title: A strand specific real-time RT-PCR method for the targeted detection of the three species (vRNA, cRNA and mRNA) of infectious salmon anaemia virus (ISAV) replicative RNA.
abstract: Three species of viral-derived RNA (vRNA, cRNA and mRNA) are produced during an infectious salmon anaemia virus (ISAV) infection. Conventional real-time RT-PCR (RT-qPCR) targeting ISAV segment 8 provides a very sensitive method for the detection of ISAV RNA, however it does not differentiate between these three individual RNA species. In this study, strand-specific tagged primers have been utilised in the RT reaction to specifically produce cDNA corresponding to each of the 3 viral RNA types produced from ISAV segment 8 for the subsequent detection by real-time PCR. The RNA species-specific assay was successfully used to specifically distinguish synthetic T7-produced RNA transcripts representing the 3 species of ISAV RNA at levels up to approximately 10(5)-fold higher than the other types. In addition, the method was applied to investigate the production of segment 8 RNA in time-course tissue culture experiments performed at optimal (15°C), sub-optimal (20°C) and inadequate (25°C) temperatures for replication or in the presence of a chemical inhibitor to vary the RNA populations and investigate its effectiveness. Variation in RNA production was observed between the optimal and sub-optimal temperatures and in the presence of the chemical inhibitor. Production of all RNA species was completely inhibited at 25°C indicating the potential usefulness of the assay as a tool in the understanding of ISAV replication and transcription dynamics.
authors.affiliation: Marine Scotland Science, Marine Laboratory, 375 Victoria Road, Aberdeen, Scotland, UK. alastair.mcbeath@scotland.gsi.gov.uk
authors.forename: Alastair
authors.initials: A
authors.lastname: McBeath
authors.forename: Nicola
authors.initials: N
authors.lastname: Bain
authors.forename: Mickael
authors.initials: M
authors.lastname: Fourrier
authors.forename: Bertrand
authors.initials: B
authors.lastname: Collet
authors.forename: Michael
authors.initials: M
authors.lastname: Snow
mesh_terms: D000818:Animals; D005393:Fish Diseases / Q000175:diagnosis / Q000821:virology; D016679:Genome, Viral; D046829:Isavirus / Q000235:genetics*; D009976:Orthomyxoviridae Infections / Q000175:diagnosis / Q000662:veterinary / Q000821:virology; D018075:RNA, Complementary / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis*; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012254:Ribavirin / Q000494:pharmacology; D020317:Salmo salar / Q000821:virology; D013696:Temperature; D014779:Virus Replication / Q000187:drug effects
chemical_list: D018075:RNA, Complementary; D012333:RNA, Messenger; D012367:RNA, Viral; D012254:Ribavirin