Increasing Clinical Severity during a Dengue Virus Type 3 Cuban Epidemic: Deep Sequencing of Evolving Viral Populations.

UNLABELLED: 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.

Serotype-specificity of recombinant fusion proteins containing domain III of dengue virus.

Here, the antigenic specificity of the recombinant fusion proteins containing aa 286-426 of the dengue envelope protein fused to P64k from Neisseria meningitidis and the cross-reactive antibody response induced in immunized mice and monkeys were evaluated. The anti-dengue mice antibodies showed a higher reactivity to the homologous recombinant proteins compared to the wide cross-reactivity observed by dot blot to the viral antigens. The immune response induced by the recombinant proteins in mice and monkeys, was highly serotype specific. The serotype-specificity associated with these recombinant proteins in addition to the high antigenicity, immunogenicity and protecting capacity suggest their advantages as possible vaccine candidates.

Immunogenicity and protective efficacy of a recombinant fusion protein containing the domain III of the dengue 1 envelope protein in non-human primates.

Recombinant fusion proteins containing the aa 286-426 of the dengue envelope protein fused to P64k protein from Neisseria meningitidis have been previously reported. Particularly, the immunogenicity and protective capacity of the dengue 2 recombinant protein was demonstrated in Macaca fascicularis monkeys. Here we evaluate the recombinant fusion protein containing the domain III of the dengue 1 envelope protein (PD10) in non-human primates (M. fascicularis and rhesus monkeys) and compare the effect of aluminum hydroxide and Freund adjuvant on the immunity induced. The PD10 protein emulsified in Freund adjuvant was highly immunogenic in M. fascicularis and rhesus monkeys. Following dengue 1 virus challenge, animals immunized with PD10 in Freund adjuvant were protected from viremia. However, monkeys receiving PD10 in aluminum hydroxide developed a poor antibody response and were not protected from viral challenge. These preliminary experiments are encouraging. Other formulations or vaccine schedules are being studied in an attempt to find regimens that enhance immunological protection.

Primary and secondary infections of Macaca fascicularis monkeys with Asian and American genotypes of dengue virus 2.

The goal of this study was to compare the immune response and the protection capacity induced by the dengue virus 2 (DENV-2) American and Asian genotypes in Macaca fascicularis monkeys. Animals were infected with American or Asian DENV-2 strains and challenged 1 year later with a DENV-2 Asian genotype strain. The viremia and monkey antibody levels were similar for the different strains after primary and secondary infection; however, the functionality of the antibody response was different. A limited viral replication was demonstrated after the secondary infection in all the monkeys. No virus was isolated in tissue culture, while reverse transcription-PCR showed a late positive reaction in four of five challenged monkeys. The immunoglobulin M response pattern and the detection of antibodies to specific proteins by Western blotting supported the protection data. Despite the demonstration of the protective effect after homologous challenge, a strong anamnestic antibody response was observed.