Genomic evidence links human dengue cases with undetermined serotypes to sylvatic lineages.

BACKGROUND: Sylvatic dengue viruses, typically maintained in non-human primate and forest mosquito cycles, have rarely been associated with human infections. However, sporadic spillovers have been reported in Southeast Asia, including Malaysia. These events are often under-detected due to the genetic divergence of sylvatic strains from endemic urban dengue viruses. During routine surveillance in Malaysia (2024-2025), a subset of clinically confirmed dengue cases yielded undetectable serotype results by commercial real-time reverse transcription polymerase chain reaction (RT-PCR) assays, prompting investigation into a possible sylvatic origin. METHODS: We investigated 22 such cases through clinical, serological, molecular, and phylogenetic analyses. NS1 antigen and broad-range RT-PCR confirmed acute dengue infection. Selected samples underwent sequencing and lineage determination. RESULTS: Most patients presented with severe dengue during early illness (mean day 3), with 95.5% NS1 positivity and predominantly primary infection profiles. Despite serotyping failure, sequencing revealed that eight of nine analyzed samples belonged to sylvatic DENV2, while one represented a divergent DENV3. Comparative amino acid analysis uncovered a unique signature in recent Malaysian sylvatic DENV2 strains, differentiating them from both urban and historical sylvatic lineages. This includes the V270 mutation in the M gene; R844, V884, and I898 in the NS1 gene; T1207 in the NS2A gene; A1597 in the NS3 gene; and D3048 and I3373 in the NS5 gene. Phylogenetic analysis clustered these strains into a distinct Malaysian clade, separate from the African sylvatic lineage. CONCLUSIONS: This study provides the first genomic evidence of a recent sylvatic DENV2 spillover into humans in Malaysia, likely undetected by standard diagnostics due to genetic divergence. These findings underscore the urgent need to enhance surveillance tools and explore the sylvatic transmission cycle's role in dengue epidemiology.