Continued RNA viral metagenomic monitoring of natural populations of Aedes aegypti reveals novel and sustained insect specific viruses

Viruses transmitted by Aedes aegypti mosquitoes are of global public health concern. Within the last decade in the United States, outbreaks of dengue, chikungunya, and Zika have occurred due to local transmission, with cases concentrated in Florida. Previous research discovered dengue virus serotype 4 in the absence of a human index case alongside numerous insect-specific viruses (ISVs) in Ae. aegypti from Florida collected in 2016 and 2017. To investigate ISV persistence, we used RNA metagenomic sequencing to characterize eukaryotic RNA viruses in ovaries of field-derived Ae. aegypti from the same location in 2018 and in a lab-maintained control. A toti-like virus highly similar to other Aedes toti-like viruses was discovered in all samples, while a partiti-like virus with < 80% identity to any known virus occurred only in field-derived ovaries, accounting for 47.1% - 75.9% of assigned viral reads. We assembled the toti-like virus genome, consisting of an RNA-dependent RNA polymerase (RdRp) and a capsid. We also assembled the RdRp gene for the partiti-like virus and four genomic segments (PA, PB1, PB2, and NP) from a low-abundance orthomyxovirus from field-derived ovaries. The presence of these viruses in ovaries alongside confirmation of each ISV in field-derived descendants strongly suggests vertical transmission as a means of ISV maintenance. This ovary virome profile establishes baseline information regarding ISV presence and persistence in a natural vector population. Given the potential impact of ISVs on vector competence, it also lays groundwork for studies that may improve our understanding of arbovirus-transmission risk in Florida.