Figure 1.
Rotavirus replication, maturity and cytopathic effect of cultured extrahepatic biliary epithelia (EHBE).
(A) Cytopathic effect (CPE) grading of cultured EHBE. The severity of EHBE injury was graded from 0 to 4 with 4 being the most severe. (B) Ultrastructural CPE. siNSP4 transfected EHBE had normal structure. The other 2 siRNAs also protected the shape of EHBE, but nuclear degeneration was noted (pointed by black arrows). (C) Rotavirus particles. Significantly less double-layered particles (DLPs, pointed by black arrows) and triple-layered particles (TLPs, pointed by black arrow heads with dash lines) existed in siNSP4 protected EHBE. siVP7 caused more synthesis of DLPs. (D) Viral particle quantification. TLPs in EHBE significantly reduced in all siRNA trasfected groups (*•♦P<0.05, compared to NC). DLPs in siVP7 group was increased significantly (▴P<0.05, compared to NC). (E) Representative gel images of reverse transcription polymerase chain reaction (RT-PCR). RT-PCR revealed the presence of 3 viral messenger RNAs (mRNAs) in NC group, but siNSP4 transfection reduced the level of NSP4 mRNA and completely inhibited the transcription of VP7 and VP4 mRNA. siVP7 caused absence of VP4 and VP7 mRNA. siVP4 reduced mRNA transcription. (F) Quantitative analysis using Gel Pro Analyzer. siNSP4 decreased the relative level of all viral mRNAs (*▴•P<0.05, compared to NC). siVP7 inhibited the transcription of VP7 and VP4 mRNAs (♦★P<0.05, compared to NC). siVP4 only inhibited the mRNA expression of VP4 (▪P<0.05, compared to NC).
Table 1.
Mean CPE percentage of different groups.
Figure 2.
Immunofluorescence for rotavirus proteins and integrin α2.
(A) All viral proteins were expressed in the cytoplasm of cultured extrahepatic biliary epithelia (EHBE). Decreased viral protein expression and less infected cells were evident in siNSP4 transfected EHBE. siVP7 transfection significantly reduced VP4 and VP7 expression while leaving NSP4 expression unchanged. siVP4 inhibited the expression of VP4, but the expression of the other 2 proteins was not influenced. Magnification, ×200. (B) NSP4 positive biliary epithelia had higher expression of integrin α2 compared to non-infected cells. Magnification, ×1000.
Figure 3.
Hepatobiliary injury, incidence of biliary atresia and intraluminal rotavirus replication in the bile ducts.
(A) Hematoxylin and eosin staining of livers. Ballooning degeneration and mononuclear cell infiltration were the basic pathologic changes in the liver of rotavirus infected mice in the NC group, but none suffered from hepatic cirrhosis. siNSP4 and siVP4 transfection decreased hepatic injury, but siVP7 transfected mice still suffered from significant hepatic injury. (B) Grading of extrahepatic bile duct injury. Grade 0: No obstruction, stenosis, necrotic epithelia or inflammatory cell infiltration. Grade 1: Mild stenosis and several inflammatory cells. Grade 2: Stenosis or obstruction caused by necrotic cells or inflammatory cells in bile duct lumens. Grade 3: Complete lumen obstruction. Black arrow pointed at the site of injury. (C) Summary of distribution of biliary injury grading. Biliary injury was significantly inhibited by siNSP4 (0/5), (P<0.05) compared to NC (5/5). Three of siVP7 transfected mice suffered mild biliary injury. Half of siVP4 transfected mice suffered BA. (D) Measurement of inner and outer diameters of bile ducts. The dashed line and black line respectively indicated the inner and outer diameters. (E) Summary of distribution of inner/outer diameter index (I/O DI). siRNA transfected mice had relatively higher index value (all P<0.05, compared to NC). siNSP4 had the highest I/O DI which was not significantly different from the blank controls (P>0.05). (F) Quantitative analysis of viral messenger RNA (mRNA) in bile ducts on 7 dpi using real-time reverse transcription polymerase chain reaction. siNSP4 decreased the level of all viral mRNAs (*▴•P<0.05, compared to NC). siVP7 inhibited the transcription of VP7 and VP4 mRNAs (♦★P<0.05, compared to NC). siVP4 only inhibited the mRNA expression of VP4 (▪P<0.05, compared to NC).
Figure 4.
Immunofluorescent assay for viral protein expression in biliary epithelia of extrahepatic bile ducts on 7 dpi.
Extrahepatic bile ducts infected by rotavirus expressed cytoplasmic viral proteins in the epithelial layer. siNSP4 transfection decreased the expression of NSP4, VP7 and VP4. In serial sections, NSP4 was positive but VP4 and VP7 were both negative in siVP7 transfected mice. siVP4 transfected mice had positive expression of VP7 and NSP4.
Table 2.
siRNA targets and sequences against NSP4, VP7 and VP4.
Table 3.
Primers for mRNA of NSP4, VP7, VP4 and β-actin.