Surveillance of West Nile Virus in Tunisia: Evidence from Human and Entomological Investigation

West Nile virus (WNV) is a mosquito-borne pathogen of the <i>Flaviviridae</i> family that poses recurring public health threats in Tunisia, where <i>Culex pipiens</i> is recognized as the primary vector. Identification of circulating strains in different mosquito species is essential for targeted prevention and control. Between November 2021 and October 2022, mosquitoes were collected at four high-risk sites, and human samples were obtained through the national meningitis surveillance program. Human serum, cerebrospinal fluid (CSF), and urine samples were tested for WNV-specific IgM and IgG antibodies using ELISA, and molecular diagnosis was performed using Real-time RT-PCR (RRT-PCR). Positive samples underwent sequencing for phylogenetic characterization. Serological investigation on human serum revealed the presence of IgM and/or IgG antibodies reactive to WNV antigens, which may indicate exposure to WNV or related flaviviruses. RNA of WNV was detected in 21 mosquito pools (10.19%) belonging to <i>Culex pipiens</i>, <i>Cx. perexiguus</i>, <i>Aedes caspius,</i> and <i>Ae. detritus</i>, as well as in three human cases. Phylogenetic analysis of positive human and mosquito samples showed that all detected WNV strains belonged to sublineage 1a. The concurrent detection of WNV in vectors and humans confirms active circulation in Tunisia and underscores the role of <i>Culex</i> spp. Mosquitoes in transmission. Sustained multidisciplinary surveillance integrating entomological and clinical data is critical for early detection, guiding control measures, and preventing future outbreaks in humans and animals.