Culex pipiens sensu lato has been documented as West Nile virus (WNV) vector in southeastern Romania. Bucharest, the densely populated capital city of Romania, and the surrounding Ilfov county are WNV hotspots. In this area, the morphologically indistinguishable biotypes of Cx. pipiens, namely pipiens and molestus , are usually differentiated by their behavioral and physiological traits. Their involvement in WNV transmission, as suggested by entomological investigations, was not previously documented for each biotype. We used a Real-Time PCR assay based on CQ11 microsatellite to identify the Cx. pipiens biotypes and their hybrids collected in various habitats in the Bucharest metropolitan area. A sympatric distribution of both biotypes was observed, with a preference of green areas for pipiens , and human settings and animal farmlands for molestus . In the latter habitats, pipiens and molestus were found in mixed aboveground populations. A low number of hybrids was found suggesting existence of reproductive isolation. In subway tunnels molestus was dominant with a higher number of hybrids recorded than aboveground. Blood-engorged mosquitoes were identified to biotype and the blood meal source identified by DNA barcoding. Overall, Cx. pipiens s.l. fed mainly on birds, commonly on house sparrows, collared doves, and blackbirds, which are potential WNV-amplifying hosts. The preference for avian hosts was expressed strongest by pipiens biotype, while molestus was substantially less specific, feeding on avian and mammal hosts with similar frequency, with humans representing 20% of the hosts. Hybrids had a host choice closer to that of molestus . These findings highlight the role of pipiens biotype as enzootic/epizootic vector, and specifically show molestus as the bridge vector for WNV. The pipiens and molestus biotypes show important differences in habitat preferences, including oviposition; these findings demonstrate that targeted mosquito control to limit WNV transmission may be possible.

Usutu virus (USUV) is an emergent arbovirus in Europe causing mortality in bird populations. Similar to West Nile virus (WNV), USUV is maintained in sylvatic cycles between mosquito vectors and bird reservoirs. Spillover events may result in human neurological infection cases. Apart from indirect evidence provided by a recent serological study in wild birds, the circulation of USUV in Romania was not assessed. We aimed to detect and molecular characterize USUV circulating in mosquito vectors collected in South-Eastern Romania—a well-known WNV endemic region—during four transmission seasons. Mosquitoes were collected from Bucharest metropolitan area and Danube Delta, pooled, and screened by real-time RT-PCR for USUV. Partial genomic sequences were obtained and used for phylogeny. USUV was detected in Culex pipiens s.l. female mosquitoes collected in Bucharest, in 2019. The virus belonged to Europe 2 lineage, sub-lineage EU2-A. Phylogenetic analysis revealed high similarity with isolates infecting mosquito vectors, birds, and humans in Europe starting with 2009, all sharing common origin in Northern Italy. To our knowledge, this is the first study characterizing a strain of USUV circulating in Romania.

Culex pipiens pipiens and Culex pipiens molestus mosquitoes are the vectors of West Nile virus in south-eastern Romania, an area of intense circulation and human transmission of this virus. The level of insecticide resistance for the mosquito populations in the region has not been previously assessed. Culex pipiens mosquitoes collected between 2018 and 2019 in south-eastern Romania from different habitats were subjected to biotype identification by real-time PCR. Substitutions causing resistance to organophosphates and carbamates (F290V and G119S in acetylcholinesterase 1) and to pyrethroids (L1014F in voltage gated Na⁺ channel) were screened by PCR or sequencing. Substitutions F290V and G119S were detected at very low frequencies and only in heterozygous state in Culex pipiens molestus biotype specimens collected in urban areas. The molestus biotype population analysed was entirely homozygous for L1014F, and high frequencies of this substitution were also found for pipiens biotype and hybrid mosquitoes collected in urban and in intensive agriculture areas. Reducing the selective pressure by limiting the use of pyrethroid insecticides only for regions where it is absolutely necessary and monitoring L1014F mutation should be taken into consideration when implementing vector control strategies.

West Nile virus (WNV) is a mosquito-borne pathogen with a worldwide distribution. Climate change and human activities have driven the expansion of WNV into new territories in Europe during the last two decades. Romania is endemic for WNV circulation since at least 1996 when the presence of lineage 1 was documented during an unprecedented outbreak. Lineage 2 was first identified in this country during a second significant human outbreak in 2010. Its continuous circulation is marked by clade replacement, and even co-circulation of different strains of the same clade was observed until 2016. The present study aims to fill the information gap regarding the WNV strains that were circulating in Romania between 2017 and 2023, providing chiefly viral sequences obtained from mosquito samples collected in the Bucharest metropolitan area, complemented by human and bird viral sequences. WNV was detected mainly in Culex pipiens mosquitoes, the vectors of this virus in the region, but also in the invasive Aedes albopictus mosquito species. Lineage 2 WNV was identified in mosquito samples collected between 2017 and 2023, as well as in human sera from patients in southern and central Romania during the outbreaks of 2017 and 2018. Both 2a and 2b sub-lineages were identified, with evidence of multiple clusters and sub-clusters within sub-lineage 2a, highlighting the complex and dynamic circulation of WNV in Romania, as a consequence of distinct introduction events from neighboring countries followed by in situ evolution.