SEQUENCE ALTERATIONS WITHIN AND DOWNSTREAM OF THE A-TYPE INCLUSION PROTEIN GENES ALLOW DIFFERENTIATION OF ORTHOPOXVIRUS SPECIES BY POLYMERASE CHAIN-REACTION

A PCR protocol was established that not only allows the detection of, but also the differentiation of species of the genus Orthopoxvirus. Th is assay was accomplished by the selection of oligonucleotides located within the gene that encodes the A-type inclusion protein of cowpox v irus. The primer pair flanked a region exhibiting distinct and specifi c DNA deletions in the corresponding sequences of vaccinia, mousepox, monkeypox and camelpox virus. For this reason, PCR resulted in DNA fra gments of different sizes. The presented PCR protocol, combined with B g/II restriction digests, allowed the unequivocal assignment of 42 ort hopoxvirus (OPV) strains and isolates to the correct OPV species. The resulting classification corresponded exactly with known biological da ta for the OPV strains investigated. Furthermore, 13 out of 22 cowpox virus isolates could be subtyped by the presence or absence of a small Bg/II fragment. DNA sequencing showed that the lack of this Bg/II fra gment was caused by a deletion of 72 nucleotides.