Mosquitoes inoculate high doses of West Nile virus as they probe and feed on live hosts

Abstract

West Nile virus (WNV) is transmitted to vertebrate hosts by mosquitoes as they take a blood meal. The amount of WNV inoculated by mosquitoes as they feed on a live host is not known. Previous estimates of the amount of WNV inoculated by mosquitoes (10(1.2)-10(4.3) PFU) were based on in vitro assays that do not allow mosquitoes to probe or feed naturally. Here, we developed an in vivo assay to determine the amount of WNV inoculated by mosquitoes as they probe and feed on peripheral tissues of a mouse or chick. Using our assay, we recovered approximately one-third of a known amount of virus inoculated into mouse tissues. Accounting for unrecovered virus, mean and median doses of WNV inoculated by four mosquito species were 10(4.3) PFU and 10(5.0) PFU for Culex tarsalis, 10(5.9) PFU and 10(6.1) PFU for Cx. pipiens, 10(4.7) PFU and 10(4.7) PFU for Aedes japonicus, and 10(3.6) PFU and 10(3.4) PFU for Ae. triseriatus. In a direct comparison, in vivo estimates of the viral dose inoculated by Cx. tarsalis were approximately 600 times greater than estimates obtained by an in vitro capillary tube transmission assay. Virus did not disperse rapidly, as >99% of the virus was recovered from the section fed or probed upon by the mosquito. Furthermore, 76% (22/29) of mosquitoes inoculated a small amount of virus ( approximately 10(2) PFU) directly into the blood while feeding. Direct introduction of virus into the blood may alter viral tropism, lead to earlier development of viremia, and cause low rates of infection in co-feeding mosquitoes. Our data demonstrate that mosquitoes inoculate high doses of WNV extravascularly and low doses intravascularly while probing and feeding on a live host. Accurate estimates of the viral dose inoculated by mosquitoes are critical in order to administer appropriate inoculation doses to animals in vaccine, host competence, and pathogenesis studies.

Figure 1. Mosquitoes Inoculate High Doses of WNV In Vivo under Various Experimental Conditions

(A) WNV doses inoculated extravascularly by mosquitoes. x-Axis labels indicate mosquito infection method (Inoc = intrathoracic inoculation, Oral = orally infected), mosquito species (Tar = Cx. tarsalis, Pip = Cx. pipiens, Jap = Ae. japonicus, Tri = Ae. triseriatus), and tissue in which the mosquito probed or fed in (Tail = mouse tail, Ear = mouse ear, Toe = chick toe). Limit of detection (LOD) of plaque assay is shown. (B) Same data as in (A) but pooled by tissue type, mosquito species, and infection method. Within each larger grouping (tissue type, mosquito species, or infection method), groups designated with different lower case letters (above graph) are significantly different from one another (p < 0.05 by Mann-Whitney or Kruskal-Wallis tests). Solid line shows mean titer, and dashed line shows median titer, for each group.

Figure 2. Movement of Virus from Mosquito Feeding Site on Tail

Most of the virus inoculated by mosquitoes was recovered from the tail section(s) that the mosquito probed and fed in (feeding site). Percent of total virus recovered from each tail section. Data from each mouse tail were shifted to align feeding sites. If mosquitoes probed or fed in two sections, those sections were summed and combined into one feeding site. Tail sections toward the tail tip in relation to the feeding site are labeled −1 and −2. Tail sections toward the body in relation to the feeding site are labeled +1 to +4. Includes data from orally and parenterally infected Cx. tarsalis, Ae. japonicus, and Ae. triseriatus feeding on a mouse tail.

Figure 3. Mosquitoes Inoculate a Small Amount of Virus Intravascularly when Blood Feeding

Amount of WNV in the serum of animals following mosquito feeding on distal tissues. Mosquitoes either probed tissue and imbibed blood (Fed) or probed without blood feeding (Unfed). Solid line indicates mean WNV titer in serum of each group. LOD of plaque assay is shown.

Figure 4. Amount of WNV Inoculated by Mosquitoes into Mouse Tails Increases with Probing Time

Included on graph are data from the probe time experiment (open circles) and from other experiments (filled triangles) in which parenterally or orally infected Cx. tarsalis probed and fed on a mouse tail. Solid line shows best-fit one-site binding model, and dotted lines show 95% confidence band of the best-fit curve. LOD of plaque assay was 5 PFU. Values less than the LOD are reported as 0.

Figure 5. Mosquitoes Inoculate Higher WNV Doses into Mouse Tails than into Glass Capillary Tubes

Intrathoracically inoculated Cx. tarsalis females (n = 15) probed/fed on a mouse tail; the tail was removed and macerated, and WNV titer was determined (Mouse Tail). Salivary secretions were collected 2–4 h later from these same mosquitoes by an in vitro capillary tube transmission assay (Capillary Tube – Fed). At the same time, salivary secretions were collected from intrathoracically inoculated female Cx. tarsalis from the same cohort that had not fed on a mouse tail (n = 44) (Capillary Tube – Unfed). Shown are the combined data from two independent studies. Mean and median WNV titers in each column are designated by solid and dashed horizontal lines. LOD of plaque assay is shown.

Figure 6. Culex Mosquitoes Contain High Amounts of WNV

Geometric mean WNV titers in head (H), thorax (T), abdomen (A), and legs (L) of orally (Oral) or parenterally (Inoc) infected Cx. tarsalis and parenterally infected Cx. pipiens. Orally infected mosquitoes were tested at day 16 PI and parenterally infected mosquitoes were tested at day 7 PI. Letters within bars designate body segment titers that are significantly different from one another (p < 0.05) within each mosquito group. Letters above bars designate the mosquito groups that differ significantly from one another (p < 0.05) when compared by body segment. Error bars show standard deviation. LOD of plaque assay was 5 PFU.

Figure 7. Clearance of WNV from the Serum following Intravenous Inoculation

Mice (n = 4) were inoculated intravenously with 10⁵ PFU and serum samples taken at various times PI. For each mouse, WNV titers in the serum were normalized to the 5-min serum titer and reported as log change in WNV serum titer. Solid line indicates best-fit linear regression (R² = 0.90, p < 0.0001); dashed lines indicate 95% confidence band for regression line.