A capripoxvirus detection PCR and antibody ELISA based on the major antigen P32, the homolog of the vaccinia virus H3L gene

• Published in: Journal of immunological methods 1999-07, Vol.227 (1), p.187-196
• Main Authors: Heine, H.G, Stevens, M.P, Foord, A.J, Boyle, D.B
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antibodies, Viral - analysis; Antibody detection ELISA; Antigens, Viral - genetics; Antigens, Viral - immunology; Biological and medical sciences; Capripoxvirus; Capripoxvirus - isolation & purification; Cloning, Molecular; DNA, Viral - chemistry; Enzyme-Linked Immunosorbent Assay; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Goatpox virus; Immunodominant antigen P32; Lumpy skin disease virus; Microbiology; Molecular Sequence Data; Polymerase Chain Reaction; Sheep; Sheeppox virus; Sheeppoxvirus; Techniques used in virology; Vaccinia virus; Vaccinia virus - genetics; Virology; Virus detection PCR
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(99)00072-1

Sheeppoxvirus (SPV), goatpoxvirus (GPV) and lumpy skin disease virus (LSDV) of cattle belong to the Capripoxvirus genus of the Poxviridae family and can cause significant economic losses in countries where they are endemic. Capripox diagnosis by classical virological methods dependent on live capripox virus is not suitable in countries such as Australia where the virus is exotic and live virus is not available. To develop diagnostic tests based on recombinant material, we cloned and sequenced a 3.7 kb viral DNA fragment of SPV that contained open reading frames homologous to the vaccinia virus J6R, H1L, H2R, H3L and H4L genes. A capripoxvirus specific PCR assay was developed that differentiated between SPV and LSDV on the basis of unique restriction sites in the corresponding PCR fragments. The vaccinia virus H3L homolog was identified as the capripoxvirus P32 antigen. The P32 proteins of SPV and LSDV were expressed in Escherichia coli as a fusion protein with a poly-histidine tag and affinity purified on metal binding resin. The full-length P32 protein contained a transmembrane region close to the carboxy terminus and was membrane associated but could be solubilised in detergent and used as trapping antigen in an antibody detection ELISA. The ELISA was specific for capripoxvirus as only sera from sheep infected with capripoxvirus but not orf or vaccinia virus reacted with the capripoxvirus P32 antigen.