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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]

Seoul orthohantavirus (SEOV) is a rat-borne zoonotic virus that is transmitted via inhalation of aerosolized infectious excreta, and can cause hemorrhagic fever with renal syndrome (HFRS) in humans worldwide. In rats, SEOV predominantly exists as a persistent infection in the absence of overt clinical signs. Lack of disease in rats is attributed to downregulation of pro-inflammatory and upregulation of regulatory host responses. As lung microvascular endothelial cells (LMECs) represent a primary target of infection in both human and rats, infections in these cells provide a unique opportunity to study the central role of LMECs in the dichotomy between pathogenicity in both species. In this study, host responses to SEOV infection in primary human and rat LMECs were directly compared on a transcriptional level. As infection of rat LMECs was more efficient than human LMECs, the majority of anti-viral defense responses were observed earlier in rat LMECs. Most prominently, SEOV-induced processes in both species included responses to cytokine stimulus, negative regulation of innate immune responses, responses to type I and II interferons, regulation of pattern recognition receptor signaling and MHC-I signaling. However, over time, in the rat LMECs, responses shifted from an anti-viral state towards a more immunotolerant state displayed by a PD-L1, B2M-, JAK2-focused interaction network aiding in negative regulation of cytotoxic CD8-positive T cell activation. This suggests a novel mechanism by which species-specific orthohantavirus-induced endothelium and T cell crosstalk may play a crucial role in the development of acute disease in humans and persistence in rodents. Author summary: Seoul orthohantavirus is a rat-borne zoonotic virus that can cause hemorrhagic fever with renal syndrome in humans worldwide while it generally leads to persistent infection in absence of clinical signs in the natural reservoir host, the Norway rat. It is believed that persistent infection in reservoir hosts is due to downregulation of pro-inflammatory immune responses and upregulation of regulatory responses, while in humans excessive immune responses contribute to disease. Endothelial cells in the lungs are of particular interest as they represent a primary target during Seoul orthohantavirus infection in both species. So far, it remains incompletely understood how the responses of these cells to Seoul orthohantavirus contribute to the development of disease in humans and the prevention of such in rats. In this study, we compared the host responses of human and rat lung endothelial cells to Seoul orthohantavirus infection and found that anti-viral responses from both species are comparable, but differ in timing. Additionally, we identified a novel protein interaction network during later phases of acute infection in rat cells, which may play a crucial role in the development of disease in humans and persistence in rodents. [ABSTRACT FROM AUTHOR]