RAPID CHARACTERIZATION OF GENETIC DIVERSITY AMONG 12 DENGUE-2 VIRUS ISOLATES BY SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS

Single-strand conformation polymorphism (SSCP) analysis was used to ch aracterize genetic polymorphisms among 12 isolates of dengue-2 virus, which were previously genetically characterized by RNase T1 oligonucle otide mapping and by sequencing the viral envelope (E) gene. Specific cDNA fragments from the dengue-2 isolates were amplified by the revers e transcriptase-polymerase chain reaction. The viral E, premembrane (p rM), and nonstructural 5 (NS5) gene cDNAs of 291 basepairs (bp), 291 b p, and 201 bp, respectively were denatured, rapidly chilled to promote intrastrand reassociation, electrophoretically separated on nondenatu ring polyacrylamide gels, and SSCP patterns were observed by silver st aining. The SSCP analysis revealed polymorphisms among a number of den gue-2 isolates from the same topotype, and these were markedly differe nt between isolates of different topotype (distinct genetic group). Co mparison of nucleotide sequence and SSCP analyses of the 291-bp E cDNA demonstrated that virus isolates that produced identical SSCP pattern s contained 0-7 nucleotide substitutions, whereas isolates that showed different SSCP patterns contained 4-25 nucleotide substitutions. Posi tive predictive value and negative predictive value as measures of cer tainty for predicting identical and different sequences were 26% and 1 00%, respectively. The SSCP patterns of the 12 dengue-2 isolates sugge sted greater genetic variation in the prM gene region than in either t he E or NS5 gene regions. The SSCP analyses should allow easy, sensiti ve, and rapid screening of dengue viruses isolates and the assessment of variation at a number of sites in the virus genome. Additionally, S SCP screening of dengue-2. virus for genetic variability may reveal th e introduction of new viral genotypes in a given geographic area. Thes e genetic variants of the virus could serve as markers of the epidemic potential of the virus strain.