Antiviral Effects Of Small Interfering RNA In Hepatitis B Virus Infected Mice

Objective To develop a simple, convenient, efficient and stable animal model of hepatitis B virus (HBV) infection, to observe the dynamic changes of virus replication as well as expression in the model, and to evaluate the inhibitory effect of the small interfering RNA (siRNA) in vivo which could effectively inhibited HBV in vitro targeting HBV S gene, C gene, or both S and C gene regions, the research on antiviral effects of siRNA in HBV infected mice was performed.Methods (1) Different doses of pcDNA3.1-HBV (pHBV) were injected into the tail vein of BALB/c hydrodynamically. After 2 days, the levels of hepatitis B surface antigen (HBsAg) were determined with time-resolved immunofluorometric assay (IFMA) to select the optimal dose of pHBV. Then the optimal dose of pHBV was injected into the mice. In a week, the levels of HBsAg, hepatitis B e antigen (HBeAg), anti-HBs, anti-HBe, and anti-HBc were determined with IFMA, and the titers of HBV DNA were analyzed by fluorogenic quantitative PCR (FQ-PCR). In addition, viral specific proteins, HBsAg and hepatitis B core antigen (HBcAg), in the liver were assayed by immunohistochemical staining. (2) Different doses of S-siRNA and pHBV were injected into the mice. The levels of HBsAg were detected to determine the optimal dose of siRNA. Then the optimal dose of S-siRNA or C-siRNA and pHBV were together injected into the mice. The levels of HBsAg were analyzed with IFMA; the titers of HBV DNA were determined by FQ-PCR; HBV S-mRNA/C-mRNA was detected by semi-quantitative RT-PCR; HBsAg and HBcAg in the liver were assayed by immunohistochemical staining.Results (1) A mouse model of acute HBV infection was developed successfully and the infection efficiency was 100%. The optimal dose of pHBV was 60 μg. In the model, HBsAg was detected at the peak (108.88±11.77 ng/ml) of expression on day 1, and dropped gradually; the concentration of HBeAg was low; anti-HBc, anti-HBe, and anti-HBs were first detected on day 4, day 5 and day 7, respectively. The titers of HBV DNA also peaked (7.36×10~4±2.55×l0~4 copies/ml) on day 1 and declined thereafter. HBsAg-positive hepatocytes scattered in the...