Not all West Nile virus lineages behave alike: vector competence and minimum infectious dose differences of lineages 1 and 2

The globally distributed arbovirus West Nile virus (WNV) continues to expand across Europe, with rising numbers of human cases and an increasingly broad geographic distribution. WNV is primarily transmitted by mosquitoes of the genus Culex . Out of nine WNV lineages, human pathogenicity has been clearly established for lineages 1 and 2, but differences in their transmission dynamics, such as minimal infectious dose and transmission efficiency, remain poorly understood. In this study, we investigated how viral lineage, mosquito species, and blood meal titer influence the vector competence of two primary WNV vectors from Europe, Cx. pipiens biotype pipiens and Cx. torrentium , as well as the invasive mosquito species Aedes albopictus . Female mosquitoes were fed with increasing blood meal titers containing either of the WNV lineages. After an incubation period of 14 days at a mean temperature of 24°C, mosquito body titers were quantified, and the presence of infectious viral particles in the saliva was assessed. Our results revealed clear differences between the two lineages. Lineage 2 resulted in higher transmission efficiencies across all three species and required lower infectious doses to cause transmission. Among the tested species, Cx. torrentium proved to be a highly competent vector (max. transmission efficiency = 30%, minimum infectious dose = 10 5 TCID 50 /mL), despite its underrepresentation in research. These findings provide detailed insights into how viral lineage, mosquito species, and blood meal titer might shape WNV transmission, informing future risk assessments and efforts to mitigate WNV transmission in Europe.