Rosmari Rodriguez-Roche

Neutralizing Antibodies after Infection with Dengue 1 Virus

Severity of disease is markedly increased when infection with dengue virus type 2 follows infection with dengue virus type 2 by an interval of 20 years.

Understanding the Dengue Viruses and Progress towards Their Control

Traditionally, the four dengue virus serotypes have been associated with fever, rash, and the more severe forms, haemorrhagic fever and shock syndrome. As our knowledge as well as understanding of these viruses increases, we now recognise not only that they are causing increasing numbers of human infections but also that they may cause neurological and other clinical complications, with sequelae or fatal consequences. In this review we attempt to highlight some of these features in the context of dengue virus pathogenesis. We also examine some of the efforts currently underway to control this “scourge” of the tropical and subtropical world.

Dengue virus type 2 in Cuba, 1997: conservation of E gene sequence in isolates obtained at different times during the epidemic

Summary.It was recently reported that disease severity increased during the 1997 Cuban dengue 2 virus epidemic and it was suggested that this might be explained by the appearance of neutralization resistant escape mutants. We investigated these observations and ideas by sequencing 20 dengue 2 virus isolates obtained during the early (low case fatality rate) and the late (high case fatality rate) phases of the outbreak. Our results showed total conservation of the E gene sequence for these isolates suggesting that the selection of envelope gene escape mutants was not the determinant of increased disease severity. Alignment of these sequences with those available in GenBank, followed by Maximum likelihood phylogenetic analysis generated a tree, which indicated that our isolates are closely related to the virus that circulated in Venezuela in 1997/98 and subsequently in Martinique in 1998. This “American/Asian” genotype has therefore gradually dispersed across the Caribbean region during the past 5 years.

Anamnestic antibody response after viral challenge in monkeys immunized with dengue 2 recombinant fusion proteins

The suitability of dengue 2 envelope domain III recombinant fusion proteins [(fusion (PD5) and insertion (PD3) variants)] for inducing functional antibodies and a protective immune response in nonhuman primates has been reported. However, the evaluation of the antibody response after immunization did not correlate with the protection data as measured by viremia detection. Here, we characterized the anamnestic immune response after viral challenge in monkeys immunized with the dengue 2 recombinant proteins in an attempt to define correlates of protection useful for vaccine studies. Monkeys immunized with PD5 (most protected group) exhibited an earlier increase in the anti-DENV-2 IgM response after challenge compared to control animals. Hemagglutination-inhibiting (HAI) antibodies were increased significantly earlier in PD5-immunized animals compared to those immunized with PD3. The fully protected monkeys showed the earliest HAI antibody response. These results underline the usefulness of the anamnestic antibody response for supporting protection data. The induction of an early HAI and IgM antibody response after challenge suggest a protective role against dengue virus (DENV) infection in monkeys, supporting their use as correlates of protection in vaccine studies.

Neutralizing antibody response variation against dengue 3 strains

To evaluate the neutralizing antibody activity of a human sera panel against seven strains of the homotypic virus. Sera were collected from DENV‐3 immune individuals. Two DENV‐3 genotypes and strains isolated at different time‐points during the 2000 and 2001–2002 Havana epidemics were included. A panel of 20 late convalescent sera collected 16–18 months after acute illness from DF and DHF patients are studied. These individuals were infected during the 2001–2002 Havana DENV‐3 epidemic. All but four sera collected from DF cases had a secondary DENV‐1/DENV‐3 infection. Sera neutralizing antibody titer against the seven DENV‐3 strains were determined by plaque reduction neutralization technique. Sera samples were tested simultaneously. Studied sera showed higher levels of neutralizing antibodies to DENV‐3 strains of genotype III compared to genotype V. Interesting, higher levels of neutralizing antibodies were detected to DENV‐3 strain isolated at the end of the epidemic 2001–2002. An increased tendency of GMT of neutralizing antibodies according to epidemic evolution was observed for the 2001–2002 outbreak. In general, antibody levels in sera collected from DF cases were higher. Differences in the neutralization capacity of immune DENV‐3 sera tested against two homologous genotypes including strains of the same genotype are demonstrated. Observed results suggest that virus changed in the course of the epidemic. The implications of this finding in terms of dengue pathogenesis and vaccine development need to be considered. J. Med. Virol. 80:1783–1789, 2008.

Population structure of the dengue viruses, Aragua, Venezuela, 2006-2007. Insights into dengue evolution under hyperendemic transmission.

Highlights ► We examine the population structure of dengue viruses in Venezuela, 2006–2007. ► Phylogenetic trees show that only one genotype was circulating for each serotype. ► Extensive viral genetic diversity indicates significant in situ evolution. ► Particularly, variations in non-structural proteins appear to have a major role. ► These results on dengue evolution are relevant in the context of vaccine design.

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

ABSTRACT 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 epidemics 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.

FIRST DENGUE HAEMORRHAGIC FEVER EPIDEMIC IN THE AMERICAS, 1981: INSIGHTS INTO THE CAUSATIVE AGENT

Historical records describe a disease in North America that clinically resembled dengue haemorrhagic fever during the latter part of the slave-trading period. However, the dengue epidemic that occurred in Cuba in 1981 was the first laboratory-confirmed and clinically diagnosed outbreak of dengue haemorrhagic fever in the Americas. At that time, the presumed source of the dengue type 2 strain isolated during this epidemic was considered controversial, partly because of the limited sequence data and partly because the origin of the virus appeared to be southern Asia. Here, we present a molecular characterisation at the whole-genome level of the original strains isolated at different time points during the epidemic. Phylogenetic trees constructed using Bayesian methods indicated that 1981 Cuban strains group within the Asian 2 genotype. In addition, the study revealed that viral evolution occurred during the epidemic – a fact that could be related to the increasing severity from month to month. Moreover, the Cuban strains exhibited particular amino acid substitutions that differentiate them from the New Guinea C prototype strain as well as from dengue type 2 strains isolated globally.

First record of natural vertical transmission of dengue virus in Aedes aegypti from Cuba

While horizontal transmission (human-mosquito-human) of dengue viruses largely determines the epidemiology of the disease, vertical transmission (infected female mosquito- infected offspring) has been suggested as a mechanism that ensures maintenance of the virus during adverse conditions for horizontal transmission to occur. The purpose of this study was to analyze the natural infection of larval stages of Aedes aegypti (Diptera: Culicidae) with the dengue virus (DENV) in Cuba. Here, we report vertical transmission of DENV-3 genotype III in natural populations of Ae. aegypti through RT-PCR detection and serotyping plus sequencing. Our report constitutes the first record of vertical transmission of DENV in Ae. aegypti from Cuba with details of its serotype and genotype.

Spatio-temporal distribution of vertically transmitted dengue viruses by Aedes aegypti (Diptera: Culicidae) from Arroyo Naranjo, Havana, Cuba

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.