We investigated the spatial and temporal distribution of West Nile virus (WNV) in organs and tissues of Culex pipiens quinquefasciatus mosquitoes for up to 27 days following oral infection. WNV antigen was detected in paraffin-embedded mosquitoes by immunohistochemistry. Immunofluorescence assays were performed on dissected salivary glands and midguts and analyzed by confocal microscopy. We evaluated the route of virus dissemination following midgut escape and the relative importance of amplifying tissues in mosquito susceptibility to infection. WNV infection was persistent in all tissues analyzed including the midgut, salivary glands, nervous system, and fat body and only declined in the cytoplasm of posterior midgut epithelial cells over time. The phenomenon of cell-to-cell spread was observed in the midgut epithelium and WNV intensely infected both circular and longitudinal muscles of the same organ. It is possible that muscle tissue serves as a conduit for virus dissemination and contributes to WNV amplification, particularly late in infection. These findings provide insight into WNV infection dynamics in a highly susceptible, epidemiologically important, North American vector. Our results pave the way for future studies to analyze physical and biological barriers to WNV dissemination in less competent mosquitoes.

Abstract. An important variable in the amplification and escape from the enzootic cycle of the arboviral encephalitides is the degree of contact between avian hosts and mosquito vectors. To analyze this interaction in detail, blood-fed mosquitoes that were confirmed vectors of eastern equine encephalomyelitis (EEE) virus were collected in 2002 from an enzootic site in central Alabama during the time this virus was actively transmitted. Avian-derived blood meals were identified to the species level of the host, and the proportion derived from each species was compared with the overall composition of the avifauna at the study site. The EEE vector mosquito species fed significantly more on some bird species and less on other species than expected given the overall abundance, biomass, or surface area of the local avifauna. When viewed collectively, these data suggest that these mosquitoes are selectively targeting particular avian species.

A site near Tuskegee, Alabama was examined for vector activity of eastern equine encephalomyelitis (EEE) virus in 2001. More than 23,000 mosquitoes representing 8 genera and 34 species were collected during a 21-week period, and five species, Culiseta melanura, Aedes vexans, Coquillettidia perturbans, Culex erraticus, and Uranotaenia sapphirina, were examined for the presence of virus using a nested reverse transcriptase-polymerase chain reaction for EEE virus. Each species was infected at various times of the mosquito season (May-September) with different minimum infection rates (MIRs). Culiseta melanura had the highest MIR (20.2) and positive pools were detected from late May to mid-September. Aedes vexans had an MIR of 2.2 and was infected early in the season (June), while Cq. perturbans exhibited a much higher field infection rate (9.9) with all positive pools collected in August. Culiseta melanura is a likely endemic vector in central Alabama, while Ae. vexans and Cq. perturbans probably function as bridge vectors. Culex erraticus, the most common mosquito in the habitat (54% of total collections), had an MIR of 3.2, and was persistently infected from mid-June to mid-September. This is the first report of high rates of EEE virus infection in this species, a member of the tropical subgenus Melanoconion. Uranotaenia sapphirina, considered to feed on amphibians and possibly reptiles, had an MIR of 5.6, with positive pools spanning a four-month period. This suggests that species other than birds may serve as a reservoir for EEE in hardwood swamps in the Southeastern United States and elsewhere. The lengthy period of mosquito infection with EEE virus, coupled with the diverse habits of the vectors and their proximity to a population center, indicate the importance of monitoring EEE virus activity in the Mid-South.

O'nyong-nyong virus (ONNV) and chikungunya virus (CHIKV) are closely related alphaviruses that cause human disease in Africa and Asia. Like most alphaviruses, CHIKV is vectored by culicine mosquitoes. ONNV is considered unusual as it primarily infects anopheline mosquitoes; however, there are relatively few experimental data to support this. In this study, three strains of ONNV and one strain of CHIKV were evaluated in Anopheles gambiae and Aedes aegypti mosquitoes and in four cell lines. As predicted, CHIKV was not infectious to An. gambiae, and we observed strain-variability for ONNV with respect to the ability of the virus to infect An. gambiae and Ae. aegypti. The species specificity in vivo was reflected by in vitro experiments using culicine and anopheline-derived cell lines.

Abstract. Transmission experiments are a critical component of vector competence studies. In this study, a real-time reverse transcriptase−polymerase chain reaction (RT-PCR) was used to enumerate the amount of West Nile virus (WNV) secreted in mosquito saliva following oral infection. Culex pipiens quinquefasciatus were allowed to feed on WNV-infected blood, and saliva was collected on days 14 and 21 post-infection (pi). The amount of virus at these two time points varied significantly, with mean equivalent plaque-forming units (pfu) of approximately 30,500 on day 14 pi and 5,800 on day 21 pi. Individual mosquitoes secreted up to 2 × 10 5 pfu of virus. Titer of whole mosquitoes and immunofluorescence assay of salivary glands from mosquitoes collected at these two time points were also used for supplemental comparison. This report describes the first use of a real-time RT-PCR to quantify the amount of WNV in mosquito saliva.

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West Nile virus (WNV) is now the predominant circulating arthropod-borne virus in the United States with >15,000 human cases and >600 fatalities since 1999. Conventionally, mosquitoes become infected when feeding on viremic birds and subsequently transmit the virus to susceptible hosts. Here, we demonstrate nonviremic transmission of WNV between cofeeding mosquitoes. Donor, Culex pipiens quinquefasciatus mosquitoes infected with WNV were fed simultaneously with uninfected ''recipient'' mosquitoes on naïve mice. At all times, donor and recipient mosquitoes were housed in separate sealed containers, precluding the possibility of mixing. Recipients became infected in all five trials, with infection rates as high as 5.8% and no detectable viremia in the hosts. Remarkably, a 2.3% infection rate was observed when 87 uninfected mosquitoes fed adjacent to a single infected mosquito. This phenomenon could potentially enhance virus survival, transmission, and dispersion and obviate the requirement for viremia. All vertebrates, including immune and insusceptible animals, might therefore facilitate mosquito infection. Our findings question the status of dead-end hosts in the WNV transmission cycle and may partly explain the success with which WNV established and rapidly dispersed throughout North America.Culex ͉ mosquito vector

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