Replication is the key barrier during the dual-host adaption of mosquito-borne flaviviruses

Mosquito-borne flaviviruses (MBFs) adapt to a dual-host transmission circle between mosquitoes and vertebrates. Dual-host affiliated insect-specific flaviviruses (dISFs), discovered from mosquitoes, are phylogenetically similar to MBFs but do not infect vertebrates. Thus, dISF-MBF chimeras could be an ideal model to study the dual-host adaption of MBFs. Using the pseudo-infectious reporter virus particle and reverse genetics systems, we found dISFs entered vertebrate cells as efficiently as the MBFs, but failed to initiate replication. Exchange of the un-translational regions (UTRs) of Donggang virus (DONV), an dISF, with those from Zika virus (ZIKV) rescued DONV replication in vertebrate cells and critical secondary RNA structures were further mapped. Essential UTR-binding host factors were screened for ZIKV replication in vertebrate cells, displaying different binding patterns. Therefore, our data demonstrate a post-entry cross-species transmission mechanism of MBFs, while UTR-host interaction is critical for dual-host adaption. Significance Statement Most viruses have a relatively narrow host range. In contrast, vector-borne flaviviruses, such as dengue virus and Zika virus, maintain their transmission cycle between arthropods and vertebrates, belonging to different phyla. How do these viruses adapt to the distinct cellular environments of two phyla? By comparing the single-host insect specific flavivirus and dual-host Zika virus, we identified three key molecular factors that determine MBF host tropism. This study will greatly increase the understanding of entry, replication, and cross-species evolution of mosquito-borne flaviviruses.