Genomic characterization and serotype distribution of dengue virus circulating in Pakistan during 2024

Dengue virus (DENV) remains a significant public health concern in Pakistan, with recurrent outbreaks necessitating continuous genomic surveillance. The 2024 dengue outbreak prompted an investigation into circulating serotypes and genomic diversity. The National Institute of Health (NIH), Pakistan, received 524 NS-1 confirmed dengue samples across multiple districts in 2024. Serotyping via real-time PCR confirmed DENV-2 in 93 % (n = 361) and DENV-1 in 7 % (n = 27) of 388 positive samples, with no co-infections detected. Males accounted for 65 % of cases, with a mean age of 30.78 ± 15.03 years. Metagenomic sequencing using Illumina MiSeq yielded 33 successful DENV genomes (DENV-2: 23; DENV-1: 10), including the first reports from Chilas and Kech. Phylogenetic analysis showed 2024 DENV-1 (genotype III) and DENV-2 (Cosmopolitan genotype, clade IV-A/2II_F.1.1) closely resembling 2022-2023 Pakistani strains (∼99 % homology). Time-scaled analysis estimated 22 of 23 DENV-2 sequences shared a most recent common ancestor (MRCA) in 2018, while a divergent strain (NIHPAK-246) traced back to 2005. DENV-2 strains from Kech exhibited >99.8 % similarity with Rawalpindi strains, suggesting transmission links. DENV-1 sequences shared an MRCA in 2016, indicating sustained circulation. Mutation analysis identified NS4B: G240S in DENV-1 from Chilas, suggesting localized adaptation. These findings highlight the need for sustained genomic surveillance, monitoring transmission dynamics, and targeted public health interventions in Pakistan.