Simultaneous Coinfections with West Nile Virus and Usutu Virus in Culex pipiens and Aedes vexans Mosquitoes

Abstract

The mosquito-borne zoonotic flaviviruses West Nile virus (WNV) and Usutu virus (USUV) are endemic in many European countries and emerged in Germany in recent years. Due to the increasing overlap of their distribution areas and their similar epidemiology, coinfections of WNV and USUV are possible. Indeed, coinfections in vertebrate hosts as a rare event have already been reported from some countries including Germany. However, it is largely unknown whether and to what extent coinfections could affect the vector competence of mosquitoes for WNV and USUV. For this purpose, the mosquito species Culex pipiens biotype pipiens, Culex pipiens biotype molestus, and Aedes vexans were orally infected in mono- and simultaneous coinfections with German strains of WNV and USUV. Mosquitoes were incubated for 14 days at 26°C, 85% relative humidity, and a 16 : 8 light-dark photocycle, before they were dissected and forced to salivate. The results showed a decrease in USUV susceptibility in Culex pipiens biotype pipiens, an increase in USUV susceptibility in Aedes vexans, and no obvious interaction between both viruses in Culex pipiens biotype molestus. Vector competence for WNV appeared to be unaffected by a simultaneous occurrence of USUV in all tested mosquito species. Coinfections with both viruses were only found in Culex mosquitoes, and cotransmission of WNV and USUV was observed in Culex pipiens biotype molestus. Overall, our results show that viral interactions between WNV and USUV vary between mosquito species, and that the interaction mainly occurs during infection and replication in the mosquito midgut. The results of this study confirm that to fully understand the interaction between WNV and USUV, studies with various mosquito species are necessary. In addition, we found that even mosquito species with a low susceptibility to both viruses, such as Ae. vexans, can play a role in their transmission in areas with cocirculation.

Figure 1

Procedure and sampling of mosquito infection. Created with https://BioRender.com.

Figure 2

Dissection and forced salivation of survived mosquitoes. Created with https://BioRender.com.

Figure 3

Comparison of vector competences of Cx. pipiens biotype pipiens (CxP), Cx. pipiens biotype molestus (CxM), and Ae. vexans (AeV) for WNV and USUV in mono-infections. (a) Infection rates in infected bodies per surviving mosquitoes. (b) Transmission efficiencies in saliva containing viral RNA per surviving mosquitoes. Error bars indicate 95% confidence interval.

Figure 4

Coinfections with WNV and USUV reduce susceptibility to USUV in Cx. pipiens biotype pipiens. (a) Infection rates in infected mosquito bodies per surviving mosquitoes. (b) Dissemination rates in positive legs/wings per infected mosquito bodies. (c) Transmission efficiencies in saliva containing viral RNA per surviving mosquitoes. (d) Amount of viral RNA in equivalent to TCID₅₀/ml in infected mosquito bodies. (e) Amount of viral RNA in equivalent to TCID₅₀/ml in infected legs/wings. Stacked bar charts present the proportion of coinfected mosquito samples. (^∗) and (^∗∗∗) indicate statistically significant differences with p < 0.05 and p ≤ 0.001, respectively.

Figure 5

No obvious interaction between WNV and USUV in Cx. pipiens biotype molestus. (a) Infection rates in infected mosquito bodies per surviving mosquitoes. (b) Dissemination rates in positive legs/wings per infected mosquito bodies. (c) Transmission efficiencies in saliva containing viral RNA per surviving mosquitoes. (d) Amount of viral RNA in equivalent to TCID₅₀/ml in infected mosquito bodies. (e) Amount of viral RNA in equivalent to TCID₅₀/ml in infected legs/wings. Stacked bar charts present the proportion of coinfected mosquito samples.

Figure 6

Coinfections with WNV and USUV increase susceptibility to USUV in Ae. vexans. (a) Infection rates in infected mosquito bodies per surviving mosquitoes. (b) Transmission efficiencies in saliva containing viral RNA per surviving mosquitoes. (^∗∗∗) indicates a statistically significant difference with p ≤ 0.001. No sample was found positive for both viruses. Because of low positivity rates, analysis of other vector competence indices and viral loads were not done.