Usutu virus promotes West Nile virus replication in Culex pipiens biotype molestus during simultaneous and sequential co-infections

The epidemiological co-circulation of the two flaviviruses West Nile virus (WNV) and Usutu virus (USUV) in several European countries poses the risk of co-infections in vertebrate hosts and mosquito vectors. WNV is an important human pathogen, whereas USUV is primarily relevant in veterinary and wildlife health. Co-infections have been detected in birds and, occasionally, in humans. To determine the potential consequences of co-exposure and co-infections in mosquitoes on viral transmission, lab-reared Culex pipiens biotype molestus were orally infected with both viruses either simultaneously or sequentially at 7-day intervals with German isolates of WNV lineage 2 and USUV lineage Europe 3. Simultaneous exposure resulted in a significantly increased WNV infection rate, while infection and transmission of USUV were inhibited at the same time. During sequential exposure, prior WNV exposure also had a negative effect on susceptibility to USUV, whereas conversely, WNV infection rates were not altered by prior USUV exposure. Furthermore, mosquitoes with established co-infections after simultaneous co-exposure exhibited higher WNV viral loads than those infected exclusively with WNV. The study reveals complex interactions between WNV, USUV, and the mosquito vector, which could influence vector competence and vector capacity of Culex pipiens biotype molestus in areas with co-circulation of WNV and USUV.