Hepatitis G virus (HGV) is a newly discovered RNA virus associated with human hepatitis and present in blood from US accepted donors. At Genelabs we are expressing the HGV putative proteins using E. coli and Baculovirus expression systems and valuating their immunoreactivity with HGV contaminated and noncontaminated sera. Promising results were obtained with some of the proteins, however, we require including more immunoreactive proteins in order to develop a sensitive immunoassay. Putative HGV envelope protein E1 was not assessed as its expression was very low. It is important to study E1 immunoreactivity as viral envelope proteins often generate strong antibody responses. We propose to improve E1 expression by substituting its own translocation signal sequence for an efficient insect signal sequence, which can function well in insect cells. Putative NS5A and NS5B proteins were expressed to a high level, however, they were mostly insoluble. We propose to express them as constituents of a larger polyprotein precursor, which will be processed to individual NS5 proteins by the HGV protease activities. In our experiments this approach worked well for the HGV NS3 protein, providing a high yield of soluble NS3. The obtained E1, NS5A and NS5B proteins will be purified and their immunoreactivity will be evaluated with panels of HGV PCR positive vs PCR negative sera and with sera from high risk groups vs control sera. As a result, proteins suitable for a commercial immunoassay will be identified.Proposed commercial application:No immunoassay for HGV or HGV-specific antibodies has been developed to date. Development of immunoassays will greatly facilitate ongoing research on the hepatitis G epidemiology. Depending on the outcome of the epidemiological studies screening of donor blood could be mandated. Therefore, the potential market for an HGV diagnostic kit could be similar to the market for diagnostic kits for detection of other blood borne agents, such as HIV, HBV and HCV.National Institute of Heart, Lung, and Blood Institute (NHLBI)
