A novel strand-specific RT-PCR for detection of hepatitis C virus negative-strand RNA (replicative intermediate): evidence of absence or very low level of HCV replication in peripheral blood mononuclear cells

Hepatitis C virus (HCV) is reported to be lymphotropic under certain circumstances. In order to evaluate viral replication in peripheral blood mononuclear cells (PBMCs), a novel strand-specific RT-PCR was developed for the determination of HCV negative-strand RNA. The detection limit of this strand-specific RT-PCR was 100 copies of HCV negative-strand RNA in the presence of 1 μg liver RNA and 10⁷–10⁸ copies of positive-strand RNA. False positive PCR signals occurred only when HCV positive-strand RNA exceeded 10⁹ copies. With this RT-PCR, the replicative-intermediates could be detected specifically in eight of ten liver tissues, but not in any samples of serum or plasma (0/65) of patients with chronic hepatitis C. When examining the PBMCs of 46 hepatitis C patients, including 12 patients who had undergone orthotopic liver transplantation, HCV negative-strand RNA was detected in only one patient (1/46). In addition, HCV replicative intermediates were not detected in PBMCs of patients using immunosuppressive agents. It is concluded that the replication of HCV in PBMCs is very unusual.     

A reliable internally controlled RT-nested PCR method for the detection of hepatitis C virus RNA

We have developed a reliable internally controlled RT-nested PCR method for the detection of hepatitis C virus (HCV) RNA using in vitro synthesized Renilla luciferase (Rluc) RNA as an internal control. Using this method, the 5'-noncoding region of HCV RNA (144 nucleotides) and Rluc RNA (276 nucleotides) were efficiently amplified in a single tube, and the sensitivity and specificity of this method were comparable to standard RT-nested PCR. This method was successfully performed on RNA specimens obtained from in vitro HCV-infected human hepatocyte PH5CH8 cells, which support HCV replication. In addition, we demonstrated that this method was useful for the evaluation of antiviral reagents by confirming the anti-HCV activity of bovine lactoferrin, which we previously found to be a new inhibitor of HCV infection. Therefore, this method may be useful for the studies of not only HCV but also of other viruses.     

Positive and negative strand of hepatitis C virus RNA sequences in peripheral blood mononuclear cells in patients with chronic hepatitis C: No correlation with viral genotypes 1b, 2a,and 2b

Hepatitis C virus (HCV) has many genotypes which are closely associated with the severity of chronic hepatitis and the response to antiviral therapy. Although HCV is essentially hepatotropic, several lines of evidence suggest that this virus can infect peripheral blood mononuclear cells (PBMC) in most patients with chronic HCV infection. However, the methods used previously to detect negative-strand HCV RNA have been questioned, and the PBMC tropism of different HCV genotypes remains unknown. A stringent method was used to investigate the prevalence of positive- and negative-strand HCV RNA in the PBMC of 106 patients with chronic hepatitis C and to analyze the influence of HCV genotype on the tropism of PBMC. HCV type 1b was the predominant strain in the patients. Positive-strand RNA in PBMC was detected in 83 (78%) and 40% had negative-strand RNA. The demographic and clinical features were comparable among different patients grouped by the replication status of HCV in the plasma and PBMC samples. In addition, there was no significant difference of PBMC tropism between type 1b and non-1b HCV. In summary, HCV does indeed infect actively the PBMC of chronic hepatitis C patients and such infection is not correlated to the pathogenesis of liver cell damage. Moreover, the genotype is not associated specifically with PBMC tropism of HCV. J. Med. Virol. 52:270–274, 1997. © 1997 Wiley-Liss, Inc.     

Sensitivity and reproducibility of HCV quantitation in chimpanzee sera using TaqMan real-time PCR assay

The availability of molecular protocols for the detection and quantitation of very low numbers of hepatitis C virus (HCV) particles in biological samples is an issue of interest in both clinical and analytical fields of HCV research. A sensitive and reproducible assay is described for HCV RNA quantitation using the TaqMan PCR fluorogenic real-time detection system to establish the levels of HCV RNA in chimpanzee plasma. Our TaqMan PCR protocol and synthetic full length HCV RNA template show that the threshold of sensitivity for our TaqMan PCR is two copies per reaction. As few as 10 genome copies per reaction could be quantitated maintaining a linear range. The accuracy of the TaqMan PCR test was comparable to commercial bDNA and Amplicor tests. The RNA standards of the laboratory were tested in parallel with a World Health Organization (WHO) International Standard for HCV RNA obtaining ratios of 2.7+/-0.7 RNA copies per HCV international unit (IU). Our method using RNA extracted from chimpanzee samples had an estimated sensitivity of 200 RNA copies/ml of plasma (approximately eight copies/reaction or 74 WHO IU/ml). Serial plasma samples from HCV-infected chimpanzees were analyzed using this methodology to evaluate its applicability, and RNA profiles were observed consistent with the evolution of the pathology in each animal. The present study therefore illustrates the high reproducibility, sensitivity and reliability of our TaqMan methodology, providing a useful method for HCV research to consistently detect and quantify viral RNA throughout a range of concentrations.     

Automated specific capture of hepatitis C virus RNA with probes and paramagnetic particle separation

We developed and evaluated a prototype automated specimen preparation instrument for the specific capture of hepatitis C virus (HCV) RNA with probes and magnetic bead-fluid separation. HCV RNA was isolated from serum by lysis of virus particles with a chaotropic agent, followed by hybridization of the RNA with biotinylated probes and capture of the hybridized RNA with streptavidin-coated paramagnetic particles. After washing of the hybrid-particle complexes to remove nonspecifically bound materials, the particles were resuspended in a specimen diluent and were then ready for amplification and detection with a fully automated PCR system (COBAS AMPLICOR; Roche Diagnostic Systems). The analytical sensitivity in the dilution series was 33 copies per ml or greater. Comparison of the test results with those obtained by a manual method based on organic extraction and precipitation of RNA (SepaGene RV-R; Sanko Junyaku Co., Ltd.) showed 93% (49 of 53 samples) sensitivity and 100% (12 of 12 samples) specificity. There was 94% overall agreement between results. When RNA was extracted by the manual method from serum containing 10(3) or 10(5) copies of HCV per ml in the presence of heparin, there was an inhibitory effect on detection of both HCV RNA and the internal control. In contrast, when RNA was extracted from the serum by the automated method, there was no inhibitory effect. This inhibitory effect of heparin on the manual method was also observed for a series of serum specimens from a hemodialysis patient, but the inhibitory effect was eliminated by the automated specimen preparation method. In summary, a fully automated RNA extraction system for PCR detection of HCV RNA by use of specific capture with probes and magnetic bead-fluid separation was shown to have performance similar to that of the conventional manual method. In addition, it successfully eliminated the inhibitory effect of the heparin in the serum and permitted the detection of HCV RNA in serum samples from a hemodialysis patient. The prototype automated RNA extraction system is suitable as a totally automated system, starting with RNA extraction to detection of HCV, if it was combined with the fully automated COBAS AMPLICOR PCR system.     

Study of the infection of human blood derived monocyte/macrophages with hepatitis C virus in vitro

Hepatitis C virus (HCV) is essentially hepatotropic, but clinical observations based on quasispecies composition in different compartments or on viral RNA detection in cells suggest that the virus is able to infect and persist in cells other than liver cells. It was shown previously that peripheral blood mononuclear cells (PBMCs) are permissive to HCV replication in vitro but at a very low rate. Since different viruses associated with chronic infections are known to persist in monocyte/macrophages, it is important to determine whether these mononuclear blood cells are susceptible preferentially to HCV. In order to study HCV interaction with monocytes/macrophages, these cells were isolated from the blood of healthy donors and incubated with HCV genotype 1b positive sera. The detection by RT-PCR of the positive- and negative-strand RNA in the cells at different times and the increase in the amount of intracellular viral RNA measured by the branched DNA assay suggest that monocyte/macrophages can support HCV RNA replication. The rate, however, is very low. The analysis of hypervariable region 1 (HVR-1) nucleotide sequences indicated that some minor variant present in the inoculum might display a specific tropism for the monocytes/macrophages. These results provide evidence that human monocytes/macrophages might represent a reservoir for HCV. This cell tropism may be a crucial factor in the pathogenesis of hepatitis C.     

Intrahepatic cytokine expression is downregulated during HCV/HIV co-infection

HIV co-infection is associated with reduced HCV treatment response rates and accelerated HCV-related liver disease. Cytokines play an important role in regulating hepatic inflammation and fibrogenesis during chronic HCV infection, yet the roles of HIV and/or its therapies on cytokine expression are unknown. Total RNA was extracted from liver biopsies of 12 HCV mono-infected and 14 HCV/HIV co-infected persons. We used real-time PCR to quantify cytokines that contribute to innate and adaptive immune responses, including IFNalpha, IFNgamma, TNFalpha, TGFbeta(1), IL-2, IL-4, IL-8, IL-10, and IL-12p40. Positive- and negative-strand HCV RNA levels were quantified using a molecular beacon approach. Detection of positive-strand HCV RNA was 100% in both groups; negative-strand HCV RNA was detected in four (33%) HCV mono-infected persons and in nine (64%) HCV/HIV co-infected persons. Median strand-specific HCV RNA levels were not significantly different between the two groups. Detection rates of cytokine mRNAs were lower for the HCV/HIV co-infected group compared to the HCV mono-infected group; the detection rates for TNFalpha, IL-8, and IL-10 were statistically significant. Overall, cytokine mRNA quantities were lower for HCV/HIV co-infected compared to HCV mono-infected persons, with the exception of TGFbeta1. These data suggest that a defect in cytokine activation may occur in HCV/HIV co-infected persons that limits efficient clearance of HCV from the liver.     

Prediction of the efficacy of antiviral therapy for hepatitis C virus infection by an ultrasensitive RT-PCR assay

The efficacy of interferon therapy for hepatitis C virus (HCV) infection improved remarkably. However, virologic relapse occurs in a substantial proportion of patients with virologic response (defined as an HCV RNA level below 50 IU/ml at the end-of-treatment). A highly sensitive RT-nested PCR assay capable of detecting almost a single copy of HCV RNA and a real-time RT-PCR assay to quantify HCV RNA down to 120 copies per ml were developed. The RT-nested PCR assay showed that 1 IU of HCV RNA is equivalent to 12.2 copies. For 28 patients with virologic response (12 relapsers and 16 sustained virologic responders), week-4 and end-of-treatment plasma samples were retested. At week 4, HCV RNA was detected by the RT-nested PCR and qualitative COBAS Amplicor HCV version 2.0 in 8/9 (89%) and 6/9 (67%) samples from relapsers, and in 4/16 (25%) and 2/16 (13%) samples from sustained virologic responders, respectively. End-of-treatment samples with HCV-negative by the qualitative COBAS Amplicor were positive by the present assay in 4/12 (25%) of relapsing patients and 0/16 (0%) of sustained virologic responders. The viral levels detected by the present assay in the Amplicor-negative samples were 3.5-17.3 copies/ml, which is below the detection limit of COBAS Amplicor. In conclusion, the highly sensitive RT-nested PCR assay can predict sustained virologic response at week 4 and virologic relapse at the end-of-treatment more accurately than COBAS Amplicor, suggesting its usefulness in monitoring antiviral therapy for HCV infection.     

中国人丙型肝炎病毒基因组3′端非编码区的研究

目的分析中国丙型肝炎病人ＨＣＶ基因组３′端非编码区（３′ＮＣＲ）,以促进对ＨＣＶ基因组复制机制的研究。方法采用两种方法,从上海地区感染ＨＣＶ的病人血清中,扩增获得ＨＣＶ基因组３′端非编码区：一是用套式ＰＣＲ直接扩增,二是先分别获得ＨＣＶ３′ＮＣＲ的前半部分和后半部分,再将两片段进行融合ＰＣＲ。ＰＣＲ产物进行测序后作同源性分析。在此基础上,建立了针对３′端非编码区的ＲＴＰＣＲ方法,并与基于５′端非编码区的ＲＴＰＣＲ方法检测ＨＣＶＲＮＡ的特异性和灵敏度比较。结果序列分析表明,中国丙型肝炎病人ＨＣＶ基因组３′非编码区由４部分组成：高度变异区、Ｐｏｌｙ（Ｕ）区、Ｐｏｌｙ（Ｕ／Ｃ）区和９８碱基区。同源性分析显示,９８碱基区在不同分离株间高度保守并与国外报道株一致,而Ｐｏｌｙ（ＵＵＣ）区存在较大差异。３′端非编码区和５′非编码区ＲＴＰＣＲ检测血清ＨＣＶＲＮＡ有较高符合率（９５％）。结论ＨＣＶ基因组３′端非编码区的３′末端（９８碱基）,在不同分离株间的高度保守性提示,该区在ＨＣＶ基因复制中起重要作用。基于３′非编码区的ＲＴＰＣＲ方法,将有助于ＨＣＶ感染的诊断。     

Comparison of TaqMan real-time PCR and p24 Elisa for quantification of in vitro HIV-1 replication

In this study, TaqMan PCR was used to assess viral replication of HIV-1 infected cells in vitro. This PCR technique was compared with p24 ELISA as a standard method to monitor HIV-1 replication in cell culture. Hut78 T-lymphoblastoid cells were infected with different titres of HIV-1(IIIb) (MOI 0.05-0.0005). The course of HIV-1 replication was monitored by determination of p24 concentrations by ELISA in cell culture supernatants and by quantitation of HIV-1 gag RNA by TaqMan RT-PCR. Additionally, the number of HIV-1 proviral copies was assessed by TaqMan PCR. Monitoring of HIV-1 replication by p24 ELISA and TaqMan RT-PCR revealed comparable kinetics of infection. Both methods provided similar data on the exponential increase and on plateauing of HIV-1 replication. Furthermore, both methods were equally sensitive. However, a 7 log linearity of TaqMan HIV-1 gag PCR was demonstrated without dilution of the specimen, in contrast to p24 ELISA, where because of its narrow range of detectable p24 concentrations, sample dilution was necessary. Although determination of the number of proviral copies by TaqMan PCR does not measure HIV-1 replication, the kinetics of proviral copy number following in vitro inoculation of cells with HIV-1 was nearly the same as the kinetics of HIV-1 RNA copy numbers. In conclusion, TaqMan real-time RT-PCR was demonstrated as a reliable and sensitive tool to quantify and monitor HIV-1 replication in cell culture. It is suggested, therefore, that this technique be an alternative method to monitor HIV-1 replication in vitro.     

中国人丙型肝炎病毒基因组3‘端非编码区的研究

目的 分析中国丙型肝炎病人ＨＣＶ基因组３‘端非编码区,以促进对ＨＣＶ基因组复制机制的研究。方法 采用两种方法,从上海地区感染ＨＣＶ的病人血清中,扩增获得ＨＣＶ基因组３’端非编码区：一是用套式ＰＣＲ直接的增,二是先分别ＨＣＶ３‘ＮＣＲ的前半部分和后半部分,再将两片段进行融合ＰＣＲ。ＰＣＲ产物进行测序后作同源性分析。     

Hepatitis C virus replication in transfected and serum-infected cultured human fetal hepatocytes

Understanding the pathogenesis of hepatitis C requires the availability of tissue culture models that sustain viral replication and produce infectious particles. We report on the establishment of a culture system of nontransformed human fetal hepatocytes that supports hepatitis C virus (HCV) replication after transfection with full-length in vitro-transcribed genotype 1a HCV RNA without adaptive mutations and infection with patient sera of diverse HCV genotypes. Transfected and infected hepatocytes expressed HCV core protein and HCV negative-strand RNA. For at least 2 months, transfected or infected cultures released HCV into the medium at high levels and usually with a cyclical pattern. Viral replication had some cytotoxic effects on the cells, which produced interferon (IFN)-beta as a component of the antiviral response. Medium from transfected cells was able to infect na?ve cultures in a Transwell system, and the infection was blocked by IFN-alpha and IFN-lambda. Viral particles analyzed by sucrose density centrifugation had a density of 1.17 g/ml. Immunogold labeling with antibody against HCV envelope protein E2 decorated the surface of the viral particles, as visualized by electron microscopy. This culture system may be used to study the responses of nontransformed human hepatocytes to HCV infection, to analyze serum infectivity, and to clone novel HCVs from infected patients.