Emergence of the Asian 1 genotype of dengue virus serotype 2 in viet nam: in vivo fitness advantage and lineage replacement in South-East Asia

Abstract

A better description of the extent and structure of genetic diversity in dengue virus (DENV) in endemic settings is central to its eventual control. To this end we determined the complete coding region sequence of 187 DENV-2 genomes and 68 E genes from viruses sampled from Vietnamese patients between 1995 and 2009. Strikingly, an episode of genotype replacement was observed, with Asian 1 lineage viruses entirely displacing the previously dominant Asian/American lineage viruses. This genotype replacement event also seems to have occurred within DENV-2 in Thailand and Cambodia, suggestive of a major difference in viral fitness. To determine the cause of this major evolutionary event we compared both the infectivity of the Asian 1 and Asian/American genotypes in mosquitoes and their viraemia levels in humans. Although there was little difference in infectivity in mosquitoes, we observed significantly higher plasma viraemia levels in paediatric patients infected with Asian 1 lineage viruses relative to Asian/American viruses, a phenotype that is predicted to result in a higher probability of human-to-mosquito transmission. These results provide a mechanistic basis to a marked change in the genetic structure of DENV-2 and more broadly underscore that an understanding of DENV evolutionary dynamics can inform the development of vaccines and anti-viral drugs.

Figure 1. Fluctuations in dengue incidence and serotype abundance in southern Viet Nam.

A) Changes in the incidence of hospitalized dengue cases (left-hand axis, open bars) and the relative virus prevalence (right hand axis) between 1996 and 2008 in the southern 20 provinces of Viet Nam. Data is from the Vietnamese Ministry of Health Dengue passive surveillance scheme and kindly provided by the Pasteur Institute, HCMC, Viet Nam. B. Case burden (left-hand axis, open bars) and DENV serotype prevalence detected by RT-PCR in children and adults (n = 3463 RT-PCR positive patients) enrolled into clinical studies at the Hospital for Tropical Diseases, Ho Chi Minh City between 1999 and 2008. The Hospital for Tropical Diseases is a tertiary referral hospital for infectious diseases. The values underneath the graph represent the number of RT-PCR positive samples each year.

Figure 2. Maximum clade credibility (MCC) tree of complete coding region sequences of DENV-2 sampled in Viet Nam.

The major genotypes of DENV-2 and their sampling times (in parentheses) are marked. The 95% credible values for the time to the most recent common ancestor of the Asian I and Asian/American genotypes in Viet Nam are shown. In all cases tip times reflect the day of sampling of each virus. The tree is automatically rooted under the assumption of a molecular clock.

Figure 3. Maximum likelihood tree of 941 DENV-2 E gene sequences sampled globally.

Viruses from Viet Nam are shaded red, those from Cambodia shaded green and those from Thailand shaded blue. The sampling years for Viet Nam, Cambodia and Thailand are annotated next to the tree. Bootstrap support values are shown at key nodes. The tree is mid-point rooted for purposes of clarity and all horizontal branch lengths are drawn to a scale of nucleotide substitutions per site.

Figure 4. Temporal pattern of DENV-2 genotype replacement in Viet Nam, Cambodia and Thailand.

The relative proportion of sampled DENV-2 viruses belonging to either Asian I or Asian/American genotypes (as determined in Fig. 3) is shown by year of sampling in A) Viet Nam, B) Cambodia and C) Thailand. The values below the graph represent the number of E gene sequences sampled per year.

Figure 5. Viraemia levels in Asian 1 and Asian/American genotype infections by day of illness.

Shown are the mean (±95% confidence intervals) viraemia levels measured at the time of study enrolment in pediatric dengue cases infected with either Asian 1 (n = 289) or Asian/American (n = 100) genotype viruses. All patients were enrolled in the same study ward and values below the graph represent the number of cases enrolled by duration of illness. Viraemia levels were significantly higher in Asian 1 infections in patients enrolled on day 2, 3 or 4 of illness (t test). Data for patients sampled on day 1 of illness is not shown because of the small sample size (N = 3).