Detection and structural characterization of bat-associated dengue virus serotype 2: A country-level first detection in Pakistan.

Bats (order Chiroptera) are recognized as reservoirs for diverse zoonotic viruses, including members of the Flavivirus genus. Despite global reports of bat-associated Dengue Virus (DENV), its presence in Pakistan has remained undocumented. This study investigates DENV circulation in bat populations across Islamabad/Rawalpindi, Lahore, Mardan, Swat, and Lower Dir. Fecal samples were screened using CprM-specific primers, yielding 14 amplified viral isolates (BatDENV01-BatDENV14). Phylogenetic analysis confirmed all sequences as Dengue Virus serotype 2 (DENV-2), clustering with bat-associated isolates from Costa Rica (KY461760-KY461769), mosquito-derived (KY461759), and human-derived strains (AY692466, AY692469), and suggests a potential cross-species viral link. Comparative amino acid analysis revealed several substitutions in the CprM region, some of which were unique to the Pakistani bat isolates. These substitutions may reflect host-specific adaptation or regional viral evolution. Secondary structure prediction using SOPMA and PSIPRED indicated conserved α-helices and β-strands, while tertiary modeling via Robetta and visualization in PyMol showed structural homology with reference DENV-2 proteins, and minor modifications were observed. This study provides the first molecular and structural evidence of DENV-2 circulation in Pakistani bats, suggesting a possible ecological link between bat, mosquito, and human transmission cycles. These findings underscore the need for expanded molecular surveillance and structural characterization of bat-associated Flaviviruses to better understand their zoonotic potential and inform public health strategies.