The goal of this project is the production of a cross-protective flavivirus vaccine, by identifying the epitope recognized by the murine monoclonal antibody (MAb) 4G2. The flaviviruses are small enveloped viruses with a single (+)-strand RNA genome. These viruses cause human diseases such as yellow fever, dengue fever, and Japanese encephalitis, which are responsible for a significant amount of morbidity and mortality worldwide. None of the few specific vaccines available confer cross-protection. Additionally, the etiologic agent of hepatitis C, HCV, appears to be distantly related to the flaviviruses, sharing a similar genomic organization and homologous viral proteins. The 4G2 epitope is a cross- protective neutralizing epitope present on the 50-55 kD envelope (E) glycoprotein of all members of the flavivirus family. Although the flaviviruses are considered to be infectious agents that should be handled under BL3 containment, we have determined that a two-minute heating cycle at 100 degrees centigrade will inactivate up to 8 logs of viral infectivity. The 4G2 epitope on Japanese encephalitis virus (JEV) is stable to heating for up to six minutes. The dengue epitope, regardless of dengue serotype, is also stable to denaturing agents, while the JEV epitope is not. JEV was chosen for these studies because large quantities of purified virus can be obtained, and a murine animal model for testing the protective efficacy of vaccine candidates is available. Western blot analysis reveals that 4G2 reactivity is retained by a 20 kD tryptic/ chymotryptic fragment of E. The E-glycoprotein is quite susceptible to trypsin cleavage, with epitope reactivity lost after exposure to 0.5 mg/ml trypsin for 4 hours at 37 degrees centigrade; a 60 minute digestion is used. Preparative polyacrylamide gel electrophoresis is then used to separate the digested fragments. A tracking dye pinpoints the region of the gel containing the 20 kD fragment, which is excised and diced, and the protein removed from the gel by electroelution. The material is further concentrated in a small-volume concentration device (Centricon). This material will be utilized for further studies, such as microsequencing, immunization of animals, etc.