West Nile virus (WNV) is endemic in southeastern Romania and, after the unprecedented urban epidemic in Bucharest in 1996 caused by lineage 1 WNV, cases of West Nile fever have been recorded every year. Furthermore, a new outbreak occurred in 2010, this time produced by a lineage 2 WNV belonging to the Eastern European clade (Volgograd 2007-like strain), which was detected in humans and mosquitoes in the following years. We report here, for the first time, the emergence, in 2015, of lineage 2 WNV belonging to the monophyletic Central/Southern European group of strains which replaced in 2016, the previously endemized lineage 2 WNV Volgograd 2007-like strain in mosquito populations. The emerged WNV strain harbors H249P (NS3 protein) and I159T (E glycoprotein) substitutions, which have been previously associated in other studies with neurovirulence and efficient vector transmission. In 2016, both early amplification of the emerged WNV and complete replacement in mosquito populations of the previously endemized WNV occurred in southeastern Romania. These events were associated with a significant outbreak of severe West Nile neuroinvasive disease in humans.

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.

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.

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.

West Nile virus (WNV) is the most widely spread arbovirus in the world. Early detection of this virus in mosquito populations is essential for implementing rapid vector control measures to prevent outbreaks. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) is a powerful tool for the detection of WNV in mosquito pools, but it is a time- and resource-consuming assay. We used a Rapid Analyte Measurement Platform (RAMP) assay in a vector surveillance program for rapid detection of WNV in mosquitoes collected in Bucharest city, Romania, in 2021. The positive mosquito pools were tested for confirmation with real-time RT-PCR. Three out of the 24 RAMP assay positive pools were not confirmed by real-time RT-PCR. We consider that RAMP assay can be used as a fast and reliable method for the screening of WNV presence in mosquito pools, but we recommend that samples with values ranging from 30 to 100 RAMP units should fall in a grey zone and should be considered for real-time RT-PCR confirmation.