Prior exposure of BALB/c mice to Aedes aegypti feeding (two or four exposures to ~17 uninfected mosquitoes over the course of one month) resulted in increased mortality due to mosquito-transmitted WNV infection (). More intense pre-exposure corresponded to enhanced mortality: with 68% and 91% mortality observed in groups previously exposed two and four times to uninfected mosquitoes, respectively, compared to 27% mortality in unexposed mice (p<0.01).
Altered WNV pathogenesis mediated by prior host exposure to uninfected mosquito feeding.
Subsequent experiments investigated the mechanism of this divergence in disease severity. The memory response to mosquito proteins could skew the immune response in a manner that compromises early anti-viral defenses. The microenvironment at the site of viral invasion and initial replication, including cytokines and immune cells present can influence the early orchestration of the response and priming of T cells. Previous research related to mosquito allergy demonstrated a TH
1 to TH
2 shift following sensitization to Ae. aegypti 
. To evaluate whether cytokine expression differed between groups, total RNA was isolated from inoculation site and draining LN for quantifying the relative levels of IL-2, IL-4, IL-10, IL-12p40, IFN-β, and IFN-γ via real-time RT-PCR 
In three separate experiments, IL-10 expression was consistently elevated in the dermis and draining LNs of sensitized mice as compared to naïve mice at 36 h post-infection (p
0.04)(). This immuno-regulatory cytokine is multifunctional. In relation to viral infections, IL-10 may create favorable conditions for viral replication by disarming the innate and adaptive responses. In human monocytes, IL-10 and IFN-γ antagonize the function of each other 
. IL-10 also down regulates MHC class II antigen expression by monocytes and inhibits antigen presentation by several types of antigen-presenting cells (APC), including epidermal LCs 
. The presence of viruses whose genomes code for IL-10 homologs, demonstrate that there is an evolutionary advantage to enhanced IL-10 levels for an invading virus 
. Studies with peripheral blood leukocytes found that during the early phase of infection with dengue virus, increased IL-10 production induces lasting T cell inactivation and decreases the control of virus infection 
. Consequently, the interaction of immunosuppressive IL-10-producing cells with T cells early during WNV infection may result in the loss of T-cell responsiveness and facilitate an enhancement of viral replication and a blunted adaptive immune response. Thus, notwithstanding further effects of prior exposure to mosquitoes, heightened IL-10 production associated with an anti-mosquito immune response may account for the altered course of infection.
No clear pattern was observed with TH
1 cytokines however, expression of IL-4 was 50-times higher in LNs of sensitized mice at 36 h post-infection. Although this difference was not statistically significant, the consistency of the increased expression in mosquito-sensitized mice suggested a trend towards an elevated TH
2 response in pre-exposed mice. The enhancement of IL-4 following Ae. aegypti
exposure parallels that observed with previous research 
Passive transfer of serum from exposed mice to naïve mice resulted in elevated mortality (), characteristic of the group previously exposed to mosquito feeding, thus implicating the humoral immune response in the deviation of pathogenesis in mosquito-sensitized mice (Methods S1
). This does not preclude the possibility that the cellular response is also important. Anti-salivary protein antibodies were readily detected in pre-exposed mice (). Western blot analysis of the serum (Methods S2
) showed six major bands recognized by the serum of Ae. aegypti
-exposed mice, with increased reactivity observed in mice exposed four times to mosquito feeding ().
Passive transfer of mosquito saliva-sensitized serum affects disease coursed.
It has been suggested that the presence of anti-saliva antibodies in blood might conceivably impair the feeding efficiency of infected mosquitoes by inhibiting salivary protein function 
. This could necessitate greater probing activity, thereby increasing the titers of WNV inoculated into the skin. Using Anopheline mosquitoes, one study demonstrated that the suppression of the mosquito salivary protein apyrase activity was associated with decreased competence locating blood, increased probing time, and presumably increased inoculation of malaria parasites 
. In the current study, there was no significant difference in the number of blood-engorged mosquitoes recovered from naïve versus pre-exposed mice. Additionally, treatment of Ae. aegypti
SGE with serum from sensitized mice caused no reduction in apyrase activity as judged by an in vitro
biochemical assay that measures the release of phosphate from adenosine triphosphate (Methods S3
; data not shown). The titer of virus inoculated into mice was estimated by dissecting mosquito salivary glands immediately following feeding on mosquito immune and non-immune mice, and by comparing with the titers in salivary glands of unfed mosquitoes. No significant difference was observed between these groups (data not shown). Therefore, the observed effect of sensitization to mosquito saliva appears not to be due to a direct effect on the efficacy of mosquito feeding, but rather to a response subsequent to probing.
Histological analysis of the skin and draining LN at 24 and 48 h post-infectious bite revealed a transient, mild inflammatory reaction in naïve mice, in contrast to significant cellular infiltrate and a >200% increase in tissue size in pre-exposed mice. The edema at the feeding site in sensitized mice was characterized by mononuclear (plasma and histocytic) cell and neutrophil infiltrate (). As few as two pre-exposures to uninfected mosquitoes were sufficient to stimulate marked LN expansion at 36 h post-exposure to infected mosquito feeding; with 3.9±0.9×107
cells per LN for mice with two and four times pre-exposures, respectively, as compared to 1.5±0.7×107
cells per LN in naïve mice exposed to an infected mosquito (; p
<0.01). One possible explanation for the increased severity of WNV infection in sensitized mice is that the immune response to salivary proteins promotes the recruitment of susceptible cell types to the inoculation site and consequently increases opportunity for virions to infect target cells. To investigate this hypothesis, cells recovered from the skin and draining LN after mosquito inoculation of WNV were stained with appropriate antibodies and analyzed by flow cytometry 
. All groups of mice exposed to WNV displayed an inflammatory response to viral replication in the form of cell influx into the effected tissues. However, in mice pre-exposed to mosquito feeding, the inflammatory response was significantly elevated in comparison to non-sensitized mice. Following WNV infection, the frequency of MHC class II-expressing skin cells was more than double in mice sensitized to Ae. aegypti
feeding (). MHC class II molecules are predominantly present on DCs, monocytes, and macrophages, cells highly susceptible to WNV infection 
. Similarly, the frequencies of CD11c+
cells, mostly APCs and granulocytic cells, were also higher after infection in the skin of mosquito-pre-exposed mice (). Predictably, influx of these cell types was also elevated in the draining LNs of mice with prior exposure to mosquitoes. (p
Cell influx into early sites of WNV replication.
Identification of differential cell influx into mosquito feed site and draining LN.
Analogous to observations in the skin, determination of the abundance of subpopulations within LNs revealed a consistent pattern: following WNV infection via mosquitoes, mice that were pre-exposed to naïve mosquitoes displayed an enhanced leukocyte level, including CD11c+
cells, concurrent with reduced lymphocyte populations, as revealed by the lowered CD3+
cell percentage (; p
<0.01). In mice sensitized to mosquito feeding prior to WNV infection, CD3+
cells comprised 69.2% of LN cells in mice exposed twice, and 64.5% of LN cells in mice exposed four times at 36 h post-infection, as compared to 73.6% of cells in non-sensitized mice illustrating that previous exposure results in a dose dependent decrease in the CD3+
population (). With respect to specific T cell populations, the CD4+
cell density was consistently lower in mosquito-sensitized mice, although this difference was not statistically significant; cells expressing CD4 on their surface were 53.6% of the cell population in naïve mice compared to 50.3% and 45.7% in mice previously exposed to mosquitoes twice and four times, respectively. CD8-expressing T cell density was marginally affected by prior exposure to mosquito feeding, which accounted for 19.2%, 18.1%, and 17.8% of the LN population in naïve, twice and four times exposed, respectively (). The observed trend in CD4+
cell numbers resulting from prior exposure to mosquito feeding could help to explain the divergence in WNV infection, although the elevated influx of leukocytes and CD3−
cells likely play a role in this proportional decline. A recent study with WNV 
illustrated the important role that CD4+
T cells play in controlling infection. Suppression or deficiency of this subpopulation during WNV infection in mice resulted in prolonged central nervous system infection and uniform lethality. Additionally, mice lacking CD4+
T cells had reduced IgG production and, later in infection, compromised WNV-specific CD8+
T cell activation and trafficking to the CNS 
. Mice lacking CD8+
T cells have higher CNS viral burdens and increased mortality rates after infection with WNV 
. Lymphocytes were similarly lowered in 3 replicates of this experiment, suggesting a possible role for this altered lymph population in the divergent disease course of mosquito-sensitized mice.
MHC class II+ cells were more abundant in mice previously exposed to mosquitoes (), making up 27.9% and 32.6% (2 and 4 exposures respectively) of the LN population, juxtaposed with the 22.7% observed in naïve mice (p<0.01). Similarly, the LN of 4-times-exposed mice was composed of 30.3% CD11b+ cells, as compared to mosquito-naïve mice, for which 20.6% of cells were CD11b+ (; p<0.05). Interestingly, cells expressing DX5, a protein associated with natural killer (NK) cells, were found at a higher density in mice previously exposed to mosquitoes (). Cells dually positive for DX5 and CD3 accounted for the majority of the difference, suggesting an increase NK T in pre-exposed mice.
Identification of cell population in draining LN.
Additional samples were extracted to determine if differences could be detected during early viral replication. RNA was isolated from the dermal bite site, draining LN, liver, and spleen at 48 h post-infection, and viral load was assessed by real-time RT-PCR. At this early time point only minimal WNV RNA was detectable, with the highest level of virus in the draining LN (). In most tissues, the level of measurable WNV was too low for robust statistical analysis, but in two of three replicates, the level of virus in the LN was significantly higher in mice previously exposed to mosquito feeding (p<0.01). These data suggest that early expansion of virus is amplified in sensitized mice, a result that is consistent with the increased APC migration to the LN and enhanced IL-10 expression. As expected in mice of this age, viremia was moderate and brief; amongst groups there was no significant difference in virus detected in the serum by TCID50 assay (day 3 pi: naïve, 4.01±0.73; pre-exposed, 4.46±0.68 log10TCID50).