Heterogeneous co-circulation of multiple DENV-1 and DENV-2 genotypes in Vung Tau during the 2023 dengue outbreak in Vietnam.

Dengue virus (DENV) remains a significant global health threat, particularly in hyper-endemic regions like Vietnam, where the co-circulation of multiple serotypes and genotypes complicates disease control. In 2023, 6.5 million dengue cases and 6,800 deaths were reported globally, marking the largest dengue outbreak in the world. Vietnam recorded 149,557 dengue cases and 36 deaths, with a notable dominance of the DENV-2 serotype. This study provides a comprehensive genomic characterization of the 2023 outbreak using isolates collected from hospitalized patients (n = 148; 66.89% adults). To ensure robust viral recovery, we propagated 90 isolates using a low-passage, antibody-dependent enhancement (ADE)-based system. Whole-genome sequencing (WGS) of 53 isolates yielded 47 consensus sequences, including 26 complete genomes. Phylogenetic analysis revealed the simultaneous co-circulation of diverse DENV-1 and DENV-2 genotypes. DENV-1 isolates were predominantly genotype I (86%), clustering closely with prior Vietnamese strains and suggesting sustained endemic maintenance, while genotype IV (14%) was detected sporadically. Among DENV-2 isolates, the Cosmopolitan genotype was predominant (98%), with a smaller proportion (2%) belonging to the Asian 1 genotype. Both genotypes clustered with previously reported Vietnamese and regional Southeast Asian strains, indicating ongoing local circulation and regional viral exchange. Comparative mutational analysis of DENV-2 Cosmopolitan isolates identified multiple non-synonymous amino acid substitutions across the viral genome. Fourteen substitutions were shared between Vietnamese historical (2019-2022) and 2023 isolates, while 24 substitutions were unique to the 2023 outbreak strains. The majority of amino acid changes were concentrated within the NS5, E, NS1, and NS3 proteins, suggesting continued genetic diversification within the circulating Cosmopolitan lineage. Overall, these findings demonstrate the persistence of established DENV lineages alongside ongoing viral evolution during the 2023 outbreak in Vietnam. This study highlights the value of whole-genome surveillance for monitoring dengue virus genetic diversity and provides insight that may support future epidemiological surveillance, vaccine evaluation, and outbreak preparedness.