PCR-RFLP for Identification of Bluetongue Virus Serotypes

Bluetongue virus is the etiological agent of a disease characterized by bleeding in domestic and wild ruminants. Different viral strains can infect a ruminant species and severity of infection is variable. Transmission of the virus under natural conditions is through the bite of a mosquito of the genus Culicoides (Ceratopogonidae), but also can be transmitted by infected semen or embryos. The genus Culicoides is a vector of other diseases such as African horse sickness. Specimens are about 0.5 to 5 mm in size. There are about 1,400 species of Culicoides, only 32 of which can act as biological vectors. This is because of the receiver with the intestinal cells where amplification occurs in viral load. A segment 10 of the viral genome from 16 viral serotypes (of approximately 26 serotypes reported) was used. The sequences were aligned, and the consensus sequence was obtained. Subsequently, a pair of oligonucleotides was designed on a highly conserved region that theoretically allows co-amplification of the strains. Sites for restriction enzymes were identified to create a specific electrophoretic pattern for each serotype. It was possible to theoretically amplify a gene segment of 16 bluetongue virus serotypes with one set of oligonucleotides and the serotypes can be differentiated with two restriction enzymes. The proposed method could become a simple, inexpensive, and practical routine tool for type-specific identification of bluetongue virus.