New PCR assays for identification of smallpox virus

Overview

Pulford D.J., Damon, I.^δ, Ulaeto, D.

Biomedical Sciences, Dstl Porton Down, Salisbury, Wiltshire. SP40JQ. ^δCDC, United States

A simple, rapid, reliable and sensitive diagnostic assay would be essential for effective medical and public health countermeasures in the event of a variola virus release. The first genetic techniques used to specifically identify variola virus used RFLP analysis of viral genomic DNA, but this approach is slow to perform.

PCR based methods offer an alternative for rapid diagnosis, and RFLP assays of PCR amplicons from the A-type inclusion protein or the haemagglutinin protein genes have been described. Orthopoxvirus (OPV) whole genome sequences are now available for the principle species of the genus including ectromelia, camelpox, monkeypox, cowpox, vaccinia and variola viruses, an important resource for designing new PCR based assays.

We describe here the results from sequencing genes from several Orthopoxvirus (OPV) species, identifying variola virus specific single nucleotide polymorphisms (SNPs) in these genes, and the development of multiplex PCR assays that can specifically identify variola virus DNA or can determine the presence of another OPV genome. We describe the performance of these assays against an extensive collection of vaiola virus DNA samples tested at the CDC. We demonstrate their use with archived clinical samples and their ability to detect and differentiate between major and minor strains of variola virus.