PCR STRATEGY FOR IDENTIFICATION AND DIFFERENTIATION OF SMALLPOX AND OTHER ORTHOPOXVIRUS

Rapid identification and differentiation of orthopoxviruses by PCR wer e achieved with primers based on genome sequences encoding the hemaggl utinin (HA) protein, an infected-cell membrane antigen that distinguis hes orthopoxviruses from other poxvirus genera. The initial identifica tion step used a primer pair of consensus sequences for amplifying an HA DNA fragment from the three known North American orthopoxviruses (r accoonpox, skunkpox, and volepox viruses), and a second pair for ampli fying virtually the entire HA open reading frame of the Eurasian-Afric an orthopoxviruses (variola, vaccinia, cowpox, monkeypox, camelpox, ec tromelia, and gerbilpox viruses). RsaI digest electropherograms of the amplified DNAs of the former subgroup provided species differentiatio n, and TaqI digests differentiated the Eurasian-African orthopoxviruse s, including vaccinia virus from the vaccinia virus subspecies buffalo pox virus. Endonuclease HhaI digest patterns distinguished smallpox va riola major viruses from alastrim variola minor viruses. For the Euras ian-African orthopoxviruses, a confirmatory step that used a set of hi gher-sequence-homology primers was developed to provide sensitivity to discern individual virus HA DNAs from cross-contaminated orthopoxviru s DNA samples; TaqI and HhaI digestions of the individual amplified HA DNAs confirmed virus identity. Finally, a set of primers and modified PCR conditions were developed on the basis of base sequence differenc es within the HA genes of the 10 species, which enabled production of a single DNA fragment of a particular size that indicated the specific species.