The absence of a cell culture system for study of the human caliciviruses associated with epidemic gastroenteritis is a major research obstacle. A collaboration was established this year with Dr. Joshua Zimmerberg and Dr. Leonid Margolis of the Laboratory of Cellular and Molecular Biophysics, NICHHD. Cells adapted to growth on microcarrier beads in the Rotating Wall Vessel (RWV) developed by NASA will be examined for their ability to support the growth of Norwalk virus. In the first phase of this study, CaCo-2 cells (human colon carcinoma), were prepared in the RWV. The cells were inoculated with Norwalk virus and the inoculum was passaged six times. Norwalk virus was no longer detectable by RT-PCR following the sixth passage. Other cell lines (particularly of human origin) are under development for growth in the RWV and will be examined for their ability to support the propagation of Norwalk virus. In addition, Norwalk virus is also undergoing serial passage in primary human and rabbit macrophage cultures by conventional cell culture methods. Progress was made this year in the mapping of nonstructural proteins in the Norwalk virus ORF1 polyprotein. Analysis of a full-length clone of the Norwalk virus ORF1 in a coupled in vitro transcription and translation system containing T7 polymerase shows that the polyprotein encoded by ORF1 is proteolytically processed by an active protease and produces three products of the correct predicted size (45, 40, and 114 kDa). Mutagenesis of the cysteine in the predicted active site of the Norwalk virus ?3C-like? proteinase inactivated the proteolytic processing that was observed in vitro. Discrete regions of the NV ORF1 that span the entire polyprotein have been expressed in the pET bacterial system and, in addition, antisera have been raised in guinea pigs in order to identify the in vitro cleavage products. The 114 kDa protein corresponds to the C-terminal part of the polyprotein and includes the viral proteinase and polymerase. The 40 kDa protein corresponds to the predicted ?2C-like? helicase and the 45 kDa protein corresponds to the N-terminal part of the ORF1 polyprotein. - diarrhea, gastroenteritis, Norwalk virus, caliciviruses
