Belgian Culex pipiens pipiens are competent vectors for West Nile virus while Culex modestus are competent vectors for Usutu virus

Abstract

Background: West Nile virus (WNV) and Usutu virus (USUV) are emerging arthropod-borne viruses (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: Female Culex mosquitoes were captured from urban and peri-urban sites in Leuven, Belgium and offered an infectious bloodmeal containing WNV lineage 2, USUV European (EU) lineage 3, or USUV African (AF) lineage 3. Blood-fed females were incubated for 14 days at 25°C after which the body, head, and saliva were collected to measure infection, dissemination, and transmission rates as well as transmission efficiency. 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. Culex pipiens pipiens were able to transmit WNV (4.3% transmission efficiency, n = 2/47) but not USUV (EU lineage: n = 0/56; AF lineage: n = 0/37). In contrast, Culex modestus were able to transmit USUV (AF lineage: 20% transmission efficiency, n = 1/5) but not WNV (n = 0/6). 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.

Fig 1. Urban and peri-urban field collection sites in Leuven, Flemish Brabant, Belgium.

Map made using QGIS v3.18.3 [QGIS Development Team (2021). QGIS Geographic Information System. Open Source Geospatial Foundation Project. http://qgis.osgeo.org]. Open map data was obtained from OpenStreetMap contributors through the Wikimedia Commons unlabeled layer (accessed: 14 July 2022).

Fig 2. Adult Culex mosquitoes captured at the urban (A) and peri-urban (B) sites in Leuven, Belgium.

Fig 3. Culex species identification by virus infection group.

Culex mosquitoes were identified morphologically to the genus level, after which Culex pipiens (p.) biotypes (pipiens, molestus, pipiens-molestus hybrids) were identified by qRT-PCR while other species (Culex torrentium and Culex modestus) were identified by sequencing the cytochrome oxidase 1 (COX1) gene.

Fig 4. Vector competence of Culex pipiens (p.) pipiens (A) and Culex modestus (B) for WNV and USUV.

The bars represent the rates of infection in the body (yellow), disseminated infection to the head, wings, and legs (orange), transmission potential in the saliva (red), and overall transmission efficiency (purple), determined by plaque assay. Grey labels above the bars indicate the number of positive mosquitoes over the total number of mosquitoes.

Fig 5. Viral genome copies in the bodies and head, wings, and legs of Culex pipiens (p.) pipiens (A-B) and Culex modestus (C-D).

The bars represent the median viral genome copies ± interquartile range; the grey dotted lines represent the limit of detection (LOD) of the qRT-PCR assays used. Statistical analysis was performed with the Mann-Whitney U test; the asterisk represents a p-value of <0.05; ns: non-significant.

Fig 6. Infectious titer per saliva sample of Culex pipiens (p.) pipiens and Culex modestus.

Infectious virus titers were determined by plaque assay (PFU: plaque forming units). The bars show the median ± interquartile range; the grey dotted line represents the limit of detection (LOD) of the plaque assay.