West Nile virus can be transmitted within mosquito populations through infectious mosquito excreta

Abstract

Understanding the transmission routes of arboviruses is key to determining their epidemiology. Here, we tested whether West Nile viruses (WNVs) are transmitted through mosquito excreta. First, we observed a high concentration of infectious units per excreta, although viruses were short lived. Second, we showed that virion excretion starts early after oral infection and remains constant for a long period, regardless of mosquito infection level. These results highlight the infectiousness of excreta from infected mosquitoes. Third, we found that both larvae and pupae were susceptible to infection, while pupae were highly permissive. Fourth, we established the proof-of-concept that immature mosquitoes can be infected by infectious excreta, demonstrating an excreta-mediated mode of transmission. Finally, by mathematically modeling excreta-mediated transmission in the field, we demonstrated that WNV can be transmitted within mosquito populations. Our study uncovers a route of transmission for mosquito-borne arboviruses, unveiling mechanisms of viral maintenance in mosquito reservoirs.

Keywords: disease; pathogenic organism; tropical medicine.

Graphical abstract

Figure 1

Detection and quantification of infectious viruses in mosquito excreta (A and B) Detection of WNV viral RNA (A) and infectious particles (B) in supernatant from cells infected with excreta pools (i.e., amplified excreta inoculum). Control (+) corresponds to RNA extracts from WNV stock. Control (−) corresponds to water. (C and D) Quantification of PFU per excreta (C) and ratio of viral genomic RNA (gRNA)/PFU in the same excreta pools collected 6 days post mosquito exposure to blood containing 5 × 10⁶ PFU/mL. Bars show means ± SEM. Each point indicates one excreta pool, collected from several experiments. (E) Correlation between PFU per excreta and gRNA/PFU ratio for the previous samples. See also Figure S1.

Figure 2

The effect of oral inoculum and days post exposure (DPE) on virus excretion (A and B) Infection intensity and infection rate in mosquitoes exposed to blood containing 10⁷ (A) or 10⁵ (B) PFU/ml of WNV and in their excreta collected every two days. Black dots show geometric mean ± SD for infection intensity. Blue bars show percentage ±95% C.I. for infection rate. N, number of samples from at least two experiments. Mixed-effects one-way ANOVA was used to compare infection intensities, but no statistical difference was found. Chi was used to compare infection rates. ∗, p < 0.05.

Figure 3

Susceptibility of aquatic stages to WNV exposure (A) Stability of WNV in rearing water. Points indicate mean ± SEM of PFU/ml in water at different time post inoculation. N, 4. (B and C) Infection rate for L4 larvae exposed to WNV at L1 stage (B) and for adult mosquitoes exposed at the pupal stage (C). Bars show percentage ±95% C.I. Chi was used to compare infection rates between different virus concentrations. Different letters indicate significant differences, p < 0.05. N, number of individual mosquitoes from at least two experiments. See also Figure S2.

Figure 4

Susceptibility of pupae to infectious excreta Bar shows infection rate +95% C.I. in adult mosquitoes exposed at the pupal stage to infectious excreta at a concentration of 4.6 × 10³ PFU/mL. N, number of individual mosquitoes.

Figure 5

Mathematical modeling of excreta-mediated infection of mosquito aquatic stages (A) Flow chart and mathematical formulation of the excreta-mediated flavivirus transmission model. V, breeding site volume. ΦV, egg laying rate. S_E, egg survival. η, egg hatching rate. S_L, immature mosquito susceptibility to infection. μm, immature mosquito mortality. βW/V, immature mosquito infection where W represents the WNV load in the breeding site (assumed well-mixed) and β the infection rate. I_L, infected immature mosquitoes. κ, adult emergence. I_A, infected adult mosquitoes. ν, adult mosquito mortality. ζ, rate of virion excretion into the breeding site. p, decay of excreted virions. Red mosquitoes indicate infection. (B) Basic reproduction number, R^d₀, as a function of larval density and proportion of excreta falling into breeding sites. Solid curves indicate the median and dashed curve the 90^th percentile. (C) The proportion of breeding sites maintaining WNV infection (R^d₀ > 1) as a function of larval density and proportion of excreta falling into breeding sites. Color of solid and dashed curves indicates the different proportions of excreta falling into a breeding sites set at 0.01, 0.1, 1, 10, and 20%.