Validation of a reverse transcriptase multiplex PCR test for the serotype determination of U.S. isolates of bluetongue virus

Abstract

Bluetongue (BT) is an arthropod-borne viral disease affecting ruminants primarily in tropical and temperate regions of the world. Of the 24 serotypes of BT virus (BTV) identified worldwide, five have been found in the United States. Serotype identification of BTV isolates is important to the epidemiology of the virus, but current methods are cumbersome. A single-tube multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) assay, previously developed for the serotype determination of U.S. BTV isolates, was evaluated. The determination of serotype was based on the size of the resultant amplified product. The procedure was evaluated using all 24 serotypes of BTV and nine serotypes of epizootic hemorrhagic disease virus (EHDV), a closely related orbivirus. Only the five U.S. serotypes of BTV were detected by the mRT-PCR. The assay was further tested using 132 BTV isolates originating from 24 western and southern states of the United States, from several different host species, spanning a period of 24 years. The serotypes of the isolates were determined by both a virus neutralization (VN) procedure and the mRT-PCR. Comparison of the mRT-PCR to the standard VN showed that the mRT-PCR successfully identified the serotypes of 130 of the isolates and was shown to be more reliable and specific than the VN assay.

Integration and expression of Bluetongue VP2 gene in somatic embryos of peanut through particle bombardment method

“…Further, the DNA was hybridized with target gene specific probe and the hybridization was detected using anti-DIG alkaline phosphatase [14] employing Hybrimax hybridization unit (Hongkong). RNA was extracted from the tissue that were found positive with southern hybridisation assay using Qiagen RNA preparation kit as per the manufacturer's protocol and was directly used for RT-PCR employing BTVP2-specific primers [15]. After RT-PCR, the samples were assayed by electrophoresis in 1.5% agarose gels for the presence of 1064 bp amplicon.…”

Isolation ofBluetongue virusfrom canine abortions

“…5 Case 2. A breeding kennel of Bulldogs in Kansas experienced 2 abortion episodes approximately 5 days apart at 8 weeks gestation in August-September of 2012.…”

VP2 Gene Based Phylogenetic Relationship of Indian Isolates of Bluetongue Virus Serotype 1 and Other Serotypes from Different Parts of the World

“…It seems likely that the origin of new variants/strains of BTV can be predicted by comparative sequence analyses of VP2 gene Ghiasi et al, 1987;Gould, 1988;Gould and Pritchard, 1990;Yamakawa et al, 1994). In order to determine the extent of genetic variation among BTV isolates, complete sequencing of every segment of each virus is impracticable; however, sequencing of full length or partial length serotype specific segment 2 (VP2 gene) could provide valuable information for eventually designing vaccine as well as for developing serotyping tools (Wilson and Chase, 1993;Johnson et al, 2000).…”