Overwintering and vertical transmission of Usutu virus in Culex pipiens , biotype pipiens mosquitoes contribute to annual re-emergence

Usutu virus (USUV) is a mosquito-borne flavivirus that re-emerges annually in Europe, with outbreaks coinciding with the seasonal activity of Culex pipiens, its primary vector. Genomic analyses suggest local overwintering of similar virus strains, possibly in diapausing Cx. pipiens. Since diapausing mosquitoes do not blood-feed, vertical transmission is required before diapause. This study investigated whether prediapause vertical transmission occurs and whether diapausing Cx. pipiens mosquitoes can serve as overwintering reservoirs. Furthermore, to assess replication and virus evolution in the mosquitoes, the virus mutation rate in non-diapausing and post-diapausing mosquitoes was evaluated. In a controlled laboratory setting, mosquito larvae were subjected to diapause-inducing (8:16 light:dark, 16 °C) or non-diapause (16:8 light:dark, 23 °C) conditions. USUV-infected adults were then held for 16 weeks at 6 °C (diapause) or 23 °C (non-diapause). Every four weeks mosquitoes were analysed for virus in bodies, legs, and saliva. Subsets of diapausing mosquitoes were incubated at spring-like conditions (16:8 light:dark, 23 °C) for 14 days to evaluate post-diapause viral development. All non-diapausing mosquitoes developed infection, with 97% showing virus in saliva. In contrast, only 1.19% of diapausing mosquitoes were virus-positive, with no dissemination or transmission. Remarkably, post-diapausing mosquitoes showed 100% body infection, and 98% saliva positivity, indicating rapid viral reactivation after diapause. Viral sequencing showed more minor variants in post-diapausing mosquitoes. Additionally, 2.9% of egg rafts from infected females tested USUV-positive, confirming vertical transmission. These findings provide experimental evidence that diapausing Cx. pipiens can act as USUV overwintering reservoirs and contribute to seasonal re-emergence of the virus.