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.