Dengue virus (DENV) remains a global health threat, with Pakistan experiencing recurrent outbreaks, yet non-structural genes (NS1, NS2A, NS5) critical for viral replication and pathogenesis remain understudied in local strains. In this study, 150 NS1-positive serum samples (2022-2024) were analyzed via nested PCR, Sanger sequencing, and phylogenetic tools (MEGA11), revealing DENV-2 as the dominant serotype (85 % of samples), with NS5 showing 97 % homology to Indian/Swat strains, while NS1 and NS2A exhibited sequence variation in functional domains. In silico modeling and docking studies identified RK-0404,678 as a strong binder to NS5 (-6.4 kcal/mol) and Galidesivir to NS5 (-7.2 kcal/mol), suggesting their potential as antiviral candidates. These findings highlight DENV-2 dominance in Pakistan and identify conserved regions in NS5 as promising drug targets, though further experimental validation is warranted.

Dengue fever, a rapidly spreading mosquito-borne viral disease, poses a major global health issue, particularly in tropical and subtropical regions. The absence of a universally effective vaccine against all four dengue virus serotypes necessitates continued exploration of novel vaccine strategies. This study evaluated a PrM-derived multiple antigenic peptide (MAP) vaccine candidate in BALB/c mice. Molecular docking analyses predicted favourable interactions of the selected PrM-derived B-cell epitope with both human (human BCR Fab region PDB ID: 5IFH) and mouse (mouse BCR PDB ID: 8EMA) B-cell receptors, supporting its antigenic potential. ChemSketch was used to develop the PrM–MAP vaccine construct, which was then commercially synthesized. Mice received single, double, and triple booster doses of the vaccine, while control groups received either PBS or adjuvant alone. Humoral immune responses were measured by ELISA, and safety was evaluated through in vitro cytotoxicity assays on HEK293 cells and the in vivo histopathological examination of major organs. Mice immunized with a single booster dose produced significant levels of PrM-specific antibodies compared to subsequent booster doses, suggesting variability in the humoral response elicited. Optimal coating concentrations in ELISA demonstrated the importance of antigen concentrations in the sensitivity of the assay. The safety evaluation indicated high cell viability, up to 150 µg/mL of PrM–MAP, in HEK293 cells, and an absence of significant histopathological damage in treated animals. The PrM–MAP vaccine construct elicited a detectable humoral immune response while demonstrating a high safety profile in vitro and in vivo. These findings indicate the potential of MAP-based vaccine platforms as promising candidates for further development against the dengue virus.