研究HCV感染及发病机制的小动物模型

丙型肝炎病毒（HCV）可导致慢性肝炎,在全球约有1.8亿人感染。当前对于HCV的预防和治疗手段是很有限的,没有有效的疫苗,临床使用干扰素和利巴韦林联用有效率只有55%,而且有明显副作用,因而HCV感染的小动物模型对于抗HCV药物开发以及HCV发病机制的研究是非常重要的;但是HCV只感染人和黑猩猩,存在天然的物种限制。重点介绍利用三种方法克服物种屏障,开发小鼠感染HCV模型的进展,以及转HCV基因小鼠模型前景。     

丙型肝炎病毒感染实验动物模型的研究进展

丙型肝炎病毒（HCV）感染是导致人类慢性病毒性肝炎、肝硬化和肝癌的最主要病因之一。由于缺乏合适的HCV感染实验动物模型,使得针对HCV感染更为有效的疗法及疫苗的研发滞后。黑猩猩是HCV感染研究的最佳实验动物,但由于其来源有限、价格昂贵及临床症状等诸多问题,其应用受限,因此发展新的实验动物模型用于HCV感染相关的基础和应用研究迫在眉睫。近年来,以啮齿类等动物为替代模型取得了不少进展,应用转基因等实验技术使替代动物感染了HCV,并成功应用于多个学科领域的研究。本文分析了HCV自然感染的实验动物、自然感染和非自然感染的替代实验动物在致病机制研究、药物评价和疫苗研发应用中的优缺点及未来研究趋势。     

Persistent replication of a hepatitis C virus genotype 1b‐based chimeric clone carrying E1, E2 and p6 regions from GB virus B in a New World monkey

The development of effective hepatitis C virus (HCV) vaccines is essential for the prevention of further HCV dissemination, especially in developing countries. Therefore the aim of this study is to establish a feasible and immunocompetent surrogate animal model of HCV infection that will help in evaluation of the protective efficacy of newly developing HCV vaccine candidates. To circumvent the narrow host range of HCV, an HCV genotype 1b‐based chimeric clone carrying E1, E2 and p6 regions from GB virus B (GBV‐B), which is closely related to HCV, was generated. The chimera between HCV and GBV‐B, named HCV/G, replicated more efficiently as compared with the HCV clone in primary marmoset hepatocytes. Furthermore, it was found that the chimera persistently replicated in a tamarin for more than 2 years after intrahepatic inoculation of the chimeric RNA. Although relatively low (<200 copies/mL), the viral RNA loads in plasma were detectable intermittently during the observation period. Of note, the chimeric RNA was found in the pellet fraction obtained by ultracentrifugation of the plasma at 73 weeks, indicating production of the chimeric virus. Our results will help establish a novel non‐human primate model for HCV infection on the basis of the HCV/G chimera in the major framework of the HCV genome.     

Non-human primate surrogate model of hepatitis C virus infection

More than 170 million people worldwide are chronically infected by HCV, which is the causative agent of chronic hepatitis C, cirrhosis, and finally liver cancer. Although animal models of viral hepatitis are a prerequisite for the evaluation of antiviral and vaccine efficacy, the restricted host range of HCV has hampered the development of a suitable small animal model of HCV infection. Use of the chimpanzee, the only animal known to be susceptible to HCV infection, is limited by ethical and financial restrictions. In this regard GBV-B, being closely related to HCV, appears to be a promising non-human surrogate model for the study of HCV infection. This review describes the characteristic of GBV-B infection of New World monkeys, and discusses current issues concerning the GBV-B model and its future directions.     

丙型肝炎核心蛋白与NS3蛋白在转基因小鼠中的表达

为研究丙型肝炎病毒核心蛋白及ＮＳ３蛋白在转基因小鼠体内的基因表达及细胞内定位,以及病毒蛋白 直接致细胞病变效应,我们采用ＲＴ－ＰＣＲ方法检测了靶基因ｍＲＮＡ在转基因小鼠各种组织中的表达,用免疫组化方法分析了核心蛋白与ＮＳ３蛋白在转基因小鼠体内的表达及细胞内定位。     

Small rodent models of hepatitis B and C virus replication and pathogenesis

The narrow host range of infection supporting the long-term propagation of hepatitis B and C viruses is a major limitation that has prevented a more thorough understanding of persistent infection and t...     

Physical and functional interaction between hepatitis C virus NS5A protein and ovarian tumor protein deubiquitinase 7B

Hepatitis C virus (HCV) NS5A protein plays crucial roles in viral RNA replication, virus assembly, and viral pathogenesis. Although NS5A has no known enzymatic activity, it modulates various cellular pathways through interaction with cellular proteins. HCV NS5A (and other HCV proteins) are reportedly degraded through the ubiquitin–proteasome pathway; however, the physiological roles of ubiquitylation and deubiquitylation in HCV infection are largely unknown. To elucidate the role of deubiquitylation in HCV infection, an attempt was made to identify a deubiquitinase (DUB) that can interact with NS5A protein. An ovarian tumor protein (OTU), deubiquitinase 7B (OTUD7B), was identified as a novel NS5A‐binding protein. Co‐immunoprecipitation analyses showed that NS5A interacts with OTUD7B in both Huh‐7 and HCV RNA replicon cells. Immunofluorescence staining revealed that HCV NS5A protein colocalizes with OTUD7B in the cytoplasm. Moreover, HCV infection was found to enhance the nuclear localization of OTUD7B. The OTUD7B‐binding domain on NS5A was mapped using a series of NS5A deletion mutants. The present findings suggest that the domain I of NS5A is important and the region from amino acid 121 to 126 of NS5A essential for the interaction. Either V121A or V124A mutation in NS5A disrupts the NS5A‐OTUD7B interaction. The results of this in vivo ubiquitylation assay suggest that HCV NS5A enhances OTUD7B DUB activity. Taken together, these results suggest that HCV NS5A protein interacts with OTUD7B, thereby modulating its DUB activity.     

GB virus B disrupts RIG-I signaling by NS3/4A-mediated cleavage of the adaptor protein MAVS

Understanding the mechanisms of hepatitis C virus (HCV) pathogenesis and persistence has been hampered by the lack of small, convenient animal models. GB virus B (GBV-B) is phylogenetically the closest related virus to HCV. It causes generally acute and occasionally chronic hepatitis in small primates and is used as a surrogate model for HCV. It is not known, however, whether GBV-B has evolved strategies to circumvent host innate defenses similar to those of HCV, a property that may contribute to HCV persistence in vivo. We show here in cultured tamarin hepatocytes that GBV-B NS3/4A protease, but not a related catalytically inactive mutant, effectively blocks innate intracellular antiviral responses signaled through the RNA helicase, retinoic acid-inducible gene I (RIG-I), an essential sensor molecule that initiates host defenses against many RNA viruses, including HCV. GBV-B NS3/4A protease specifically cleaves mitochondrial antiviral signaling protein (MAVS; also known as IPS-1/Cardif/VISA) and dislodges it from mitochondria, thereby disrupting its function as a RIG-I adaptor and blocking downstream activation of both interferon regulatory factor 3 and nuclear factor kappa B. MAVS cleavage and abrogation of virus-induced interferon responses were also observed in Huh7 cells supporting autonomous replication of subgenomic GBV-B RNAs. Our data indicate that, as in the case of HCV, GBV-B has evolved to utilize its major protease to disrupt RIG-I signaling and impede innate antiviral defenses. These data provide further support for the use of GBV-B infection in small primates as an accurate surrogate model for deciphering virus-host interactions in hepacivirus pathogenesis.     

Molecular biology and pathogenesis of hepatitis E virus

The hepatitis E virus (HEV) is a small RNA virus and the etiological agent for hepatitis E, a form of acute viral hepatitis. The virus has a feco-oral transmission cycle and is transmitted through environmental contamination, mainly through drinking water. Recent studies on the isolation of HEV-like viruses from animal species also suggest zoonotic transfer of the virus. The absence of small animal models of infection and efficient cell culture systems has precluded virological studies on the replication cycle and pathogenesis of HEV. A vaccine against HEV has undergone successful clinical testing and diagnostic tests are available. This review describes HEV epidemiology, clinical presentation, pathogenesis, molecular virology and the host response to HEV infection. The focus is on published literature in the past decade. Equal contribution     

Role of lipid metabolism in hepatitis C virus assembly and entry

HCV (hepatitis C virus) represents a major global health problem. A consistent body of evidence has been accumulating, suggesting a peculiar overlap between the HCV life cycle and lipid metabolism. This association becomes evident both for the clinical symptoms of HCV infection and the molecular mechanisms underlying the morphogenesis and entry process of this virus. The HCV core–lipid droplets association seems to be central to the HCV morphogenesis process. Moreover, the biogenesis pathway of very‐low‐density lipoproteins has been shown to be involved in HCV morphogenesis with MTP (microsomal triacylglycerol transfer protein), ApoB (apolipoprotein B) and ApoE (apolipoprotein E) as essential elements in the production of infectious HCV particles. HCV infectivity also correlates with the lipidation status of the particles. Furthermore, some HCV cellular receptors and the regulation of the entry process are also connected to lipoproteins and lipid metabolism. Specifically, lipoproteins modulate the entry process and the cholesterol transporter SR‐BI (scavenger receptor class B type I) is a cellular entry factor for HCV. The present review aims to summarize the advances in our understanding of the HCV–lipid metabolism association, which may open new therapeutic avenues.     

Role of the cytosolic domain of occludin in trafficking and hepatitis C virus infection

The role of the tight‐junction (TJ) protein occludin (OCLN) in hepatitis C virus (HCV) entry remains elusive. Here, we investigated the OCLN C‐terminal cytosolic domain in HCV infection. We expressed a series of C‐terminal deletion mutants in Huh‐7 cells KO for OCLN and characterized their functionality in HCV infection and trafficking. Deleting the OCLN cytosolic domain led to protein instability and intracellular retention. The first 15 residues (OCLN‐C15 mutant) of the cytosolic domain were sufficient for OCLN stability, but led to its accumulation in the trans‐Golgi network (TGN) due to a deficient cell surface export after synthesis. In contrast, the OCLN‐C18 mutant, containing the first 18 residues of the cytosolic domain, was expressed at the cell surface and could mediate HCV infection. Point mutations in the context of C18 showed that I279 and W281 are crucial residues for cell surface expression of OCLN‐C18. However, in the context of full‐length OCLN, mutation of these residues only partially affected infection and cell surface localization. Importantly, the characterization of OCLN‐C18 in human‐polarized hepatocytes revealed a defect in its TJ localization without affecting HCV infection. These data suggest that TJ localization of OCLN is not a prerequisite for HCV infection in polarized hepatocytes.     

Virus-host interactions during hepatitis C virus entry — implications for pathogenesis and novel treatment approaches

Hepatitis C virus (HCV) is a member of the Flaviviridae family and causes acute and chronic hepatitis. Chronic HCV infection may result in severe liver damage including liver cirrhosis and hepatocellular carcinoma. The liver is the primary target organ of HCV, and the hepatocyte is its primary target cell. Attachment of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between the virus and the target cell that is required for successful entry into the cell and initiation of infection. This step is an important determinant of tissue tropism and pathogenesis; it thus represents a major target for antiviral host cell responses, such as antibody-mediated virus neutralization. Following the development of novel cell culture models for HCV infection our understanding of the HCV entry process and mechanisms of virus neutralization has been markedly advanced. In this review we summarize recent developments in the molecular biology of viral entry and its impact on pathogenesis of HCV infection, development of novel preventive and therapeutic antiviral strategies.      

Hepatitis C virus–apolipoprotein interactions: molecular mechanisms and clinical impact

Introduction: Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis, hepatocellular carcinoma and liver failure. Moreover, chronic HCV infection is associated with liver steatosis and metabolic disorders. With 130–150 million people chronically infected in the world, HCV infection represents a major public health problem. One hallmark on the virus is its close link with hepatic lipid and lipoprotein metabolism.

Areas covered: HCV is associated with lipoprotein components such as apolipoproteins. These interactions play a key role in the viral life cycle, viral persistence and pathogenesis of liver disease. This review introduces first the role of apolipoproteins in lipoprotein metabolism, then highlights the molecular mechanisms of HCV-lipoprotein interactions and finally discusses their clinical impact.

Expert commentary: While the study of virus-host interactions has resulted in a improvement of the understanding of the viral life cycle and the development of highly efficient therapies, major challenges remain: access to therapy is limited and an urgently needed HCV vaccine remains still elusive. Furthermore, the pathogenesis of disease biology is still only partially understood. The investigation of HCV-lipoproteins interactions offers new perspectives for novel therapeutic approaches, contribute to HCV vaccine design and understand virus-induced liver disease and cancer.     

Virus-host Interactions during Hepatitis C Virus Entry - Implications for Pathogenesis and Novel Treatment Approaches

Hepatitis C virus (HCV) is a member of the Flaviviridae family and causes acute and chronic hepatitis. Chronic HCV infection may result in severe liver damage including liver cirrhosis and hepatocellular carcinoma. The liver is the primary target organ of HCV, and the hepatocyte is its primary target cell. Attachment of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between the virus and the target cell that is required for successful entry into the cell and initiation of infection. This step is an important determinant of tissue tropism and pathogenesis; it thus represents a major target for antiviral host cell responses, such as antibody-mediated virus neutralization. Following the development of novel cell culture models for HCV infection our understanding of the HCV entry process and mechanisms of virus neutralization has been markedly advanced. In this review we summarize recent developments in the molecular biology of viral entry and its impact on pathogenesis of HCV infection, development of novel preventive and therapeutic antiviral strategies.     

GB virus C infection in Indonesian HIV‐positive patients

GB virus C (GBV‐C), a human virus of the Flaviviridae family that is structurally and epidemiologically closest to hepatitis C virus (HCV), has been reported to confer beneficial outcomes in HIV‐positive patients. However, the prevalence of GBV‐C in HIV‐positive individuals in Indonesia is unknown. Since GBV‐C is more prevalent in anti‐HCV positive patients than in anti‐HCV negative subjects, transmission of GBV‐C and HCV could be by the same method. This study examined the prevalence and molecular characteristics of GBV‐C infection in HIV patients in Yogyakarta, Indonesia. The prevalence of GBV‐C among HIV patients (n = 125, median age 31 years) based on the 5′UTR region was 111/125 (88.8%), including 39/48 (81.3%) and 72/77 (93.5%) HIV‐infected patients with and without HCV infection, respectively. GBV‐C isolates were of genotype 2a, 3 and 6 in 58.3%, 12.6% and 28.4% of patients, respectively. Patients with genotype 3 were significantly younger than those with genotypes 2a or 6 (P = 0.001 and P = 0.012, respectively). Genotypes 3 and 6 were significantly associated with injection drug use (P = 0.004 and P = 0.002, respectively) and HCV co‐infection (P < 0.001 for both genotypes), indicating a shared transmission route with HCV. In conclusion, the prevalence of GBV‐C among HIV‐positive patients in Indonesia is high, and three genotypes were detected, namely genotype 2a, 3 and 6.     

Inhibitory effect of presenilin inhibitor LY411575 on maturation of hepatitis C virus core protein,production of the viral particle and expression of host proteins involved in pathogenicity

Hepatitis C virus (HCV) core protein is responsible for the formation of infectious viral particles and induction of pathogenicity. The C‐terminal transmembrane region of the immature core protein is cleaved by signal peptide peptidase (SPP) for maturation of the core protein. SPP belongs to the family of presenilin‐like aspartic proteases. Some presenilin inhibitors are expected to suppress HCV infection and production; however, this anti‐HCV effect has not been investigated in detail. In this study, presenilin inhibitors were screened to identify anti‐HCV compounds. Of the 13 presenilin inhibitors tested, LY411575 was the most potent inhibitor of SPP‐dependent cleavage of HCV core protein. Production of intracellular core protein and supernatant infectious viral particles from HCV‐infected cells was significantly impaired by LY411575 in a dose‐dependent manner (half maximum inhibitory concentration = 0.27 μM, cytotoxic concentration of the extracts to cause death to 50% of viable cells > 10 μM). No effect of LY411575 on intracellular HCV RNA in the subgenomic replicon cells was detected. LY411575 synergistically promoted daclatasvir‐dependent inhibition of viral production, but not that of viral replication. Furthermore, LY411575 inhibited HCV‐related production of reactive oxygen species and expression of NADPH oxidases and vascular endothelial growth factor. Taken together, our data suggest that LY411575 suppresses HCV propagation through SPP inhibition and impairs host gene expressions related to HCV pathogenicity.     

树鼩肝炎动物模型的研究进展

肝炎的的大范围流行已成为国内外关注的重要公共卫生问题之一。甲肝、乙肝虽已有疫苗,但不能忽视未来由于病毒变异所带来的威胁。丙肝目前尚未发现非常有效的疫苗,且丙肝的病理学机制也尚未完全清晰,主要原因是缺乏理想的动物模型。树胸属于低等灵长类动物。研究发现,他对很多人类疾病易感,所以建立肝炎树胸动物模型成为现在肝炎研究的一个热点。本文介绍了各型肝炎主要是甲、乙、丙型肝炎研究中建立树胸动物模型的自内外进展情况及存在的问题。     

The challenge of using experimental infectivity data in risk assessment for Ebola virus: why ecology may be important

Analysis of published data shows that experimental passaging of Zaire ebolavirus (EBOV) in guinea pigs changes the risk of infection per plaque‐forming unit (PFU), increasing infectivity to some species while decreasing infectivity to others. Thus, a PFU of monkey‐adapted EBOV is 10⁷‐fold more lethal to mice than a PFU adapted to guinea pigs. The first conclusion is that the infectivity of EBOV to humans may depend on the identity of the donor species itself and, on the basis of limited epidemiological data, the question is raised as to whether bat‐adapted EBOV is less infectious to humans than nonhuman primate (NHP)‐adapted EBOV. Wildlife species such as bats, duikers and NHPs are naturally infected by EBOV through different species giving rise to EBOV with different wildlife species‐passage histories (heritages). Based on the ecology of these wildlife species, three broad ‘types’ of EBOV‐infected bushmeat are postulated reflecting differences in the number of passages within a given species, and hence the degree of adaptation of the EBOV present. The second conclusion is that the prior species‐transmission chain may affect the infectivity to humans per PFU for EBOV from individuals of the same species. This is supported by the finding that the related Marburg marburgvirus requires ten passages in mice to fully adapt. It is even possible that the evolutionary trajectory of EBOV could vary in individuals of the same species giving rise to variants which are more or less virulent to humans and that the probability of a given trajectory is related to the heritage. Overall the ecology of the donor species (e.g. dog or bushmeat species) at the level of the individual animal itself may determine the risk of infection per PFU to humans reflecting the heritage of the virus and may contribute to the sporadic nature of EBOV outbreaks.     

Hepatitis C: A mouse at the end of the tunnel

Since its discovery in 1989, researchers strive after a small animal model for Hepatitis C virus infection, so far with very limited success. A study recently published in Nature now for the first time reports the recapitulation of the complete life cycle of this virus in inbred mice with a functional adaptive immune system.Worldwide, over 130 million people are chronically infected with Hepatitis C virus (HCV). Acute infection goes along with mild and generalized symptoms, and therefore mostly remains undiagnosed; in more than half of the patients, however, the infection persists and, over the years, can cause liver damage such as fibrosis, cirrhosis or hepatocellular carcinoma.HCV is a positive strand RNA virus of the family Flaviviridae. Studies of this virus have made huge leaps forward since the implementation of efficient cell culture systems^1,2; nonetheless, our knowledge of HCV-associated pathogenesis is scarce owing to the lack of a practicable animal model. So far, chimpanzees are the only animals fully susceptible to HCV infection; however, legal, ethical, economical let alone practical reasons dictate the establishment of small animal models as an alternative. Most efforts have been put into various mouse models³, but in general, mice are resistant to HCV infection. Only individual steps of the lifecycle could be reproduced in mouse cells: expression of human variants of the entry receptors CD81 and occludin mediates virus uptake into mouse cells^4,5; selectable replicons demonstrated that HCV RNA can be replicated in mouse cells, albeit inefficiently; and assembly as well as secretion of HCV particles can be achieved in mouse hepatocytes³. In contrast, for studying the full replication cycle in living animals, systems had to be developed, in which mouse hepatic tissue was inducibly or constitutively deteriorated to allow repopulation by human hepatocytes³. This inter-species chimerism naturally required the animals to be immuno-deficient to avoid graft rejection. Nonetheless, particularly the uPA-SCID model has become very popular, especially for pre-clinical drug testing and validation and for studying passive immunization strategies against HCV infection. Still, for research on vaccine development and pathogenesis, these models are of limited to no use, as such studies require robust immune responses.The teams of Alexander Ploss and Charles Rice now report a breakthrough on the way to an immune-competent mouse model⁶. They had shown previously that adenoviral expression of human CD81 and occludin in mouse liver cells rendered fully immune-competent mice susceptible to HCV infection; however, replication was abortive⁵. In their present study, the authors take this approach two steps further: (1) by establishing mice that express the entry factors transgenically; (2) by additionally incapacitating the innate antiviral response. It had been suggested earlier that this response severely impacts HCV replication in mouse cells^7,8, and indeed, its blocking in an otherwise fully immune-competent background sufficed to increase viral replication to detectable levels in entry factor transgenic (EFT) mice. By testing knock-outs of several factors involved in innate antiviral defense, STAT1^−/− EFT mice were found to be best to support HCV replication, which was sustained for up to 11 weeks, with viral genomes detectable in both liver tissue and serum. The authors could corroborate that this viremia relied on authentic viral replication, as it could be inhibited by neutralizing antibodies or an HCV-specific antiviral compound; moreover, a drastic reduction in viral replication was observed in mice that additionally carried a knock-out of the ppia (Cyclophilin A) gene, a well-known HCV host dependency factor in humans⁹. On the down side, one has to acknowledge that the infection rate in the liver was stunningly low with only 0.4% of hepatocytes infected at a given time. In contrast, in human livers HCV antigen has been detected in around 20% of cells on average. This low rate of infection in mice is coherent with comparatively low infectivity titers of < 100 infectious units per ml mouse serum; somewhat in contrast, however, are the high RNA titers of 10⁴-10⁶ copies per ml, arguing for a high excess of non-infectious RNA-containing structures. The mechanism by which they are released into the serum of infected mice and their biophysical properties remain to be determined. Regardless of the rather sparse hepatic infection, strikingly, the authors found clear evidence for the mounting of an immune response, such as splenomegaly with increased relative frequencies of NK- and B-cells as well as infiltration of infected livers by CD8⁺ T-cells. HCV infection was apparently cleared by the T-cell response, which at late stages of infection shifted towards a memory phenotype.These results are remarkable and highlight the perspective of such an animal model possibly also for studies on (immune-mediated) pathogenesis and eventually development of T-cell activating vaccines. A few caveats, however, still need to be overcome on this front: none of the animals in the study developed a lasting, chronic infection, but rather cleared the virus after about 80 days; this might be resolved by adaptation of the virus to its new host by prolonged, serial passaging in mice. Additionally, the used transgenic mice lack STAT1, which is not only responsible for the immediate intrinsic antiviral response, but also required for signaling in response to all types of interferons (I, II and III). This deficiency for one deprives CD8⁺ T-cells of their antiviral activity through interferon-γ secretion, which has been reported to be more important in controlling HCV than direct cytolytic effects¹⁰, and, secondly, is likely to also affect the phenotypic differentiation and activation of various immune cells. One possible way to overcome the latter issue could be to use a tissue-specific knock-out of the stat1 gene, specifically targeting hepatocytes. Alternatively, a combined knock-out of different interferon effector genes with antiviral activity against HCV might still allow for HCV replication, while restoring general responsiveness to interferons.Even with the mentioned limitations, the presented strategy denotes a milestone for HCV research and is the first system that can truly be called a small animal model for HCV infection. As it does not rely on xenografts, it is not only more practicable and much cheaper than previous models, but also more robust as it is completely independent from variations of human graft donors. Albeit still in need for optimizations, finally one can see the long-awaited HCV-susceptible mouse at the end of the tunnel.