Ex vivo midgut cultures of Aedes aegypti are efficiently infected by mosquito-borne alpha- and flaviviruses

Aedes aegypti mosquitoes are known vectors of several arboviruses, including the chikungunya virus (CHIKV), dengue virus (DENV), and Zika virus (ZIKV). When blood-feeding on a virus-infected human, the mosquito ingests the virus into the midgut (stomach), where it must replicate and overcome the midgut barrier to disseminate to other organs (e.g., salivary glands) and ultimately be transmitted via the saliva. Tools to study mosquito-borne viruses (MBVs) include two-dimensional cell culture systems and in vivo mosquito infection models, which offer great advantages, yet have some limitations. Here, we describe for the first time a long-term ex vivo culture of Ae. aegypti midguts. Cultured midguts were viable for 7 days in a 96-well plate at 28°C and displayed peristaltic movements. Several representative MBVs efficiently replicated in the midguts: ZIKV, DENV, Ross River virus (RRV) and CHIKV, as measured by qRT-PCR and plaque assay. Fluorescence microscopy revealed replication and spread of a fluorescent reporter virus (DV2/mCherry) in the midgut. Finally, we evaluated the antiviral activity of the nucleoside analogs β-D-N4-hydroxycytidine (NHC) and 7-deaza-2’- C -methyladenosine (7DMA), in the ex vivo midgut model. At 50 µM, NHC reduced the infectious virus levels at day 3 post-infection (p.i.) for both RRV and CHIKV by 4.5- and 9.4-fold, respectively. Likewise, 7DMA at 100 µM reduced ZIKV RNA viral loads by 20-fold at day 7 p.i..Together, our results show that ex vivo midguts can be efficiently infected with mosquito-borne alpha- and flaviviruses. Furthermore, the cultured midguts could potentially be used to evaluate the antiviral activity of inhibitors. Ex vivo midgut cultures could thus be a new model to study MBVs, offering the advantage of reduced biosafety measures compared to infecting living mosquitoes. ### Competing Interest Statement The authors have declared no competing interest.