A major impediment in the molecular characterization of the Norwalk- like human caliciviruses, viruses associated with epidemic gastroenteritis, has been the inability to identify a cell culture system that will support virus replication. A major goal of this project is to develop a reverse genetic system for the cultivatable feline calicivirus (FCV) that can serve as a model for study of the basic molecular biology and replication of the caliciviruses. This year, we completed the production of the panel of region-specific sera for FCV open reading frames (ORF) 1, 2, and 3. The sera specific to FCV nonstructural proteins (ORF1) will help us to characterize these proteins and to analyze the cascade of proteolytic events that takes place during virus replication in cells. Using the proteinase and polymerase specific sera we were able to characterize proteolytic processing of the C-terminal part of the ORF1 polyprotein and to detect minor cleavage products resulting from autocatalytic processing of a proteinase-polymerase protein when the region was overexpressed in bacterial cells. Studies on the functional characterization of cleavage products as well as the full-length protease-polymerase protein are in progress. An approximately 15 kDa protein was detected when virions were probed with sera raised to the VPg region. Direct sequencing of the 15 kDa protein purified from virus particles confirmed its origin from ORF1. Antisera raised against the purified capsid precursor leader sequence allowed us to detect this polypeptide in infected cells, however, it did not react with proteins present in purified virions. Sera raised to the protein encoded in ORF3 recognized a 9 kDa protein in purified virions. The function of this protein is not known, but the availability of tools such as specific antisera and an infectious full- length cDNA clone will enable future studies of the ORF3 product, as well as other viral proteins. - calicivirus, gastroenteritis, diarrhea, RNA
