17Background: West Nile virus (WNV) is a zoonotic arbovirus of great medical and veterinary 18 importance, threatening the health of humans and equines worldwide. Mosquitoes belonging 19 to the Culex (Cx.) pipiens complex are major vectors but numerous other mosquito species 20 have also been implicated as vectors of WNV. Due to variations in blood-feeding behaviour, 21 the different biotypes and hybrids of Cx. pipiens influence the transmission of WNV, from 22 enzootic cycles (between mosquitoes and birds), to spill-over transmission to humans and 23 equines.
24Methods: In this study, mosquitoes were collected and analysed from two regional units 25 (RUs) of Greece with reported cases of WNV within the past 4 years; Palaio Flairo and 26 Argolida (in Attica and Peloponnese regions, respectively). Collections using different types 27 2 of mosquito surveillance traps were undertaken in May-June 2018 during the early period of 28 the WNV transmission season.29 Results: A total of 1062 mosquitoes were collected, with Biogents Sentinel traps (BG traps) 30 collecting both a greater number of mosquitoes across all species and Cx. pipiens complex 31 individuals than Centres for Disease Control miniature light traps (CDC traps) or Heavy Duty 32 Encephalitis Vector Survey traps (EVS traps). Identification of collected mosquitoes (using 33 both morphological keys and molecular barcoding) confirmed the presence of additional 34 species including Aedes (Ae.) albopictus, Ae. caspius and Culiseta (Cs.) longiareolata. The 35 prevalence of Cx. pipiens biotypes in the RU of Palaio Faliro was 54.5% pipiens type, 20.0% 36 molestus type and 25.5% hybrids. In the RU of Argolida, the collection comprised 68.1% 37 pipiens type, 8.3% molestus type and 23.6% hybrids. Screening individual unfed female38 mosquitoes for WNV (molecular xenomonitoring) resulted in detection in three females of the 39 pipiens type and in one hybrid; all collected from the RU of Argolida.40 Conclusions: As hybrids play an important role in spill-over transmission of WNV to 41 humans and equines, these findings highlight the importance of undertaking entomological 42 surveillance programs incorporating molecular xenomonitoring at the onset of the 43 transmission season to provide an early warning system for health authorities aiming to 44 prevent WNV outbreaks in Greece. 45 46 Keywords 47 52 genus [1-4]. Natural transmission of WNV mainly occurs in enzootic cycles between birds 53 and competent ornithophilic mosquito vectors, with avian species being the principal 54 3 maintenance and amplifying hosts of WNV as many species develop sufficient viremia for 55 onward transmission. This allows transmission to continue where competent mosquitoes are 56 present in a specific area under suitable environmental conditions [5]. Additionally, spill-over 57 transmission can occur when competent vectors feed on humans or horses. During natural 58 transmission these mammalian species are considered dead-end hosts since they cannot 59 sustain sufficient viraemia for further vec...