Suitable Mouse Model to Study Dynamics of West Nile Virus Infection in Culex quinquefasciatus Mosquitoes

Abstract

West Nile Virus (WNV) poses a significant global public health threat as a mosquito-borne pathogen. While laboratory mouse models have historically played a crucial role in understanding virus biology, recent research has focused on utilizing immunocompromised models to study arboviruses like dengue and Zika viruses, particularly their interactions with Aedes aegypti mosquitoes. However, there has been a shortage of suitable mouse models for investigating WNV and St. Louis encephalitis virus interactions with their primary vectors, Culex spp. mosquitoes. Here, we establish the AG129 mouse (IFN α/β/γ R^-/-) as an effective vertebrate model for examining mosquito-WNV interactions. Following intraperitoneal injection, AG129 mice exhibited transient viremia lasting several days, peaking on the second or third day post-infection, which is sufficient to infect Culex quinquefasciatus mosquitoes during a blood meal. We also observed WNV replication in the midgut and dissemination to other tissues, including the fat body, in infected mosquitoes. Notably, infectious virions were present in the saliva of a viremic AG129 mouse 16 days post-exposure, indicating successful transmission capacity. These findings highlight the utility of AG129 mice for studying vector competence and WNV-mosquito interactions.

Keywords: AG129 mouse; Culex quinquefasciatus; West Nile Virus (WNV); vector competence.

Figure 1

Mortality of immunocompromised AG129 (IFNα/β/γR^−/−) mice following WNV inoculation. (A) Experimental Design: Three or eight-week-old AG129 mice were intraperitoneally inoculated with WNV, with injections performed in the lower right quadrant. (B) Kaplan–Meier survival plot depicting the survival probability of three-week-old AG129 mice inoculated with 8.6 × 10⁸ plaque-forming units (p.f.u.) of WNV over eight days.

Figure 2

Arbovirus viremia in AG129 mice. (A) Experimental setup: Three-week-old AG129 mice were given intraperitoneal injections of WNV in the lower right quadrant. Blood samples were taken every 24 h for eight days to quantify viral RNA using RT-qPCR. Each data point represents a blood sample from an individual mouse, with samples from at least two different mice collected at each time point. Mice were observed daily for eight days and euthanized after one blood collection. (B) Viral RNA levels in the blood of AG129 mice inoculated with 8.6 × 10⁸ plaque-forming units (p.f.u.) of WNV.

Figure 3

Viremic AG129 Mice as a competent vertebrate animal model for WNV infection in Culex quinquefasciatus. (A) Experimental design scheme. (B) The WNV RNA levels of abdomen of each mosquito tested are shown 4, 8, and 16 days post-feeding (d.p.f.) are shown. Detection of WNV in the abdomen was utilized as an indicator of infection. (C) WNV RNA levels in the head + thorax of each tested mosquito at 4, 8, and 16 d.p.f. are shown. Detection of WNV in the head + thorax was utilized as an indicator of dissemination efficiency. The number of mosquitoes with detected WNV RNA out of the total tested is indicated below the bar charts. WNV load quantification was performed using the 2^−ΔCt (delta Ct) method. The same individuals from the cohort were used to obtain results in both the abdomen and head + thorax. Empty circles indicate samples where viral RNA was not detected.

Figure 4

WNV tissue tropism following oral infection in Culex quinquefasciatus. (A) Midgut of a female mosquito that was fed only on blood. WNV is detected in midgut epithelial enterocytes of female mosquitoes at 4 (B) and 8 (C) days after feeding on blood from an infectious AG129 mouse. (D) Fat body from a female mosquito fed solely on blood. (E) WNV was identified in the fat body of female mosquitoes 8 days after feeding on blood from an infectious AG129 mouse. Panels (A–E) show merged images from triple-stained immunofluorescence assays of adult female tissues. The tissues were stained with an antibody against WNV (magenta), actin marked with phalloidin (cyan), and DNA marked with Hoechst (yellow). (A′–E′) show WNV immunostaining. (A″–E″) show DNA staining. (A‴–E‴) show actin staining.

Figure 5

Culex quinquefasciatus mosquitoes are capable of transmitting WNV 16 days post-feeding on an AG129 viremic mouse. (A) Experimental design scheme illustrating how the infectivity of mosquito saliva was tested. Saliva samples were collected from female mosquitoes (A) that had previously taken an infectious blood meal from a WNV-viremic AG129 mouse at 16 d.p.f. The collected saliva from each individual mosquito was then injected into five naïve mosquitoes, which were subsequently tested for WNV infection by RT-qPCR five days post-injection. (B) Levels of WNV RNA detected in mosquitoes injected with saliva from 16 d.p.f. females. Mosquitoes that became infected are shown as solid dots, while uninfected mosquitoes are depicted as empty dots. Each letter on the X-axis represents a single saliva sample.