Development of large-scale mosquito densovirus production by in vivo methods

Mosquito-borne diseases (MBDs) cause a significant proportion of the global infectious disease burden. Vector control remains the primary strategy available to reduce the transmission of MBDs. However, long-term, wide-scale and large-scale traditional chemical pesticide application has caused significant and increased negative effects on ecosystems and broader emerging insecticide resistance in vectors; therefore, the development of a novel alternative approach is urgently needed. Mosquito densoviruses (MDVs) are entomopathogenic viruses that exhibit a narrow host range and multiple transmission patterns, making MDVs a great potential bioinsecticide. However, the application process has been relatively stagnant over the past three decades. The major obstacle has been that viruses must be produced in mosquito cell lines; therefore, the production process is both expensive and time-consuming. In our study, two wild-type (wt) MDVs, AaeDV and AalDV-3, and a recombinant rAaeDV-210 were used to infect the Aag2 and C6/36 mosquito cell lines and the 1st–2nd-instar and 3rd–4th-instar larvae of Ae. albopictus, Ae. aegypti and Cx. quinquefasciatus. Viral titers and yields in cells, media, larvae and rearing water and total viral yield were evaluated. Three kinds of virus displayed higher maximum virus titers in vivo than in vitro, and they displayed higher maximum viral yields in rearing water. The three viruses displayed higher total maximum viral yields in C6/36 cells than in Aag2 cells. The three viruses displayed higher total maximum viral yields in Aedes mosquitoes than in Culex mosquitoes. Higher viral yields were produced by 1st–2nd-instar larvae compared to 3rd–4th-instar larvae. The recombinant viruses did not display significantly lower yields than wt viruses in nearly all samples. In summary, by using 100 1st–2nd-instar Aedes mosquito larvae in 200 ml of rearing water, more than 1013 genome equivalents (geq) MDV yield can be obtained. Considering the lower production cost, this in vivo method has great potential for the large-scale production of MDVs. MDVs exhibit promising prospects and great potential for mosquito control in the future.