1Defence Research Development Organization, Gwalior, India.
2Centre for Animal Disease Research and Diagnosis, IVRI, Izatnagar.
Bluetongue is an infectious, non-contagious and Culicoides midge-borne viral disease of domestic and wild ruminants. It occurs throughout the tropical semitropical and temperate regions of the world. It is primarily a disease of sheep and wild ruminants whereas in cattle, buffaloes and goats it is in sub-clinical form. Cattle mostly serve as the reservoir and amplifying host of bluetongue virus. The staggering economic losses to animal husbandry around the world are mainly attributed to high morbidity and mortality and reduced production of milk, meat and wool. The disease is endemic in India and lots of outbreaks are reported throughout the country around the year. Based on the serosurveillance studies, 21 out of 24 serotypes have been reported in India. Out of 10 viral proteins very little or no information has been available about the genetic makeup of the bluetongue virus isolates. Outer capsid protein VP2 coded by L2 gene is of prime importance as it exhibits serotype specificity, elicits neutralizing antibodies and confers protection against the disease. The PCR amplification of the bluetongue virus dsRNA was carried out on two stages (1) cDNA from dsRNA by reverse transcriptase (2) cDNA amplification by PCR. Initially the genome of the bluetongue virus was confirmed by using the VP7 serogroup specific primers followed by using L2 gene (VP2) specific primers. A PCR product of 658 bp in length was amplified where L2 gene specific primers were used. The amplicon was subsequently cloned in pGEM-T vector using DNA ligase enzyme followed by transformation of E. coli JM 109 by PEG method and plated on LB agar plates containing ampicillin, IPTG and x-gal. The white colonies were picked up, grown in LB agar and plasmid DNA was extracted. The extracted DNA was digested with Apa I and Not I RE and insert of desired length visualized in the agarose gel under UV transilluminator indicating the authenticity of the part of the L2 gene of bluetongue virus serotype 23.
