# Systemic infection with insect-specific viruses does not affect Plasmodium sporozoite formation in Anopheles mosquitoes

## TL;DR

This study found that insect-specific viruses do not hinder the development of malaria parasites in mosquitoes, suggesting they may not be useful for malaria control.

## Contribution

The study systematically tested insect-specific viruses for their potential to interfere with Plasmodium development in Anopheles mosquitoes.

## Key findings

- Four insect-specific viruses efficiently replicated in Anopheles mosquitoes without causing high mortality.
- Coinfection with these viruses did not affect Plasmodium sporozoite formation or infection rates.
- The study identified viable viruses for studying Anopheles immunity but found no impact on malaria transmission.

## Abstract

Malaria remains a leading cause of morbidity and mortality in large parts of the world. Resistance threatens current preventative and curative strategies, highlighting the need for novel complementary strategies. The opportunities that the mosquito virome may offer to reduce pathogen transmission have not been systematically explored for malaria control. In this study, we assessed whether insect-specific viruses affect Plasmodium development in mosquitoes. A panel of 15 viruses was tested for in vitro and in vivo replication in anopheline cells and mosquitoes. From this panel, the RNA viruses Flock House virus, Wallerfield virus, Agua Salud alphavirus, Herbert herbevirus and Gouléako goukovirus and the dsDNA virus invertebrate iridescent virus 6 efficiently replicated in two Anopheles gambiae cell lines and were further assessed in in vivo experiments. Intrathoracic injection of these viruses in Anopheles stephensi mosquitoes resulted in efficient viral RNA replication for Herbert herbevirus, Agua Salud alphavirus and Gouléako goukovirus and high levels of infectious viral particle production for invertebrate iridescent virus 6. In contrast, Wallerfield virus showed poor replication, whereas Flock House virus replicated efficiently but caused high mosquito mortality. Subsequently, we performed in vivo coinfections of Plasmodium falciparum with the four efficiently replicating insect-specific viruses that were not associated with high mosquito mortality. Sporozoite formation and Plasmodium infection rates did not differ between virus-infected mosquitoes and non-infected controls. While none of the tested viruses significantly affected Plasmodium development, our study identified multiple viruses that efficiently infect Anopheles stephensi mosquitoes, providing a useful resource to study Anopheles immunity. Future studies may address whether native Anopheles-specific viruses affect Plasmodium parasite transmission.

Malaria continues to be a major health issue in many endemic countries, and current prevention and treatment methods are not fully effective. This study explores a new way to combat malaria transmission using insect-specific viruses to reduce Plasmodium development in mosquitoes. A panel of 15 different insect viruses was tested for replication in mosquito cells and adult female mosquitoes. Four efficiently replicating viruses were analysed in co-infection experiments, where mosquitoes were first infected with the viruses and subsequently exposed to malaria parasites in an infectious bloodmeal. No significant differences were observed in parasite development, suggesting that persistent infection with these viruses does not affect malaria transmission. Whether other, Anopheles specific viruses affect Plasmodium parasite transmission remains to be investigated.