Differentiation of sheeppox and goatpox viruses by polymerase Chain reaction-restriction fragment length polymorphism
Gnanavel Venkatesan1, Vinayagamurthy Balamurugan, Revaniah Yogisharadhya
1Division of Virology, Indian Veterinary Research Institute, Nainital, Mukteswar 263138, Uttarakhand, India.
Abstract:
In the present study, the partial gene sequences of P32 protein, an immunogenic envelope protein of Capripoxviruses (CaPV), were analyzed to assess the genetic relationship among sheeppox and goatpox virus isolates, and restriction enzyme specific PCR-RFLP was developed to differentiate CaPV strains. A total of six goatpox virus (GTPV) and nine sheeppox virus (SPPV) isolates of Indian origin were included in the sequence analysis of the attachment gene. The sequence analysis revealed a high degree of sequence identity among all the Indian SPPV and GTPV isolates at both nucleotide and amino acid levels. Phylogenetic analysis showed three distinct clusters of SPPV, GTPV and Lumpy skin disease virus (LSDV) isolates. Further, multiple sequence alignment revealed a unique change at G120A in all GTPV isolates resulting in the formation of Dra I restriction site in lieu of EcoR I, which is present in SPPV isolates studied. This change was unique and exploited to develop restriction enzyme specific PCR-RFLP for detection and differentiation of SPPV and GTPV strains. The optimized PCR-RFLP was validated using a total of fourteen (n=14) cell culture isolates and twenty two (n=22) known clinical samples of CaPV. The Restriction Enzyme specific PCR-RFLP to differentiate both species will allow a rapid differential diagnosis during CaPV outbreaks particularly in mixed flocks of sheep and goats and could be an adjunct/supportive tool for complete gene or virus genome sequencing methods.
Insights
Genetic analysis of Capripoxviruses (CaPV) reveals high similarity between sheeppox virus (SPPV) and goatpox virus (GTPV) in India. A new PCR-RFLP method effectively differentiates SPPV and GTPV strains for rapid diagnosis.
Area of Science:
- Veterinary Virology
- Molecular Biology
- Genetics
Background:
- Capripoxviruses (CaPV) cause significant diseases in sheep and goats.
- Distinguishing between sheeppox virus (SPPV) and goatpox virus (GTPV) is crucial for effective disease management.
- Previous methods for CaPV strain differentiation were often time-consuming.
Purpose of the Study:
- To analyze the genetic relationship between Indian SPPV and GTPV isolates.
- To develop a rapid and accurate method for differentiating SPPV and GTPV strains.
- To aid in the differential diagnosis of CaPV outbreaks.
Main Methods:
- Partial P32 protein gene sequencing of six GTPV and nine SPPV isolates.
- Phylogenetic analysis of nucleotide and amino acid sequences.
- Development and validation of a restriction enzyme specific PCR-RFLP assay.
Main Results:
- High sequence identity observed between Indian SPPV and GTPV isolates at nucleotide and amino acid levels.
- Phylogenetic analysis revealed distinct clusters for SPPV, GTPV, and Lumpy Skin Disease Virus (LSDV).
- A unique G120A mutation in GTPV created a Dra I restriction site absent in SPPV, enabling differentiation via PCR-RFLP.
Conclusions:
- The developed PCR-RFLP method accurately differentiates SPPV and GTPV strains.
- This technique offers a rapid diagnostic tool for CaPV outbreaks, especially in mixed-species flocks.
- PCR-RFLP serves as a valuable adjunct to full genome sequencing for CaPV diagnostics.
