Our research program has been organized in 3 specific areas: i) Studies on cell entry and pathogenesis of HAV: We have identified a cellular receptor for HAV and are interested in understanding how HAV enters the cells and its role in pathogenesis. ii) Studies on replication of HCV: We have recently developed a research program to identify cellular factors required for HCV replication. iii) Studies on cell entry of Ebola virus: In recognition of the need to develop expertise at CBER in CT pathogens, we have recently initiated a program to study Ebola virus cell entry and develop biologics to prevent infection. Replication and Pathogenesis of HCV. Hepatitis C virus (HCV) is a major human bloodborne pathogen. Growth of HCV in cell culture is controversial, and the lack of an "in vitro" infection system is an obstacle to study HCV. Subgenomic replicons of HCV have been developed to study viral replication. However, the growth of these subgenomic replicons is restricted to specific cell lines. To identify cell factors required for HCV replication, we are using an expression cloning strategy. cDNA libraries of cell lines permissive to HCV-replicon replication will be transfected into non-permissive cell lines. Stable transfectants will be selected with puromycin, which resistance gene is coded in the expression cloning vector. HCV replicons coding for neomycin-resistance gene will be transfected into the puromycin resistant cells. Cells that gained susceptible to HCV-replicon replication will be selected using neomycin. Plasmids will be rescued from neomycin resistant cells. cDNAs that confer susceptibility to HCV-replicon replication will be further analyzed. Development of a small animal model to study pathogenesis of HAV. We are interested in developing a small animal model to study pathogenesis of HAV. To do so, we produced transgenic mice carrying the hepatitis A virus cellular receptor-1 (havcr-1). Inoculation of HAV into the liver of havcr-1 transgenic and nontransgenic mice resulted in the production of anti-HAV antibodies. Since antibody production cleared the virus without apparent signs of disease, we decided to increase the virulence of HAV by adapting it to grow in a mouse liver cells. We hypothesized that a virus adapted to grow in mouse liver cells would replicate faster in the liver and have a better chance to cause disease. To test our hypothesis, we transfected mouse liver cells with HAV virion RNA and showed that the virus grew in these cells. Transient transfection of the cDNA coding for the HAV cellular receptor 1 (havcr-1) into mouse liver cells followed by virus infection showed that HAV grew in these cells. However, HAV replicated to low levels in mouse liver cells. Serial passages of HAV in the mouse liver cells resulted in the adaptation of HAV to grow more efficiently in the mouse liver cells. Changes that occur in the HAV genome leading to adaptation to the mouse system will be characterized, and the mouse liver adapted HAV will be used to develop a mouse model for HAV. Development of Biologics against Ebola virus. Ebola virus (EBO) is a "Category A" BT agent that causes hemorrhagic fever and results in a high mortality rate. Research in EBO has been hampered by the strict biosafety containment procedures required for handling the infectious agent. EV cell entry is mediated by the interaction of a cellular receptor with the GP1 subunit of the viral envelope. Studies using recombinant EBO envelope are feasible in BL-2 containment facilities at CBER. Although little is known about the mechanism of cell entry of EBO, the folate receptor (FR ) and the dendritic cell-specific ICAM-3 grabbing non-integrin receptor CD209 (DC-SIGN) and its homolog L-SIGN have recently been identified as cofactors for cellular entry of EV in certain cell types. It is likely that other not yet identified cellular receptors play a role in EBO cell entry. To identify cellular receptors for EBO and develop biologics capable of preventing cell entry, we will overexpress the EBO envelope in insect cells and CHO cells. Purified recombinant EBO envelope will be used to screen human cDNA libraries using an expression cloning strategy to identify novel ligans. Soluble forms of DC-SIGN, L-SIGN, FR, and newly identified ligands will be produced to determine whether they could block binding of the EBO envelope to cells. Humanized monoclonal antibodies against the recombinant EV envelope will be produced using antibody displayed phage libraries. Soluble receptors and monoclonal antibodies that block binding of EBO glycoprotein to cells will be further characterized and evaluated as potential treatment for EBO infection. This project incorporates FY2002 projects 1Z01BP004019-02, 1Z01BP004020-02, and 1Z01BP004021-01.