A total of 10 wart samples from affected cattle were aseptically collected. The Majority of cutaneous warts were of variable sizes, rough or coarse in texture, grayish or flesh-colored, irregular in shape (dome or button), or resembling cauliflower-like masses, grossly and elevated from the skin surface by a broad base. The wart samples were stored at –20 °C until used in further research. The details of the samples are presented in Table 1.
Extraction of viral DNA and Detection by Polymerase Chain Reaction and Sequencing
DNA was extracted from wart tissue samples using the Genomic DNA Mini Kit (Qiagen) according to the manufacturer’s protocol. PCR was performed to detect the presence of BPV serotype 1 and 2. Primers were used for BPV-1(Forward: 5’-gga gcg cct gct aac tat agg a-3’; Reverse: 5’-atc tgt tgt ttg ggt ggt gac-3’) of 301 bp and BPV-2 (Forward: 5’-gttata cca ccc aaa gaa gac cct-3’; Reverse; 5’-ctg gtt gcaaca gct ctc ttt ctc-3’) of 165 bp as described earlier (Leishangthem et al., 2008). The reaction was set up as follows: 2 µl Template DNA, 10 µl 5X PCR buffer , 3 µl MgCl2 , 1 µl dNTPs, 1 µl Taq polymerase, 2 µl Forward Primer, 2 µl Reverse Primer, 29 µl Nuclease Free Water to make a volume of 50 µl. All these ingredients were mix properly by vortexing. The PCR reaction was done as per the following: Initial denaturation at 94 °C for 5 minutes, followed by 30 cycles of 1 minute at 94 °C, 2 minutes annealing at 50 °C, 2 minutes extension at 72 °C, and 10 minutes final extension at 72 °C.5 µl of PCR product was then mixed with 3 µl of bromophenol blue (6X) and the PCR product was run via gel electrophoresis using 1.5% agarose gel and visualized by using a UV trans illuminator (Syngene G box, UK). The obtained PCR product was purified and sequenced for the confirmation of the PCR product.
Gene Sequencing and Phylogenetic Analysis
The PCR products were directly sequenced from both ends by Cellbiosis Pvt. Ltd. for the confirmation of the BPV-1 and phylogenetic analysis. The obtained sequences from the field sample were subjected to manual analysis and BLAST analysis to find out the sequence similarity. The obtained sequence is further aligned with reference sequences by the CLUSTAL W pairwise and multiple alignment method. The nucleotide sequences of the BPV-1 gene fragment of papillomavirus were aligned using MEGA 6.0 software and a phylogeny was constructed.