Adventitious viruses persistently infect three commonly used mosquito cell lines
Mosquito cell lines were first established in the 1960s and have been used extensively in research to isolate and propagate arthropod-borne (arbo-) viruses, study the invertebrate immune system, and understand virus-vector interactions. Despite their utility as an in vitro tool, these cell lines are poorly defined and may harbor insect-specific viruses that could impact experimental results. Accordingly, we screened four commonly-used mosquito cell lines, C6/36 and U4.4 cells from Aedes albopictus, Aag2 cells from Aedes aegypti, and Hsu cells from Culex quinquefasciatus, for the presence of adventitious viruses. All four cell lines stained positive for double-stranded RNA by immunofluorescence, indicative of RNA virus replication. We subsequently identified viruses infecting Aag2, U4.4 and Hsu cell lines using untargeted next-generation sequencing, but not C6/36 cells. Sequences from viruses in the families Birnaviridae, Bunyaviridae, Flaviviridae, and Rhabdoviridae were abundant in the mosquito cell lines. PCR confirmation revealed that these sequences stem from active viral replication and/or integration into the cellular genome. Our results show that these commonly-used mosquito cell lines are persistently-infected with several viruses. This finding may be critical to interpreting data generated in these systems.