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• Mosquitoes belonging to seven species were collected in 2022 in western Iran. • WNV viral RNA was detected in Culiseta longiareolata and Culex hortensis. • Both species are added to vectors of the virus in Iran. • To the best of our knowledge herein we report WNV in Cx.hortensis for the first time. 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. [Display omitted] [ABSTRACT FROM AUTHOR]

During July-October 2017 a WNV outbreak took place in the Peloponnese, Southern Greece with five confirmed deaths. During routine monitoring survey in the Peloponnese, supported by the local Prefecture, we have confirmed the presence of all three Culex pipiens biotypes in the region, with a high percentage of Culex pipiens/molestus hybrids (37.0%) which are considered a highly competent vector of WNV. Kdr mutations related to pyrethroid resistance were found at relatively low levels (14.3% homozygosity) while no mosquitoes harboring the recently identified chitin synthase diflubenzuron-resistance mutations were detected in the region. As an immediate action, following the disease outbreak (within days), we collected a large number of mosquitoes using CO 2 CDC traps from the villages in the Argolis area of the Peloponnese, where high incidence of WNV human infections were reported. WNV lineage 2 was detected in 3 out of 47 Cx. pipiens mosquito pools (detection rate = 6.38%). The virus was not detected in any other mosquito species, such as Aedes albopictus , sampled from the region at the time of the disease outbreak. Our results show that detection of WNV lineage 2 in Cx. pipiens pools is spatially and chronologically associated with human clinical cases, thus implicating Cx. pipiens mosquitoes as the most likely WNV vector. The absence of diflubenzuron resistance mutations and the low frequency of pyrethroid ( kdr ) resistance mutations indicates the suitability of these insecticides for Cx. pipiens control, in the format of larvicides and/or residual spraying applications respectively, which was indeed the main (evidence based) response, following the disease outbreak. [ABSTRACT FROM AUTHOR]

• Screening of mosquito-borne viruses in Culex pipiens mosquitoes by high-throughput chip. • Detection of anti-WNV antibodies in the sera of Moroccan horses by three techniques. • Detection, for the first time in Morocco, of WNV from Culex pipiens mosquitoes. West Nile virus (WNV) is one of the most widely distributed mosquito-borne viruses in the world. In North Africa, it causes human cases of meningoencephalitis with fatalities in Algeria and in Tunisia, whereas only horses were affected in Morocco. The aims of this study were to detect WNV in mosquitoes and to determine seroprevalence of WNV in Moroccan horses by the detection of IgG antibodies. A total of 1455 mosquitoes belonging to four different species were grouped by collection site, date, and sex with 10 specimens per pool and tested for 38 arboviruses using a high-throughput chip based on the BioMark Dynamic array system. Out of 146 mosquito pools tested, one pool was positive for WNV. This positive pool was confirmed by real time RT-PCR. The serosurvey showed that 33.7% (31/92) of horses were positive for competitive enzyme-linked immunosorbent assay (cELISA) test. The flavivirus-sphere microsphere immnoassay (MIA) test, targeting three flaviviruses (WNV, Usutu virus (USUV) and Tick borne encephalitic virus (TBEV)) showed that 23 sera out of 31 were positive for WNV, two for USUV, two for USUV or WNV, and four for an undetermined flavivirus. Virus neutralization tests with USUV and WNV showed that 28 of 31 sera were positive for WNV and all sera were negative for USUV. This study reports, for the first time, the detection of WNV from Culex pipiens mosquitoes in Morocco and its circulation among horses. This highlights that the detection of arboviruses in mosquitoes could serve as an early warning signal of a viral activity to prevent future outbreaks in animals and humans. This study describes, for the first time, the detection of WNV in Culex pipiens mosquitoes from Morocco. We used high-throughput to screening mosquito-borne viruses. Image, graphical abstract [ABSTRACT FROM AUTHOR]