Targeting the Dengue Genome with a Hepatitis Delta Virus Ribozyme

The mosquito-vectored Dengue virus (DENV) causes a potentially fatal infection found mostly in tropical regions of the world. Nearly 2.5 billion people are at risk of infection and an estimated 50 million cases occur annually. There is no vaccine and the only way to fight the spread of Dengue is to combat the mosquito vector. An emerging strategy for preventing dengue transmission involves genetic modification of the mosquito with anti-viral agents. RNA enzymes known as ribozymes hold great potential in this area due to their autocatalytic properties and minimal cofactor requirements. The Hepatitis Delta Virus (HDV) ribozyme is a promising candidate for dengue inactivation because it can be easily altered to cleave a target of choice and is both active and stable at physiological pH and magnesium concentrations. We have investigated the potential of an HDV-based ribozyme to function as a means of inducing DENV refractivity in mosquitoes. We have modified the HDV ribozyme to target a portion of the DENV genome that is conserved among all four Dengue serotypes. The ability of the ribozyme to cleave this conserved sequence was examined in cell culture using a 5’ DENV/firefly luciferase fusion target and luciferase assays, RT-PCR, western dot-blot, and in-vitro cleavage assays. Our analyses provide validation of this ribozyme as a candidate effector gene for Dengue virus refractivity in mosquito cells, although future work is necessary to determine the extent of target cleavage. We also propose further development of the ribozyme for use as an RNA drug.