Rapid chemical mapping of dengue virus variability using RNA isolated directly from cells.

Osmium tetroxide and hydroxylamine (in combination with piperidine) have previously been shown to cleave mismatched T and C bases, respectively, in DNA.DNA heteroduplexes. In this work we report that mismatched T and C bases were similarly cleaved in DNA.RNA heteroduplexes of nucleic acids derived from different strains of dengue virus type 2. Further, some matched T or C bases one or two bases from mismatches were also chemically reactive and thus cleavable as detected by minor bands. Cleavages both at and near mismatches combined to generate a simply obtained pattern of difference between virus strains that could be used as a fingerprint of a given virus relative to another. The patterns obtained using viral RNA of one strain hybridized with the cDNA of another were similar for RNA prepared from purified virions and for total RNA extracted from infected cells. Use of probes of both senses should detect all differences. Two sequenced (NGC and PUO-218) and one unsequenced (D80-100) strains of virus were compared in these studies. The analyses allowed proof reading of the differences between NGC and PUO-218, ascertained from nucleotide sequencing, and demonstrated that D80-100 is more similar to PUO-218 than to NGC.