Genetic characterization of Orf virus isolates targeting PACR gene: Optimization of a diagnostic PCR for host-specific differentiation of goat and sheep origin isolates.

Abstract

Parapoxviruses are known for its unique feature of carrying a number of virulence genes in their large genome and deploy them as a weapon to evade host immune response. One of its unique gene is Poxviral anaphase promoting complex regulator (PACR) from the left terminal of the genome named as ORF014. This PACR protein basically mimics the subunit, APC11 of anaphase promoting complex (APC)/cyclosome of the host cell. Orf virus is the prototypic member of Parapoxvirus genera and causes mostly localized infection in animals as well as humans. Considering the importance of gene in ORFV replication and virulence, and the endemicity of the disease in the country affecting sheep and goats, we designed a study to genetically characterize different ORFV isolates from different geographical parts of India. Interestingly, multiple sequence alignment of the gene revealed that sheep and goat isolates encoded different ORF lengths of gene i.e. 282bp and 288bp respectively with an additional two amino acids namely valine and lysine at 7th and 8th position in ORFV isolates of goat origin only. Likewise, the difference was also reflected in phylogenetic tree as the sheep and goat isolates of ORFV were clustered into two groups. Based on these salient findings, the difference in sequences was exploited to design species-specific primers for the development of diagnostic PCR to differentiate sheep and goats ORFV isolates. On evaluation of clinical samples and cell culture isolates of ORFV, PCR clearly distinguished them as sheep and goat isolates. The comparable relative specificity and sensitivity of the ORF014 based diagnostic PCR with in-use PCR also revealed a refine potential for epidemiological insights of parapoxviruses. This study seems to have proposed for the first time a basis for differentiating the ORFV isolates by their origin of species.