Rapid characterization of genetic diversity among twelve dengue-2 virus isolates by single-strand conformation polymorphism analysis.
Single-strand conformation polymorphism (SSCP) analysis was used to characterize genetic polymorphisms among 12 isolates of dengue-2 virus, which were previously genetically characterized by RNase T1 oligonucleotide mapping and by sequencing the viral envelope (E) gene. Specific cDNA fragments from the dengue-2 isolates were amplified by the reverse transcriptase-polymerase chain reaction. The viral E, premembrane (prM), and nonstructural 5 (NS5) gene cDNAs of 291 basepairs (bp), 291 bp, and 201 bp, respectively, were denatured, rapidly chilled to promote intrastrand reassociation, electrophoretically separated on nondenaturing polyacrylamide gels, and SSCP patterns were observed by silver staining. The SSCP analysis revealed polymorphisms among a number of dengue-2 isolates from the same topotype, and these were markedly different between isolates of different topotype (distinct genetic group). Comparison of nucleotide sequence and SSCP analyses of the 291-bp E cDNA demonstrated that virus isolates that produced identical SSCP patterns contained 0-7 nucleotide substitutions, whereas isolates that showed different SSCP patterns contained 4-25 nucleotide substitutions. Positive predictive value and negative predictive value as measures of certainty for predicting identical and different sequences were 26% and 100%, respectively. The SSCP patterns of the 12 dengue-2 isolates suggested greater genetic variation in the prM gene region than in either the E or NS5 gene regions. The SSCP analyses should allow easy, sensitive, and rapid screening of dengue viruses isolates and the assessment of variation at a number of sites in the virus genome. Additionally, SSCP screening of dengue-2 virus for genetic variability may reveal the introduction of new viral genotypes in a given geographic area. These genetic variants of the virus could serve as markers of the epidemic potential of the virus strain.
Farfan
J A
JA
Department of Microbiology, Colorado State University, Fort Collins 80523, USA.
Olson
K E
KE
Black
W C
WC
Gubler
D J
DJ
Beaty
B J
BJ
D018076:DNA, Complementary / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D003716:Dengue Virus / Q000145:classification / Q000235:genetics*; D014644:Genetic Variation; D005838:Genotype; D018807:Polymorphism, Single-Stranded Conformational; D015203:Reproducibility of Results
D018076:DNA, Complementary; D004279:DNA, Viral