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. 2016 Apr 20:2:88-94.
doi: 10.1016/j.onehlt.201604001. eCollection 2016 Dec.

Culex pipiens and Culex torrentium populations from Central Europe are susceptible to West Nile virus infection

Affiliations

Culex pipiens and Culex torrentium populations from Central Europe are susceptible to West Nile virus infection

Mayke Leggewie et al. One Health. .

Abstract

West Nile virus (WNV), a Flavivirus with an avian primary host, is already widespread in Europe and might also pose an infection risk to Germany, should competent mosquito vectors be present. Therefore, we analysed the ability of WNV to infect German Culex mosquitoes with special emphasis on field collected specimens of Culex torrentium and Culex pipiens biotype pipiens. We collected egg rafts of Culex mosquitoes over two subsequent seasons at two geographically distinct sampling areas in Germany and differentiated the samples by molecular methods. Adult females, reared from the various egg rafts, were challenged with WNV by feeding of artificial blood meals. WNV infection was confirmed by real-time RT-PCR and virus titration. The results showed that field collected C. pipiens biotype pipiens and C. torrentium mosquitoes native to Germany are susceptible to WNV infection at 25 °C as well as 18 °C incubation temperature. C. torrentium mosquitoes, which have not been established as WNV vector so far, were the most permissive species tested with maximum infection rates of 96% at 25 °C. Furthermore, a disseminating infection was found in up to 94% of tested C. pipiens biotype pipiens and 100% of C. torrentium. Considering geographical variation of susceptibility, C. pipiens biotype pipiens mosquitoes from Southern Germany were more susceptible to WNV infection than corresponding populations from Northern Germany. All in all, we observed high infection and dissemination rates even at a low average ambient temperature of 18 °C. The high susceptibility of German Culex populations for WNV indicates that an enzootic transmission cycle in Germany could be possible.

Keywords: Culex mosquitoes; Infection assay; West Nile virus.

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Figures

Fig. 1
Fig. 1
Temperature dependence of infection rates for field populations C. pipiens biotype pipiens and C. torrentium. (A–B) Adult females were sorted into small plastic containers 5–10 days after emergence and feed over night with human blood containing 1–1.5 ×ばつ 107 PFU WNV lineage 1 strain NY99. Fully engorged females were separated and incubated at 25 °C or 18 °C and 80% humidity in a climate chamber for 14 to 35 days. Graphs represent the infection rates (in percentage) of C. pipiens biotype pipiens (panel A) and C. torrentium (panel B), at 25 °C (dark grey bars) and 18 °C (light grey bars) as determined via WNV-specific qRT-PCR (cut-off = 35 cycles). Detailed numbers of individuals and numbers of independent experiments used for analysis are listed in Table 1. Data presented in these graphs are pooled data from North and South populations collected in 2013. Statistical analyses were performed using the GraphPad Prism software and Fischer's exact test (p < 0.05).
Fig. 2
Fig. 2
C. pipiens biotype pipiens and C. torrentium mosquitoes display high dissemination rates at 18 and 25 °C incubation temperature. (A–B) Adult females were sorted into small plastic containers 5–10 days after emergence and feed over night with human blood containing 1–1.5 ×ばつ 107 PFU WNV lineage 1 strain NY99. Fully engorged females were separated and incubated at 25 °C or 18 °C and 80% humidity in a climate chamber for 14 to 35 days. Graphs represent the dissemination rate (in percentage) of C. pipiens biotype pipiens (panel A) and C. torrentium (panel B), at 25 °C (dark grey bars) and 18 °C (light grey bars) as determined via WNV-specific qRT-PCR (cut-off = 35 cycles). Detailed numbers of individuals and numbers of independent experiments used for analysis are listed in Table 1. Data presented in these graphs are pooled data from North and South populations collected in 2013. Statistical analyses were performed using the GraphPad Prism software and Fischer's exact test (p < 0.05).
Fig. 3
Fig. 3
Spatial and temporal variation in vector competence of wild C. pipiens biotype pipiens and C. torrentium populations. (A–C) For comparison of infection rates between two years and two geographically distinct mosquito populations of the same species, the data obtained through infection experiments described in Table 1 and Fig. 1 of 2013 were analysed together with data obtained in 2012. (A) Infection rates for 2012 are indicated by dark grey bars and infection rates for 2013 are represented by light grey bars. Statistical analysis was performed using GraphPad Prism and Fisher's exact test (p < 0.05). Detailed numbers of individuals and numbers of independent experiments used for analysis are listed in Table 1. (B and C) Infection rates are determined via WNV-specific qRT-PCR (cut-off = 35 cycles). Statistical analysis was performed using GraphPad Prism and Fisher's exact test (p < 0.05). Detailed numbers of individuals and numbers of independent experiments used for analysis are listed in Table 1. (B) Infection rates for C. pipiens biotype pipiens at 21 days post-infection and two temperatures, 18 °C and 25 °C, for the North population (N) and South population (S) respectively. (C) Infection rates for C. torrentium at 21 days post-infection and two temperatures, 18 °C and 25 °C, for the North population (N) and South population (S) respectively.
Fig. S1
Fig. S1
Infection rates for Cx. quinquefasciatus and Cx. pipiens biotype molestus laboratory strains. (A-B) Adult females were sorted into small plastic containers 5–10 days after emergence and feed over night with human blood containing 1–1.5 ×ばつ 107 PFU WNV lineage 1 strain NY99. Fully engorged females were separated and incubated at 25 °C or 18 °C and 80% humidity in a climate chamber for 14 to 35 days. Graphs represent the infection rate (in percentage) of Cx. pipiens quinquefasciatus (panel A) and Cx. pipiens biotype molestus (panel B), at 25 °C (dark grey bars) and 18 °C (light grey bars) as determined via WNV-specific qRT-PCR (cut-off = 35 cycles). Detailed numbers of individuals and numbers of independent experiments used for analysis are listed in Table 1. Data presented in these graphs are pooled data from North and South populations. Statistical analyses were performed using the GraphPad Prism software and Fischer's exact test (p < 0.05).

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