Inter- and intra-host sequence diversity reveal the emergence of viral variants during an overwintering epidemic caused by dengue virus serotype 2 in southern Taiwan

Abstract

Purifying selection during dengue viral infection has been suggested as the driving force of viral evolution and the higher complexity of the intra-host quasi-species is thought to offer an adaptive advantage for arboviruses as they cycle between arthropod and vertebrate hosts. However, very few studies have been performed to investigate the viral genetic changes within (intra-host) and between (inter-host) humans in a spatio-temporal scale. Viruses of different serotypes from various countries imported to Taiwan cause annual outbreaks. During 2001-2003, two consecutive outbreaks were caused by dengue virus serotype 2 (DENV-2) and resulted in a larger-scale epidemic with more severe dengue cases in the following year. Phylogenetic analyses showed that the viruses from both events were similar and related to the 2001 DENV-2 isolate from the Philippines. We comprehensively analyzed viral sequences from representative dengue patients and identified three consensus genetic variants, group Ia, Ib and II, with different spatio-temporal population dynamics. The phylodynamic analysis suggested group Ib variants, characterized by lower genetic diversity, transmission rate, and intra-host variant numbers, might play the role of maintenance variants. The residential locations among the patients infected by group Ib variants were in the outer rim of case clusters throughout the 2001-2003 period whereas group Ia and II variants were located in the centers of case clusters, suggesting that group Ib viruses might serve as "sheltered overwintering" variants in an undefined ecological niche. Further deep sequencing of the viral envelope (E) gene directly from individual patient serum samples confirmed the emergence of variants belonging to three quasi-species (group Ia, Ib, and II) and the ancestral role of the viral variants in the latter phase of the 2001 outbreak contributed to the later, larger-scale epidemic beginning in 2002. These findings enhanced our understanding of increasing epidemic severity over time in the same epidemic area. It also highlights the importance of combining phylodynamic and deep sequencing analysis as surveillance tools for detecting dynamic changes in viral variants, particularly searching for and monitoring any specific viral subpopulation. Such subpopulations might have selection advantages in both fitness and transmissibility leading to increased epidemic severity.

Fig 1. Spatio-temporal distributions of DENV-2 isolated and sequenced from patients in Taiwan, during 2001–2003.

(A) The number of laboratory-confirmed, indigenous dengue cases and their major viral serotype found in Kaohsiung city from January 2000 to April 2015. The total numbers of laboratory-confirmed dengue cases, cases diagnosed with dengue fever (DF, blue) or dengue hemorrhagic fever (DHF, red) are shown separately. (B) The isolated DENV-2 strains at each of the respective time points (upper panel), compared to the total numbers of confirmed dengue cases (5305, including 5064 DF and 241 DHF cases) embedded in the epidemic curve for the same period (lower panel). Two consecutive outbreaks occurred in the period from the 35th week, 2001 to the 11th week, 2003: 232 confirmed cases, including 222 DF and 10 DHF cases in 2001; and 5073 confirmed cases, including 4842 DF and 231 DHF cases. (C)(D) Spatial distributions of dengue cases in 2001 (C) and 2002 (D). Red dots represent viral isolates that were investigated in this study; gray plot represents dengue cases reported from Taiwan-CDC.

Fig 2. Maximum clade credibility (MCC) tree of the E and ORF genes.

(A) A MCC tree based on the E genes of 104 DENV-2 viruses isolated during 2001–2003 in southern Taiwan together with those from the Philippines as a reference group. Based on the tree and nucleotide signatures, group I [nt-1073C; threonine (T) at E-46] and group II [nt-1073T; isoleucine (I) at E-46] were identified. Posterior probabilities were labeled on distinct nodes. Heatmap next to the tree indicates three corresponding nucleotide positions in the E gene for each of the different isolates. (B) MCC tree based on ORF of 43 Taiwan DENV-2 isolates. Two distinguishable virus variants were further noted in group I viruses: Ia viruses have isoleucine (I) at the NS5-271 position and valine/glutamate (V/E) at the NS5-357 position, whereas Ib viruses have threonine (T) at NS5-271 and glutamate (E) at NS5-375.

Fig 3. Temporal distributions of the three groups of DENV-2 viruses.

Dynamic changes of the three groups of DENV-2 variants isolated from patients over time. At the peak of 2001 outbreak, the group Ia and Ib variants accounted for about 75.86% and 24.14% of the isolates, respectively. The earliest Ib variant was identified in November 2001, whereas the first confirmed dengue case occurred in July 2001. The group Ib variants remained circulating after 2001 outbreak, whereas the group II variants became dominant viruses during the outbreak period from February 2002 to September 2002, accounting for more than 85.19% of the isolates. After October 2002, 95.24% of the isolates belonged to group II.

Fig 4. Differences in the pattern of geographical distribution of group Ia, Ib and II variants.

Dots on the map indicate locations of patients’ residential areas. The intensity of color for each dot represents the chronology for the three groups of viral variants during their circulating period (A-C). The arrow on the map indicates the direction of virus spread based on the locations of MRCAs inferred in the MCC trees (A and B). Compared with groups Ia and Ib, the group II viral variants do not show a simple linear spreading route but did spread from the center of the clusters. Total isolates were plotted as dots in the indicated period, in colors blue, green and purple representing the groups Ia, Ib and II, respectively. The red shaded area illustrates all laboratory-confirmed dengue cases in the respective periods; the darker shading reflects higher case numbers.

Fig 5. Genetic distances by isolation date among three groups of DENV-2 viral variants.

Genetic distance, based on the E gene of each group of viral variants, was analyzed by linear regression. Solid lines show the estimated regression intervals. Group Ib variants had lower evolution rates (substitutions/site/year), with fewer genetic changes, compared with those of group Ia and II variants. The difference in estimated rates between the E protein and the ORF (S2 Fig) of Ia variants may be due to insufficient sample size and the fact that most genetic variations of group Ia viruses were found in the E gene.

Fig 6. Viral transmissibility of the three groups of DENV-2 variants measured by effective reproductive number (R).

The R (solid line) values and 95% HPD interval (shaded area) from 2001 to 2003 of three DENV-2 viral variants were estimated by birth-death skyline serial analysis. The plot clearly demonstrates that the two peaks of viral transmissibility corresponded well with the two peaks of confirmed DENV-2 cases from 2001 to 2003. The R values of both group Ia and Ib variants surpassed 1.00 during 2001. They were replaced by group II variants when R surged in early 2002, accompanied by the recurrence of group Ib variants.

Fig 7. Deep sequencing of evolving viral populations.

Proportions of quasi-species variants of each virus isolate (x-axis) are illustrated by three nucleotide positions in E-1073, -1227 and -2064. Proportions of Thymine (T) at position 1073 (upper), Adenine (A) at position 1227 (middle) and Cytosine (C) at position 2064 (lower) are shown separately in three panels, respectively, by black dots. Their corresponding putative nucleotide substitution counterparts - 1073C, 1227T, and 2064T are shown in gray dots. Three phylogenetic groups of DENV-2 variants are highlighted by different colors (Ia-blue, Ib-green, and II-purple) on the x-axis. Viruses are arranged by the chronological order of their isolation dates within each group. The vertical dashed line separates viruses isolated in 2001 (left side) versus those in 2002 (right side).

Fig 8. Factors associated with viral diversity: Population sizes, associated cases and population densities and secondary infection.

(A) Distributions in the numbers of three groups of DENV-2 variants. The number of variants in group Ib was significantly lower than in group Ia viral variants. (B) Distribution of Li-specific population densities. (C) Li-specific numbers of dengue cases. Numbers of dengue cases associated with the group Ia and Ib variants were significantly lower than with group II. The Mann–Whitney–Wilcoxon test was used and significant differences (p<0.05) were labeled with an asterisk. The horizontal bar in the middle of the violin plots indicates the median values of the three groups. Proportions of primary and secondary DENV infections within each group of viruses classified according to the numbers of the two groups: (1) low variant (< 20) (D), and (2) high variant (≥20) (E), boundary by medium value (medium is 20). (E). Primary DENV infection accounted for percentages of dengue cases in the low variant group, particularly in Ia variants. However, secondary DENV infection increased its percentages in the low variant groups of Ib and II viruses and eventually accounted for 100% of the secondary infection group for all the Ia, Ib and II groups with a high variant number.