Members of the California serogroup of bunyaviruses are a cause of arthropod-borne encephalitis in the United States and Europe. Mice provide an excellent model to study the pathogenesis of this infection. The bunyavirus genome is divided into 3 segments and genetic reassortants can be made between the 14 viruses in the California serogroup. We have been using this system to dissect the genetic determinants of virulence in mice. Prior studies have shown that the middle-sized RNA segment is an important determinant of neuroinvasiveness, and recent studies have implicated the large and small RNA segments as determinants of neurovirulence. A single complete cDNA clone of the middle-sized RNA segment has been constructed and sequenced, and is being expressed in vaccinia virus. A panel of monoclonal antibodies against the G1 and N proteins have been made, and the G1 antibodies have been used to select a panel of monoclonal antibody- resistant variant viruses. Our future goals include: (1) EXPRESSION. We will express in vaccinia the full ORF of the cDNA clone of the middle sized RNA segment, and of truncated clones carrying the G1 or the G2 glycoprotein sequence. These clones will be used to determine domains required for expression of the fusion and receptor-binding functions of the glycoproteins. (2) RECEPTOR. The G1 protein acts as the ligand which binds to the viral receptor, and this protein will be used as a probe to search for the putative viral receptor on susceptible cells. In addition, virus variants which do and do not replicate in myocytes will be used as a probe for putative receptor on striated muscle cells. (3) IMMUNITY. The role of the G1 and G2 glycoproteins in protective immunity will be probed by using vaccinia virus expressing these proteins as an immunogen. We will use the vaccinia expression system to try to raise monoclonal antibodies against the G2 glycoprotein, and will compare G2 and G1 monoclonal antibodies to identify protective epitopes. C. Hackett, our collaborator, will use our parent and reassortant viruses to develop a cytolytic T cell assay, and to determine which proteins and epitopes are major determinants for T cell responses. (4) L RNA SEGMENT. We will complete biological characterization of reassortants between a neuro-attenuated clone, B.5, and a neurovirulent clone, B1-1a to confirm that the large RNA segment is the major determinant of neurovirulence. We will begin to sequence the large RNA segment, and compare the sequence of the large RNA segment in attenuated clone B.5 and in temperature sensitive revertants which have regained virulence. (5) VECTOR. Our collaborator, B. Beaty, will use our newly constructed panel of viral reassortants to determine the gene segments responsible for replication in neural tissues of Aedes triseriatus, the natural vector of California serogroup viruses. (6) HOST GENETICS. We will survey of inbred mouse strains for susceptibility to La Crosse virus and, if differences are found, initiate a genetic study of susceptibility/resistance.
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