Determination of bovine rotavirus G and P serotypes by polymerase chain reaction

Among bovine rotaviruses there are two major G serotypes (G6 and G10) and three P serotypes (P1, P5, and P11, each of which is defined on the basis of the VP4 antigenic specificity of NCDV, UK and KK-3, respectively). The nucleotide sequence of a P11 gene (KK-3) was determined. The predicted KK-3 VP4 contained 772 amino-acids and showed 96% amino-acid identity with B223 VP4, an American prototype of P11 bovine rotavirus. Comparative analysis of the genes determining the G and P serotypes of bovine rotaviruses allowed us to develop polymerase chain reaction (PCR)-based assays which distinguished two G serotypes and three P serotypes commonly found in bovine rotaviruses. For determination of G serotypes, a 1013 bp fragment of the VP7 gene was first reverse-transcribed and then amplified with a pair of generic primers. In a second PCR amplification, the 5' generic primer and two different typing primers (either G6- or G10-specific) were used to generate fragments whose sizes served to identify the G serotype. Similarly, for determination of P serotypes, an 864 bp fragment of the VP4 gene was first reverse-transcribed and then amplified with another pair of generic primers. In a second PCR amplification, the 5' generic primer and three different typing primers, each one specific to one of the three P serotypes, were used to generate fragments whose sizes served to identify the P serotype. These assays were able to identify the G and P serotypes of six reference bovine rotavirus strains.