Belgian Culex pipiens pipiens are competent vectors for West Nile virus but not Usutu virus

Background West Nile virus (WNV) and Usutu virus (USUV) are emerging arboviruses in Europe transmitted by Culex mosquitoes. In Belgium, it is currently unknown which Culex species are competent vectors for WNV or USUV and if these mosquitoes carry Wolbachia, an endosymbiotic bacterium that can block arbovirus transmission. The aims of our study were to measure the vector competence of Belgian Culex mosquitoes to WNV and USUV and determine if a naturally acquired Wolbachia infection can influence virus transmission. Methodology/Principal Findings We captured 876 non-engorged female Culex mosquitoes from urban and peri-urban sites in Leuven, Belgium. We provided females with an infectious bloodmeal containing WNV lineage 2, USUV European (EU) lineage 3, or USUV African (AF) lineage 3. Blood-fed females (n=154) were incubated for 14 days at 25°C after which the body, head, and saliva were collected to measure infection (IR), dissemination (DR), and transmission (TR) rates, respectively. Mosquito species were identified by qRT-PCR or Sanger sequencing, the presence of infectious virus in mosquitoes was confirmed by plaque assays, and viral genome copies were quantified by qRT-PCR. We found that Culex pipiens pipiens were able to transmit WNV (11% IR, 40% DR, 100% TR) but not USUV (EU lineage: 13% IR, 0% DR; AF lineage: 16% IR, 17% DR, 0% TR). In contrast, Culex modestus was able to transmit USUV (AF lineage: 60% IR, 67% DR, 50% TR), but not WNV (0% IR). We found that the presence or absence of Wolbachia was species-dependent and did not associate with virus transmission. Conclusions/Significance This is the first report that Belgian Culex mosquitoes can transmit both WNV and USUV, forewarning the risk of human transmission. More research is needed to understand the potential influence of Wolbachia on arbovirus transmission in Culex modestus mosquitoes. Author Summary West Nile virus and Usutu virus can cause seasonal epidemics in humans. They are maintained in a transmission cycle between wild birds and Culex mosquitoes, and humans that are bitten by infected mosquitoes can develop life-threatening neurological disease. Certain Culex species carry the symbiotic bacterium Wolbachia which can block virus transmission in mosquitoes. In Belgium, it is currently unknown which Culex species can transmit West Nile virus and/or Usutu virus, or if they carry Wolbachia bacteria. In our study, we captured wild mosquitoes from Belgium and infected them with West Nile virus or Usutu virus. We found that a common European species (Culex pipiens pipiens, the Northern House mosquito) could transmit West Nile virus, whereas a lesser known species (Culex modestus) could transmit Usutu virus. Wolbachia bacteria could be found in almost all Culex pipiens pipiens, but not in Culex modestus, suggesting that Wolbachia prevalence is species-specific. More research is needed to understand if Wolbachia can block virus transmission in Culex modestus. This is the first report on the ability of Culex mosquitoes to transmit West Nile virus and Usutu virus in Belgium, forewarning the risk of transmission to humans.