The tissues were analyzed for the presence of replication-competent virus by focus-forming selleck kinase inhibitor assay (FFA). Cell Lines and Virus A mouse cholangiocyte cell line, mCl, derived from primary cholangiocytes, harvested from BALB/c, mice and immortalized with SV40 large T-cell antigen was provided by the laboratory of Dr. James Boyer (Yale Liver Care Center, Hartford, CT). These cells express gamma-glutamyl transpeptidase (GGT) and cytokeratin-7, consistent with their biliary epithelium origin (data not shown). H2.35 cells, a hepatocyte cell line derived from BALB/c mice, were purchased from the American Type Culture Collection (Manassas, VA). The cell lines were maintained in DMEM (Cellgro, Herndon, VA) with 10% heat-inactivated FBS (Invitrogen, Carlsbad, CA), penicillin (10,000 U/ml)-streptomycin (10,000 ��g/ml) (Invitrogen), 1% l-glutamine (Invitrogen), and amphotericin B (250 ��g/ml; Cellgro).

The rotavirus strain RRV, obtained from Dr. Harry Greenberg (Stanford University, Palo Alto, CA), was maintained in MA104 cells. Triple-layered RRV particles were purified by cesium chloride centrifugation and were used in all in vitro assays. Viral Assays Measurement of replication-competent virus by using FFA. Virus within samples was enumerated by the formation of foci in MA104 cells as previously described (1, 25). Measurement of the ability of rotavirus to infect cell lines in vitro. Cells were seeded in culture tubes at a quantity of 5 �� 105 in DMEM and were incubated at 37��C for 3 days. Tubes were washed with Earle’s balanced salt solution and were infected with RRV at varying multiplicities of infection (MOI) at 37��C for 1 h.

At an MOI of 1, the amount of virus equals the number of cells. The cultures were washed and incubated with serum-free DMEM + 4 ��g trypsin/ml at 37��C for 24 h. Cultures were monitored for the development of cell lysis [cytopathic effect (CPE)]. CPE was determined by visual inspection of the cell layer and graded based on a previously described scale (17), and viral yield was assessed by FFA. Measurement of viral binding by using attachment and blocking assays. Cells were grown to confluence in 24-well plates. The cells, medium, and inoculating virus were cooled to 4��C. Viral binding was Dacomitinib assessed by using previously described attachment assays (17). For each assay, the sample size consisted of at least three wells of cells per experimental condition, and the experiment was repeated between three and six times.