Re-emergence of DENV-3 in Paraguay after two decades: a genomic and epidemiological investigation.

Dengue is one of the most rapidly expanding mosquito-borne viral diseases worldwide, posing a persistent public health challenge across tropical and subtropical regions. Here, we report the re-emergence of dengue virus serotype 3 (DENV-3) in Paraguay following approximately 15 years without confirmed detection. In early 2025, twelve laboratory-confirmed cases were identified in the metropolitan region of Asunción through routine epidemiological surveillance and molecular diagnostics. To investigate the origin and evolutionary context of these infections, whole-genome sequencing and phylogenetic reconstruction were performed. Genomic analyses classified the viruses within DENV-3 genotype III, lineage B.3.2, supporting the hypothesis of a recent external introduction of an emerging lineage rather than persistence or renewed transmission of previously circulating local strains. Temporal and phylogeographic inferences further indicated recent introduction events consistent with regional viral movement across South America, highlighting the dynamic nature of dengue transmission in highly connected urban settings. These findings provide early evidence of DENV-3 re-establishment in Paraguay and emphasize the epidemiological relevance of continuous molecular surveillance, particularly in countries experiencing intense and recurrent dengue activity driven by multiple serotypes. Strengthening genomic monitoring frameworks is essential to enable rapid detection of lineage introductions, characterize transmission pathways, and support timely public health decision-making. Enhanced integration of genomic, epidemiological, and entomological data will be critical to anticipate outbreak risk and guide targeted control strategies aimed at reducing dengue burden in Paraguay and the broader region.