The role of mosquito saliva on host immune response and pathogenesis of West Nile virus\r\n

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West Nile virus is a positive sense single-stranded RNA virus in the family Flaviviridae that emerged globally following the appearance of a more neurotropic subtype. Recently outbreaks of WNV disease have occurred in the Middle East, Europe, Africa, South America, and North America. Vertebrates typically become infected when an infectious mosquito pierces the host epidermis to take a blood meal, depositing virus principally in the extravascular tissue. Accumulating evidence has demonstrated that the mosquito saliva, which carries WNV into the vertebrate, is not simply a transport medium, but can have a profound effect on vertebrate immunity, pathogen transmission efficiency, pathogenesis, and disease course. In the past most small animal models of arbovirus disease have used needle-inoculation, but recent evidence suggests that because of the potential effects of mosquito saliva on the immune system, it is important to re-evaluate the pathogenesis of these infections in the presence of mosquito saliva. A central question of this dissertation project was to determine if mosquito feeding or mosquito saliva could impact WNV disease. To evaluate the potential for mosquito saliva to alter WNV infection, the mouse model of disease employed in this dissertation revealed a potentiation of WNV disease when mosquitoes were allowed to feed at the virus inoculation site immediately before injection of WNV. Further, with higher titer inoculations of WNV subsequent to the feeding of mosquitoes more progressive infection, higher viremia, and accelerated neuroinvasion developed than the mice inoculated with an equivalent titer of WNV alone. To determine the mechanism of this potentiation, the in vivo expression of key TH1, TH2, inflammatory, and antiviral cytokines was quantified during peripheral arbovirus infection in the presence or absence of mosquito saliva. Data clearly showed that during early arbovirus infection mosquito salivary proteins down-modulates specific antiviral cytokines while enhancing production of TH2 and immunosuppressive cytokines. To understand the source of these shifts in immune signalling, the in vitro response of DCs and macrophages was investigated. Following exposure to Ae. aegypti SGE, APCs recently exposed to arbovirus displayed reductions in IFN-? and iNOS expression and transient amplification of IL-10 mRNA levels. Macrophages appeared to be more susceptible to the modulating effects of mosquito saliva than DCs. The influence of mosquito saliva on immune cell migration patterns both into the dermal site of WNV inoculation and the draining lymph node was also evaluated. The principal observation from this study was that the inclusion of mosquito saliva/feeding at the inoculation site of WNV leads to a suppression of lymphocytes, particularly CD4+ T cells, and a corresponding increase in DCs. Finally, although mosquito exposure and sensitization to it is widespread, the effect of prior exposure to mosquitoes on subsequent arbovirus infection had up until this point been unexplored. Accordingly, the potential for an immune response directed against mosquito salivary proteins to have a protective or confounding effect on naturally transmitted WNV infection was investigated by comparing early WNV pathogenesis in mosquito naïve and sensitized populations of mice. Previous exposure to Ae. aegypti feeding results in significantly higher mortality rates associated with elevation of inflammation, APC recruitment, and IL-4 expression concurrent with a decrease in lymphocytes mainly the CD4+ subtype. Mosquito sensitization-mediated amplification of WNV disease is facilitated by the humoral response to mosquito salivary proteins. This dissertation confirms that by ignoring the possible effects of the vector we may misinterpret the early immune response to arboviruses and also possibly aspects of the overall pathogenesis of arboviral infections. The information from the present study provides insight into early host responses to arbovirus infection, and suggests further determinants of WNV virulence.

west nile virus, vector, saliva, mosquito, immunomodulation, arbovirus