Application of genetic tools to identify the determinants of arbovirus infection of the Aedes aegypti midgut

The midgut epithelial cells of the mosquito Aedes aegypti are known to be selectively susceptible to arbovirus infection and constitute a barrier to infection. This barrier is thought to be a result of the genetics of the mosquito and the arbovirus. The arboviral genetic determinants of midgut infection are very poorly understood despite the extreme importance of this organ in arboviral transmission cycles. This dissertation intends to investigate the viral genetic determinants of midgut infection through the use of infectious clone (ic) technology. Four arboviral genomes were investigated for their ability to differentially infect Ae. aegypti midguts: Sindbis virus (SINV) strains TE/5'2J, TR339, MRE16 and DENV-2 strain 1409.; Through the use of the SINV TE/5'2J is as a backbone, the TR339 genetic determinants of midgut infection were associated with the SINV E2 glycoprotein at as positions E2-55 and E2-70. These sites affected the midgut infection rate (MIR) independently and in combination with each other. Structurally, theses sites were found in predicted loop regions of the protein.; Also through the use of the SINV TE/5'2J ic, the MRE16 genetic determinants of midgut infection were associated with the E2 glycoprotein at as positions E2-95-6 and E2-116-119. These sites were also in loop regions of E2 working collectively to enhance the MIR. Interestingly, a conserved as motif (i.e. PPF/.GDS) as well as a common structural configuration was identified among the envelope proteins of the alpha- and flaviviruses. Moreover, the specific genetic determinants of MRE16 and TR339 led to the hypothesis that envelope proteins with protruding loop regions, which have variable as sequences, was a common structure involved in alpha- and flavivirus infections of midguts.; The arbovirus DENV-2 strain 1409 is proposed to be an early isolate of the newly established American/Asian DENV-2 genotype that has caused significant outbreaks of disease in the tropical Americas. The 1409 isolate has been well characterized in the laboratory for its infection of Ae. aegypti midgets and represents an ideal candidate for is construction and future genetic determinant assays. This dissertation describes the technical hurdles overcome to successfully generate and characterize a full-length is of DENV-2 1409.