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 (∼10² 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. Since it was first introduced into the United States in 1999, West Nile virus (WNV) has caused significant disease in humans, horses, and other animals. WNV is transmitted to humans and other vertebrate hosts by female mosquitoes as they take a blood meal. Currently, the amount of virus inoculated by mosquitoes while feeding on live hosts is unknown, and accurate estimates are critical so that appropriate challenge doses can be used in vaccine and viral pathogenesis studies. Here, we use a novel technique to determine the dose of WNV inoculated by mosquitoes as they probe and feed on the peripheral tissues of live animals. We found that mosquitoes inoculate high doses of virus into host tissues; these doses are 10 to 1,000 times higher than previous estimates obtained with assays that do not allow mosquitoes to probe or feed naturally. We also found that mosquitoes inoculate low doses of virus directly into the blood while blood feeding. Direct introduction of virus into the blood may alter viral tropism and cause low rates of infection in co-feeding mosquitoes. Our study provides new insights into the transmission of an emerging viral pathogen and the interaction of virus with its mosquito vector and vertebrate host.