Increasing clinical severity during a dengue virus type 3 Cuban epidemic: deep sequencing of evolving viral populations

During the DENV-3 epidemic occurred in Havana in 2001-2002, severe disease was associated with the infection sequence DENV-1/DENV-3, whilst the sequence DENV-2/DENV-3 was associated with mild/asymptomatic infections. To determine the role of the virus in the increasing severity demonstrated during the epidemic serum samples collected at different point times were studied. A total of 22 full-length sequences were obtained using a deep sequencing approach. Bayesian phylogenetic analysis of consensus sequences revealed that two DENV-3 lineages were circulating in Havana at that time, both grouped within genotype III. The predominant lineage is closely related to Peruvian and Ecuadorian strains, whilst the minor lineage is related to Venezuelan strains. According to consensus sequences, relatively few non-synonymous mutations were observed; only one was fixed during the epidemic at position 4380 in the NS2B gene. Intra-host genetic analysis indicated that a significant minor population was selected and became predominant towards the end of the epidemic. In conclusion, greater variability was detected during the epidemic's progression in terms of significant minority variants, particularly in the non-structural genes. An increasing trend of genetic diversity towards the end of the epidemic was only observed for synonymous variant allele rates, with higher variability in secondary cases. Remarkably, significant intra-host genetic variation was demonstrated within the same patient during the course of secondary infection DENV-1/DENV-3, including changes in the structural proteins PrM and E. Therefore, the dynamic of evolving viral populations in the context of heterotypic antibodies could be related to the increasing clinical severity observed during the epidemic.