Development of Entebbe Bat Virus 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 arthropod 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, the complete 3' untranslated region, which was not previously available, was sequenced, and an infectious clone of ENTV was developed 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 the Aedes aegypti (Ae. aegypti) and Aedes albopictus (Ae. albopictus) mosquito cell lines, but there was no replication or infection in Culex tarsalis 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 Aedes cells, Ae. aegypti and Ae. albopictus were orally exposed to ENTV to evaluate infection. The ENTV blood-fed mosquitoes were all negative for infection; however, when ENTV was intrathoracically (IT) inoculated, bypassing the initial midgut infection and escape barriers, it replicated to high levels in the body without disseminating infectious virus into the saliva. These findings suggest that despite demonstrating high molecular compatibility at the cellular level in Aedes mosquitoes, Ae. aegypti and Ae. albopictus are unlikely to serve as competent vectors for ENTV transmission because of strong midgut infection barriers. The clone presented in this manuscript should help clarify the mechanisms for transmission and maintenance of ENTV, which remain poorly understood.