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

Sheeppoxvirus (SPV), goatpoxvirus (GPV) and lumpy skin disease virus (LSDV) of cattle belong to the Capripoxvirus genus of the Poxviridae family and c an cause significant economic losses in countries where they are endemic. C apripox diagnosis by classical virological methods dependent on live caprip ox 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 o n 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 d eveloped that differentiated between SPV and LSDV on the basis of unique re striction sites in the corresponding PCR fragments. The vaccinia virus H3L homolog was identified as the capripoxvirus P32 antigen. The P32 proteins o f 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 t erminus and was membrane associated but could be solubilised in detergent a nd used as trapping antigen in an antibody detection ELISA. The ELISA was s pecific for capripoxvirus as only sera from sheep infected with capripoxvir us but not orf or vaccinia virus reacted with the capripoxvirus P32 antigen . (C) 1999 Elsevier Science B.V. All rights reserved.