Use of PCR for detection of positive (genomic) and negative (replicative) RNA chains of hepatitis C virus in the serum, lymphocytes, and liver of patients with chronic hepatitis C

The presence of viral RNA in liver tissue and peripheral blood serum and lymphocytes of patients with chronic hepatitis C (CHC) was studied by polymerase chain reaction with nested primers on the 5'-untranslated region of hepatitis C virus (HCV) genome. Positive (genome) RNA was more often detected in the liver (81% cases) than in the peripheral blood serum (55%) or lymphocytes (64%). Active replication of HCV (presence of negative RNA chains) was observed only in the liver (in 37% cases). Correlation between the frequency of HCV RNA detection in the liver, blood cells and sera and parameters of the inflammatory process activity (SGPT level, histologic activity index and sclerosis index) was investigated. No relationship between the studied parameters was revealed. Positive correlation between the presence of HCV genome RNA in lymphocytes and serum was detected. A tendency to a decrease in the incidence of replicative RNA of the virus in liver tissue with increase in the activity of chronic hepatitis C was observed.     

Quantitation of replication of the HCV genome in human livers with end‐stage cirrhosis by strand‐specific real‐time RT‐PCR assays: Methods and clinical relevance

HCV replicates in liver via an intermediate negative strand RNA. To study the relevance of HCV genome replication, quantitative strand‐specific HCV real‐time RT‐PCR assays were developed and applied to livers explanted because of end‐stage cirrhosis. The assays have broad ranges of determination and a high reproducibility and accuracy. Analysis of five different samples showed an even distribution of HCV genomes in four livers. Hepatic concentrations of positive (PS)‐ and negative (NS)‐strand RNA did correlate with each other, with PS/NS ratios ranging between 3 and 340. Hepatic concentrations of HCV‐PS or ‐NS RNA did not correlate with serum HCV‐RNA levels or with genotypes. A high HCV envelope‐2 protein expression correlated with a low NS concentration. HCV‐PS and ‐NS levels, E2 protein expression and genotype did not correlate with biochemical tests or with histological changes in the explanted liver, but the ratio NS/PS, a marker of viral replication, correlated with the severity of the recurrent post‐transplant hepatitis caused by HCV. This suggests the existence of an extra‐hepatic location of HCV with comparable viral replication rate being responsible for the infection of the newly transplanted liver. J. Med. Virol. 81:1569–1575, 2009. © 2009 Wiley‐Liss, Inc.     

Hepatitis C virus NS2/3 protease regulates HCV IRES-dependent translation and NS5B RdRp activity

Chronic hepatitis C virus (HCV) infection often leads to liver cancer. NS2/3 protease is the first of two virally encoded proteases required for HCV polyprotein processing. In this report, we investigated the function of NS2/3 protease on HCV replication and translation. Cells transfected with plasmids encoding wild-type or mutant NS2/3 and a dual-luciferase reporter construct containing an HCV internal ribosome entry site (IRES) were used to examine the effect of NS2/3 protease on translation of HCV RNA. Cells transfected with plasmids encoding wild-type or mutant NS2/3, pcDNA-NS5B and a reporter plasmid were used to examine the effect of NS2/3 protease on HCV replication. The results showed that both autocleavage processing and the uncleaved form of NS2/3 protease specifically decrease HCV IRES-directed translation, while the uncleaved form of NS2/3 protease decreases HCV NS5B RdRp activity (replication), indicating that autoregulation by NS2/3 protease of HCV replication and translation may play an important role in persistent HCV infection.     

丙型肝炎病毒体外感染人肝癌细胞株HepG2的研究

目的：研究人肝癌细胞株HepG2体外对丙型肝炎病毒（hepatitis C virus,HCV)的易感性。方法：慢性丙型肝炎患者的血清与HepG2共同孵育后，以逆转录－聚合酶链反应、免疫组化法、原位杂交法分别检测细胞和（或）培养上清中的HCV正、负链RNA、HCV抗原表达和HCV RNA在细胞内的定位。结果：感染血清和细胞共同孵育后的第2－40天，从细胞内和培养上清中均可间断地检测出HCV正、负链HCV RNA，HCV NS3抗原在细胞内能稳定表达，原位杂交证实HCV RNA阳性物质多位于细胞质中。结论：HepG2细胞在体外不但对HCV易感，而且可以支持HCV体外复制。     

Inhibition of hepatitis C virus nonstructural protein, helicase activity, and viral replication by a recombinant human antibody clone

Hepatitis C virus (HCV) nonstructural protein 3 (NS3), with its protease, helicase, and NTPase enzymatic activities, plays a crucial role in viral replication, and therefore represents an ideal target for the development of anti-viral agents. We have developed a recombinant human antibody (Fab) that reacts with the helicase domain of HCV NS3. The affinity-purified Fab antibody completely inhibited the helicase activity of HCV NS3 at equimolar concentration. To evaluate the effect of the Fab on HCV replication, the clone encoding the Fab gene was put into an expression vector, which converts Fab into a complete IgG1 antibody. Using a DNA-based transfection model, we demonstrated that intracellular expression of this antibody resulted in significant reduction of HCV-negative strand RNA synthesis. Intracellular expression of this antibody into either a stable cell line replicating subgenomic RNA, or a transient full-length HCV replication model, reduced both HCV RNA and viral protein expression. These results support the use of recombinant antibody fragments to inhibit NS3 enzyme as a novel, feasible, and effective approach for inhibiting HCV replication.     

丙型肝炎病毒体外感染原代人胎肝细胞的研究

目的　研究丙型肝炎病毒 (HCV)体外细胞培养方法。方法　原代人胎肝细胞与HCV感染血清共孵育后 ,用逆转录 聚合酶链反应、原位杂交、免疫组化分别检测细胞和培养上清中的HCVRNA和HCV抗原表达。结果　从感染血清和细胞共同孵育后的 2～ 2 5d ,细胞内和 /或培养上清中可间断检出HCV正、负链RNA ;HCVNS3抗原在细胞内能稳定表达 ;原位杂交显示HCV负链RNA阳性物质多位于细胞浆。结论　原代人胎肝细胞对HCV易感 ,可用作HCV体外感染和复制的靶细胞     

Polypyrimidine tract-binding protein (PTB) inhibits Hepatitis C virus internal ribosome entry site (HCV IRES)-mediated translation,but does not affect HCV replication

Summary. Polypyrimidine tract-binding protein (PTB) has previously been shown to affect Hepatitis C virus (HCV) IRES-mediated translation. In the present study we investigated the functional role of PTB for HCV translation, replication and chronic HCV infection. Bicistronic HCV IRES reporter plasmids and two different subgenomic replicons (bicistronic: pHCVrep1bBB7 (s1179I); monocistronic: pFK1-389/hyg-ubi/NS3-3/5.1) were used to analyze the effects of PTB. Following transfection of plasmids expressing PTB RNA in sense or antisense orientation, translational activity and HCV RNA were analyzed by luciferase assay, quantitative real-time RT-PCR and northern blot analysis. Additionally, in liver tissue (n=53) intrahepatic PTB RNA levels were determined by quantitative real-time RT-PCR. Significant inhibition of HCV IRES activity up to 42.6% was observed upon PTB sense RNA expression for HCV IRES reporter plasmids, while translational activity was enhanced up to 63.8% for PTB antisense RNA expression. In the HCV replicons PTB did not affect replication and no correlation was found between intrahepatic PTB mRNA levels and serum HCV RNA or histological changes in liver tissue of HCV infected patients. Although PTB inhibits HCV IRES-mediated translation from bicistronic reporter constructs, data obtained from two subgenomic HCV replicons and liver tissue do not indicate a significant role of PTB for HCV replication and chronic HCV infection.     

Lack of hepatitis C virus replication intermediate RNA in diseased skin tissue of chronic hepatitis C patients.

The extent of extrahepatic hepatitis C virus (HCV) replication seems to be low-level and confined to cells of hematopoietic lineage. However, given the spectrum of extrahepatic manifestations associated with HCV, several tissues other than the liver have been suggested as targets of HCV replication and damage. The presence and level of HCV RNA were examined in 19 skin tissue samples from patients chronically infected with HCV and referred for lichen ruber planus (n = 11) or cutaneous vasculitis associated with mixed cryoglobulinemia (n = 8). Serum HCV RNA was quantitated and genotyped by assays that are available commercially. Tissue HCV RNA of genomic- and minus-strand polarity was titrated by a strand-specific semiquantitative RT-PCR. Low titers of genomic-strand HCV RNA were found in three skin specimens from patients with cutaneous vasculitis due to mixed cryoglobulinemia, but in none with lichen ruber planus. The replication intermediate HCV RNA was not detected in any of the skin tissues examined, independent of the serum HCV RNA level or genotype. It is concluded that the occurrence of cutaneous vasculitis and lichen ruber planus in chronic hepatitis C patients is unlikely to be due to HCV replication in the skin.     

Cell culture-adaptive NS3 mutations required for the robust replication of genome-length hepatitis C virus RNA

We recently established a genome-length HCV RNA-replicating cell line (O strain of genotype 1b; here called O cells) using cured cells derived from sO cells, in which HCV subgenomic replicon RNA with an adaptive NS5A mutation (S2200R) is replicated. Characterization of the O cells revealed a second adaptive NS3 mutation (K1609E) required for genome-length HCV RNA replication. To clarify the role of adaptive mutation in genome-length HCV RNA replication, we newly established one and three kinds of genome-length HCV RNA-replicating cell lines possessing the cell background of sO and O cells, respectively, and found additional adaptive NS3 mutations (Q1112R, P1115L, and E1202G) required for the robust replication of genome-length HCV RNA. We further found that specific combinations of adaptive NS3 mutations drastically enhanced HCV RNA replication, regardless of the cell lines examined. These findings suggest that specific viral factors may affect the replication level of genome-length HCV RNA.     

Longitudinal use of phenotypic resistance testing to HIV-1 protease inhibitors in patients developing HAART failure

Hepatitis C virus (HCV) can infect and propagate in humans and chimpanzees. Whereas the chimpanzee has been used as an animal model for infection, ethical considerations, conservation, and the prohibitively high cost preclude progress for experimental research on the biology of the virus. The development of a small animal model for HCV infection is thus desirable to facilitate studies on the infectious cycle of the virus and for the evaluation of drugs for the treatment of HCV infections in humans. As an alternative to the chimpanzee model, we have established a model based on ex vivo infection of orthotopically-implanted human hepatocellular carcinoma cells (HCC) in athymic nude mice. The results show that up to 42 days post-infection, HCV RNA was present in the tumor cells as well as in the liver and serum of infected mice. Furthermore, a direct correlation between size of the tumor and the presence of HCV RNA in the liver was observed, which is concordant with the finding that HCV RNA was detectable only in mice harboring human tumor. Immunohistochemistry analysis of infected liver specimens showed cells expressing the HCV encoded NS5B protein. A few mice developed a humoral response against the nonstructural viral proteins, providing further evidence for expression of these proteins during viral infection. In summary, these results suggest that mice harboring orthotopic tumors support a basal level of HCV replication in vivo.     

Hepatitis C virus infection in dialysis and chronic liver patients: Viraemia dependent anti-E2-antibody response

Cryptic hepatitis C virus (HCV) infection relates to patients infected chronically with HCV that are seronegative but have HCV-RNA. These patients are not identified by the standard serological tests for HCV, which are based on detection of antibodies to core, NS3 and NS5 antigens. They will, therefore, be wrongly diagnosed as non-infected, and are considered as a potential risk for others. Cryptic HCV infection in dialysis units occurs frequently and, due to medical procedures, is a major factor for contracting the virus when unrecognised. This study was conducted in order to assess the humoral immune responses to E2-antigen in sera of patients infected chronically with HCV. Recombinant E2 protein in enzyme linked immunosorbent assay (ELISA) and Western blot (WB) were used to test the occurrence of anti-E2 antibodies in the sera of patients from the liver clinic and of dialysis patients. The presence of E2 antibodies was found to be correlated with the presence of HCV-RNA and with viral load. Antibodies to the E2 protein could be detected in as many as 30% of the sera from dialysis patients with cryptic HCV infection (HCV-RNA only). The results suggest that detection of anti-E2 antibodies may enhance significantly HCV serological standard testing; especially among patients on dialysis, and that antibodies to envelope E2 protein appear to depend on and correlate with the presence of HCV particles.     

Comparison of hepatitis C virus markers in patients with NANB hepatitis.

10 different HCV-specific assays and RT-PCR of the 5' untranslated region of HCV RNA were used to analyze sixty-four patients with chronic NANB liver disease. Po, CP-9 and C22 antigens are located in the putative core; C33c in the putative NS3; C100-3 in the putative NS3/4; KCL in the putative NS4/5 and C825 is located in the putative NS5. GOR protein is not part of the HCV genome, but antibodies to it appear to be present in response to a hepatitis C infection. Positive rates were 91% for Po, 89% for CP-9, 94% for C22, 97% for C33c, 88% for C100-3 (Ortho, EIA), 86% for C100-3 (Abbott, EIA), 84% for C100-3 (Ohtsuka, RIA), 88% for KCL, 59% for C825, 58% for GOR, and 83% for RT-PCR. There were 8 cases which were negative by all anti-C100 tests. 7 of these cases were positive by other anti-HCV markers and/or PCR suggesting the need for improved blood screening assays. There is a variation in the relative reactivity for different markers with different samples. Of the tests employed, anti C33c shows the highest positivity rate.     

Hepatitis C virus is frequently coinfected with serum marker-negative hepatitis B virus: Probable replication promotion of the former by the latter as demonstrated by in vitro cotransfection

Patients with hepatitis C have been reported occasionally to be coinfected with serum marker-negative (silent) hepatitis B virus (HBV). The frequency and significance of such coinfection were investigated. Thirty patients with hepatitis C virus (HCV) infections (10 acute, 10 chronic, 10 cirrhotic) were selected randomly; the acute cases were without serum hepatitis B surface antigen (HBsAg) and anti-hepatitis B core IgM, and the chronic cases were without HBsAg. A nested polymerase chain reaction for the X open reading frame was used to amplify HBV DNA in serum, and immunoperoxidase staining was carried out on liver biopsy specimens. Nucleotide sequencing was carried out to characterize the amplified HBV DNAs. In order to clarify the possibility that the silent HBV mutant promotes HCV replication in the liver, the full-length HCV RNA and the cloned silent HBV DNA dimer were cotransfected into an established cell line, HuH-7, and the amount of secreted HCV RNA was quantified serially. The target HBV DNA was amplified in 26 (86.7%) of the 30 patients. Subsequent direct nucleotide sequencing in 9 selected patients revealed an 8-nucleotide deletion, characteristic of a silent HBV mutant. Immunostaining revealed hepatitis B surface antigen in 15 (50.0%). Cotransfected silent HBV DNA augmented the secretion of HCV RNA by up to 5-fold in comparison with HCV RNA transfection alone. In conclusion, HCV is coinfected frequently with the silent HBV mutant and the latter probably promotes the replication of the former in the liver. J. Med. Virol. 52:399–405, 1997. © 1997 Wiley-Liss, Inc.     

检测丙型肝炎患者NS5区抗体的临床意义探讨

目的检测慢性丙型肝炎患者和１４例ＨＣＶ原发感染后ＮＳ５抗体长达一年半的动态变化，探讨ＮＳ５抗体的临床意义。方法应用重组ＮＳ５抗原，建立ＥＩＡ方法进行检测。结果慢性丙型肝炎患者抗－ＮＳ５抗体阳性率为６０．４８％，ＨＣＶ感染后一月抗－ＮＳ５抗体阳性率为１６．３５％，三月为７５％。结论抗－ＮＳ５抗体无早期诊断价值。抗－ＮＳ５抗体持续阳性者，血清ＡＬＴ多明显升高，抗－ＮＳ５抗体与肝脏疾病活动性相关。丙型肝炎患者中存在抗－Ｃ、抗－ＮＳ３、抗－ＮＳ４抗体阴性，而抗－ＮＳ５抗体单独阳性，表明检测抗－ＮＳ５抗体具有独特的诊断价值