Microevolution of DENV in Kamphaeng Phet.
To determine the extent and structure of genetic diversity during the 2001 dengue season, we sequenced the E genes of all available viruses collected during this year (Table ). In total, all nine DENV-1 virus samples, collected from students in schools 8, 11, and 12 from July to November 2001, were available. All were assigned to genotype I, which is common in South East Asia, especially in Thailand. Similarly, 40/52 DENV-2 viruses, from 9 of the 12 schools, were available for sequencing, all of which represented the Asian I genotype, which is common in Thailand. The period of collection of these viruses was diverse, ranging from February to November and hence covering periods of both low and high DENV transmission. A total of 18/25 DENV-3 viruses, obtained from three schools in 2001 and largely from school 11 between June and November, were available for sequencing. All DENV-3 viruses were assigned to genotype II, which is again common in South East Asia. Viruses that were unavailable for sequencing were unavailable because of a lack of clinical material, a failure to grow a viral stock during the study, or insufficient genetic material for sequencing.
ML phylogenetic trees for the DENV-1, DENV-2, and DENV-3 E-gene sequences from Kamphaeng Phet, combined with the background isolates, are presented in Fig. , , and , respectively. In the case of DENV-1, viral isolates fall into two distinct clades, separated by other Asian viruses. That the DENV-1 isolates from school 11 fall in both clades provides compelling evidence for the independent entry of genetically distinct viruses into this spatially restricted region. A similar pattern was observed in DENV-2. Here, those viruses sampled from schools 1, 5, 8, 9, and 10 formed school-specific clusters (although only those from school 1 were clearly phylogenetically distinct from those from the other schools), while multiple genetic variants were seen in schools 2, 4, 11, and 12 (with one highly divergent variant observed in school 11). Finally, in the case of DENV-3, single clades were observed in schools 4 and 10, while two clades were again observed in school 11, although with weak bootstrap support. Notably, children from school 11 come from a densely populated urban environment at the center of the study area, close to the major transportation throughway and a public health clinic, which may have contributed to the appearance of multiple viral variants in this school.
FIG. 2. ML tree of selected isolates of genotype I of DENV-1. Different schools from within Kamphaeng Phet are designated by different colors. All horizontal branch lengths are drawn to scale according to the numbers of nucleotide substitutions per site, with (more ...)
FIG. 3. ML tree of selected isolates of the Asian I genotype of DENV-2. Different schools from within Kamphaeng Phet are designated by different colors. All horizontal branch lengths are drawn to scale according to the numbers of nucleotide substitutions per (more ...)
FIG. 4. ML tree of selected isolates of genotype II of DENV-3. Different schools from within Kamphaeng Phet are designated by different colors. All horizontal branch lengths are drawn to scale according to the numbers of nucleotide substitutions per site, with (more ...)
Overall, these phylogenetic results reveal a marked clustering by school (reflected in the grouping of colors in Fig. to ), indicating that multiple genetic variants of DENV circulate within a spatially restricted area during a single dengue season but that individual schools represent distinct evolutionary entities that have experienced clear population subdivision. In only a relatively small number of cases do multiple variants of individual serotypes cocirculate, most notably in some cases involving school 11. Further, the observation that these distinct clusters are often separated by viruses isolated from outside Kamphaeng Phet (and in the cases of DENV-1 and DENV-2 outside Thailand) indicates that there were multiple introductions of DENV into this region during 2001. We believe that this is the first report of evolution on a localized scale for DENV, and this report indicates that even viruses sampled from spatially restricted regions may harbor extensive genetic diversity.
To determine the extent of spatial clustering more rigorously, we examined DENV-2 in more detail as this represented our best-sampled serotype. A parsimony-based analysis confirmed that there is a highly significant clustering of viral isolates by school (P < 0.005). However, despite this strong genetic segregation, there is also clear evidence (P < 0.005) of viral migration between schools 8 and 9. Notably, these two schools are geographically the closest in the study population, separated by only 5 km, and the furthest from the city, at distances of 36 and 33 km from the field site, respectively (Fig. ). Further, the villages that feed these schools have low population densities, are highly rural, and possess poor road infrastructure, making access to this area more difficult than access to other regions of Kamphaeng Phet. As such, our data suggest that patterns of viral gene flow are determined by local geographic and economic variables. The villages that make up the Kamphaeng Phet region are diverse, with the most developed and densely populated villages lying close to the city center, where frequent travel into and out of the city is common. In contrast, villages become less developed and densely populated further from the city center, in turn restricting travel and resulting in the genetic isolation of viruses from schools 8 and 9.
In contrast, these DENV-2 sequence data exhibited no significant clustering by month of sampling, indicating that there was little replacement of viral lineages at this level of spatial and temporal resolution. Similarly, at the level of the E-gene sequences analyzed, there was little evidence for in situ evolution, such that viruses sampled from the same school over time periods ranging from days to months exhibited few mutational differences (in some cases only one mutation over a 6-month sampling period). Such relative intraschool stability sits in marked contrast to the often strong genetic differentiation among schools. This observation, coupled with the strong population subdivision exhibited by each school, indicates that the importation of DENV into the study population, rather than in situ evolution within school catchment areas, was the most important factor shaping viral genetic diversity on this spatial scale.
To determine the selection pressures acting on the DENV E-gene sequences sampled from Kamphaeng Phet, we compared the relative numbers of nonsynonymous (dN
) and synonymous (dS
) substitutions per site. This analysis provided no evidence for positive selection acting on any nucleotide site, although all dN
methods are inherently conservative. Indeed, the overall picture obtained was that of strong purifying selection, with mean dN
values of 0.084, 0.065, and 0.008 for DENV-1, DENV-2 and DENV-3, respectively. Such strong purifying selection has previously been observed for these serotypes in Thailand (21
), highlighting the predominance of genetic drift over natural selection in shaping DENV substitution dynamics in the short term. However, the reasons for the greater selective constraints in DENV-3, which is characterized by very low dN
values, are unclear. Further, as random genetic drift is the most common process determining substitution dynamics, allele frequencies are not expected to change greatly over the period of sampling, even given the high mutation rate of DENV (3
Overall, these results have mixed implications for how DENV populations might respond to imperfect vaccination in the near future. DENV sampled on a short spatial and temporal scale evidently exhibits remarkably high levels of genetic diversity, thereby providing the raw material for adaptive evolution. However, this variation is more often generated by migration than by mutation accumulation, with purifying selection the dominant evolutionary force.