Interaction of hepatitis C virus with the type I interferon system

Hepatitis C virus (HCV) needs to tightly manipulate host defences in order to establish infection. The innate immune response slows down viral replication by activating cytokines such as the type I interferons (IFN-α/ β), which trigger the synthesis of antiviral proteins and modulate the adaptive immune system. HCV has therefore developed a number of countermeasures to stay ahead of the IFN system. Here, I will attempt to summarize the current state of research regarding IFN responses against HCV and the viral escape strategies. Particular emphasis will be put on the newly discovered mechanisms HCV employs to avoid the induction of IFN in infected cells.     

Interaction of hepatitis C virus with the type Ⅰ interferon system

Hepatitis C virus (HCV) needs to tightly manipulate host defences in order to establish infection. The innate immune response slows down viral replication by activating cytokines such as the type Ⅰ interferons (IFN-α/β), which trigger the synthesis of antiviral proteins and modulate the adaptive immune system. HCV has therefore developed a number of countermeasures to stay ahead of the IFN system. Here, I will attempt to summarize the current state of research regarding IFN responses against HCV and the viral escape strategies.Particular emphasis will be put on the newly discovered mechanisms HCV employs to avoid the induction of IFN in infected cells.     

Interaction of hepatitis C virus with the type Ⅰ interferon system

Hepatitis C virus (HCV) needs to tightly manipulate host defences in order to establish infection. The innate immune response slows down viral replication by activating cytokines such as the type Ⅰ interferons (IFN-α/β), which trigger the synthesis of antiviral proteins and modulate the adaptive immune system. HCV has therefore developed a number of countermeasures to stay ahead of the IFN system. Here, I will attempt to summarize the current state of research regarding IFN responses against HCV and the viral escape strategies.Particular emphasis will be put on the newly discovered mechanisms HCV employs to avoid the induction of IFN in infected cells.     

Dendritic cells in hepatitis C virus infection: Key players in the IFNL3-genotype response

Recently, single nucleotide polymorphisms, in the vicinity of the interferon lambda 3 (IFNL3) gene have been identified as the strongest predictor of spontaneous and treatment induced clearance of hepatitis C virus (HCV) infection. Since then, increasing evidence has implicated the innate immune response in mediating the IFNL3 genotype effect. Dendritic cells (DCs) are key to the host immune response in HCV infection and their vital role in the IFNL3 genotype effect is emerging. Reports have identified subclasses of DCs, particularly myeloid DC2s and potentially plasmacytoid DCs as the major producers of IFNL3 in the setting of HCV infection. Given the complexities of dendritic cell biology and the conflicting current available data, this review aims to summarize what is currently known regarding the role of dendritic cells in HCV infection and to place it into context of what is know about lambda interferons and dendritic cells in general.     

Human hepatocytes transplanted into genetically immunocompetent rats are susceptible to infection by hepatitis B virus in situ

Immune tolerance of human cells without generalized immunosuppression was created in groups of normal fetal rats at 17 days of gestation by inoculation ip with primary human hepatocytes in utero. One day after birth, suspensions of human hepatocytes were transplanted via intrasplenic injection and one week later groups of rats were inoculated with hepatitis B virus (HBV). Tolerized rats that were transplanted with human hepatocytes and subsequently infected with HBV produced hepatitis B surface antigen (HBsAg) in serum beginning on day 3. Levels rose fivefold and remained stable at 0.75 pg/ml through at least 60 days. Of cells that stained positive for human serum albumin, approximately 30% were found to be also positive for HBsAg by immunohistochemistry. Serum HBV DNA was detectable from 1 to 15 weeks postinfection. Finally, covalently closed circular DNA, reflecting HBV replication, was found in liver and serum. Controls that were tolerized and not transplanted, but inoculated with HBV, as well as untreated controls, had no evidence of HBV gene expression or replication under identical conditions. The data support the conclusion that primary human hepatocytes transplanted into genetically immunocompetent rodent hosts, survive and maintain sufficient differentiation to produce human serum albumin and be infected by HBV.     

Different hepatitis C virus dynamics of free-virions and immune-complexes after initiation of interferon-alpha in patients with chronic hepatitis C

BACKGROUND/AIMS: To elucidate the mechanisms of action of interferon (IFN) against hepatitis C virus (HCV), we studied the serum HCV dynamics of free-virions (FV) and immune-complexes (IC) in patients treated with IFN. METHODS: FV and IC were separated by immunoprecipitation using anti-human immunoglobulin and quantified serially using real-time detection-polymerase chain reaction. RESULTS: Initially [1st phase (0-24 h)], the FV decreased more rapidly compared to IC [exponential decay slope (EDS)=1.78+/-0.42 vs. 0.99+/-0.31 log10/day, P<0.001; half-life=5.65+/-2.02 vs. 12.5+/-2.83 h, P<0.0001], but at the 2nd phase (1-14 days), half-life of FV was significantly longer than that of IC (101+/-117 vs. 14.2+/-1.08 h, P<0.005). Regarding response to IFN, the decline slope was not significantly different at the 1st phase, but at the 2nd phase, the FV-HCV RNA decreased more slowly in non-responders than in sustained responders to IFN (EDS=0.05+/-0.02 vs. 0.34+/-0.19 log10/day, P<0.005; half-life=186+/-112 vs. 15.3+/-1.85 h, P<0.005). CONCLUSIONS: The presence of escape mutants from the neutralizing antibodies may be involved in resistance to IFN. Analyzes of FV- and IC-HCV dynamics are useful for predicting the IFN efficacy and understanding the mechanism of IFN action in chronic hepatitis patients.     

The role of HBV DNA quantitative PCR in monitoring the response to interferon treatment in chronic hepatitis B virus infection

BACKGROUND/AIMS: To investigate whether the measurement of HBV DNA by quantitative polymerase chain reaction (PCR) is helpful in monitoring response to interferon treatment in chronic hepatitis B virus infection, we have determined sequentially serum levels of HBV DNA during and up to 18 months after treatment, in 10 patients with a sustained response (all anti-HBe positive, five also HBsAg negative and anti-HBs positive) and, as controls, in 12 non-responders. METHODS: Serum HBV DNA was measured by standard hybrisation assay (Genostics, Abbott) and by quantitative PCR (Amplicor HBV Monitor test, Roche Diagnostic Systems). RESULTS: A clear difference in HBV viral load between responders and non-responders was observed from the fourth week of treatment and was maintained throughout the study period. At the last follow up 16-26 (median 21) months after starting treatment, all the 10 responders were HBV DNA negative by hybridisation. By PCR, however, five (one anti-HBs and four anti-HBe positive) were still HBV DNA positive. In addition, one anti-HBs positive patient HBV DNA negative by PCR at last follow up, had fluctuating levels of HBV DNA by PCR during the observation period, only intermittently falling below the threshold of the assay. CONCLUSIONS: The measurement of HBV DNA by quantitative PCR provides early prediction of response to interferon, allowing prompt modification of treatment. With this technique, HBV DNA is detected in a high proportion of sustained responders, suggesting that HBV may never be completely eliminated by interferon treatment, even after anti-HBs seroconversion.     

Evolutionary dynamics of hepatitis C virus NS3 protease domain during and following treatment with narlaprevir,a potent NS3 protease inhibitor

Narlaprevir, a hepatitis C virus (HCV) NS3/4A serine protease inhibitor, has demonstrated robust antiviral activity in a placebo‐controlled phase 1 study. To study evolutionary dynamics of resistant variants, the NS3 protease sequence was clonally analysed in thirty‐two HCV genotype 1–infected patients following treatment with narlaprevir. Narlaprevir monotherapy was administered for one week (period 1) followed by narlaprevir/pegylated interferon‐alpha‐2b combination therapy with or without ritonavir (period 2) during two weeks, interrupted by a washout period of one month. Thereafter, all patients initiated pegylated interferon‐alpha‐2b/ribavirin combination therapy. Longitudinal clonal analysis was performed in those patients with NS3 mutations. After narlaprevir re‐exposure, resistance‐associated mutations at position V36, T54, R155 and A156 were detected in five patients in >95% of the clones. Narlaprevir retreatment resulted in a 2.58 and 5.06 log₁₀ IU/mL viral load decline in patients with and without mutations, respectively (P = <0.01). After treatment, resistant variants were replaced with wild‐type virus within 2–24 weeks in three patients. However, the R155K mutation was still observed 3.1 years after narlaprevir dosing in two patients in 5% and 45% of the viral population. Resistant variants could be detected early during treatment with narlaprevir. A slower viral load decline was observed in those patients with resistance‐associated mutations detectable by direct population sequencing. These mutations disappeared within six months following treatment with the exception of R155K mutation, which persisted in two patients.     

Viral elimination reduces incidence of malignant lymphoma in patients with hepatitis C

Purpose

A high prevalence of malignant lymphoma among patients with hepatitis C virus (HCV) infection has been reported. The aim of this retrospective study was to determine the incidence of malignant lymphoma and the relationship between malignant lymphoma and viral elimination in patients with HCV.

Method

We studied 501 consecutive HCV-infected patients who had never received interferon therapy and 2708 consecutive HCV-infected patients who received interferon therapy.

Results

In the non-interferon group, the cumulative rates of malignant lymphoma development were 0.6% at the 5th year, 2.3% at the 10th year, and 2.6% at the 15th year. The cumulative rates of malignant lymphoma development in interferon-treated patients with sustained virologic response were 0% at the 5th year, 0% at the 10th year, and 0% at the 15th year. The cumulative rates of malignant lymphoma development with persistent infection were 0.4% at the 5th year, 1.5% at the 10th year, and 2.6% at the 15th year. The malignant lymphoma development rate was higher in patients with persistent infection than in patients with sustained virologic response (P = .0159). The hazard ratio of lymphomagenesis in 1048 patients with sustained virologic response was significantly lower than in patients with persistent infection (hazard ratio: 0.13; P = .049).

Conclusion

Our retrospective study is the first to determine the annual incidence of malignant lymphoma among patients with HCV at 0.23%. Our results indicate that sustained virologic response induced by interferon therapy protects against the development of malignant lymphoma in patients with chronic HCV.     

A case-control study of the relationship between hepatitis B virus DNA level and risk of hepatocellular carcinoma in Qidong, China

AIM：To investigate the role of hepatitis B virus （HBV） replication in the development of hepatocellular carcinoma （HCC）, a nested case-control study was performed to study the relationship between HBV DNA level and risk of HCC.
METHODS：One hundred and seventy cases of HCC and 276 control subjects free of HCC and cirrhosis were selected for this study. Serum HBV DNA level was measured using fluorescein quantitative polymerase chain reaction at study entry and the last visit.
RESULTS：In a binary unconditional logistic regression analysis adjusted for age, cigarette smoking, alcohol consumption and family history of chronic liver diseases, the adjusted odds ratios （95% confidence intervals） of HCC in patients with increasing HBV DNA level were 2.834 （1.237-6.492）, 48.403 （14.392-162.789）, 42.252 （14.784-120.750）, and 14.819 （6.992-31.411） for HBV DNA levels ≥ 10^4 to 〈 10^5; ≥ 10^5 to 〈 10^6; ≥ 10^6 to 〈 10^7; ≥ 10^7 copies/mL, respectively. Forty-six HCC cases were selected to compare the serums viral loads of HBV DNA at study entry with those at the last visit. The HBV DNA levels measured at the two time points did not differ significantly.
CONCLUSION：The findings of this study provide strong longitudinal evidence of an increased risk of HCC associated with persistent elevation of serum HBV DNA level in the 10^4-10^7 range.     

Regulation of microRNA by hepatitis B virus infection and their possible association with control of innate immunity

Hepatitis B virus (HBV) chronically infects more than 350 million people worldwide. HBV causes acute and chronic hepatitis, and is one of the major causes of cirrhosis and hepatocellular carcinoma. There exist complex interactions between HBV and the immune system including adaptive and innate immunity. Toll-like receptors (TLRs) and TLR-signaling pathways are important parts of the innate immune response in HBV infections. It is well known that TLR-ligands could suppress HBV replication and that TLRs play important roles in anti-viral defense. Previous immunological studies demonstrated that HBV e antigen (HBeAg) is more efficient at eliciting T-cell tolerance, including production of specific cytokines IL-2 and interferon gamma, than HBV core antigen. HBeAg downregulates cytokine production in hepatocytes by the inhibition of MAPK or NF-κB activation through the interaction with receptor-interacting serine/threonine protein kinase. MicroRNAs (miRNAs) are also able to regulate various biological processes such as the innate immune response. When the expressions of approximately 1000 miRNAs were compared between human hepatoma cells HepG2 and HepG2.2.15, which could produce HBV virion that infects chimpanzees, using real-time RT-PCR, we observed several different expression levels in miRNAs related to TLRs. Although we and others have shown that HBV modulates the host immune response, several of the miRNAs seem to be involved in the TLR signaling pathways. The possibility that alteration of these miRNAs during HBV infection might play a critical role in innate immunity against HBV infection should be considered. This article is intended to comprehensively review the association between HBV and innate immunity, and to discuss the role of miRNAs in the innate immune response to HBV infection.     

NS3 protease polymorphisms and genetic barrier to drug resistance of distinct hepatitis C virus genotypes from worldwide treatment‐naïve subjects

Hepatitis C virus (HCV) NS3 protease inhibitors have been primarily designed against genotype 1, the one with the lowest response to dual therapy. However, less evidence of their efficacy on non‐1 genotypes is available, and any such information is mostly concentrated on genotypes 2‐4. This study evaluated HCV protease resistance profiles in the major six HCV genotypes and identified genetic barrier (GB) profiles to each available protease inhibitor across HCV strains from different locations worldwide. We obtained 15 099 HCV sequences from treatment‐naïve subjects retrieved at the Los Alamos HCV Sequence Database. The wild‐type codons of different HCV genotypes were used to analyse the smallest number of nucleotide substitution steps required for changing that codon to the closest one associated with drug resistance. The 36L and 175L RAVs were found as genetic signatures of genotypes 2‐5, while the 80K RAV was found in all genotype 5 sequences. Genotypes 4 and 6 showed a higher GB to RAV mutations conferring resistance to telaprevir, while genotypes 2‐5 presented baseline resistance to that drug, carrying the 36L mutation. Genotype 4 had a higher GB to simeprevir resistance, requiring three substitutions to acquire the 155K mutation. Subtype 1b showed a higher GB than subtype 1a to resistance for most PIs, with RAVs at codons 36 and 155. Geographic disparities were also found in frequencies of certain RAVs in genotypes 2 and 3. Under a scenario of unprecedented evolution of anti‐HCV direct‐acting agents, the genetic composition of the circulating HCV sequences should be evaluated worldwide to choose the most appropriate/feasible therapeutic schemes with the highest genetic barriers to resistance.     

Nonstructural protein 3-4A: the Swiss army knife of hepatitis C virus

Hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) is a complex composed of NS3 and its cofactor NS4A. It harbours serine protease as well as NTPase/RNA helicase activities and is essential for viral polyprotein processing, RNA replication and virion formation. Specific inhibitors of the NS3-4A protease significantly improve sustained virological response rates in patients with chronic hepatitis C when combined with pegylated interferon-α and ribavirin. The NS3-4A protease can also target selected cellular proteins, thereby blocking innate immune pathways and modulating growth factor signalling. Hence, NS3-4A is not only an essential component of the viral replication complex and prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. This review provides a concise update on the biochemical and structural aspects of NS3-4A, its role in the pathogenesis of chronic hepatitis C and the clinical development of NS3-4A protease inhibitors.     

商陆抗病毒蛋白对感染细胞模型中HCV复制的影响

目的探讨商陆抗病毒蛋白(PAP)对丙型肝炎病毒(HCV)感染细胞模型中 HCV 的抑制作用。方法用 HCV 阳性血清感染人肝癌细胞株 HepG2细胞.制成 HCV感染细胞模型,用不同浓度的 PAP 干预该模型;利用荧光定量 PCR 法分别于药物干预后48h、96h、144h 检测培养细胞及上清液中 HCV RNA 含量,同时以不同浓度干扰素(IFN)处理该模型作为对照。结果 PAP 处理 HepG2感染 HCV细胞模型后,细胞内 HCV RNA 含量在48h、96h、144h 各组间与对照组相比有显著差异(P<0.01)。在第48h,HCVRNA 含量100μg/ml 组和10μg/ml 组<1μg/ml 组<0.1μg/ml 组<0.01μg/ml 组及对照组;在第96h,HCV RNA含量100μg/ml 组和10μg/ml 组<1μg/ml 组<0.1μg/ml 组和0.01μg/ml 组<对照组;在第144h,HCVRNA含量100μg/ml 组及10μg/ml 组<1μml 组<0.1μg/ml 组、0.01μg/ml组及对照组。培养上清液中 HCV RNA 含量各组间在48h时与对照组相比差异无显著性(P>0.05),第96h,培养上清液中 HCV RNA 含量各组间差异显著(P<0.05)。100μg/ml组及10μg/ml 组<1μg/ml 组<0.1μg/ml 组、0.01μg/ml 组及对照组;第144h 时,培养上清液中 HCV RNA 含量各组间差异显著(P<0.01),100μg/ml 组、10μg/ml 组及1μg/ml 组<0.1μg/ml 组、0.01μg/ml 组及对照组。PAP 对 HCV 的抑制作用随 PAP 浓度的增加而增强,以100μg/ml 组对 HCV的抑制作用最强。IFN 干预该模型亦得出相似结果,以3.0×10~4IU/ml 组对 HCV 抑制作用最强。PAP 与 IFN 均以第48h 的抑制效果最好,在48h、96h 及144h时,PAP 对 HCV的抑制作用显著高于 IFN(P<0.01)。实验浓度的 PAP 未引起细胞死亡或脱壁,对细胞的形态、生长也无明显影响。结论 PAP 对 HCV 复制有明显的抑制作用,且作用强于IFN。实验浓度的 PAP 对细胞形态及生长无明显影响。     

Establishment and primary application of a mouse model with hepatitis B virus replication

AIM： To establish a rapid and convenient animal model with hepatitis B virus （HBV） replication.
METHODS： A naked DNA solution of HBV-replicationcompetent plasmid was transferred to BALB/C mice via the tail vein, using a hydrodynamic in vivo transfection procedure. After injection, these mice were sacrificed on d 1, 3, 4, 5, 7 and 10. HBV DNA replication intermediates in the liver were analyzed by Southern blot hybridization. The expression of hepatitis B core antigen （HBcAg） and hepatitis B surface antigen （HBsAg） in the liver was checked by immunohistochemistry. Serum HBsAg and hepatitis B e antigen （HBeAg） was detected by enzyme- linked immunosorbent assay （ELISA）. Inhibition of HBV replication was compared in HBV replication model mice treated intraperitoneally with polyinosinic-polytidylin acid （polyIC） or phosphate-buffered saline （PBS）.
RESULTS： After hydrodynamic in vivo transfection, HBV DNA replication intermediates in the mouse liver were detectable on d 1 and abundant on d 3 and 4, the levels were slightly decreased and remained relatively stable between d 5 and 7, and were almost undetectable on d 10. The expression patterns of HBcAg and HBsAg were similar to that of HBV replication intermediate DNA, except that they reached a peak on d 1 after injection. No obvious differences in HBV DNA replication intermediates were observed in the left, right and middle lobes of the liver. After treatment with polyIC, the level of HBV intermediate DNA in the liver was lower than that in the control mice injected with PBS.
CONCLUSION： A rapid and convenient mouse model with a high level of HBV replication was developed and used to investigate the inhibitory effect of polyIC on HBV replication, which provides a useful tool for future functional studies of the HBV genome.     

Open reading frame 3 protein of hepatitis E virus: Multi-function protein with endless potential

Hepatitis E virus (HEV), a fecal-orally transmitted foodborne viral pathogen, causes acute hepatitis in humans and is responsible for hepatitis E outbreaks worldwide. Since the identification of HEV as a zoonotic agent, this virus has been isolated from a variety of hosts with an ever-expanding host range. HEV-open reading frame (ORF) 3, the smallest ORF in HEV genomes, initially had been perceived as an unremarkable HEV accessory protein. However, as novel HEV-ORF3 function has been discovered that is related to the existence of a putative third virion structural form, referred to as “quasi-enveloped” HEV particles, HEV is challenging the conventional virion structure-based classification scheme, which assigns all viruses to two groups, “enveloped” or “non-enveloped”. In this review, we systematically describe recent progress that has identified multiple pathogenic roles of HEV-ORF3, including roles in HEV virion release, biogenesis of quasi-enveloped virus, regulation of the host innate immune response, and interference with host signaling pathways. In addition, implications of HEV-ORF3-associated quasi-enveloped virions are discussed to guide future development of improved vaccines against zoonotic HEV infection.     

一种新的免疫活性细胞(IPCs)在慢性乙型肝炎患者中的检测

目的近年来研究发现了一种在人的外周血中专职产生α/β干扰素的免疫活性细胞,即“干扰素产生细胞(IPCs)。IPCs在外周血中产生干扰素的量是其他产生干扰素细胞的200～1000倍。因此可以说IPCs是体内IFN的专职产生细胞。 IPCs在慢性乙型肝炎患者体内数量的多少和功能的强弱,决定着患者体内IFN的产量,并直接影响着HBV的清除和病程的转归。本文初步检测了干扰素产生细胞(IPCs)在慢性乙型肝炎患者体内的变化特点,以期进一步明确HBV持续感染的患者中是否存在着IPCs数量和功能的缺失及其对慢性肝炎发病机制的影响。方法随机选取了解放军第三○二医院2001年7月～8月住院的25例慢性乙型肝炎患者,男性19例,女性6例,年龄12～49岁。对照组14例为健康供血员。新鲜分离的抗凝外周全血3ml,用PBS等倍稀释,混合均匀后轻轻加在淋巴细胞分离液面上,血液与淋巴细胞分离液的体积比为2:3,2 500rpm离心25min,轻轻吸界面细胞到一干净离心管中,加PBS(含2％胎牛血清和0.5mMEDTA)悬浮细胞,以1500rpm、1 000rpm离心洗涤细胞2次,洗尽血小板。1×10~6PBMC用荧光标记鼠抗人CD_4-FITC,CD3,CD14,CD16,CD20和CD11c—PE单抗染色25min,1％的多聚甲醛固定,上流式细胞仪检测。按照国外Liu YJ(Blood2001,98(4):906-912)所采用的方法,在前向角和侧向角散点图中找出所有可见的PBMC,先设门R1,记数10~5个PBMC。再以PE标记的CD3,CD14,CD16,CD20,CD11c为横轴,以FITC标记的CD4为纵轴作散点图,设门2,门2内的细胞即我们所要检测的IPCs。记数门2内所有的细胞所占10~5个PBMC的百分数,然后比较慢性乙型肝炎患者和健康人外周血中IPCs数量的变化。结果 25例慢性乙型肝炎患者外周血中IPCs的百分数为0.093±0.078,较正常人(0.326±0.092)明显降低,两者相差3.5倍,有显著差异(P<0.001)。同时发现在HBV DNA(+)的患者中其IPCs的百分数为0.081±0.054,明显高于HBV DNA(-)患者(0.040±0.031),差异显著(P<0.01)。IPCs数量与慢性乙型肝炎患者ALT水平无相关性。结论本文通过检测25例慢性乙型肝炎患者和14例正常人外周血IPCs的数量发现,在慢性肝炎患者外周血中存在着明显的IPCs数量的降低,平均只占其外周血PBMC的0.093％,而明显低于正常人的0.326％(P<0.001)。因此,在临床中我们观察到慢性肝炎患者体内HBV病毒总是在潜伏或增殖,导致病情迁延反复以及在实验研究中发现患者体内IFN的量低于正常人等现象,这些现象最直接的原因可能是其体内IPCs数量的下降所致。同时我们也观察到,虽然在慢性乙型肝炎患者体内存在着IPCs数量的降低,机体的免疫反应受到抑制,但并不是机体处于无反应状态。因为在有HBV复制的病例中,其IPCs为0.081±0.054,与无HBV复制的病例组相比(0.040±0.031),P<0.05。由此可见在病毒的刺激作用下,机体自身的细胞免疫应答也是增强的,但可能由于数量上的不足而不足以抑制病毒的复制,因而HBV DNA呈阳性。本研究只是初步检测了慢性肝炎患者IPCs的数量及其临床意义,有关IPCs的研究还存在着许多待阐明的问题,例如IPCs数量和T细胞亚群变化之间的关系;IPCs水平的高低与患者HBV病毒载量动态变化以及IPCs与慢性肝炎病程之间的关系等问题,有待我们去进一步探讨。