors.lastname: Wadowska
authors.forename: David B
authors.initials: DB
authors.lastname: Groman
authors.forename: Frederick S B
authors.initials: FS
authors.lastname: Kibenge
mesh_terms: D000818:Animals; D002460:Cell Line; D004912:Erythrocytes / Q000276:immunology / Q000821:virology*; D006387:Hemagglutination, Viral; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D046829:Isavirus / Q000472:pathogenicity* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020317:Salmo salar / Q000821:virology*; D014779:Virus Replication
chemical_list: D016898:Interferon-alpha