The applicants propose to study the interactions of Sindbis virus with Aedes aegypti. To do this they will use molecular clones of SIN that have been engineered to express phenotypic characteristics of interest and which bear convenient marker genes. They will examine two infectious DNA clones of SIN, one of which infects Aedes midguts inefficiently (the 2J virus) and the other which infects midguts efficiently and causes disseminated infections in the mosquito (the MRE1001 virus). The MRE1001 virus is a chimera between he nonstructural genes of the 2J virus and the structural genes from a midgut infecting strain, the MRE16 strain. The 2J strain and the chimeric MRE1001 strains have both been engineered to contain a second sub-genomic RNA 3-prime to the normal capsid region which encodes GFP (green fluorescent protein) They will first compare growth curves of these three viruses when given by the oral or injection routes, and compare the organ tropisms, extrinsic incubation periods, and transmission potentials of the viruses. They will then create additional chimeras, gene by gene, of the structural genes of the MRE1001 and 2J strains to define the genetic determinants of midgut tropism and dissemination. Finally, they will introduce site specific mutations and deletions to further refine the genetic determinants of viral gut replication and dissemination. In a related line of research they propose to create a new infectious DNA clone of SIN from the MRE16 strain. This strain will differ from the MRE1001 strain in that all the non-structural genes as well as the structural genes will derive from a mid-gut replicating, disseminating virus strain.
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