ABSTRACT
To determine the variability of the NS3/NS3A gene of field strains of BTV contained in Culicoides sonorensis collected from a single site in California (CA), the NS3/NS3A gene was directly amplified and sequenced from 22 pools of C. sonorensis and compared with those of previously characterized field isolates from CA, as well as to viruses that caused recent outbreaks of bluetongue disease in ruminants in CA. Phylogenetic analysis established that the NS3/NS3A gene of strains of BTV contained in C. sonorensis collected from the site exists as a heterogeneous population. The two most divergent nucleotide sequences of the NS3/NS3A genes of these viruses differed by 2.5% (18 nucleotides). Comparison with the NS3/NS3A gene sequences from viruses that caused recent instances of bluetongue disease in ruminants in CA indicated that BTV strains from different geographic regions can exhibit a higher degree of genetic heterogeneity (up to 6.6%; 0-48 nucleotide differences) than those contained in C. sonorensis collected from a single site.
Subject(s)
Bluetongue/genetics , Ceratopogonidae/virology , Genes, Viral , Genetic Variation , Viral Nonstructural Proteins/genetics , Animals , Bluetongue/classification , California , Cell Line , Chick Embryo , Cricetinae , PhylogenyABSTRACT
A one-tube real-time TaqMan reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of equine arteritis virus (EAV). The test was validated using the seminal plasma and nasal secretions of infected horses that were proven to contain EAV by traditional virus isolation in rabbit kidney thirteen (RK-13) cells, as well as a variety of cell culture-propagated European and North American strains of EAV. The primers and a fluorogenic TaqMan probe were designed to amplify and detect a highly conserved region of open reading frame 7 (ORF7) of EAV. The real-time TaqMan PCR assay detected EAV RNA in all samples that were confirmed to contain infectious EAV by virus isolation. The assay had an analytical sensitivity of 10 molecules of EAV RNA allowing the detection of EAV in clinical samples or tissue culture fluid (TCF) containing at least 200 viral RNA copies per ml. Thus, the one-tube real-time TaqMan RT-PCR assay provides a rapid, accurate, quantitative, convenient and high sample throughput system for diagnosis of EAV infection, in a closed-tube format that minimizes the risk of cross-contamination.
