Sequence analysis of C18L gene of buffalopox virus: PCR strategy for specific detection and differentiation of buffalopox from orthopoxviruses

Abstract

The C18L gene of buffalopox virus (BPXV), a homologue of Vaccinia virus (VACV), which encodes the ankyrin repeat protein was sequenced and analyzed to elucidate its genetic relationship with VACVs and also to devise a PCR strategy for the diagnosis of buffalopox. PCR amplification and sequencing of the C18L gene of BPXV-BP4 revealed the truncated ankyrin protein with a coding region consisting of only 50 amino acids (aa) as against a 150-aa-long peptide expressed by VACV (Copenhagen strain). BPXV-specific primers were designed and employed for sequence determination of six Indian BPXV isolates. Comparative sequence analyses of the C18L gene of BPXV isolates with that of published data of the genus orthopox viruses (OPXVs) revealed 71.2–77.3% homology at the nucleotide (nt) and 35.5–67.1% at the aa levels with VACVs. Phylogenetic analyses based on deduced aa sequences of all BPXVs showed clustering in a single group which is distinct from VACVs. Furthermore, PCR performed on the C18L gene (conventional and TaqMan) and duplex PCR based on C18L and DNA polymerase genes were developed using purified viral DNA for the specific detection and differentiation of BPXV from other OPXVs. This resulted in a specific amplicon of 368 bp from the C18L gene of BPXV. Duplex PCR resulted in 96 and 368 bp products from DNA Pol and C18L genes of BPXV and only a 96-bp amplicon of the DNA pol gene in other OPXVs. These assays were employed successfully for the differentiation of BPXV from Orthopox, Capripox and Parapox viruses as it was found to be specific only for BPXV. The authenticity of the amplicons was confirmed based on their size in agarose gel electrophoresis and sequence analysis. In contrast to the conventional PCR, the TaqMan assay was found to be rapid, specific and 100 times more sensitive with a detection limit as low as 5 pg of viral DNA. In addition, the assays were evaluated with DNA extracted from suspected clinical scab materials obtained from buffaloes, cows and human beings.