title: Strand-specific, real-time RT-PCR assays for quantification of genomic and positive-sense RNAs of the fish rhabdovirus, Infectious hematopoietic necrosis virus.
abstract: The fish rhabdovirus, Infectious hematopoietic necrosis virus (IHNV), is an important pathogen of salmonids. Cell culture assays have traditionally been used to quantify levels of IHNV in samples; however, real-time or quantitative RT-PCR assays have been proposed as a rapid alternative. For viruses having a single-stranded, negative-sense RNA genome, standard qRT-PCR assays do not distinguish between the negative-sense genome and positive-sense RNA species including mRNA and anti-genome. Thus, these methods do not determine viral genome copy number. This study reports development of strand-specific, qRT-PCR assays that use tagged primers for enhancing strand specificity during cDNA synthesis and quantitative PCR. Protocols were developed for positive-strand specific (pss-qRT-PCR) and negative-strand specific (nss-qRT-PCR) assays for IHNV glycoprotein (G) gene sequences. Validation with synthetic RNA transcripts demonstrated the assays could discriminate the correct strand with greater than 1000-fold fidelity. The number of genome copies in livers of IHNV-infected fish determined by nss-qRT-PCR was, on average, 8000-fold greater than the number of infectious units as determined by plaque assay. We also compared the number of genome copies with the quantity of positive-sense RNA and determined that the ratio of positive-sense molecules to negative-sense genome copies was, on average, 2.7:1. Potential future applications of these IHNV strand-specific qRT-PCR assays are discussed.
authors.affiliation: Western Fisheries Research Center, 6505 Northeast 65th Street, Seattle, WA 98115, USA; School of Aquatic and Fishery Sciences, University of Washington, Box 355020, Seattle, WA 98195, USA. maureenp@u.washington.edu
authors.forename: Maureen K
authors.initials: MK
authors.lastname: Purcell
authors.forename: S Alexandra
authors.initials: SA
authors.lastname: Hart
authors.forename: Gael
authors.initials: G
authors.lastname: Kurath
authors.forename: James R
authors.initials: JR
authors.lastname: Winton
mesh_terms: D000818:Animals; D005393:Fish Diseases / Q000821:virology; D016679:Genome, Viral; D029142:Infectious hematopoietic necrosis virus / Q000235:genetics / Q000302:isolation & purification*; D008099:Liver / Q000821:virology; D017686:Oncorhynchus mykiss / Q000821:virology; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D018353:Rhabdoviridae Infections / Q000662:veterinary / Q000821:virology; D012680:Sensitivity and Specificity; D014759:Viral Envelope Proteins / Q000235:genetics
chemical_list: C104040:IHNVG protein, Infectious hematopoietic necrosis virus; D012367:RNA, Viral; D014759:Viral Envelope Proteins