Molecular genetic investigations of dengue epidemic potential and virulence

Abstract

Epidemic dengue and dengue hemorrhagic fever shock syndrome (DHF-SS) have emerged as major public health problems throughout the tropical and subtropical world. One of the underlying reasons for this emergence is the rapid trafficking and hyperendemic circulation of dengue viruses. Introduction of virulent dengue viruses into new geographic regions has been associated with dramatic increases in DHF-SS. Development of new molecular tools to rapidly characterize genetically dengue viruses and to detect virulent genotypes which could condition severe dengue disease was the central theme of this research. A RT-PCR SSCP (reverse transcription-polymerase chain reaction, single strand conformation polymorphism) technique was developed to assay rapidly for genetic variability and virulence biomarkers in dengue virus genomes. Distinct regions of the genome were analyzed as genetic markers, and a sequence of the prM protein gene was the more informative region to reveal genetic diversity in dengue viruses in SSCP analyses. RT-PCR SSCP was used to characterize dengue viruses from the Yucatan Peninsula in Mexico. Multiple haplotypes were documented in each one of the serotypes that circulated in the Yucatan Peninsula. Interestingly, dengue-3 virus exhibited the lowest genetic diversity, which is probably due to its recent introduction into the Yucatan. RT PCR and sequence analyses were then used to investigate the effect of microenvironment on dengue virus haplotypes. Sequences of dengue virus amplified directly from a patient serum and from a virus isolate in mosquito C6/36 cells were compared. Nucleotide changes detected in virus cDNAs obtained by direct amplification from serum were predominantly synonymous substitutions. In contrast, passage in C6/36 cells yielded nucleotida changes that were nonsynonymous, perhaps reflecting the selective pressures of passage in a new microenvironment. RT-PCR was also used to investigate the possibility that virus variants that were not detectable by conventional virus bioassays could condition DHF-SS. The composition of virus populations from patients with DF and DHF were analyzed by RT-PCR SSCP. Genetic diversity was detected in each patient specimens, but there did not seem to be associations between any specific haplotype and severe dengue disease. RT-PCR SSCP analysis is a relatively inexpensive and rapid technique for detecting genetic variability in dengue viruses. It provides a new surveillance tool to rapidly detect introduction of new and potentially virulent dengue viruses into an area. Such information could be used by resource limited public health agencies to focus control efforts in on areas posing greatest risk for severe dengue disease.