During the dengue virus type 3 (DENV-3) epidemic that occurred in Havana in 2001 to 2002, severe disease was associated with the infection sequence DENV-1 followed by DENV-3 (DENV-1/DENV-3), while 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 time points 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, while the minor lineage is related to Venezuelan strains. According to consensus sequences, relatively few nonsynonymous mutations were observed; only one was fixed during the epidemic at position 4380 in the NS2B gene. Intrahost genetic analysis indicated that a significant minor population was selected and became predominant toward 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 nonstructural genes. An increasing trend of genetic diversity toward the end of the epidemic was observed only for synonymous variant allele rates, with higher variability in secondary cases. Remarkably, significant intrahost genetic variation was demonstrated within the same patient during the course of secondary infection with DENV-1/DENV-3, including changes in the structural proteins premembrane (PrM) and envelope (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.IMPORTANCE Based on the evidence that DENV fitness is context dependent, our research has focused on the study of viral factors associated with intraepidemic increasing severity in a unique epidemiological setting. Here, we investigated the intrahost genetic diversity in acute human samples collected at different time points during the DENV-3 epidemic that occurred in Cuba in 2001 to 2002 using a deep-sequencing approach. We concluded that greater variability in significant minor populations occurred as the epidemic progressed, particularly in the nonstructural genes, with higher variability observed in secondary infection cases. Remarkably, for the first time significant intrahost genetic variation was demonstrated within the same patient during the course of secondary infection with DENV-1/DENV-3, including changes in structural proteins. These findings indicate that high-resolution approaches are needed to unravel molecular mechanisms involved in dengue pathogenesis.

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Objective To study the distribution of vertical transmission of dengue viruses in field‐collected Aedes aegypti larvae in the municipality of Arroyo Naranjo in Havana, Cuba. Methods Aedes aegypti larvae and pupae were collected monthly between September 2013 and July 2014 in the seven Municipal Health Areas of Arroyo Naranjo. Pools formed of 30–55 larvae were examined through PCR and sequencing to detect the presence of each serotype. Results We analysed 111 pools of larvae and pupae (4102 individuals) of which 37 tested positive for at least one DENV. More than one DENV type was observed in 10 of the 37 positive pools. Infected pools were detected every month, except in January, suggesting a sustained circulation of DENV in the vector populations. DENV‐1 and DENV‐3 were the most frequent and dispersed, though all four DENV types were detected. Nucleotide sequencing from positive pools confirmed RT‐PCR results for DENV‐1 (genotype V), DENV‐3 (genotype III) and DENV‐4 (genotype II). DENV‐2 was detected by RT‐PCR but could not be confirmed by nucleotide sequencing. Conclusion Our study of the distribution of natural vertical transmission of dengue virus types highlights extrinsic virus activity patterns in the area and could be used as a new surveillance tool.

Until now, three genotypes of Zika virus (ZIKV) have been detected (two African lineages and one Asian lineage). After the declaration of Public Health Emergency of International Concern issued by The Pan American Health Organization and the World Health Organization authors from some Latin American countries have identified the Asian genotype as the lineage responsible for the Zika epidemic in the western hemisphere. However, data from the Caribbean are sparse, and there is no published data regarding the genotypes that produced isolated outbreaks in Cuba. Aiming to realize the molecular characterization of ZIKV in Cuba, we will sequence by next-generation sequencing the full genome of the ZIKV identified in samples from Cuban patients of different provinces in which ZIKV produced outbreaks. All samples required for this study have been collected during the molecular surveillance of Arboviral diseases conducted at the National Reference Laboratory at Pedro Kourí Tropical Medicine Institute. Viral RNA will be purified from urine and serum samples collected from patients with confirmed ZIKV infection by real time PCR. Using evolutionary dynamics studies, we will map the spread of a virus or of particular variants in time and space in order to understand how frequently ZIKV has been introduced into Cuba. Moreover, we will evaluate the amino acid diversity of each ZIKV proteins. Further, we will evaluate the population dynamics of ZIKV in samples from patients with varying clinical outcomes. The results will allow us to characterize the ZIKV genome and its evolution into the Cuban population that would also have impact for vaccine development, diagnosis, and pathogenesis studies.