Spatio-Temporal Tracking and Phylodynamics of an Urban Dengue 3 Outbreak in São Paulo, Brazil

The dengue virus has a single-stranded positive-sense RNA genome of ∼10.700 nucleotides with a single open reading frame that encodes three structural (C, prM, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. It possesses four antigenically distinct serotypes (DENV 1–4). Many phylogenetic studies address particularities of the different serotypes using convenience samples that are not conducive to a spatio-temporal analysis in a single urban setting. We describe the pattern of spread of distinct lineages of DENV-3 circulating in São José do Rio Preto, Brazil, during 2006. Blood samples from patients presenting dengue-like symptoms were collected for DENV testing. We performed M-N-PCR using primers based on NS5 for virus detection and identification. The fragments were purified from PCR mixtures and sequenced. The positive dengue cases were geo-coded. To type the sequenced samples, 52 reference sequences were aligned. The dataset generated was used for iterative phylogenetic reconstruction with the maximum likelihood criterion. The best demographic model, the rate of growth, rate of evolutionary change, and Time to Most Recent Common Ancestor (TMRCA) were estimated. The basic reproductive rate during the epidemics was estimated. We obtained sequences from 82 patients among 174 blood samples. We were able to geo-code 46 sequences. The alignment generated a 399-nucleotide-long dataset with 134 taxa. The phylogenetic analysis indicated that all samples were of DENV-3 and related to strains circulating on the isle of Martinique in 2000–2001. Sixty DENV-3 from São José do Rio Preto formed a monophyletic group (lineage 1), closely related to the remaining 22 isolates (lineage 2). We assumed that these lineages appeared before 2006 in different occasions. By transforming the inferred exponential growth rates into the basic reproductive rate, we obtained values for lineage 1 of R0 = 1.53 and values for lineage 2 of R0 = 1.13. Under the exponential model, TMRCA of lineage 1 dated 1 year and lineage 2 dated 3.4 years before the last sampling. The possibility of inferring the spatio-temporal dynamics from genetic data has been generally little explored, and it may shed light on DENV circulation. The use of both geographic and temporally structured phylogenetic data provided a detailed view on the spread of at least two dengue viral strains in a populated urban area.Author Summary: Most of the molecular phylogeny studies of dengue fever, an important public health problem, use convenience samples for their analysis, and they do not evaluate the spatial and temporal features involved in the spread of the different serotypes (and genotypes) circulating in urban settings during an outbreak. Our study describes the patterns of spread of different lineages of dengue 3 virus circulating in a medium-sized city from Brazil, and we also analyzed the dynamics and microevolution of the disease during the 2006 outbreak. We used both geographic and temporally structured phylogenetic data, which provided a relatively detailed view on the spread of at least two dengue viral lineages circulating in an urban area. The pattern of dengue virus circulation might be similar to many other settings all over the world, and the information provided by our study can help a better understanding of dengue outbreaks, providing important information for public-health systems. We could identify at least two lineages, which were introduced in different occasions. They circulated and spread at different rates within the city, and this differential spread and the role of socioeconomic features in this phenomenon are discussed.