The Nipah and Hendra viruses are a new genus of paramyxovirus (Henipahvirus), and are currently the only members of the paramyxoviridae family that are classified as BSL-4 pathogens. The Nipah virus (NiV) causes fatal encephalitis in 40% of infected patients and is classified as a Category C priority pathogen in the NIAID Biodefense Research Agenda. A replication incompetent minigenome system amendable to reverse genetics experimentation would expand the opportunities to study the biology of this emerging pathogen. While plasmid based transfection systems for recovering recombinant paramyxoviruses have been recently established, the extremely pathogenic nature of the NiV requires additional safeguards if reverse genetics experiments are to be performed at less than BSL-4 conditions. To ensure the complete lack of key portions in the viral genome required for competent replication, and to introduce a series of silent mutations in order to facilitate downstream reverse genetics experiments, we have chemically synthesized almost the entire genome of the NiV (18 kb) in a manner that allows for the generation of viral-like particles carrying a minigenome of the NiV. All expression constructs and reagents directly related to the NiV genome have been registered in compliance with the Public Health Security and Bioterrorism Preparedness and Response Act of 2002: We propose to use our system to (1) establish the safety and utility of our minigenome system at less than BSL-4 conditions, and (2) to use our codon-optimized envelope glycoproteins of Nipah virus for functional and immunogenicity studies. Novel elements in our proposal include the ability to produce functional envelope proteins in the absence of vaccinia augmentation. The increased expression of our codon-optimized genes over their wild-type counterparts may also lead to greater immunogenicity in the novel electroporation-based genetic immunization strategy that we have proposed. In addition, we have noticed unique elements in the 3'UTR of the Nipah N gene that can be studied in our minigenome system. This application represents a concerted effort to expand the opportunities to study this group of emerging pathogens. This application includes the initial steps to study the biology of this new pathogen and to determine the potential of our reagents for vaccine development. In addition, the investigator has established collaborative efforts with a BSL-4 facility and a company specializing in a patented method of DNA vaccination (letters of support included). If funded, it will also represent a successful attempt to attract new investigators into our nation's response to Bioterrorism preparedness as encouraged by PA-03-080 (Biodefense and Emerging Infectious Disease Research Opportunities).
