Abstract
Greece is one of the countries in Europe most affected by West Nile virus (WNV), and since 2010, when the virus caused a large outbreak with 197 human neuroinvasive cases, outbreaks occur almost every year. Mosquito surveillance is an indirect sign of virus circulation; therefore, the purpose of the study was the molecular detection of WNV in 45,988 C. pipiens s.l. mosquitoes collected during 2024 in four Regions of Greece and the genetic characterization of the virus strains. WNV was detected in 41 of 1316 (3.12%) Culex spp. mosquito pools. Next-generation sequencing was applied to the WNV-positive samples that had a high viral load. All WNV sequences belong to Cluster B of the sub-lineage Europe WNV-2A presenting a temporal clustering. The WNV infection rates varied highly across the Regions, regional units and months, being higher in Thessaly and Central Macedonia Regions, especially in July and September. All mosquito pools were also tested for Usutu virus (USUV), and one pool was found positive, with sequence clustering into the EU-2 lineage. A subset of mosquitoes (737 pools) was tested for additional viruses, and bunya-like viruses were detected in 6 pools with sequences clustering into four distinct subclades. The prompt detection of pathogenic viruses is helpful for the design of control measures, while the detection of insect-specific viruses provides insights into viral diversity and evolution.