We plan to explore changes of the specific sequence space of genomes of arboviruses that have evolved to replicate efficiently in cells of two taxa (mammals and insects) at two different temperatures (27 and 37 C) and with a delicately balanced codon pair bias (CPB) accommodating CPB differences in mammals and insects. We have chosen for our studies dengue virus (DENV) that is the leading cause of arthropod-borne human diseases in the world. This is a truly multidisciplinary project that brings together investigators from a leading laboratory studying dengue virus pathogenesis and molecular biology (Berkeley, CA), from an Arbovirus Laboratory (Albany, NY) studying the biology of insect borne viruses, from a Department of Computer Sciences (University of Miami) specializing in encodings of biological system, and from a Department of Molecular Genetics and Microbiology (Stony Brook University, NY) known for work on plus stranded viruses. With the aid of computers and specifically tailored algorithms we will recode the genome of DENV such that it will contain large segments (encoding either the proteins E, NS3, or NS5) of codon pairs dis-favored in mammals but normal in insects. We predict that these genomes will encounter severe restrictions of replication in mammalian cells, but will replicate in insects cells with wild type kinetics. To avoid the possibility that the recoded viruses could be a biohazard for the environment (via Ae. aegypti, their natural vectors), the recoded viruses will be tested in mosquitoes for replication phenotypes. The recoded DENV genomes, in turn, will also be tested for their virulence and attenuation in experimental animals (mice and monkeys). Since the basic strategy allows us tailoring the virulence of the agent by specifically down-regulating the expression of virus- encoded proteins, our project offers a variety of possibilities to study replication phenotypes of DENV tissue culture cells and experimental animals. At the same time it may yield candidate strains that may be suitable for further development as vaccines. Vaccine development, however, is not the primary goal pursued in this application.
Human infections by dengue viruses are a rapidly growing global health concern, with 2.5 billion people at risk, 100,000,000 annual infections, and tens of thousands of deaths. The studies outlined in this grant deal with basic issues of dengue virus genetics (encoding of specific viral proteins) and gene expression. Because we will construct dengue virus variants that have large segments of their genome recoded with a 'non-human design' but unchanged 'insect design' we propose to study the replication of the dengue variants also in its natural host and vector, Aedes aegypti. Since the basic strategy of computer-designed recoding allows us tailoring the virulence of the agent by specifically down-regulating the expression of virus-encoded proteins, our project offers a variety of possibilities to study replication phenotypes of dengue virus in tissue culture cells and experimental animals. There are no vaccines licensed that protect humans from dengue virus infection. Ultimately, we believe that our work proposed here will lead to a new class of unique vaccines that will prevent infection with, and disease caused by, all four dengue virus serotypes.
