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 cDNA was prepared using a process called reverse
transcription. The cDNA was 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 BTV serotype 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 USA isolates. 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 USA isolates
placed in a single clad.
Conclusion: A novel BTV isolate was isolated and
confirmed as BTV serotype 10. Upon molecular analysis Indian BTV10 isolate was
found closer to that of USA isolates than other global isolates.
Keywords:
bluetongue, BTV10, PCR, restriction enzyme analysis, vp2 gene
