Design of primers and use of RT-PCR assays for typing European bluetongue virus isolates: differentiation of field and vaccine strains

Abstract

Bluetongue virus (BTV) is the causative agent of bluetongue, a disease of ruminant livestock that occurs almost worldwide between latitudes 356 S and 506 N. There are 24 serotypes of BTV (currently identified by serum neutralization assays). Since 1998, eight strains of six BTV serotypes (1, 2, 4, 8, 9 and 16) have invaded Europe. The most variable BTV protein is major outer-capsid component VP2, encoded by segment 2 (Seg-2) of the double-stranded RNA virus genome. VP2 represents the major target for neutralizing (and protective) antibodies that are generated in response to BTV infection, and is therefore the primary determinant of virus serotype. RT-PCR primers and assays targeting Seg-2 have been developed for rapid identification (within 24 h) of the six European BTV types. These assays are sensitive, specific and show perfect agreement with the results of conventional virus-neutralization methods. Previous studies have identified sequence variations in individual BTV genome segments that allow different isolates to be grouped on the basis of their geographical origins (topotypes). The assays described in this paper can detect any of the BTV isolates of the homologous serotype that were tested from different geographical origins (different Seg-2 topotypes). Primers were also identified that could be used to distinguish members of these different Seg-2 topotypes, as well as field and vaccine strains of most of the European BTV serotypes. The serotype-specific assays (and primers) showed no cross-amplification when they were evaluated with multiple isolates of the most closely related BTV types or with reference strains of the remaining 24 serotypes. Primers developed in this study will be updated periodically to maintain their relevance to current BTV distribution and epidemiology (http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm).

Serotypic identification of local Bluetongue virus isolates using Plaque Reduction Neutralization (PRN) and Reverse Transcriptase Polymerase Chain Reaction (RTPCR) Techniques

“…This is most probably caused by incompatibility between primer and target gene sequences as designated primers belonged to Mediterranean Basin isolates, and so BTV-4 primers did not match the target cDNA of isolate TR-23. This observation was found in concordance to several studies in which serotypic discrimination of BTV-4 European isolates and vaccine strains has been performed (16,28).…”

“…Generic RT-PCR was performed on segment 10 using primers designed previously (17). On the other hand, sRT-PCR was carried on segment 2 with primer sets, two of which had been designed previously (16) while the remaining two (BTV-9 and BTV-16) were designed within this study.…”

Bluetongue virus: virology, pathogenesis and immunity

“…These geographical variations define eastern and western VP2 topotypes within individual serotypes. Oligonucleotide primers can be designed targeting Seg-2, that can be used in RT-PCR assays to facilitate typing of BTV field and vaccine isolates of each serotype and topotype [69]. Despite the overall sequence variability, some features of VP2 appeared to be conserved across serotypes, including the hydrophobicity profile, charge distribution and the position of certain cysteine residues [58].…”

Bluetongue: Virus proteins and recent diagnostic approaches

“…Serogroup-specific RT-PCR, sequencing, restriction enzyme profile analysis (REPA) and phylogenetic analyses (targeting conserved genome segments) are now available in an increasing number of laboratories for the identification of BTV. Serotypespecific RT-PCR assays (targeting genome segments 2 or 6) have also been used to identify different BTV serotypes (Maan et al, , 2008Mertens et al, 2007).…”