Development and Evaluation of Real Time RT-PCR Assays for Detection and Typing of Bluetongue Virus

Abstract

Bluetongue virus is the type species of the genus Orbivirus, family Reoviridae. Bluetongue viruses (BTV) are transmitted between their vertebrate hosts primarily by biting midges (Culicoides spp.) in which they also replicate. Consequently BTV distribution is dependent on the activity, geographic distribution, and seasonal abundance of Culicoides spp. The virus can also be transmitted vertically in vertebrate hosts, and some strains/serotypes can be transmitted horizontally in the absence of insect vectors. The BTV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in order of decreasing size (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable BTV protein and the primary target for neutralising antibodies. Consequently VP2 (and Seg-2) determine the identity of the twenty seven serotypes and two additional putative BTV serotypes that have been recognised so far. Current BTV vaccines are serotype specific and typing of outbreak strains is required in order to deploy appropriate vaccines. We report development and evaluation of multiple ‘TaqMan’ fluorescence-probe based quantitative real-time type-specific RT-PCR assays targeting Seg-2 of the 27+1 BTV types. The assays were evaluated using orbivirus isolates from the ‘Orbivirus Reference Collection’ (ORC) held at The Pirbright Institute. The assays are BTV-type specific and can be used for rapid, sensitive and reliable detection / identification (typing) of BTV RNA from samples of infected blood, tissues, homogenised Culicoides, or tissue culture supernatants. None of the assays amplified cDNAs from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures.

Identification and genomic characterization of the first isolate of bluetongue virus serotype 5 detected in Australia

“…In the context of this study, it is noteworthy that the levels of nucleotide identity for Seg‐2 between the South African prototype strain of BTV‐9 (RSArrrr/09) isolated in 1942 and several Eastern topotype BTV‐9 isolates (<70% nt) (Table ) are the lowest so far observed for intra‐serotypic variation within a BTV serotype (Maan et al . ). In addition, some Indian isolates of BTV‐9 failed to be neutralized by reference antiserum raised to BTV‐9 RSA (Rao et al .…”

“…The collective BTV‐5 and BTV‐9 sequence data presented in this study and the earlier work of others (Maan et al . , ; Rao et al . ) have further highlighted the apparent discrepancies between the current internationally recognized serological tests for BTV serotype designation (Huismans & Erasmus ; Erasmus ) and the range of percentage nucleotide identities that have subsequently been found to exist between the Seg‐2 genes of different isolates of the same serotypes.…”

“…At least 27 serotypes have been documented to date (Erasmus ; Maan et al . ). However, the rapid output from next‐generation nucleic acid sequencing technologies has enabled the development of BTV‐specific quantitative reverse transcriptase polymerase chain reaction (RT‐PCR) assays which, together with the application of phenotyping software algorithms, play an increasingly significant role in initial BTV detection and serotype confirmation (Maan et al .…”

Concurrent infection of Bluetongue and Peste-des-petits-ruminants virus in small ruminants in Haryana State of India

“…Twenty‐seven serotypes have been recognized for this virus so far (Hofmann, Griot, Chaignat, Perler, & Thur, ; Maan et al., ; Zientara et al., ). There is evidence of two additional putative serotypes (Maan et al., ). The BTV genome consists of ten segments of double‐stranded RNA surrounded by three concentric protein layers without any lipid envelope (Mertens, Ross‐smith, Diprose, & Attoui, ).…”

Microfluidic System for Detection of Viral RNA in Blood Using a Barcode Fluorescence Reporter and a Photocleavable Capture Probe

“…In addition, the processing throughput of the SPM needs to be improved to match high throughput techniques such as qPCR. 30 …”