Ixodes ricinus as potential vector for Usutu virus.

Usutu virus (USUV) is an emerging flavivirus that is maintained in an enzootic cycle with mosquitoes as vectors and birds as amplifying hosts. In Europe, the virus has caused mass mortality of wild birds, mainly among Common Blackbird (Turdus merula) populations. While mosquitoes are the primary vectors for USUV, Common Blackbirds and other avian species are exposed to other arthropod ectoparasites, such as ticks. It is unknown, however, if ticks can maintain and transmit USUV. We addressed this question using in vitro and in vivo experiments and field collected data. USUV replicated in IRE/CTVM19 Ixodes ricinus tick cells and in injected ticks. Moreover, I. ricinus nymphs acquired the virus via artificial membrane blood-feeding and maintained the virus for at least 70 days. Transstadial transmission of USUV from nymphs to adults was confirmed in 4.9% of the ticks. USUV disseminated from the midgut to the haemocoel, and was transmitted via the saliva of the tick during artificial membrane blood-feeding. We further explored the role of ticks by monitoring USUV in questing ticks and in ticks feeding on wild birds in the Netherlands between 2016 and 2019. In total, 622 wild birds and the Ixodes ticks they carried were tested for USUV RNA. Of these birds, 48 (7.7%) carried USUV-positive ticks. The presence of negative-sense USUV RNA in ticks, as confirmed via small RNA-sequencing, showed active virus replication. In contrast, we did not detect USUV in 15,381 questing ticks collected in 2017 and 2019. We conclude that I. ricinus can be infected with USUV and can transstadially and horizontally transmit USUV. However, in comparison to mosquito-borne transmission, the role of I. ricinus ticks in the epidemiology of USUV is expected to be minor. Author summary: Usutu virus (USUV) is a virus primarily circulating between birds and mosquitoes. Other blood-feeding arthropods, such as ticks, feed on birds. However, it is unknown whether ticks can be infected with USUV and transmit the virus to birds or other animals. Therefore, we infected a tick cell line and ticks collected from the field with USUV. We showed that the virus replicates in tick cells, as well as in field-collected ticks. Moreover, we showed that USUV disseminated through the tick body and was excreted with the saliva of ticks during artificial feeding. We further investigated if ticks are infected with USUV while feeding on wild birds and when searching for a host in the vegetation. We found that 7.7% of tick-infested birds carried USUV positive ticks, while USUV was not detected in questing ticks. Our findings provide evidence that ticks can be infected with USUV and might transmit the virus to another host, but the role of ticks in the transmission of USUV is expected to be minor. [ABSTRACT FROM AUTHOR]