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In the present study plaque purification of BTV-12 isolated from bluetongue outbreaks in 2016 of Kurnool was done along with studying its infection kinetics in Vero cell line. BTV-12 isolate adapted to Vero cell line had a titre of 106.116/ml. Plaque purification was carried for three times using agarose overlaying method in 6 well plate. The plaque purified virus revealed 10 segmented genome on agarose gel electrophoresis. Plaque purification was confirmed since RT-PCR amplified only a product of 750 bp with BTV-12 specific primers. The purified virus was not amplified by other primers specific for BTV-1, 2, 4, 9, 10, 16, 21, 23 and 24. Furthermore, cytopathic effect of plaque purified BTV12 in Vero cell line was studied by H&E staining of infected Vero cells at 24, 36, 48 and 72 hrs post infection for understanding the kinetics of infection. The thus plaque purified virus can be used either to study pathogenesis or to raise hyper immune serum which can be used in neutralization assays to determine the serotype of unknown BTV.

Key words: Orbivirus, Plaque purification, BTV serotypes, Infection kinetic, Hyper immune serum.