Segment 2 based characterization of a novel Indian bluetongue virus isolate

Abstract

Aim: The study was conducted to characterize and serotype the novel isolate of bluetongue virus (BTV) isolated from India. Materials and Methods: The BTV isolate was propagated in BHK-21 cell line. Nucleic acid (dsRNA) was extracted using Trizol method and cDNAwas prepared using a process called reverse transcription. The cDNAwas subjected to group specific PCR using ns1 gene specific primer to confirm the isolate as BTV. The type specific PCR was conducted to confirm the serotype of the virus using vp2 gene specific primers for all the BTV serotype including BTV10. The vp2 gene specific PCR amplicon was sequenced and in-silico restriction enzyme analysis and phylogenetic analysis was conducted. Results: Group specific PCR using ns1 gene specific primers showed a single 274bp amplicon in agarose gel electrophoresis confirmed the sample as BTV. The type specific PCR using BTV10 vp2 gene specific primer showed a single amplicon of 647bp. Remaining BTVserotype specific primers didn't show any amplification. The vp2 gene PCR amplicon was sequenced. The in-silico restriction enzyme analysis of vp2 gene of Indian BTV10 isolate along with other isolates from GenBank database using HindIII, XhoII and ApoI showed a common pattern between Indian and USAisolates. Similarly, phylogenetic analyses using vp2 gene nucleotide as well as deduced amino acid sequence of Indian BTV10 isolate and global isolates showed that Indian and most of the USAisolates placed in a single clad. Conclusion: Anovel BTV isolate was isolated and confirmed as BTV serotype 10. Upon molecular analysis Indian BTV10 isolate was found closer to that of USAisolates than other global isolates.