Culex pipiens, an experimental efficient vector of West Nile and Rift Valley fever viruses in the Maghreb region

Abstract

West Nile fever (WNF) and Rift Valley fever (RVF) are emerging diseases causing epidemics outside their natural range of distribution. West Nile virus (WNV) circulates widely and harmlessly in the old world among birds as amplifying hosts, and horses and humans as accidental dead-end hosts. Rift Valley fever virus (RVFV) re-emerges periodically in Africa causing massive outbreaks. In the Maghreb, eco-climatic and entomologic conditions are favourable for WNV and RVFV emergence. Both viruses are transmitted by mosquitoes belonging to the Culex pipiens complex. We evaluated the ability of different populations of Cx. pipiens from North Africa to transmit WNV and the avirulent RVFV Clone 13 strain. Mosquitoes collected in Algeria, Morocco, and Tunisia during the summer 2010 were experimentally infected with WNV and RVFV Clone 13 strain at titers of 10(7.8) and 10(8.5) plaque forming units/mL, respectively. Disseminated infection and transmission rates were estimated 14-21 days following the exposure to the infectious blood-meal. We show that 14 days after exposure to WNV, all mosquito st developed a high disseminated infection and were able to excrete infectious saliva. However, only 69.2% of mosquito strains developed a disseminated infection with RVFV Clone 13 strain, and among them, 77.8% were able to deliver virus through saliva. Thus, Cx. pipiens from the Maghreb are efficient experimental vectors to transmit WNV and to a lesser extent, RVFV Clone 13 strain. The epidemiologic importance of our findings should be considered in the light of other parameters related to mosquito ecology and biology.

Figure 1. Localization of Culex pipiens samples collected in 2010 in the Maghreb (Morocco, Algeria and Tunisia).

Figure 2. Transmission rate and mean titer of infectious viral particles present in saliva of Culex pipiens at different days after ingestion of an infectious blood-meal containing WNV (A) and RVFV (B).

We exposed a Culex pipiens colony, Tabarka (Tunisia) to an infectious blood-meal containing 10^7.8 PFU/mL of WNV or 10^8.5 PFU/mL of RVFV. At day 3, 6, 9, 14 and 21 post-infection, 20 females were analyzed. Saliva were collected using the forced salivation technique. After removing wings and legs, the proboscis of mosquitoes was inserted into 20 µL tip filled with 5 µL of Fetal Bovine Serum (FBS). After 45 min, medium containing the saliva was collected into 45 µL of L15 medium. The number of infectious particles per saliva was estimated by titration on Vero cells and expressed as log₁₀PFU/saliva. Lines refer to TR and bars to Log10 pfu/saliva.

Figure 3. Disseminated infection rate (A), Transmission rate (B) and mean titer of infectious viral particles present in saliva (C) of Culex pipiens challenged with WNV.

F1 mosquitoes (autogenous AU and anautogenous AN) were orally challenged with WNV at a titer of 10^7.8 PFU/mL using an artificial feeding system. After completion of the blood-meal, mosquitoes were maintained in BSL-3 insectaries at 28°C. At day 14 pi, saliva was collected from surviving females using the forced salivation technique. The number of infectious viral particles present in saliva was estimated by plaque assay on Vero cells. After salivation, females were tested for the presence of WNV on head squashes by IFA. p<0.05, Fisher’s exact test. In brackets, the number of mosquitoes tested. Error bars show the confidence interval (95%) for DIR and TR, and the standard deviation for Log10 pfu/saliva.

Figure 4. Disseminated infection rate, Transmission rate and mean titer of infectious viral particles present in saliva of Culex pipiens at day 14 (A,B and C) and 21 (D,E and F) post-infection with RVFV.

F1 mosquitoes (autogenous AU and anautogenous AN) were orally challenged with RVFV at a titer of 10^8.5 PFU/mL using an artificial feeding system. After completion of the blood-meal, mosquitoes were maintained in BSL-3 insectaries at 28°C. At day 14 pi and day 21 pi, saliva was collected from surviving females using the forced salivation technique. The number of infectious viral particles present in saliva was estimated by plaque assay on Vero cells. After salivation, females were tested for the presence of RVFV on head squashes by IFA. In brackets, the number of mosquitoes tested. Error bars show the confidence interval (95%) for DIR and TR, and the standard deviation for Log10 pfu/saliva.