S.21. VP3 gene based RT-PCR for the detection of bluetongue virus in laboratory and blood samples

Bluetongue is a Culicoides-transmitted disease of wild and domestic ruminants, caused by bluetongue virus (BTV). BTV is the prototype member of the Orbivirus genus within the family Reoviridae. The BTV serogroup contains 24 serotypes of which 21 are reported to be present in India. Being the List ‘A’ disease of OIE and endemic in India, it imposes trade restrictions. As a result there is a demand for increasingly rapid and reliable methods of BTV detection and RT-PCR is proving to be the method of choice and prescribed by OIE. Moreover, RT-PCR coupled with sequencing based on serogroup specific genes such as VP3 and NS3 may be used not only to detect the presence of viral nucleic acid, but also provide information on possible geographical origin (topotype) of BTV isolates within few days of receipt of infected blood samples.

Keeping in view the above facts, 648 bp (1026 nt-1673 nt) region of VP3 gene based RT-PCR has been standardized using laboratory and blood samples for subsequent sequencing to topotype BTV isolates. The standardized RT-PCR has been evaluated for specificity by detecting 648 bp PCR product from known Indian isolates of various BTV serotypes (1, 18 and 23) in infected BHK-21 cell culture fluid. The RT-PCR then has been applied for detection of BTV in 42 blood samples, 4 infected cell culture fluids and 2 chicken embryo homogenates which were found negative for characteristic BTV induced CPE by conventional virus isolation in cell culture. Of which 6 blood samples and 1 cell culture fluid were found positive by RT-PCR which are 14% and 25% more sensitive in detecting BTV from blood samples and infected cell culture fluid respectively. Further screening of samples using nested primers is needed to use this RT-PCR as routine diagnostic tool.