Development of ENTV reverse genetics system and phenotypic evaluation of rescued virus reveals host-specific replication patterns in mosquitoes.

Entebbe bat virus (ENTV) is a bat-associated flavivirus with no known vector. Research into the biology of this virus, including assessment of the possibility that it may be vector-transmitted, is hindered by a lack of molecular tools and robust genetic systems. Therefore, we sequenced the complete 3' untranslated region, which was not previously available, and developed an infectious clone of ENTV to facilitate further investigation of the virus. Virus derived from the clone replicated similarly to the parental virus isolate in various vertebrate cells. Surprisingly, ENTV replicated to high titers in and mosquito cell lines, but there was no replication or infection in cells. In addition, phylogenetic and bioinformatics analyses strongly suggested that ENTV may be associated with a mosquito host. Given the bioinformatics support and efficient growth in cells, we orally exposed and to ENTV to evaluate infection. The ENTV blood-fed mosquitoes were all negative for infection; however, when ENTV was intrathoracically inoculated, bypassing the initial midgut infection and escape barriers, it replicated to high levels in the body, without dissemination of infectious virus into the saliva. These findings suggest that, despite demonstrating high molecular compatibility at the cellular level in mosquitoes, and are unlikely to serve as competent vectors for ENTV transmission due to strong midgut infection barriers. The clone presented in this manuscript should help to clarify the mechanisms for transmission and maintenance of ENTV, which remain poorly understood.