Contents lists available at ScienceDirect Acta Tropica journal homepage: www.elsevier.com/locate/actatropica Detection of West Nile Virus – Lineage 2 in Culex pipiens mosquitoes, associated with disease outbreak in Greece, 2017 Konstantinos Mavridis a, ⁎ , Emmanouil A. Fotakis b , Ilias Kioulos b , Spiridoula Mpellou c , Spiros Konstantas c , Evangelia Varela d , Sandra Gewehr e , Vasilis Diamantopoulos f , John Vontas a,b a Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, 70013, Greece b Pesticide Science Laboratory, Department of Crop Science, Agricultural University of Athens, 11855, Athens, Greece c Bioefarmoges Eleftheriou LP -Integrated Mosquito Control, Marathon, 19007, Greece d Inseco LP -Integrated Mosquito Control, Elefsina, 19200, Greece e EcoDevelopment SA-Integrated Mosquito Control, Thessaloniki, 57010, Greece f Public Health Director, Region of Peloponnese, Tripoli, 22100, Greece ARTICLE INFO Keywords: WNV Greece Culex pipiens Culex pipiens/molestus Pyrethroid resistance Kdr Diflubenzuron resistance Chitin synthase Vector control Insecticide resistance Larvicides Residual spraying Evidence-based response Vector-borne infections ABSTRACT 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 mos- quitoes 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. 1. Introduction During July-October 2017, a West Nile Virus (WNV) outbreak un- raveled in Southern Greece, in the Peloponnese region, the largest pe- ninsula in Greece covering 21 439 km 2 and hosting 1 100 071 people. Until October 25, 2017 the outbreak has resulted in five human fatal- ities and a total of forty-eight confirmed WNV-infection cases in Greece. Twenty-eight of these cases (58.3%) presented West Nile neuroinvasive disease (WNND) with severe clinical manifestations. The suspected prefectures of exposure were Argolis (municipalities of Argos-Mykines and Nafplio) and Arcadia (municipality of North Kynouria) (HCDCP, 2017). WNV is thought to circulate in Europe at least since the 1960s and WNV Lineage 2 is responsible for one of the deadliest outbreaks in Europe occurring in Northern Greece during the summer of 2010, resulting in 262 clinical human cases and 35 fatalities. Successive outbreaks occurred over the next four years mainly in Central and Northeastern Greece (Gossner et al., 2017; Patsoula et al., 2016; Hernandez-Triana et al., 2014). WNV is an RNA virus belonging to the Flaviviridae family, which is transmitted mainly by the mosquito species Culex pipiens. Aedes albo- pictus can also transmit the virus, but is considered a secondary vector (Hernandez-Triana et al., 2014; Fortuna et al., 2015; Giatropoulos et al., 2012). Birds are amplifying hosts of WNV while mammals, including humans, are incidental and “dead-end” hosts. Among the three Cx. pi- piens biotypes, Cx pipiens pipiens, Cx. pipiens molestus and the hybrid form Cx. pipiens pipiens/molestus, the latter is considered a highly competent vector of WNV due to its opportunistic biting behavior which maintains the virus among avian hosts and favors its https://doi.org/10.1016/j.actatropica.2018.02.024 Received 11 November 2017; Received in revised form 14 February 2018; Accepted 17 February 2018 ⁎ Corresponding author. E-mail address: konstantinos_mavridis@imbb.forth.gr (K. Mavridis). Acta Tropica 182 (2018) 64–68 Available online 21 February 2018 0001-706X/ © 2018 Elsevier B.V. All rights reserved. T