Establishment of a model of tree shrew primary small intestinal epithelial cells infected with human rotavirus G1P[8] / 中国实验动物学报

Objective To explore the proliferation characteristics of primary small intestinal epithelial cells of tree shrews and the characteristics of human rotavirus(RV) G1P[8] infection to these cells,and establish a model of tree shrew primary small intestinal epithelial cells infected with human rotavirus G1P[8].Methods The primary small intestinal epithelial cells were obtained by collagenase Ⅺ and dispase I digestion from tree shrew.After purification and identification,the obtained primary small intestinal epithelial cells were infected with RV.Then,culture supernatants of infected cells were collected every 12 hours after infection.Viral titer and viral load were subsequently determined.Western blot and indirect immunofluorescence observation were used to detect the expression of RV protein VP6 in the primary cells.The infectivity of RV to the tree shrew primary cells was finally evaluated.Results After purification and identification of primary epithelial cells from the tree shrew,high purity above 90% primary tree shrew small intestinal epithelial cells was obtained.These primary small intestinal epithelial cells could be infected with RV virus by comparing the virus infectivity to primary renal cells,HCT116 cells and MA104 cells.The virus titer reached to 2.0×105TCID 50/mL at 72 h after infection.Using Western blot and indirect immunofluorescence observation,the specific viral protein of VP6 was determined to be expressed in the tree shrew primary small intestinal epithelial cells,and were located in the cytoplasm from days 1 to 5.Conclusions The separation,purification and cultivation methods of tree shrew primary small intestinal epithelial cells are successful,and the tree shrew model of RV-infected the tree shrew primary small intestinal epithelial cells is successfully established.

Establishment of a EV71 virus infection model of tree shrew primary renal cells / 中国实验动物学报

Objective To establish an enterovirus 71(EV71) infection model of tree shrew primary renal cells.Methods Tree shrew primary renal cells were obtained by trypsin digestion.After subculture and purification,EV71 virus was used to infect these primary cells.The culture supernatant of these EV71-infected cells was collected for virus titer detection at 1,2,4,6 and 8 days post-infection.The cells were collected for detection of EV71 VP1 protein by Western blot assay.Furthermore,the expression and location of VP1 protein in the infected cells were detected by indirect immunofluorescence assay.Vero cells were taken as positive control to evaluate the infectivity of EV71 virus to tree shrew primary renal cells.Results Morphologically,the cultured cells were proved to be majorly consisted of the primary renal cells after subculture and purification.The obtained primary cells were infected by EV71 virus.The virus titer was up to 1.3×106 TCID 50/mL during 48-96 h post-infection,proving that EV71 virus infected and proliferated in the tree shrew primary renal cells.Western blot showed that the viral VP1 protein was detected from infected primary cells at 2 to 8 d post infection.VP1 protein was also observed in the cytoplasm at 2 to 6 d post infection by indirect immunofluorescence.Compared with Vero cells,the infectivity of EV71 virus to tree shrew primary renal cells and its proliferation were confirmed.Conclusions Based on the successful establishment of cell culture of tree shrew primary renal cells,the infectivity to the obtained cells and proliferation of EV71 virus in the cells are confirmed.The model of EV71 virus-infected tree shrew primary renal cells is initially established.