Dengue 1 diversity and microevolution, French Polynesia 2001-2006: connection with epidemiology and clinics

Abstract

Background: Dengue fever (DF) is an emerging infectious disease in the tropics and subtropics. Determinants of DF epidemiology and factors involved in severe cases-dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS)-remain imperfectly characterized. Since 2000, serotype 1 (DENV-1) has predominated in the South Pacific. The aim of this study was (i) to determine the origin and (ii) to study the evolutionary relationships of DENV-1 viruses that have circulated in French Polynesia (FP) from the severe 2001 outbreak to the recent 2006 epidemic, and (iii) to analyse the viral intra-host genetic diversity according to clinical presentation.

Methodology/principal findings: Sequences of 181 envelope gene and 12 complete polyproteins of DENV-1 viruses obtained from human sera in FP during the 2001-2006 period were generated. Phylogenetic analysis showed that all DENV-1 FP strains belonged to genotype IV-"South Pacific" and derived from a single introduction event from South-East Asia followed by a 6-year in situ evolution. Although the ratio of nonsynonymous/synonymous substitutions per site indicated strong negative selection, a mutation in the envelope glycoprotein (S222T) appeared in 2002 and was subsequently fixed. It was noted that genetic diversification was very significant during the 2002-2005 period of endemic DENV-1 circulation. For nine DF sera and eight DHF/DSS sera, approximately 40 clones/serum of partial envelope gene were sequenced. Importantly, analysis revealed that the intra-host genetic diversity was significantly lower in severe cases than in classical DF.

Conclusions/significance: First, this study showed that DENV-1 epidemiology in FP was different from that described in other South-Pacific islands, characterized by a long sustained viral circulation and the absence of new viral introduction over a 6-year period. Second, a significant part of DENV-1 evolution was observed during the endemic period characterized by the rapid fixation of S222T in the envelope protein that may reflect genetic drift or adaptation to the mosquito vector. Third, for the first time, it is suggested that clinical outcome may be correlated with intra-host genetic diversity.

Figure 1. Map of French Polynesia (FP).

Sampling of DENV-1 sera was conducted in the five FP archipelagos: Society, Tuamotu, Gambier, Austral and Marquesas.

Figure 2. Phylogenetic tree based on 240 DENV-1 nucleotide sequences of 1,759 bp including the E-gene (Neighbor-Joining method, Kimura 2 algorithm).

The 181 sequences generated in FP are condensed in the branch named “FP 2001–2006”. Taxon names of GenBank sequences correspond to D1.country/last two digits of year of isolation and GenBank accession number. In this condensed tree, branch length is not proportional to genetic distance. Numbers on branches represent bootstrap support for each branch. Five DENV-1 genotypes were identified. The validity of these genotypes, in particular genotype II “Thailand” and genotype III “sylvatic/Malaysia”, is supported by previous phylogenetic analyses based on maximum likelihood method ,.”

Figure 3. Phylogenetic tree based on 53 DENV-1 complete genome amino acid sequences (maximum likelihood phylogenetic analysis using Bayesian method).

Taxon names of FP sequences correspond to D1_FP_sample number_year_month_geographical origin_clinical presentation. Taxon names of GenBank sequences correspond to D1_country_last two digits of year of isolation_GenBank accession number. Posterior probabilities (percent) are shown for values >80 only. All horizontal branch lengths are drawn to a scale of substitutions per site. The tree was rooted using a DENV-3 strain (D3_SriLanka_00_NC001475, not shown for purposes of clarity only).

Figure 4. Variability of 181 protein sequences of DENV-1 during epidemic and endemic periods in FP from 2001 to 2006.

Ten amino acid changes newly occurred during the 2001 outbreak, 23 during the 2002–2005 endemic period, and eight during the 2006 outbreak. S222T appeared in August 2002 and was subsequently fixed by viral evolution.