The emergence of West Nile virus and Usutu virus in Europe poses a significant risk to public health. In the absence of efficient antiviral therapy or vaccine candidates, the only strategy to control these arboviruses is to target the Culex (Diptera: Culicidae) mosquito vector. However, the selection pressure caused by exposure to insecticides for vector control or agricultural pest control can lead to insecticide resistance, thereby reducing the efficacy of insecticide-based vector control interventions. In Culex mosquitoes, two of the most common amino acid substitutions associated with insecticide resistance are the kdr L1014F in voltage gated sodium channels and G119S in acetylcholinesterase. In this study, Culex pipiens biotype pipiens, Culex torrentium, and Culex modestus were sampled from 2019 to 2021 in three distinct environmental habitats (urban, peri-urban, and agricultural) in and around the city of Leuven, Belgium. Individual mosquitoes were screened for two mutations resulting in L1014F and G119S amino acid substitutions. Both mutations were observed in Cx. pipiens and Cx. modestus but not in Cx. torrentium mosquitoes across the four collection sites. Furthermore, multi-resistance or cross-resistance in Cx. pipiens could be a threat in these areas, as both mutations were observed at low frequencies. These results provide the first report of kdr L1014F and ace-1 G119S resistance mutations in Cx. pipiens and Cx. modestus mosquitoes from Belgium, highlighting the importance of mosquito surveillance to design effective arbovirus outbreak control strategies.

The impact of the host microbiome on arbovirus infections is currently not clearly understood. Arboviruses are viruses transmitted through the bites of infected arthropods, predominantly mosquitoes or ticks. The first site of arbovirus inoculation is the biting site in the host skin, which is colonized by a complex microbial community that could possibly influence arbovirus infection. We demonstrated that pre-incubation of arboviruses with certain components of the bacterial cell wall, including lipopolysaccharides (LPS) of some Gram-negative bacteria and lipoteichoic acids or peptidoglycan of certain Gram-positive bacteria, significantly reduced arbovirus infectivity in vitro. This inhibitory effect was observed for arboviruses of different virus families, including chikungunya virus of the Alphavirus genus and Zika virus of the Flavivirus genus, showing that this is a broad phenomenon. A modest inhibitory effect was observed following incubation with a panel of heat-inactivated bacteria, including bacteria residing on the skin. No viral inhibition was observed after pre-incubation of cells with LPS. Furthermore, a virucidal effect of LPS on viral particles was noticed by electron microscopy. Therefore, the main inhibitory mechanism seems to be due to a direct effect on the virus particles. Together, these results suggest that bacteria are able to decrease the infectivity of alphaviruses and flaviviruses.

The emergence of West Nile virus and Usutu virus in Europe is a significant risk to public health. In the absence of efficient antiviral therapy or vaccine candidates, the only strategy to control these arboviruses is to target the Culex mosquito vector. However, the selection pressure caused by exposure to insecticides in vector control or agricultural pest control can lead to the development of insecticide resistance reducing the efficacy of insecticide-based vector control interventions. In Culex mosquitoes, two of the most common amino acid substitutions associated with insecticide resistance are the kdr L1014F in the voltage gated sodium channel and G119S in the acetylcholinesterase. In this study, Culex pipiens biotype pipiens, Culex torrentium, and Culex modestus were sampled from 2019 to 2021 in three distinct environmental habitats (urban, peri-urban, and agricultural) in and around the city of Leuven in Belgium. Two mutations resulting in L1014F and G119S amino acid substitutions were screened for in individual mosquitoes. Both mutations were observed in Culex pipiens and Culex modestus mosquitoes across the four collection sites, but not in Culex torrentium. Furthermore, multi-resistance or cross-resistance could be a developing threat, since both mutations were observed at low frequencies (resistant allele frequency <30%) and always in heterozygosity. These results provide the first report of kdr L1014F and ace-1 G119S resistance in Culex pipiens and Culex modestus mosquitoes from Belgium, highlighting the importance of mosquito surveillance for the development of effective arbovirus control strategies in the event of an outbreak in Belgium.