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Persistent West Nile virus (WNV) infection in the mosquito Culex quinquefasciatus Say (Diptera: Culicidae) is associated with pathological changes in the salivary glands, including apoptotic cell death and a corresponding reduction in virus transmission over time. The vector host response to WNV infection and the molecular basis of WNV pathogenesis in Cx. quinquefasciatus was investigated using oligonucleotide microarrays designed to detect differences in the salivary gland transcriptome between WNV-infected mosquitoes and uninfected controls. Transcripts with increased abundance in infected salivary glands included those related to immunity, transcription, protein transport and degradation, amino acid and nucleotide metabolism, signal transduction, and cellular detoxification. Microarray-based analysis detected a decrease in transcript levels of a Culex inhibitor of apoptosis gene (IAP-1) and a decrease in abundance of 11 transcripts encoding salivary gland proteins. Transcript levels for an endonuclease, a proline-rich mucin, and several D7 protein family members also decreased. Transcripts with the greatest change in abundance during infection had either no similarity to sequences found in GenBank, VectorBase, and FlyBase, or were similar to sequences with uncharacterized protein products. These transcripts represent exciting targets for future analysis. Results from this study suggest that WNV infection influences transcriptional changes in an invertebrate host target tissue that may confer an advantage to the replicating virus, induce a host defense response, and alter the composition of vector saliva. The ramifications of these changes are discussed in terms of mosquito vector competence and WNV pathogenesis.

Arthropod-borne (arbo-) viruses have emerged as a major human health concern. Viruses transmitted by mosquitoes are the cause of the most serious and widespread arbovirus diseases worldwide and are ubiquitous in both feral and urban settings. Arboviruses, including dengue and West Nile virus, are injected into vertebrates within mosquito saliva during mosquito feeding. Mosquito saliva contains anti-haemostatic, anti-inflammatory and immunomodulatory molecules that facilitate the acquisition of a blood meal. Collectively, studies investigating the effects of mosquito saliva on the vertebrate immune response suggest that at high concentrations salivary proteins are immmunosuppressive, whereas lower concentrations modulate the immune response; specifically, T(H)1 and antiviral cytokines are downregulated, while T(H)2 cytokines are unaffected or amplified. As a consequence, mosquito saliva can impair the antiviral immune response, thus affecting viral infectiousness and host survival. Mounting evidence suggests that this is a mechanism whereby arbovirus pathogenicity is enhanced. In a range of disease models, including various hosts, mosquito species and arthropod-borne viruses, mosquito saliva and/or feeding is associated with a potentiation of virus infection. Compared with arbovirus infection initiated in the absence of the mosquito or its saliva, infection via mosquito saliva leads to an increase in virus transmission, host susceptibility, viraemia, disease progression and mortality.

Background: The global emergence of West Nile virus (WNV) has highlighted the importance of mosquito-borne viruses. These are inoculated in vector saliva into the vertebrate skin and circulatory system. Arthropod-borne (arbo)viruses such as WNV are transmitted to vertebrates as an infectious mosquito probes the skin for blood, depositing the virus and saliva into the skin and circulation. Growing evidence has demonstrated that arthropod, and recently mosquito, saliva can have a profound effect on pathogen transmission efficiency, pathogenesis, and disease course. A potentially important aspect of natural infections that has been ignored is that in nature vertebrates are typically exposed to the feeding of uninfected mosquitoes prior to the mosquito that transmits WNV. The possibility that pre-exposure to mosquito saliva might modulate WNV infection was explored., Principal Findings: Here we report that sensitization to mosquito saliva exacerbates viral infection. Prior exposure of mice to mosquito feeding resulted in increased mortality following WNV infection. This aggravated disease course was associated with enhanced early viral replication, increased interleukin-10 expression, and elevated influx of WNV-susceptible cell types to the inoculation site. This exacerbated disease course was mimicked by passive transfer of mosquito-sensitized serum., Significance: This is the first report that sensitization to arthropod saliva can exacerbate arthropod-borne infection, contrary to previous studies with parasite and bacteria infections. This research suggests that in addition to the seroreactivity of the host to virus, it is important to take into account the immune response to vector feeding.

To evaluate the potential for nonviremic transmission (NVT) of West Nile virus (WNV) to occur in nature, we examined the effect of increasing spatial and temporal separation between co-feeding mosquitoes on the efficiency of nonviremic transmission and the potential of a West Nile virus bridge vector species, Aedes albopictus, to be infected via nonviremic transmission. West Nile virus-infected (donor) Culex pipiens quinquefasciatus were allowed to feed on a mouse for 5 minutes followed by non-infected (recipient) mosquitoes with increasing spatial (0, 10, 20, 30, 40, or 50 mm) or temporal (0, 15, 30, 45, or 60 min) separation from the site or time of donor feeding, respectively. Recipients became infected when feeding up to 40 mm from the donor and up to 45 minutes after donor feeding. Additionally, nonviremic transmission of West Nile virus from Cx. p. quinquefasciatus to Ae. albopictus was observed.

The effect of long-term West Nile virus (WNV) infection on Culex salivary gland morphology and viability was evaluated by transmission electron microscopy during a four week period post-blood feeding. These studies showed that apoptosis and other cytopathologic changes occurred more frequently in WNV-infected mosquitoes compared with uninfected controls. The effect of long-term infection on WNV transmission was evaluated by titering virus in saliva over the same time period. Although the mean titer of WNV in mosquito saliva did not change significantly over time, the percentage of saliva samples containing WNV decreased. Because of the importance of saliva in blood meal acquisition and virus delivery, salivary gland pathology has the potential to affect mosquito feeding behavior and virus transmission. Results from this study add to a growing body of evidence that arbovirus infections in mosquito vectors can be cytopathic, and offer a potential mechanism for virus-induced cell death in mosquitoes.

Mosquitoes infect human beings with arboviruses while taking a blood meal, inoculating virus with their saliva. Mosquito saliva contains compounds that counter host hemostatic, inflammatory, and immune responses. Modulation of these crucial defensive responses may facilitate virus infection. Using a murine model we explored the potential for mosquitoes to impact the course of West Nile virus (WNV) disease by determining whether differences in pathogenesis occurred in the presence or absence of mosquito saliva. Mice inoculated intradermally with 10(4) pfu of WNV subsequent to the feeding of mosquitoes developed more progressive infection, higher viremia, and accelerated neuroinvasion than the mice inoculated with WNV alone. At a lower dose of WNV (10(2) pfu), mice fed upon by mosquitoes had a lower survival rate. This study suggests that mosquito feeding and factors in mosquito saliva can potentiate WNV infection, and offers a possible mechanism for this effect via accelerated infection of the brain.

The introduction of West Nile virus (WNV) into North America has been associated with relatively high rates of neurological disease and death in humans, birds, horses, and some other animals. Previous studies identified strains in both genetic lineage 1 and genetic lineage 2, including North American isolates of lineage 1, that were highly virulent in a mouse neuroinvasion model, while other strains were avirulent or significantly attenuated (D. W. C. Beasley, L. Li, M. T. Suderman, and A. D. T. Barrett, Virology 296:17-23, 2002). To begin to elucidate the basis for these differences, we compared a highly virulent New York 1999 (NY99) isolate with a related Old World lineage 1 strain, An4766 (ETH76a), which is attenuated for mouse neuroinvasion. Genomic sequencing of ETH76a revealed a relatively small number of nucleotide (5.1%) and amino acid (0.6%) differences compared with NY99. These differences were located throughout the genome and included five amino acid differences in the envelope protein gene. Substitution of premembrane and envelope genes of ETH76a into a NY99 infectious clone backbone yielded a virus with altered in vitro growth characteristics and a mouse virulence phenotype comparable to ETH76a. Further site-specific mutagenesis studies revealed that the altered phenotype was primarily mediated via loss of envelope protein glycosylation and that this was associated with altered stability of the virion at mildly acidic pH. Therefore, the enhanced virulence of North American WNV strains compared with other Old World lineage 1 strains is at least partly mediated by envelope protein glycosylation.

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.

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 x 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.