Evidence for inter- and intra-genotypic variations in dengue serotype 4 viruses representing predominant and non-predominant genotypes co-circulating in Thailand from 1977 to 2001.

In order to characterize viral genetic variation among predominant and non-predominant genotypes of Thai dengue serotype 4 viruses (DENV-4) and follow mutations that occur during virus evolution, we performed a comparative analysis of the complete genomic sequences of six DENV-4 isolates representing three genotypes (I, IIA, and III) co-circulating in Thailand over a 24-year period. The results revealed [1] remarkable genetic variation in the viral genome between predominant and non-predominant genotypes; [2] inter-genotype-specific amino acid and nucleotide mutations in most regions of the viral genome; [3] more amino acid and nucleotide substitutions in later as compared to earlier isolates for predominant genotype I strains; [4] a single nucleotide substitution at nucleotide position 77 of the 5-'NTR of two non-predominant genotype III strains that disrupted a small conserved 3'stem-loop (SL) in the cyclization sequence required for virus replication; [5] a high degree of conservation of PrM/M and NS2B proteins, and the 5'-NTR in predominant genotype I strains with no mutations observed over the 24-year period of observation; and [6] no molecular markers that appeared to correlate with disease severity. Several mutations identified in this study might have a significant impact on the persistence of virus in the population, including one in the 5'-NTR that disrupted a small, highly conserved 3'SL2 structure at the terminus of the cyclized 5'-3' RNA sequences in two genotype III strains, and three amino acid (aa) charge change mutations in the E and NS5 proteins of genotype I strains. The conserved 3'-SL structure may be a target for antiviral drug development.