West Nile virus in adults and larvae of Culiseta longiareolata and Culex hortensis (Diptera: Culicidae) captured in Hamedan, western Iran

West Nile virus (WNV) is an emerging arbovirus transmitted by mosquitoes. Although it is considered the most widespread mosquito-borne arbovirus in Iran, vectors of this zoonotic pathogen remain unknown in many regions. This study aimed to assess the presence of WNV in mosquitoes collected in the western city of Hamedan in 2022. Adult mosquitoes were captured using light traps, and mosquito larvae were collected by dipping technique from 45 diverse habitats, including urban, suburban, and rural sites. Specimens were identified and pooled into 69 batches based on their species for viral RNA extraction and Real-Time PCR. In total, 3243 mosquitoes (2209 larvae and 1034 adults) were captured and identified as Culiseta longiareolata, Culex hortensis, Anopheles maculipennis s.l., Culex theileri, Culex pipiens, Anopheles claviger, and Anopheles superpictus s.l. in decreasing order. Molecular screening revealed seven WNV-positive pools of Culiseta longiareolata and Culex hortensis in rural (n = 5) and urban areas (n = 2). Detection of WNV RNA indicates active circulation in mosquitoes and risk of transmission to humans and animals in Hamadan. These findings identify putative vectors in Hamadan, though vectors likely vary regionally in Iran. Further surveillance is needed to elucidate local WNV epidemiology and transmission dynamics fully. Nonetheless, this study provides important baseline evidence of WNV activity to guide prevention strategies in this area.