Investigation on insect-specific viruses - West Nile virus interactions: focus on European Culex pipiens mosquitoes

Abstract

Arboviruses such as West Nile virus (WNV) and Usutu virus (USUV) represent an increasing public health concern in Europe, where mosquitoes of the Culex pipiens complex and Culex torrentium serve as principal vectors. Despite their epidemiological relevance, the molecular determinants underlying arbovirus infection and vector competence in Culex mosquitoes remain insufficiently understood. In parallel, insect-specific viruses (ISVs), which are restricted to insect hosts, have emerged as important components of the mosquito virome with the potential to modulate arbovirus replication and transmission. Using a combination of in vitro infection assays, small RNA sequencing, virome analysis, and in vivo mosquito infection experiments, this study evaluated viral replication kinetics, antiviral RNA interference (RNAi) responses, and potential interference effects between ISVs and medically relevant arboviruses. Two newly established Cx. pipiens-derived cell lines (CPE/LULS50 and CPL/LULS56) were characterized using representative viruses from the families Flaviviridae, Togaviridae, and Peribunyaviridae. Both cell lines supported replication of a broad range of arboviruses and ISVs and exhibited robust virus-derived siRNA responses, confirming functional RNAi activity. In contrast to many established mosquito cell lines, no evidence of persistent ISV infections was detected, highlighting their suitability as controlled experimental systems. Co-infection experiments in cell culture using the insect-specific viruses Eilat virus (EILV) and Niénokoué virus (NIEV) revealed no consistent modulation of arbovirus replication, including WNV and USUV, under both acute and persistent infection conditions. Similarly, in vivo experiments demonstrated that prior infection with the insect-specific Agua Salud alphavirus (ASALV) did not significantly alter WNV infection or transmission dynamics in Cx. pipiens biotype pipiens mosquitoes. Infection studies further indicated that ISV susceptibility is highly dependent on infection route, with efficient infection following intrathoracic injection but limited infection via oral exposure, suggesting midgut-associated barriers. Collectively, these findings demonstrate that ISV-arbovirus interactions are highly complex and context-dependent, influenced by factors such as virus strain, infection timing, and host background. While ISVs remain promising candidates for innovative vector control strategies, the lack of consistent interference observed in this study highlights the need for comprehensive evaluation across diverse experimental and ecological settings. Furthermore, this work characterized Cx. pipiens sensu stricto cell lines as valuable tools for advancing research on arbovirus-vector interactions in temperate mosquito species.