Article history: Received: 21-04-2018, Accepted: 13-06-2018, Published online: 01-08-2018
Corresponding author: Awadh Bihari Pandey
E-mail: firstname.lastname@example.orgCitation: Saxena A, Biswas SK, Chand K, Naskar J, Chauhan A, Mohd G, Tewari N, Ain KU, Ramakrishnan MA, Pandey AB (2018) Genetic and phylogenetic analysis of the outer capsid protein genes of Indian isolates of bluetongue virus serotype-16, Veterinary World, 11(8): 1025-1029.
Aim: The aim of the study was to characterize bluetongue virus serotype 16 (BTV-16), recently isolated from different states of India. The evolutionary relationship of newly isolated BTV-16 and previously reported Indian and global BTV-16 isolates were compared using molecular analysis.
Materials and Methods: In the present study, five (n=5) BTV-16 isolates were used to amplify gene segment-2 and segment-6 encoding the outer capsid proteins VP2 and VP5, respectively. The amplified products were purified and sequenced by the Sanger sequencing method. The phylogenetic relationship and nucleotide identity of all five BTV-16 isolates were compared with previously reported Indian and global BTV-16 isolates. Nucleotide sequence data were aligned using the CLUSTAL W algorithm implemented in the MegAlign of DNASTAR program package (MegAlign 5.00, DNASTAR Inc., Madison, USA). Phylogenetic analyses were carried out using MEGA version 6.0 software with the best nucleotide substitution model.
Results: Phylogenetic analysis based on the VP2 and VP5 encoding genes, segregates Indian BTV-16 isolates in a distinct cluster with proximity to the Eastern topotype. Indian isolates make a monophyletic cluster with Eastern topotypes with Western topotype BTV-16 (BTV-16/NIG/AJ586694) occupying a separate cluster. Indian isolates were found to share 91.5%- 97.5% and 96.5%-98.9% identity at the nucleotide and deduced amino acid (aa) level, respectively, to the global BTV-16 isolates. There is a high degree of variation with the Nigerian isolate with 27.0-27.7% and 26.0-26.9% at the nucleotide and aa sequence level, respectively. These data suggest that Indian BTV-16 isolates might have evolved separately within the Eastern BTV topotype.
Conclusion: Phylogenetic analyses and nucleotide identity of BTV-16 isolates at the VP2 and VP5 gene encoded level indicate that isolates used in the present study might have evolved from a common Eastern topotype ancestor. The data presented in this study will be helpful for future selection of reference strains in a serological and molecular epidemiology study.
Keywords: bluetongue virus, phylogenetic analysis, VP2 gene, VP5 gene.
1. MacLachlan, N.J. (1994) The pathogenesis and immunology of bluetongue virus infection of ruminants. Comp. Immunol. Microbiol. Infect Dis., 17(3-4): 197-206. [Crossref]
2. Rushton, J. and Lyons, N. (2015) Economic impact of bluetongue: A review of the effect on production. Vet. Ital., 51(4): 401-406. [PubMed]
3. Belhouchet, M., Jaafar, F.M., Firth, A.E., Grimes, J.M., Mertens, P.P. and Attoui, H. (2011) Detection of a fourth orbivirus non-structural protein. PLoS One, 6(10): e25697. [Crossref]
4. Ratinier, M., Caporale, M., Golder, M., Franzoni, G., Allan, K., Nunes, S.F. and Palmarini, M. (2011) Identification and characterization of a novel non-structural protein of bluetongue virus. PLoS Pathog., 7(12): e1002477. [Crossref]
5. Huismans, H., Van der Walt, N.T., Cloete, M. and Erasmus, B.J. (1987) Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep. Virology, 157(1): 172-179. [Crossref]
7. Huismans, H., Van der Walt, N.T. and Erasmus, B.J. (1985) Immune response against the purified serotype-specific antigen of bluetongue virus and initial attempts to clone the gene that codes for the synthesis of this protein. Prog. Clin. Biol. Res., 178: 347-353. [PubMed]
9. Shafiq, M., Minakshi, P., Bhateja, A., Ranjan, K. and Prasad, G. (2013) Evidence of genetic reassortment between Indian isolate of bluetongue virus serotype 21 (BTV-21) and bluetongue virus serotype 16 (BTV-16). Virus. Res., 173(2): 336-343. [Crossref] [PubMed]
10. Maan, S., Maan, N.S., Van Rijn, P.A., Van Gennip, R.G., Sanders, A., Wright, I.M. and Potgieter, A.C. (2010) Full genome characterization of bluetongue virus serotype 6 from the Netherlands 2008 and comparison to other field and vaccine strains. PloS one, 5(4): e10323. [Crossref] [PubMed] [PMC]
11. He, C.Q., Ding, N.Z., He, M., Li, S.N., Wang, X.M., He, H.B. and Guo, H.S. (2010) Intragenic recombination as a mechanism of genetic diversity in bluetongue virus. J. Virol., 84(21): 11487-11495. [Crossref] [PubMed] [PMC]
12. Chand, K., Biswas, S.K., Pandey, A.B., Muthuchelvan, D. and Mondal, B. (2015) Bluetongue in India: A review. Adv. Anim. Vet. Sci., 3(11): 605-612. [Crossref]
13. Chand, K., Biswas, S.K. and Mondal, B. (2018) Isolation and characterization of bluetongue Virus recovered from blood samples by immunoaffinity purification. Indian J. Microbiol., available from: https://link.springer.com/article/10.1007/s12088-018-0735-z. Accessed on 08-05-2018. [Crossref]
14. Attoui, H., Billoir, F., Cantaloube, J.F., Biagini, P., de Micco, P. and de Lamballerie, X. (2000) Strategies for the sequence determination of viral dsRNA genomes. J. Virol. Methods, 89(1-2): 147-158. [Crossref]
16. Thompson, J.D., Higgins, D.G., Gibson, T.J. and Clustal, W. (1994) Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic. Acids Res., 22(22): 4673-4680. [Crossref]
17. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013), MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol., 30(12): 2725-2729. [Crossref] [PubMed] [PMC]
19. Ranjan, K., Prasad, G., Kumar, P., and Prasad, M. (2014) Molecular characterization of segment 6 of bluetongue serotype 16 of sheep origin from India. Adv. Anim. Vet. Sci., 2(2): 98-103. [Crossref]
20. Koushlesh, R., Minakshi, P., Hari, M., Vishwaradhaya, T.M. and Gaya, P. (2012) Nucleotide sequence comparison of VP5 gene of Indian isolates of bluetongue virus serotype 2. Haryana Vet., 51: 29-33.
21. Kumar, P., Minakshi, P., Ranjan, K., Dalal, R. and Prasad, G. (2013) Evidence of reassortment between eastern and Western topotype strains of bluetongue virus serotype 16 (BTV-16) from India. Adv. Anim. Vet. Sci., 1(supplement 4): 14-19.22. Maan, S., Maan, N.S., Ross-Smith, N., Batten, C.A., Shaw, A.E., Anthony, S.J. and Singh, K.P. (2008) Sequence analysis of bluetongue virus serotype 8 from the Netherlands 2006 and comparison to other European strains. Virology. 377(2): 308-318. [Crossref] [PubMed]