pRNA介导的RNA干扰抑制HBV表达和复制的研究

为了研究由pRNA携带的siRNA(HBVsi18-42)所介导的RNAi过程能有效地抑制HBV的基因表达和病毒复制,我们利用细胞模型和高压注射小鼠模型评价HBVsi18-42对HBV复制和基因表达的抑制作用。通过Western印迹检测细胞内的HBsAg含量,用ELISA检测细胞培养上清和小鼠血清中的HBsAg水平,采用Southern印迹检测HBV的复制中间体,通过免疫组织化学检测肝组织切片中HBcAg的表达情况。试验结果显示,HBVsi18-42能以剂量依赖的方式在293T细胞中抑制HBsAg的表达以及在HepG2细胞中下调病毒HBsAg和HBeAg的表达和病毒复制中间体的水平。在小鼠模型中,注射后的3d内HBVsi18-42使小鼠血清中HBsAg的水平分别下降了98.98%、77.07%和60.73%,免疫组织化学检测显示,在注射后的第3天小鼠肝组织内HBcAg阳性细胞数减少了79.1%。初步结果显示HBVsi18-42无论是在细胞或是在小鼠模型中都能下调HBV的复制和基因的表达。本研究为我们下一步实现由pRNA介导的靶向RNAi及基因治疗提供了理论和技术支持。     

pRNA介导的RNA干扰抑制HBV表达和复制的研究

为了研究由pRNA携带的siRNA(HBVsi18-42)所介导的RNAi过程能有效地抑制HBV的基因表达和病毒复制,我们利用细胞模型和高压注射小鼠模型评价HBVsi18-42对HBV复制和基因表达的抑制作用.通过Western印迹检测细胞内的HBsAg含量,用ELISA检测细胞培养上清和小鼠血清中的HBsAg水平,采用Southern印迹检测HBV的复制中间体,通过免疫组织化学检测肝组织切片中HBcAg的表达情况.试验结果显示,HBVsi18-42能以剂量依赖的方式在293T细胞中抑制HBsAg的表达以及在HepG2细胞中下调病毒HBsAg和HBeAg的表达和病毒复制中间体的水平.在小鼠模型中,注射后的3d内HBVsi18-42使小鼠血清中HBsAg的水平分别下降了98.98%、77.07%和60.73%,免疫组织化学检测显示,在注射后的第3天小鼠肝组织内HBcAg阳性细胞数减少了79.1%.初步结果显示HBVsi18-42无论是在细胞或是在小鼠模型中都能下调HBV的复制和基因的表达.本研究为我们下一步实现由pRNA介导的靶向RNAi及基因治疗提供了理论和技术支持.     

Replication Inhibition of Hepatitis B Virus and Hepatitis C Virus in Co-Infected Patients in Chinese Population

Background

Hepatitis B virus (HBV) and hepatitis C virus (HCV) co-infections contributes to a substantial proportion of liver disease worldwide. The aim of this study was to assess the clinical and virological features of HBV-HCV co-infection.

Methods

Demographic data were collected for 3238 high-risk people from an HCV-endemic region in China. Laboratory tests included HCV antibody and HBV serological markers, liver function tests, and routine blood analysis. Anti-HCV positive samples were analyzed for HCV RNA levels and subgenotypes. HBsAg-positive samples were tested for HBV DNA.

Results

A total of 1468 patients had chronic HCV and/or HBV infections. Among them, 1200 individuals were classified as HCV mono-infected, 161 were classified as HBV mono-infected, and 107 were classified as co-infected. The HBV-HCV co-infected patients not only had a lower HBV DNA positive rate compared to HBV mono-infected patients (84.1% versus 94.4%, respectively; P<0.001). The median HCV RNA levels in HBV-HCV co-infected patients were significantly lower than those in the HCV mono-infected patients (1.18[Interquartile range (IQR) 0–5.57] versus 5.87[IQR, 3.54–6.71] Log₁₀ IU/mL, respectively; P<0.001). Furthermore, co-infected patients were less likely to have detectable HCV RNA levels than HCV mono-infected patients (23.4% versus 56.5%, respectively; P<0.001). Those HBV-HCV co-infected patients had significantly lower median HBV DNA levels than those mono-infected with HBV (1.97[IQR, 1.3–3.43] versus 3.06[IQR, 2–4.28] Log₁₀ IU/mL, respectively; P<0.001). The HBV-HCV co-infection group had higher ALT, AST, ALP, GGT, APRI and FIB-4 levels, but lower ALB and total platelet compared to the HBV mono-infection group, and similar to that of the HCV mono-infected group.

Conclusion

These results suggest that co-infection with HCV and HBV inhibits the replication of both viruses. The serologic results of HBV-HCV co-infection in patients suggests more liver injury compared to HBV mono-infected patients, but is similar to HCV mono-infection.     

人工microRNA抗乙型肝炎病毒效果初探

目的:设计靶向乙型肝炎病毒(HBV)基因保守区的人工microRNA(amiRNA),考察其对HBV基因表达的抑制作用。方法:比对HBV全基因组现有序列,选择保守区设计amiRNA,定向克隆到pcDNA6.2-GW/EmGFP-miR载体,将amiRNA载体与HBV复制载体pHBV1.31共转染HepG2细胞,72 h后收取细胞上清,ELISA检测HBV表面抗原(HBsAg)及e抗原(HBeAg)的含量,荧光定量PCR检测HBV DNA含量。结果:amiRNA可显著抑制细胞上清HBsAg、HBeAg和HBV DNA的水平。结论:amiRNA作为防治HBV感染的潜在有效手段之一值得进一步深入研究。     

mTOR Signaling: The Interface Linking Cellular Metabolism and Hepatitis B Virus Replication

Virologica Sinica - Mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that includes mTOR complex (mTORC) 1 and mTORC2. The mTOR pathway is activated in viral hepatitis, including...     

RNA干扰抑制人乙型肝炎病毒复制的研究

采用表达shRNA的载体构建了表达针对病毒HBsAg mRNA保守区的shRNA的质粒psiHBs,利用细胞模型和高压注射小鼠模型评价RNA干扰对HBV复制和基因表达的抑制作用.通过Western印迹检测细胞内的HBsAg,用ELISA检测细胞培养上清和血清中的HBsAg,采用Southern印迹检测HBV的复制中间体,最后通过免疫组化的方法检测肝组织切片中HBcAg的表达情况.结果显示pHBV1.3和psiHBs共转染HepG2后,与对照组相比病毒HBsAg和HBeAg的表达和病毒复制中间体的水平下降了90%以上,并且shRNA的作用效率存在序列特异性和剂量依赖性.在高压注射小鼠模型中,psiHBs表达的shRNA使小鼠血清中HBsAg的水平下降了80%以上,免疫组化检测显示,小鼠肝组织内HBcAg阳性细胞数减少了75.1%,而且shRNA的抑制作用至少能持续4d.研究显示载体表达的shRNA无论是在细胞或是在小鼠模型中都能对HBV的复制和基因的表达发挥序列特异性的抑制作用.本研究为我们下一步实现由RNAi介导的基因治疗提供了理论和技术支持.     

RNA干扰抑制人乙型肝炎病毒复制的研究

采用表达shRNA的载体构建了表达针对病毒HBsAgmRNA保守区的shRNA的质粒psiHBs,利用细胞模型和高压注射小鼠模型评价RNA干扰对HBV复制和基因表达的抑制作用。通过Western印迹检测细胞内的HBsAg,用ELISA检测细胞培养上清和血清中的HBsAg,采用Southern印迹检测HBV的复制中间体,最后通过免疫组化的方法检测肝组织切片中HBcAg的表达情况。结果显示pHBV1.3和psiHBs共转染HepG2后,与对照组相比病毒HBsAg和HBeAg的表达和病毒复制中间体的水平下降了90%以上,并且shRNA的作用效率存在序列特异性和剂量依赖性。在高压注射小鼠模型中,psiHBs表达的shRNA使小鼠血清中HBsAg的水平下降了80%以上,免疫组化检测显示,小鼠肝组织内HBcAg阳性细胞数减少了75.1%,而且shRNA的抑制作用至少能持续4d。研究显示载体表达的shRNA无论是在细胞或是在小鼠模型中都能对HBV的复制和基因的表达发挥序列特异性的抑制作用。本研究为我们下一步实现由RNAi介导的基因治疗提供了理论和技术支持。     

Inhibition of Hepatitis B Virus Replication during Adenovirus and Cytomegalovirus Infections in Transgenic Mice

We have previously demonstrated that hepatitis B virus (HBV) replication and gene expression are abolished in the livers of HBV transgenic mice by cytotoxic T lymphocytes (CTLs) and during lymphocytic choriomeningitis virus (LCMV) infection, stimuli that trigger the production of alpha/beta interferon, gamma interferon, and tumor necrosis factor alpha in the liver. We now report that hepatic HBV replication and gene expression are inhibited by the local induction of these cytokines during adenovirus- and murine cytomegalovirus (MCMV)-induced hepatitis. Further, we show that MCMV also blocks HBV replication and gene expression in the proximal convoluted tubules of the kidney by causing interstitial nephritis and inducing the same cytokines in the renal parenchyma. These results suggest that inflammatory cytokines probably contribute to viral clearance during acute viral hepatitis in humans, and they imply that induction of these cytokines in the liver and other infected tissues of chronically infected patients might have therapeutic value.     

Minimum Requirements for Bluetongue Virus Primary Replication In Vivo

The replication mechanism of bluetongue virus (BTV) has been studied by an in vivo reverse genetics (RG) system identifying the importance of certain BTV proteins for primary replication of the virus. However, a unique in vitro cell-free virus assembly system was subsequently developed, showing that it did not require the same set of viral components, which is indicative of differences in these two systems. Here, we studied the in vivo primary replicase complex more in-depth to determine the minimum components of the complex. We showed that while NS2 is an essential component of the primary replication stage during BTV infection, NS1 is not an essential component but may play a role in enhancing BTV protein synthesis. Furthermore, we demonstrated that VP7, a major structural protein of the inner core, is not required for primary replication but appears to stabilize the replicase complex. In contrast, VP3, the other major structural core protein, is an essential component of the complex, together with the three minor enzymatic proteins (VP1, VP4, and VP6) of the core. In addition, our data have demonstrated that the smallest minor protein, VP6, which is known to possess an RNA-dependent helicase activity, may also act as an RNA translocator during assembly of the primary replicase complex.     

RNA干扰抑制乙型肝炎病毒复制及基因表达研究进展

RNA干扰(RNAinterference,RNAi)是指由21～23个核苷酸组成的双链RNA(dsRNA)所引发的生物细胞内同源基因转录后沉默的现象,是生物体在进化过程中普遍存在的一种基因调控机制。目前对由乙型肝炎病毒(HBV)引起的病毒性肝炎尚无令人满意的治疗效果,而RNA干扰技术的出现为各类慢性HBV感染的治疗开辟了新的途径。本文对RNA干扰抑制HBV复制及基因表达的研究现状、存在问题及应用前景进行了综述。     

Cell-Free Transmission and In Vivo Replication of Marek''s Disease Virus

Marek's disease virus recovered from the feather follicle of infected chickens was found to be infectious for chickens in cell-free preparations. The virus replicated in epithelial cells of the germinative layer of the feather follicle epidermis, producing both intranuclear and round or diffuse cytoplasmic inclusion bodies in the infected cells. It was found at this site 2 weeks postinoculation and prior to the development of tumor or other gross lesions. In the nucleus, many naked and a few enveloped herpesvirions were found, whereas the cytoplasm contained predominantly enveloped herpesvirions, which were usually within the cytoplasmic inclusion bodies. Approximately 80% of the extracellular virions were enveloped. Studies with both virulent and avirulent strains of the virus revealed a relationship between virulence, contagiousness, and replication of the virus in the feather follicle.     

Interference of Hepatitis B Virus with Cellular Signaling

The presence of hepatitis B virus (HBV) proteins leads to changes in the cellular gene expression. As a consequence, the cellular signaling processes are influenced by the actions of HBV proteins. It has been shown that HBV nucleocapsid protein and the amino-terminal part of polymerase termed as terminal protein (TP) could inhibit interferon signaling. Further, the global gene expression profiles differ in hepatoma cells with and without HBV gene expression and replication. The expression of interferon (IFN) stimulated genes (ISGs) was differently regulated in cells with HBV replication and could be modulated by antiviral treatments. The HBV TP has been found to modulate the ISG expression and enhance the HBV replication. The modulation of the cellular signaling processes by HBV may have significant implications for pathogenesis.     

Herpes Simplex Virus Type 1 Glycoprotein gC Mediates Immune Evasion In Vivo

Many microorganisms encode proteins that interact with molecules involved in host immunity; however, few of these molecules have been proven to promote immune evasion in vivo. Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) binds complement component C3 and inhibits complement-mediated virus neutralization and lysis of infected cells in vitro. To investigate the importance of the interaction between gC and C3 in vivo, we studied the virulence of a gC-null strain in complement-intact and C3-deficient animals. Using a vaginal infection model in complement-intact guinea pigs, we showed that gC-null virus grows to lower titers and produces less severe vaginitis than wild-type or gC rescued virus, indicating a role for gC in virulence. To determine the importance of complement, studies were performed with C3-deficient guinea pigs; the results demonstrated significant increases in vaginal titers of gC-null virus, while wild-type and gC rescued viruses showed nonsignificant changes in titers. Similar findings were observed for mice where gC null virus produced significantly less disease than gC rescued virus at the skin inoculation site. Proof that C3 is important was provided by studies of C3 knockout mice, where disease scores of gC-null virus were significantly higher than in complement-intact mice. The results indicate that gC-null virus is approximately 100-fold (2 log₁₀) less virulent that wild-type virus in animals and that gC-C3 interactions are involved in pathogenesis.     

Hepatitis B and Hepatitis C Virus Replication Upregulates Serine Protease Inhibitor Kazal,Resulting in Cellular Resistance to Serine Protease-Dependent Apoptosis

Hepatitis B and C viruses (HBV and HCV, respectively) are different and distinct viruses, but there are striking similarities in their disease potential. Infection by either virus can cause chronic hepatitis, liver cirrhosis, and ultimately, liver cancer, despite the fact that no pathogenetic mechanisms are known which are shared by the two viruses. Our recent studies have suggested that replication of either of these viruses upregulates a cellular protein called serine protease inhibitor Kazal (SPIK). Furthermore, the data have shown that cells containing HBV and HCV are more resistant to serine protease-dependent apoptotic death. Since our previous studies have shown that SPIK is an inhibitor of serine protease-dependent apoptosis, it is hypothesized that the upregulation of SPIK caused by HBV and HCV replication leads to cell resistance to apoptosis. The evasion of apoptotic death by infected cells results in persistent viral replication and constant liver inflammation, which leads to gradual accumulation of genetic changes and eventual development of cancer. These findings suggest a possibility by which HBV and HCV, two very different viruses, can share a common mechanism in provoking liver disease and cancer.Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are serious worldwide health problems, with more than 500 million people believed to be chronically infected with at least one of these viruses (36). HBV is a DNA virus belonging to the Hepadnaviridae family (21), while HCV is an RNA virus belonging to the Flaviviridae family (7). Despite the fact that they are two very different viruses, they share a common pathology in the ability to cause chronic hepatitis, liver cirrhosis, and ultimately, hepatocellular carcinoma (HCC) (34). It remains unclear why these two viruses, which are fundamentally so different, can both lead to similar disease states and the development of HCC.Numerous studies suggest that in chronic viral hepatitis, the host''s immune system is unable to clear infected cells (34). The persistent viral replication further stimulates liver inflammation, and prolonged inflammation and viral persistence result in a gradual accumulation of genetic changes which can subsequently lead to transformation and development of HCC (3, 13). It is possible that part of this failure of the host to clear infected cells results from an inability to induce apoptosis in these cells. For example, persistent HBV/HCV infection suppresses cytotoxic-T-lymphocyte (CTL)-induced apoptosis (3, 4). Apoptosis, or programmed cell death, plays a critical role in embryonic development, immune system function, and the overall maintenance of tissue homeostasis in multicellular organisms. It is also important in the host''s control of viral infection (4). The execution of the apoptotic program has traditionally been considered the result of the activation of a family of proteases known as caspases. Caspase-dependent cell apoptosis (CDCA) usually initiates by activating caspases 8 and 10 through proteolysis of their proenzymes, which further activates the executioner caspases, such as caspase 3 and caspase 7, resulting in the degradation of chromosomal DNA and cell death (28, 29). Recent evidence, however, has suggested that apoptotic cell death can also be promoted and triggered by serine proteases in a caspase-independent manner (5, 6, 39). Serine protease-dependent cell apoptosis (SPDCA) differs from CDCA in that serine proteases, not caspases, are critical to the apoptotic process (1, 6, 39). Interestingly, certain viral infections have been shown to induce SPDCA (27, 39).Failure of the immune-mediated removal of malignant cells through apoptosis may be due to the upregulation of apoptosis inhibitors in these cells (12, 18). We recently demonstrated that SPDCA can be inhibited by a small, 79-amino-acid protein called serine protease inhibitor Kazal (SPIK) (22). SPIK, which is also known as SPINK1, TATI (tumor-associated trypsin inhibitor), and PSTI (pancreas secretory trypsin inhibitor) (8, 24, 38), was first discovered in the pancreas as an inhibitor of autoactivation of trypsinogen (9). The expression of SPIK in normal tissue is limited or inactivated outside the pancreas, but expression of SPIK is elevated in numerous cancers, such as colorectal tumors, renal cell carcinoma, gastric carcinoma, and intrahepatic cholangiocarcinoma (ICC) (16, 19, 24, 31, 40, 41). It remains unknown, however, what role SPIK may play in cancer formation and development. Additionally, overexpression of SPIK was also found in HBV/HCV-infected human livers (32), and an even higher level of expression of SPIK was found in HBV/HCV-associated HCC tissue (19, 31). This implies that SPIK may be closely associated with hepatitis virus infection and development of HCC.Here we show direct evidence that HBV/HCV replication does in fact upregulate expression of the apoptosis inhibitor SPIK, resulting in resistance to SPDCA, which could ultimately lead to the development of chronic hepatitis and liver cancer.     

针对不同基因靶位的锤头状核酶对HBV的抑制作用研究

前基因组mRNA是HBV(Hepatitis Bvirus)基因表达和复制的重要中间产物,全长的前基因组mRNA分子具有复杂易变的二级结构,是设计抑制HBV的核酶时所必须考虑的因素。我们使用多个最新的计算机软件对HBV前基因组mRNA二级结构进行模拟、分析,在全面分析核酶的可作用位点的基础上设计三个针对不同基因靶位的锤头状核酶,并对它们在细胞中对HBV的抑制作用进行研究。结果表明在HBV前基因组mRNA上存在几个高度复杂二级结构的区域,可能对核酶完全不敏感,而S、C、X基因的编码区是合适的核酶作用位点,都可达到对HBV的有效抑制,而且X基因位点的核酶对HBV的各种mRNA的抑制作用最为明显,是设计针对HBV核酶时应该优先考虑的位点。     

针对不同基因靶位的锤头状核酶对HBV的抑制作用研究

前基因组mRNA是HBV(Hepatitis B virus)基因表达和复制的重要中间产物,全长的前基因组mRNA分子具有复杂易变的二级结构,是设计抑制HBV的核酶时所必须考虑的因素.我们使用多个最新的计算机软件对HBV前基因组mRNA二级结构进行模拟、分析,在全面分析核酶的可作用位点的基础上设计三个针对不同基因靶位的锤头状核酶,并对它们在细胞中对HBV的抑制作用进行研究.结果表明在HBV前基因组mRNA上存在几个高度复杂二级结构的区域,可能对核酶完全不敏感,而S、C、X基因的编码区是合适的核酶作用位点,都可达到对HBV的有效抑制,而且X基因位点的核酶对HBV的各种mRNA的抑制作用最为明显,是设计针对HBV核酶时应该优先考虑的位点.     

Inhibition of Hepatitis B Virus Replication by Rheum palmatum L. Ethanol Extract in a Stable HBV-producing Cell

Hepatitis B virus(HBV) infection is a severe health problem in the world.However,there is still not a satisfactory therapeutic strategy for the HBV infection.To search for new anti-HBV agents with higher efficacy and less side-effects,the inhibitory activities of traditional Chinese medicine Rheum palmatum L.ethanol extract(RPE) against HBV replication were investigated in this study.Quantitative real-time polymerase chain reaction(PCR) was employed to analyze the inhibitory activity of RPE against HBV-DNA replication in a stable HBV-producing cell line HepAD38; the expression levels of HBV surface antigen(HBsAg) and e antigen(HBeAg) were also determined by enzyme linked immunosorbent assay(ELISA) after RPE treatment.RPE could dose-dependently inhibit the production of HBV-DNA and HBsAg.The concentration of 50% inhibition(IC50) was calculated at 209.63,252.53 μg/mL,respectively.However,its inhibitory activity against HBeAg expression was slight even at high concentrations.RPE had a weak cytotoxic effect on HepAD38 cells(CC50 = 1 640 μg/mL) and the selectivity index(SI) was calculated at 7.82.Compared with two anthraquinone derivatives emodin and rhein,RPE showed higher ability of anti-HBV and weaker cytotoxicity.So Rheum palmatum L.might possess other functional agents which could effectively inhibit HBV-DNA replication and HBsAg expression.Further purification of the active agents,identification and modification of their structures to improve the efficacy and decrease the cytotoxicity are required.