Zika virus (ZIKV) is an arthropod-borne flavivirus predominantly transmitted by Aedes aegypti mosquitoes and poses a global human health threat. All flaviviruses, including those that exclusively replicate in mosquitoes, produce a highly abundant, noncoding subgenomic flavivirus RNA (sfRNA) in infected cells, which implies an important function of sfRNA during mosquito infection. Currently, the role of sfRNA in flavivirus transmission by mosquitoes is not well understood. Here, we demonstrate that an sfRNA-deficient ZIKV (ZIKVΔSF1) replicates similar to wild-type ZIKV in mosquito cell culture but is severely attenuated in transmission by Ae. aegypti after an infectious blood meal, with 5% saliva-positive mosquitoes for ZIKVΔSF1 vs. 31% for ZIKV. Furthermore, viral titers in the mosquito saliva were lower for ZIKVΔSF1 as compared to ZIKV. Comparison of mosquito infection via infectious blood meals and intrathoracic injections showed that sfRNA is important for ZIKV to overcome the mosquito midgut barrier and to promote virus accumulation in the saliva. Next-generation sequencing of infected mosquitoes showed that viral small-interfering RNAs were elevated upon ZIKVΔSF1 as compared to ZIKV infection. RNA-affinity purification followed by mass spectrometry analysis uncovered that sfRNA specifically interacts with a specific set of Ae. aegypti proteins that are normally associated with RNA turnover and protein translation. The DEAD/H-box helicase ME31B showed the highest affinity for sfRNA and displayed antiviral activity against ZIKV in Ae. aegypti cells. Based on these results, we present a mechanistic model in which sfRNA sequesters ME31B to promote flavivirus replication and virion production to facilitate transmission by mosquitoes.

Usutu virus (USUV) is a mosquito-borne flavivirus of African origin. Over the past decades, USUV has spread through Europe causing mass die-offs among multiple bird species. The natural transmission cycle of USUV involves Culex spp. mosquitoes as vectors and birds as amplifying hosts. Next to birds and mosquitoes, USUV has also been isolated from multiple mammalian species, including humans, which are considered dead-end hosts. USUV isolates are phylogenetically classified into an African and European branch, subdivided into eight genetic lineages (Africa 1, 2, and 3 and Europe 1, 2, 3, 4, and 5 lineages). Currently, multiple African and European lineages are co-circulating in Europe. Despite increased knowledge of the epidemiology and pathogenicity of the different lineages, the effects of co-infection and transmission efficacy of the co-circulating USUV strains remain unclear. In this study, we report a comparative study between two USUV isolates as follows: a Dutch isolate (USUV-NL, Africa lineage 3) and an Italian isolate (USUV-IT, Europe lineage 2). Upon co-infection, USUV-NL was consistently outcompeted by USUV-IT in mosquito, mammalian, and avian cell lines. In mosquito cells, the fitness advantage of USUV-IT was most prominently observed in comparison to the mammalian or avian cell lines. When Culex pipiens mosquitoes were orally infected with the different isolates, no overall differences in vector competence for USUV-IT and USUV-NL were observed. However, during the in vivo co-infection assay, it was observed that USUV-NL infectivity and transmission were negatively affected by USUV-IT but not vice versa.