Expressing the Pro-Apoptotic Reaper Protein via Insertion into the Structural Open Reading Frame of Sindbis Virus Reduces the Ability to Infect <i>Aedes aegypti</i> Mosquitoes

Arboviruses continue to threaten a significant portion of the human population, and a better understanding is needed of the determinants of successful arbovirus infection of arthropod vectors. Avoiding apoptosis has been shown to be one such determinant. Previous work showed that a Sindbis virus (SINV) construct called MRE/rpr that expresses the <i>Drosophila</i> pro-apoptotic protein Reaper via a duplicated subgenomic promoter had a reduced ability to orally infect <i>Aedes aegypti</i> mosquitoes at 3 days post-blood meal (PBM), but this difference diminished over time as virus variants containing deletions in the inserted <i>reaper</i> gene rapidly predominated. In order to further clarify the effect of midgut apoptosis on disseminated infection in <i>Ae. aegypti</i>, we constructed MRE/rprORF, a version of SINV containing <i>reaper</i> inserted into the structural open reading frame (ORF) as an in-frame fusion. MRE/rprORF successfully expressed Reaper, replicated similarly to MRE/rpr in cell lines, induced apoptosis in cultured cells, and caused increased effector caspase activity in mosquito midgut tissue. Mosquitoes that fed on blood containing MRE/rprORF developed significantly less midgut and disseminated infection when compared to MRE/rpr or a control virus up to at least 7 days PBM, when less than 50% of mosquitoes that ingested MRE/rprORF had detectable disseminated infection, compared with around 80% or more of mosquitoes fed with MRE/rpr or control virus. However, virus titer in the minority of mosquitoes that became infected with MRE/rprORF was not significantly different from control virus. Deep sequencing of virus populations from ten mosquitoes infected with MRE/rprORF indicated that the <i>reaper</i> insert was stable, with only a small number of point mutations and no deletions being observed at frequencies greater than 1%. Our results indicate that expression of Reaper by this method significantly reduces infection prevalence, but if infection is established then Reaper expression has limited ability to continue to suppress replication.