Members of the California serogroup of bunyaviruses are a cause of arthropod borne encephalitis in the United States and Europe. Infection of mice and rats provides an excellent experimental model to study the pathogenesis of this infection. The bunyavirus genome is divided into 3 segments and genetic reassortants can be made between certain members of the group, for instance some of the California serogroup viruses. This system has already been used to show that the middle genome segment, coding for the viral glycoproteins and a nonstructural protein, is an important determinant of virulence (neuroinvasiveness). We plan to exploit this system to extend the genetic approach to viral pathogenesis. Proposed work includes: (l) REASSORTANTS. We will complete our studies of reassortants between a virulent virus (LAC/original) and an avirulent, non-neuroinvasive virus (TAH/l8l-57). (2) BIOLOGIC VARIANTS. We will select two prototype strains differing in neurovirulence (in contrast to neuro-invasiveness) and will construct reassortants between these. (3) NONOCLONAL ANTIBODY VARIANTS. We will complete the selection and characterization of variants selected by monoclonal antibodies against the G1 protein, to determine which epitopes are associated with altered virulence, in variants of virulent LAC/original and avirulent TAH/l8l-57 viruses. (4) PATHOGENESIS. Descriptive pathogenesis studies will be undertaken of prototype biological and antibody-selected variants to determine the in vivo correlates of virulence and avirulence. (5) FUSION FUNCTION. We have already demonstrated that bunyaviruses mediate fusion from without (FFWO) and from within (FFWI). We will now survey our panel of anti-G1 monoclonal antibodies to determine their ability to block fusion, select fusion variant viruses, and correlate altered fusion with virulence. (6) MONOCLONAL ANTIBODIES. We will continue our construction and characterization of anti-LAC monoclonal antibodies, with emphasis on anti-G2 antibodies, epitope mapping of anti-Gl antibodies, and in vivo protection with anti-G1 antibodies. (7) MOLECULAR STUDIES. We will initiate studies of our reassortants and variants, with emphasis on oligonucleotide fingerprinting; synthesis of DNA oligonucleotide primers, and RNA sequencing by chain termination.
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