A PCR-based assay for detection of emerging vaccinia-like viruses isolated in Brazil

We report 2 strategies to identify Brazilian vaccinia virus (VACV) isolates related to Cantagalo virus (CTGV) based on the amplification of the hemagglutinin (HA) gene by the polymerase chain reaction (PCR). One PCR protocol was combined with restriction analysis using the endonuclease SnaB I, generating a unique digestion pattern for CTGV amplicons. The restriction profile could identify 41 CTGV-related isolates in 43 clinical specimens and clearly differentiated them from other orthopoxviruses and strains of VACV. Alternatively, we used a 1-step PCR assay with primers that specifically targeted CTGV HA sequence. This protocol produced similar results more rapidly than the 1st strategy, eliminating post-PCR procedures. The results were supported by Western blot analysis of the viral protein profile in infected cells. Both PCR-based methods enabled a fast, sensitive, and cost-effective detection of new isolates of VACV related to CTGV directly from clinical samples without requiring virus isolation.