Detection of West Nile Virus Lineage 2 in Eastern Romania and First Identification of Sindbis Virus RNA in Mosquitoes Analyzed using High-Throughput Microfluidic Real-Time PCR

Abstract

The impact of mosquito-borne diseases on human and veterinary health is being exacerbated by rapid environmental changes caused mainly by changing climatic patterns and globalization. To gain insight into mosquito-borne virus circulation from two counties in eastern and southeastern Romania, we have used a combination of sampling methods in natural, urban and peri-urban sites. The presence of 37 mosquito-borne viruses in 16,827 pooled mosquitoes was analyzed using a high-throughput microfluidic real-time PCR assay. West Nile virus (WNV) was detected in 10/365 pools of Culex pipiens (n = 8), Culex modestus (n = 1) and Aedes vexans (n = 1) from both studied counties. We also report the first molecular detection of Sindbis virus (SINV) RNA in the country in one pool of Culex modestus. WNV infection was confirmed by real-time RT-PCR (10/10) and virus isolation on Vero or C6/36 cells (four samples). For the SINV-positive pool, no cytopathic effectwas observed after infection of Vero or C6/36 cells, but no amplification was obtained in conventional SINV RT-PCR. Phylogenetic analysis of WNV partial NS5 sequences revealed that WNV lineage 2 of theCentral-Southeast European clade, has a wider circulation in Romania than previously known.

Spatial patterns ofHyalomma marginatum-borne pathogens in the Occitanie region (France), a focus on the intriguing dynamics ofRickettsia aeschlimannii

“…CCHFV detection on samples was performed apart from this study . See Michelet et al 2014;Gondard et al 2018b;Crivei et al 2023; for more details.…”

Belgian Culex pipiens pipiens are competent vectors for West Nile virus while Culex modestus are competent vectors for Usutu virus

“…Conversely, no Culex modestus in the WNV-fed group (n = 6) developed a WNV infection. There is substantial evidence of WNV RNA detected in field-captured Culex modestus from other countries [14,[27][28][29][30][31][32], and there is data on the vector competence of laboratory colonies showing that Culex modestus is an efficient WNV vector [12,33]. Therefore, it is possible that our sample size was not high enough to obtain WNV-infected Culex modestus.…”

“…Of the two biotypes, pipiens is considered the more efficient vector for both WNV [21,26] and USUV [25]. In field-captured Culex modestus mosquitoes, WNV and USUV RNA have been detected [14,15,[27][28][29][30][31][32], but currently the only evidence of WNV vector competence in live mosquitoes comes from laboratory colonies [12,33]. So far, there are no measures of USUV vector competence in Culex modestus using field or laboratory mosquitoes.…”

Serologic and Genomic Investigation of West Nile Virus in Kosovo

“…This suggests a dynamic circulation sustained by the local evolution and transit of migratory bird hosts between Africa and Eurasia. Over the past few years, there has been an increasing number of serological and genomic studies on WNV epidemiology in southeast Europe [ 18 , 20 , 21 , 22 , 23 , 24 ]. However, there is no information about the impact of WNV on human health in Kosovo, while only scarce data are available from surveillance of animal hosts [ 7 , 8 ].…”