Competition between Usutu virus and West Nile virus during simultaneous and sequential infection of Culex pipiens mosquitoes

Abstract

Usutu virus (USUV) and West Nile virus (WNV) are closely related mosquito-borne flaviviruses that are mainly transmitted between bird hosts by vector mosquitoes. Infections in humans are incidental but can cause severe disease. USUV is endemic in large parts of Europe, while WNV mainly circulates in Southern Europe. In recent years, WNV is also frequently detected in Northern Europe, thereby expanding the area where both viruses co-circulate. However, it remains unclear how USUV may affect the future spread of WNV and the likelihood of human co-infection. Here we investigated whether co-infections with both viruses in cell lines and their primary mosquito vector, Culex pipiens, affect virus replication and transmission dynamics. We show that USUV is outcompeted by WNV in mammalian, avian and mosquito cells during co-infection. Mosquitoes that were exposed to both viruses simultaneously via infectious blood meal displayed significantly reduced USUV transmission compared to mosquitoes that were only exposed to USUV (from 15% to 3%), while the infection and transmission of WNV was unaffected. In contrast, when mosquitoes were pre-infected with USUV via infectious blood meal, WNV transmission was significantly reduced (from 44% to 17%). Injection experiments established the involvement of the midgut in the observed USUV-mediated WNV inhibition. The competition between USUV and WNV during co-infection clearly indicates that the chance of concurrent USUV and WNV transmission via a single mosquito bite is low. The competitive relation between USUV and WNV may impact virus transmission dynamics in the field and affect the epidemiology of WNV in Europe.

Keywords: Usutu virus; West Nile virus; competition; mosquito; vector competence.

Figure 1.

Co-circulation of Usutu virus (USUV) and West Nile virus (WNV) in European countries. The map shows European countries where USUV, WNV or co-infection cases are reported according to the data provided by European Centre for Disease prevention and control (ECDC) and literatures. Countries where both USUV and WNV co-circulate are represented in green. Countries where only USUV or WNV circulates are represented in blue or orange, respectively. The map was generated by using the free online tools https://mapchart.net/europe.html.

Figure 2.

Growth kinetics of USUV (the Netherlands 2016) and WNV (lin2 Greece 2010). (A) Vero (green monkey), (B) DF-1 (chicken) and (C) C6/36 (Aedes albopictus mosquito) and (D) Cx.t (Culex tarsalis mosquito) cells were infected with either USUV or WNV at a multiplicity of infection (MOI) of 0.1. Virus titers were determined by end point dilution assay on Vero cells. Error bars represent the standard deviation (SD).

Figure 3.

Co-infection of USUV (the Netherlands 2016) and WNV (Greece 2010) in cells of different origin. Vero, DF-1, C6/36 and Cx.t cells were co-infected with both USUV and WNV at multiplicity of infection of either 0, 0.1 or 5. At 3 days post infection (dpi), total RNA of the cells was extracted using TRIzol reagent. Five hundred nanogram of total RNA per sample was used for TaqMan qPCR. Error bars represent the standard deviation (SD). The blue and orange dash lines represent the cut-off value for USUV and WNV at Ct value of 34, respectively.

Figure 4.

Culex pipiens selectively transmit WNV when exposed to both viruses through blood meal. (A) Schematic overview of the experimental design. (B, C) Bar graphs show the percent of USUV and WNV positive mosquito bodies and saliva of the total tested engorged mosquitoes at 14 days after blood meal. “U”, “W” and “UW” represent USUV, WNV and co-infectious blood meal, respectively. (+) and (n) indicate the numbers of viral positive samples and total numbers of the tested engorged mosquitoes, respectively. The results present cumulative numbers from three independent experiments (Table S4). Statistics were performed using Fisher's exact test. Asterisks (*) and (***) indicate significance at P < 0.05 and < 0.001, respectively; ns indicates no significant difference. (D) Viral genome copies of USUV and E, WNV in the infected mosquito bodies after either a single or co-infectious blood meal exposure. One-way ANOVA with Tukey's multiple comparison was used to compare the mean of the genome copies. Asterisk (*) indicates a significant difference (P < 0.05); ns indicates no significant difference. The cut-off value for USUV and WNV genomes copies was indicated by black dash lines.

Figure 5.

Culex pipiens pre-exposed to USUV infectious blood meal show a decreased WNV infection and transmission rate. (A) Schematic overview of the sequential blood meal experiment design. (B) Bar graph shows the percent of WNV positive mosquito bodies and saliva of the total engorged mosquitoes at 14 days after the WNV blood meal. “C”, “U” and “W” represent virus-free, USUV and WNV infectious blood meal, respectively. (+) and (n) indicate the numbers of WNV positive mosquito bodies/saliva and the total numbers of the engorged mosquitoes, respectively. The results present cumulative numbers from four independent experiments (Table S5). Fisher’s exact test was performed on the cumulative data. Asterisks (*) and (***) indicate significance at P < 0.05 and < 0.001, respectively. (C) WNV genome copies in mosquito bodies and (D) saliva after a sequential blood meal exposure. Kruskal-Wallis with Dunn's multiple comparison or t test was used to compare the mean of the genome copies among three or two data sets, respectively. ns indicates no significant difference. Black dash lines represent the cut-off value for WNV genome copies which corresponds to a Ct value of 34.

Figure 6.

Culex pipiens pre-infection with USUV via injection did not inhibit subsequent WNV transmission rate. (A) Schematic overview of the sequential infection experiment design. (B) Bar graph shows the percent of WNV positive mosquito saliva of the total engorged mosquitoes at both 7 and 14 days after the WNV blood meal. “C” and “U” represent virus-free and USUV injection, respectively; “W” represents WNV infectious blood meal. (+) and (n) indicate the numbers of WNV positive mosquito saliva and the total numbers of the tested engorged mosquitoes, respectively. The results present cumulative numbers from three independent experiments (Table S6). WNV transmission rate was compared using Fisher's exact test. (C) WNV genome copies in mosquito saliva at 7 days and (D) 14 days after blood meal exposure. One-way ANOVA with Tukey's multiple comparison was used to compare the mean of the WNV genome copies among each groups. ns indicates no significant difference. Black dash lines represent the cut-off value for WNV genome copies.