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

Abstract

Aedes aegypti is one of the most important mosquito species in medical entomology today. It is a known vector of dengue (DENV) and yellow fever viruses as well as being susceptible to a number of other arboviruses capable of causing disease in humans and domestic animals. It has very rapidly achieved an expansive distribution throughout most of the tropical and subtropical regions on the world. Ae. aegypti has also evolved into a "domesticated" species by living and breeding within the human communities increasing its contact frequency with people. Arbovirus infection of Ae. aegypti begins with the acquisition of an infectious bloodmeal into the mosquito's midgut. Midgut epithelial cells then become infected eventually leading to midgut escape and the potential to retransmit the arbovirus via an infected salivary gland. However, the midgut epithelial cells are known to be selectively susceptible to arbovirus infection and constitute a barrier to arbovirus 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 ic as a backbone, the TR339 genetic determinants of Ae. aegypti midgut infection were associated with the E2 glycoprotein, specifically at amino acid (aa) positions E2-55 and E2-70. These sites were identified to affect the midgut infection rate (MIR) independently and in combination with each other. Structurally, theses sites were found in predicted loop regions of the protein. Phenotypically, I observed that mammalian derived viruses more efficiently established an infection of midgut cells than mosquito derived viruses independently of the TR339 genetic determinants. Also through the use of the SINV TE/5'2J ic, the MRE16 genetic determinants of Ae. aegypti midgut infection were associated with the E2 glycoprotein as well, specifically at aa 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 aa 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 aa 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 number of disease outbreaks in the tropical Americas. The 1409 isolate has been well characterized in the laboratory for its infection potential in Ae. aegypti midguts and represents an ideal candidate for ic construction and future genetic determinant assays. This dissertation describes the technical hurdles overcome to successfully generate and characterize a full-length infectious clone of DENV-2 1409. The work and data described in this dissertation adds to the knowledge of mosquito midgut infections by presenting insights into the specific residues responsible for midgut infections. It also provides a new tool for further investigations into infection determinants of arboviruses. Moreover, future research projects can be designed from the information in this dissertation to better identify the determinants of mosquito midgut infections with the ultimate goal of applying such information for the control and elimination of arboviral diseases.