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Isolation and Characterization of Bluetongue Virus Recovered from Blood Samples by Immunoaffinity Purification

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Abstract

An immunoaffinity chromatography (IAC) method was optimized for the selective capture of bluetongue virus (BTV) from blood samples and isolation of the virus in cell culture. The antibody against BTV core particles (lacking the outer capsid proteins VP2 and VP5) was used for the optimization of IAC technique. The antibody against BTV core particle was conjugated with Protein A-virus complex and the complex was dissociated using elution buffer (4 M MgCl2 with 75 mM HEPES, pH 6.5). The optimized IAC method specifically purified the BTV without capturing other commonly infecting small ruminant’s viruses like gaotpox virus (GTPV), sheeppox virus (SPPV), Peste des petits ruminants virus (PPRV) and Foot and mouth disease virus (FMDV). The blood samples (n = 22), positive for BTV antigen in sandwich-ELISA were attempted for virus isolation in the BHK-21 cell using the optimized IAC method. A total of seven BTV were isolated by selective capturing of the virion particles. The isolated viruses were characterized by RNA-PAGE, sequence analysis and serum neutralization test (SNT). Electropherotypic analysis of viral dsRNA in the RNA-PAGE revealed the presence of ten dsRNA segments characteristic of BTV. Out of seven isolates, four isolates were identified as BTV-1 and three isolates were identified as BTV-16 based on nucleotide sequences of segment-2. Phylogenetic analysis of segment-2 nucleotide sequence segregated BTV-1 and BTV-16 isolates to monophyletic cluster at close proximity to other eastern topotype. In SNT, hyperimmune serum (HIS) against BTV-1 neutralized the four BTV-1 isolates up to a titer > 256 and HIS against BTV-16 neutralized the three BTV-16 isolates up to a titer > 128. The IAC technique will be useful for the selective capture of BTV from mixed infection (BTV with other small ruminant’s viruses) and isolation from blood sample having low viral load by enrichment.

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Acknowledgements

This work has been supported by an ICAR (No. F.13 (1)/2000-ASRIV) project—All India Network Programme on Bluetongue. The authors wish to thank the Director ICAR-IVRI; Head, Division of Virology and the Coordinator, AINP-BT project for providing the facilities.

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Authors and Affiliations

  1. Division of Virology, ICAR-Indian Veterinary Research Institute, Mukteswar Campus, Nainital, Uttarakhand, 263 138, India

    Karam Chand & Sanchay K. Biswas

  2. Eastern Regional Station, ICAR-Indian Veterinary Research Institute, 37, Belgachia Road, Kolkata, West Bengal, 700 037, India

    Bimalendu Mondal

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  1. Karam Chand
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  2. Sanchay K. Biswas
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  3. Bimalendu Mondal
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Correspondence to Karam Chand.

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Chand, K., Biswas, S.K. & Mondal, B. Isolation and Characterization of Bluetongue Virus Recovered from Blood Samples by Immunoaffinity Purification. Indian J Microbiol 58, 433–439 (2018). https://doi.org/10.1007/s12088-018-0735-z

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  • DOI: https://doi.org/10.1007/s12088-018-0735-z

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