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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 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.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.

SUBMITTER: Rodriguez-Roche R

PROVIDER: S-EPMC4836355 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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

Rodriguez-Roche Rosmari R Blanc Hervé H Bordería Antonio V AV Díaz Gisell G Henningsson Rasmus R Gonzalez Daniel D Santana Emidalys E Alvarez Mayling M Castro Osvaldo O Fontes Magnus M Vignuzzi Marco M Guzman Maria G MG

Journal of virology 20160414 9

<h4>Unlabelled</h4>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 obtaine ...[more]

PMID: 26889031

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Project description:Please see Additional file 1 for translations of the abstract into the five official working languages of the United Nations.Currently, in Brazil, there is a co-circulation of the four dengue (DENV-1 to DENV-4) serotypes. This study aimed to assess whether different serotypes and antibody response patterns were associated with the severity of the disease during a dengue outbreak, which occurred in 2012/2013 in centre of Brazil.We conducted a prospective study with 452 patients with laboratory confirmed dengue in central Brazil, from January 2012 to July 2013. The clinical outcome was the severity of cases: dengue, dengue with warning signs, and severe dengue. The patients were evaluated at three different moments. Blood sampling for laboratory testing and confirmatory tests for dengue infection were performed. We performed a multinomial analysis considering the three categories of the dependent variable, as outlined above. The odds ratios (ORs) were calculated. A multinomial logistic regression model was applied for variables with a P-value <0.20. Statistical analysis was performed with STATA 12.0 software.Four hundred fifty-two patients (452/632, 71.5%) were diagnosed with dengue. The dengue virus (DENV) serotypes were identified in 243 cases. DENV-4 was detected in 135 patients (55.6%), DENV-1 in 91 (37.4%), DENV-3 in 13 (5.3%), and DENV-2 in 4 (1.6%). Patients with the DENV-1 serotype were more prone to present with several clinical and laboratory features as compared with DENV-4 patients, including spontaneous bleeding (P = 0.03), intense abdominal pain (P = 0.004), neurological symptoms (P = 0.09), and thrombocytopenia (P = 0.01). Secondary infection was more predominant among DENV-4 cases (80.0%) compared with DENV-1 cases (62.3%) (P = 0.03). The univariate analysis showed that females (OR = 2.12; 95% CI: 1.44-3.13; P < 0.01) had a higher risk of having dengue with warning signs. The multinomial analysis showed that severe dengue cases with secondary infection had an adjusted OR of 2.80 (95% CI: 0.78-10.00; P = 0.113) as compared with dengue fever with primary infection when adjusted for age and sex.The current data show that 5.8% of patients recruited for treatment in healthcare centres and hospitals during the study period had severe dengue. DENV-4 was the predominant serotype, followed by DENV-1, in a large outbreak of dengue in central Brazil. Our findings contribute to the understanding of clinical differences and immune status related to the serotypes DENV-1 and DENV-4 in central of Brazil.

Project description:BackgroundDeep-sequencing allows for an in-depth characterization of sequence variation in complex populations. However, technology associated errors may impede a powerful assessment of low-frequency mutations. Fortunately, base calls are complemented with quality scores which are derived from a quadruplet of intensities, one channel for each nucleotide type for Illumina sequencing. The highest intensity of the four channels determines the base that is called. Mismatch bases can often be corrected by the second best base, i.e. the base with the second highest intensity in the quadruplet. A virus variant model-based clustering method, ViVaMBC, is presented that explores quality scores and second best base calls for identifying and quantifying viral variants. ViVaMBC is optimized to call variants at the codon level (nucleotide triplets) which enables immediate biological interpretation of the variants with respect to their antiviral drug responses.ResultsUsing mixtures of HCV plasmids we show that our method accurately estimates frequencies down to 0.5%. The estimates are unbiased when average coverages of 25,000 are reached. A comparison with the SNP-callers V-Phaser2, ShoRAH, and LoFreq shows that ViVaMBC has a superb sensitivity and specificity for variants with frequencies above 0.4%. Unlike the competitors, ViVaMBC reports a higher number of false-positive findings with frequencies below 0.4% which might partially originate from picking up artificial variants introduced by errors in the sample and library preparation step.ConclusionsViVaMBC is the first method to call viral variants directly at the codon level. The strength of the approach lies in modeling the error probabilities based on the quality scores. Although the use of second best base calls appeared very promising in our data exploration phase, their utility was limited. They provided a slight increase in sensitivity, which however does not warrant the additional computational cost of running the offline base caller. Apparently a lot of information is already contained in the quality scores enabling the model based clustering procedure to adjust the majority of the sequencing errors. Overall the sensitivity of ViVaMBC is such that technical constraints like PCR errors start to form the bottleneck for low frequency variant detection.

Dataset Information

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

Publications

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

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