Negative-strand Hepatitis C Virus RNA Research Articles

Dendritic cell susceptibility to hepatitis C virus genotype 1 infection.

In vitro infection of human monocyte-derived dendritic cells was carried out to study their susceptibility to hepatitis C virus (HCV) infection. Immature dendritic cells and mature dendritic cells were incubated overnight at 37 degrees C with HCV-positive (genotype 1) serum samples; the presence of the viral genome associated with the production of its replicative intermediate was used as evidence of infection. In immature dendritic cells, HCV RNA was detectable from days 1-10 post-infection (p.i.), and de novo synthesis of negative-strand HCV RNA could be demonstrated by a strand-specific rTth reverse transcription-polymerase chain reaction at day 2. In mature dendritic cells, the positive-strand form was detectable from days 1-5 p.i., while the negative-strand HCV RNA appeared at days 1 and 2 p.i. Quasispecies present in the inoculum and 6 days p.i. were analyzed by sequencing hypervariable region 1 of the E2 protein. Only two of seven HVR variants present in the inoculum were found in HCV-infected immature dendritic cells. Another two HVR variants not found in the inoculum were recovered from infected immature dendritic cells, suggesting serum minor variants selection or virus evolution during in vitro replication. Analysis by single-strand conformation polymorphism assay of 5' untranslated region of HCV sequences showed that the patterns obtained from the inoculum and infected immature dendritic cells and mature dendritic cells differed slightly. These findings indicate that both immature dendritic cells and mature dendritic cells are susceptible to HCV genotype 1 infection, supporting at least HCV RNA replication. This model should be a valuable tool for the study of modulation of dendritic cell functions in HCV infection.

Immunological and virological effects of ribavirin in hepatitis C after liver transplantation.

Hepatitis C recurring after liver transplant may cause progressive liver dysfunction, and available treatment regimens are unsatisfactory. A better understanding of the mechanisms of action of drugs currently used to manage hepatitis C would be helpful. In a pilot, uncontrolled clinical trial, we treated 12 patients with post-liver transplantation hepatitis C with 1000-1200 mg qd of ribavirin, given as a monotherapy. We measured the transaminases levels, the liver disease grading and staging scores, the intrahepatic interferon-gamma, tumor necrosis factor-alpha, interleukin (IL)-4 and IL-10 mRNA levels, the serum and liver hepatitis C virus (HCV) RNA titers, and the intrahepatic HCV envelope 2 protein staining score before and after 12 weeks of ribavirin monotherapy. Ribavirin induced a significant amelioration of the transaminases levels. This biochemical response was not associated with a distinct change in the intrahepatic T helper 1/T helper 2 cytokine mRNA profile. Furthermore, some histological parameters, such as the portal inflammation and the fibrosis scores, worsened significantly even in the short term. A slight, albeit not significant, decrease of serum HCV RNA level and intrahepatic HCV antigen staining score was observed. Intrahepatic genomic-strand (but not negative-strand) HCV RNA titer decreased significantly (P=0.024). Contrary to what is suggested by experimental data, administration of ribavirin alone to patients with recurrent hepatitis C after liver transplantation is not accompanied by a specific change of the intrahepatic interferon-gamma, tumor necrosis factor-alpha, IL-4, or IL-10 mRNA transcription profile.

Search for hepatitis C virus negative-strand RNA sequences and analysis of viral sequences in the central nervous system: evidence of replication.

Patients with chronic hepatitis C are more likely to have significant changes in their physical and mental well-being than patients with liver disease of other etiology, and hepatitis C virus (HCV) has been occasionally implicated in diseases of the central nervous system. We analyzed the presence of the HCV negative-strand RNA sequence, which is the viral replicative intermediary, in autopsy brain tissue samples from six HCV-infected patients. Negative-strand HCV RNA was searched for by a strand-specific Tth-based reverse transcriptase PCR, and viral sequences amplified from brain tissue and serum were compared by single-strand conformational polymorphism analysis and direct sequencing. HCV RNA negative strands were detected in brain tissue in three patients. In two of these patients, serum- and brain-derived viral sequences were different and classified as belonging to different genotypes. In one of the latter patients, HCV RNA negative strands were detected in lymph node and, while being different from serum-derived sequences, were identical to those present in the brain. The results of the present study suggest that HCV can replicate in the central nervous system, probably in cells of the macrophage/monocyte lineage.

The hepatitis C virus (HCV)-Trimera mouse: a model for evaluation of agents against HCV.

The lack of small-animal models that are suitable for evaluation of agents used to treat infection with hepatitis C virus (HCV) severely hinders the assessment of potential new therapies for the disease. This study created such a model, termed the "HCV-Trimera" model. The HCV-Trimera model was developed by using lethally irradiated mice, reconstituted with SCID mouse bone marrow cells, in which human liver fragments infected ex vivo with HCV had been transplanted. Viremia (positive-strand HCV RNA levels) in HCV-Trimera mice peaked at approximately day 18 after liver transplantation, and an infection rate of 85% was reached. Viral replication in liver grafts was evidenced by the presence of specific negative-strand HCV RNA. The usefulness of this model for evaluation of anti-HCV agents was demonstrated by the ability of a small molecule (an HCV internal ribosomal entry site inhibitor) and an anti-HCV human monoclonal antibody (HCV AB(XTL)68) to reduce virus loads in HCV-Trimera mice in a dose-dependent manner.

Detection of the negative-strand hepatitis C virus RNA in tissues: implications for pathogenesis

The replication of hepatitis C virus (HCV) RNA is believed to occur via its transcription into a complementary, genomic-length RNA, the so-called negative-strand HCV RNA. This is based on the comparison with the replication of other members of the Flaviviridae family. Detection of the negative-strand HCV RNA in human tissues by semi-quantitative, strand-specific RT-PCR has contributed to the understanding of the HCV cell tropism and of the pathogenesis of HCV-associated disease manifestations. In particular, it was shown that the levels of intrahepatic HCV RNA are not correlated to the extent of the necroinflammation, but that a significant correlation was found with the liver steatosis. These results suggest that most liver disease associated with HCV infection is mediated by the host immune response. However, in some patients, most notably those infected with HCV genotype 3, HCV may cause a cytopathic effect, consisting in the lipid accumulation within hepatocytes.

Acquisition of susceptibility to hepatitis C virus replication in HepG2 cells by fusion with primary human hepatocytes: Establishment of a quantitative assay for hepatitis C virus infectivity in a cell culture system

Hepatitis C virus (HCV) replicates in human and chimpanzee hepatocytes. To characterize the nature of HCV and evaluate antiviral agents, the development of an HCV replication system in a cell culture is essential. We developed a cell line derived from human hepatocytes by fusing them with a hepatoblastoma cell line, HepG2, and obtained several clones. When we tested the clones for their ability to support HCV replication by nested RT-PCR, we found 1 clone (IMY-N9) that was more susceptible to HCV replication than HepG2. The negative-strand HCV RNA was detected in IMY-N9 by strand-specific RT-PCR, and viral RNA was identified in culture supernatant during the culture. Then we monitored HCV RNA titers in IMY-N9 and HepG2, respectively, by real-time detection PCR throughout the culture. A significant increase in the HCV RNA titer was observed only in IMY-N9. Serial passages of HCV culture supernatant were shown in the culture system. Furthermore, we tested several infectious materials for viral infectivity by monitoring HCV RNA titers and/or 50% tissue culture infectious dose (TCID50) of HCV on IMY-N9. In each material, HCV showed various growth patterns and a different TCID50 even though the PCR titer in each material was identical. The results showed that HCV in each material served various growth patterns and different TCID50 even though PCR titer in each material was identical. This cell line is useful for estimating viral activity and for studying cellular factors that may be necessary to HCV replication in human hepatocytes.

Specific interaction of hepatitis C virus protease/helicase NS3 with the 3'-terminal sequences of viral positive- and negative-strand RNA.

The hepatitis C virus (HCV)-encoded protease/helicase NS3 is likely to be involved in viral RNA replication. We have expressed and purified recombinant NS3 (protease and helicase domains) and Delta pNS3 (helicase domain only) and examined their abilities to interact with the 3'-terminal sequence of both positive and negative strands of HCV RNA. These regions of RNA were chosen because initiation of RNA synthesis is likely to occur at or near the 3' untranslated region (UTR). The results presented here demonstrate that NS3 (and Delta pNS3) interacts efficiently and specifically with the 3'-terminal sequences of both positive- and negative-strand RNA but not with the corresponding complementary 5'-terminal RNA sequences. The interaction of NS3 with the 3'-terminal negative strand [called 3'(-) UTR(127)] was specific in that only homologous (and not heterologous) RNA competed efficiently in the binding reaction. A predicted stem-loop structure present at the 3' terminus (nucleotides 5 to 20 from the 3' end) of the negative-strand RNA appears to be important for NS3 binding to the negative-strand UTR. Deletion of the stem-loop structure almost totally impaired NS3 (and Delta pNS3) binding. Additional mutagenesis showed that three G-C pairs within the stem were critical for helicase-RNA interaction. The data presented here also suggested that both a double-stranded structure and the 3'-proximal guanosine residues in the stem were important determinants of protein binding. In contrast to the relatively stringent requirement for 3'(-) UTR binding, specific interaction of NS3 (or Delta pNS3) with the 3'-terminal sequences of the positive-strand RNA [3'(+) UTR] appears to require the entire 3'(+) UTR of HCV. Deletion of either the 98-nucleotide 3'-terminal conserved region or the 5' half sequence containing the variable region and the poly(U) and/or poly(UC) stretch significantly impaired RNA-protein interaction. The implication of NS3 binding to the 3'-terminal sequences of viral positive- and negative-strand RNA in viral replication is discussed.

Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction.

To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. HCV may exist and remain functional in a cultured cell line for a long period.

Hepatitis C–induced hepatic allograft injury is associated with a pretransplantation elevated viral replication rate

Hepatitis C virus (HCV) allograft infection after liver transplantation follows a variable but accelerated course compared with the nontransplantation population. Predictors of outcome and mechanisms of reinfection remain elusive. The accelerated HCV-induced allograft injury associated with a 10- to 20-fold increase in serum viral quantity posttransplantation was hypothesized to be the result of elevated intrahepatic viral replication rates. Patients (N = 23) with HCV-induced end-stage liver disease who underwent liver transplantation between October 1995 and December 1998 were prospectively studied. HCV-induced allograft injury was defined by posttransplantation persistent biochemical hepatitis or allograft fibrosis not explained by other diagnoses. Liver biopsies (N = 92) were obtained by protocol and when clinically indicated. Negative-strand HCV RNA (putative intermediate for replication) was detected by a strand-specific reverse-transcription polymerase chain reaction (RT-PCR) assay and semiquantatively compared with constitutively expressed 18S rRNA. Recipients with increased pretransplantation replication were at increased risk for the development of posttransplantation biochemical hepatitis (P = .03), an increased rate of allograft fibrosis (P = .006), and increased mortality rate (40.0% vs. 0.0%; P = .02). There was no correlation with quantities of genomic HCV RNA in the serum with relative intrahepatic viral replication either before or after liver transplantation. The relative rate of HCV replication within the allograft was not elevated in the posttransplantation period compared with that seen within the explanted liver. Accelerated allograft injury caused by HCV may be predicted by viral replication rates within the explanted liver. The stable intrahepatic replication rate after transplantation suggests that elevated serum viral loads are the result of decreased viral clearance, possibly secondary to immunosuppressive therapy. (Hepatology 2000;32:418-426.)

Hepatitis C Virus from the Hearts of Patients with Myocarditis and Cardiomyopathy

The myocardium may be the target of several types of viral infections. The importance of hepatitis C virus (HCV) infection has been recently noted in patients with myocarditis and in patients with dilated or hypertrophic cardiomyopathy. The present study sought to detect HCV genomes in formalin-fixed paraffin sections of autopsied hearts from patients with myocarditis and patients with dilated or hypertrophic cardiomyopathy. Paraffin sections were deparaffinized, RNA was extracted, and the positive and negative strands of HCV RNA were detected by performing reverse transcription and nested polymerase chain reaction. The polymerase chain reaction products were cloned and sequenced. β-actin gene was used as a control for the successful amplification of a housekeeping gene. Among 106 hearts examined, β-actin gene was amplified in 61 hearts (57.5%). Among the latter, HCV RNA was detected in 13 hearts (21.3%), and negative strands in 4 hearts (6.6%). HCV RNA was found in 4 hearts (33.3%) with myocarditis, in 3 hearts (11.5%) with dilated cardiomyopathy, and in 6 hearts (26.0%) with hypertrophic cardiomyopathy. The sequences recovered from nine patients were highly homologous to the standard strain of HCV. HCV genomes were not found in either 35 hearts from patients with myocardial infarction or 20 hearts from patients with noncardiac diseases. These HCV RNA positive samples were obtained from 1 heart in 1979, 7 hearts between 1980 and 1989, and 5 hearts since 1990, indicating that HCV RNA can be amplified from paraffin-embedded hearts preserved for many years. This method of detecting HCV genomes in formalin-fixed paraffin cardiac specimens has enabled us to widen our research into HCV infection and has been helpful in identifying the presence of HCV infection in cardiac myopathic disorders.

Effects of delayed freezing of liver biopsies on the detection of hepatitis C virus RNA strands

Background/Aims: There are no data about the influence of handling conditions of liver biopsies on the integrity of viral RNAs. We studied the influence of the time delay between obtaining and freezing the liver biopsy on the stability of intrahepatic positive and negative hepatitis C virus RNA (HCV-RNA) strands.Methods: Liver samples from 30 anti-HCV patients were included. For each case, one portion of the liver biopsy (first sample) was immediately frozen (20–28 s), while the other section (second sample) was kept at room temperature (1–30 min) before freezing. Each experimental time point was performed in triplicate using liver samples from three different patients. Semi-quantitative analysis of the positive and negative HCV-RNA strands and of the α1-antitrypsin mRNA was performed by a Tth-based reverse-transcription polymerase chain reaction.Results: A significant time-related decrease in both positive (r=−0.8412, p=0.001) and negative (r= −0.8539, p=0.001) HCV-RNA strand titres was found in the second liver fractions. There were no appreciable changes in RNA titres in those samples frozen after less than 3 min. The RNA titres decreased in all but two samples incubated for 4–30 min. Thus, 3/15 (20%) and 7/11 (64%) of these samples lost positive and negative HCV-RNA strands, respectively. Alpha-1-antitrypsin mRNA titres decreased significantly (r=−0.8935, p=0.01) in those samples kept at room temperature for more than 4 min.Conclusion: Freezing of liver samples immediately after extraction is crucial to avoid false negative HCV-RNA detection results, especially for the anti-genomic RNA strand.

Detection of hepatitis C virus RNA sequences in B-cell non-Hodgkin lymphoma.

Serologic testing shows that hepatitis C virus (HCV) may have a role in the pathogenesis of B-cell non-Hodgkin lymphomas (B-cell NHLs). We tried to demonstrate HCV RNA sequences in paraffin-embedded tissue from B-cell NHLs by reverse-transcription double polymerase chain reaction (RT-PCR) and Southern blotting. We studied 31 consecutive cases of B-cell NHLs; lymph nodes from 32 patients with diseases other than B-cell NHL were negative controls. Positive-strand HCV RNA was tested with primers for the 5' untranslated region. Replicative negative strand HCV RNA was tested with strand-specific RT-PCR for the 5' untranslated region. Immunohistochemical staining for HCV was done using an antibody to HCV core protein. Positive-strand HCV RNA was detected in 8 patients with B-cell NHL; negative-strand HCV RNA was detected in 6 of these cases, indicating viral replication. All control cases were negative for HCV RNA. Immunohistochemistry showed no staining of lymphoma cells for HCV core proteins in any case. HCV and B-cell NHLs may be associated. RT-PCR on paraffin-embedded lymphoma tissue is an alternative method of testing for HCV. The value of immunohistochemistry could not be ascertained. The exact role of HCV in the pathogenesis of B-cell NHL needs to be studied further.

Hepatitis C virus in lymphoid cells of patients coinfected with human immunodeficiency virus type 1: evidence of active replication in monocytes/macrophages and lymphocytes.

It has been reported that hepatitis C virus (HCV) may be lymphotropic in the setting of human immunodeficiency virus type 1 (HIV-1) coinfection. The present study was undertaken to determine the phenotype of lymphoid cells harboring replicating HCV in HIV-1-positive subjects. By means of highly strand-specific thermostable enzyme Tth-based reverse-transcriptase polymerase chain reaction, the presence of viral RNA-negative strand was sought in different subpopulations of peripheral blood mononuclear cells from 10 HIV-positive patients. HCV RNA-negative strand was most commonly present in monocytes/macrophages (4 cases), followed by CD8+ and CD4+ lymphocytes (2 cases) and CD19+ cells (1 case). In 2 cases that were further analyzed, viral-negative strand remained detectable in monocytes/macrophages cultured for 3 weeks. Moreover, monocyte/macrophage- and serum-derived viral sequences differed in the 5' untranslated region. These findings imply that, in HIV-infected subjects, HCV may replicate in the same cells as HIV-1, which raises the possibility of direct interactions between these pathogens.

Long-term follow-up of hepatitis G virus/GB virus C replication in liver during and after interferon therapy in patients coinfected with hepatitis C and G viruses.

We investigated changes in the titers of positive and negative strands of hepatitis G virus (HGV) RNA and hepatitis C virus (HCV) RNA in serum and liver during and 1 to 3 years after interferon therapy in patients with chronic hepatitis C coinfected with HGV/ GBV-C. Eight (6%) of 134 patients with chronic hepatitis C treated with interferon were positive for HGV RNA and were examined retrospectively. Titers of positive and negative strands of HGV RNA and HCV RNA were determined by strand-specific reverse transcriptase-polymerase chain reaction. Before therapy, HGV RNA titers in liver were lower than those in serum (P = 0.0169), while HCV RNA titers in liver were significantly higher than those in serum (P = 0.0074). No negative strands of HGV RNA were detected in serum, liver, or peripheral blood mononuclear cells in any patients. With interferon therapy, 5 of the 8 patients lost HCV RNA from serum and liver, with sustained normal liver biochemical values and significant histological improvement. HGV RNA disappeared transiently from serum and liver at the end of therapy in 5 patients, but reappeared again after therapy in 4 of them. Two of the 8 patients naturally lost HGV RNA after completion of therapy. These findings suggest that: (1) HGV/GBV-C does not appear to replicate in liver, serum, or peripheral blood mononuclear cells, (2) detection of HGV RNA in liver and peripheral blood mononuclear cells may be a mere reflection of serum HGV RNA, and (3) the long-term clinical outcome of chronic HGV/GBV-C infection appeared to be benign.

Liver and peripheral blood mononuclear cells are not major sites for GB virus-C/hepatitis G virus replication.

The pathogenesis and replication sites of GB virus-C/hepatitis G virus (GBV-C/HGV) in humans remain unclear. The presence of GBV-C/HGV and hepatitis C virus (HCV) RNA sequences in matched serum, peripheral blood mononuclear cells (PBMC) and liver samples in 10 patients with GBV-C/HGV infection, 8 of whom were coinfected with HCV was explored. Positive- and negative-strand GBV-C/HGV and HCV RNA were detected by strand-specific reverse-transcription polymerase chain reaction (RT-PCR), and virus titers were quantified by competitive PCRs. Positive-strand GBV-C/HGV RNA was detected in 7 of 10 PBMC samples of the patients with serum GBV-C/HGV RNA, but negative-strand GBV-C/HGV RNA was not found in these cells. Positive-strand GBV-C/HGV RNA was found in 9 liver samples, and 2 (22%) of them also had negative strand. In contrast, negative-strand HCV RNA was frequently found in PBMC and liver samples. A positive correlation between the titer of viral RNA in liver tissue and that in serum sample was demonstrated in HCV infection, but not in GBV-C/HGV infection. These findings suggest that liver and PBMC are not the major replication sites for GBV-C/HGV and that GBV-C/HGV is not a primary hepatotropic virus.

Hepatitis C Virus Infection Involves CD34+Hematopoietic Progenitor Cells in Hepatitis C Virus Chronic Carriers

AbstractAlthough hepatitis C virus (HCV) mainly affects hepatocytes, infection is widespread and involves immunologically privileged sites. Whether lymphoid cells represent further targets of early HCV infection, or whether other cells in the hematopoietic microenvironment may serve as a potential virus reservoir, is still unclear. We studied whether pluripotent hematopoietic CD34+ cells support productive HCV infection and can be used to establish an in vitro infection system for HCV. Six patients were selected as part of a cohort of HCV chronic carriers who developed a neoplastic disease. Reverse transcriptase-polymerase chain reaction (RT-PCR) and branched DNA signal amplification assays were used to detect and quantitate HCV RNA in extracted nucleic acids from purified bone marrow and peripheral blood CD34+ cells. Direct in situ RT-PCR, flow cytometry analysis, and immunocytochemistry were applied to demonstrate specific viral genomic sequences and structural and nonstructural virus-related proteins in intact cells. Results indicated that both positive and negative HCV RNA strands and viral proteins were present in CD34+ cells from all HCV-positive patients and in none of the controls. Additional experiments showed that a complete viral cycle took place in CD34+ cells in vitro. Spontaneous increases in viral titers indicated that virions were produced by infected hematopoietic progenitor cells. To further define the cellular tropism, we attempted to infect CD34+ cells in vitro. We were unable to demonstrate viral uptake by cells. These findings suggest that HCV replication can occur in the early differentiation stages of hematopoietic progenitor cells, and that they may be an important source of virus production.© 1998 by The American Society of Hematology.

Hepatitis C Virus Infection Involves CD34+Hematopoietic Progenitor Cells in Hepatitis C Virus Chronic Carriers

Although hepatitis C virus (HCV) mainly affects hepatocytes, infection is widespread and involves immunologically privileged sites. Whether lymphoid cells represent further targets of early HCV infection, or whether other cells in the hematopoietic microenvironment may serve as a potential virus reservoir, is still unclear. We studied whether pluripotent hematopoietic CD34+ cells support productive HCV infection and can be used to establish an in vitro infection system for HCV. Six patients were selected as part of a cohort of HCV chronic carriers who developed a neoplastic disease. Reverse transcriptase-polymerase chain reaction (RT-PCR) and branched DNA signal amplification assays were used to detect and quantitate HCV RNA in extracted nucleic acids from purified bone marrow and peripheral blood CD34+ cells. Direct in situ RT-PCR, flow cytometry analysis, and immunocytochemistry were applied to demonstrate specific viral genomic sequences and structural and nonstructural virus-related proteins in intact cells. Results indicated that both positive and negative HCV RNA strands and viral proteins were present in CD34+ cells from all HCV-positive patients and in none of the controls. Additional experiments showed that a complete viral cycle took place in CD34+ cells in vitro. Spontaneous increases in viral titers indicated that virions were produced by infected hematopoietic progenitor cells. To further define the cellular tropism, we attempted to infect CD34+ cells in vitro. We were unable to demonstrate viral uptake by cells. These findings suggest that HCV replication can occur in the early differentiation stages of hematopoietic progenitor cells, and that they may be an important source of virus production.© 1998 by The American Society of Hematology.

The presence of active hepatitis C virus replication in lymphoid tissue in patients coinfected with human immunodeficiency virus type 1.

The existence of extrahepatic replication sites of hepatitis C virus (HCV) remains controversial. Highly strand-specific Tth-based reverse transcriptase-polymerase chain reaction was used to search for the presence of viral RNA negative strand in lymph nodes from 16 patients with AIDS and in peripheral blood mononuclear cells (PBMC) from 14 other human immunodeficiency virus (HIV)-positive patients. Negative-strand HCV RNA was detected in lymph node samples from 10 patients (63%) and in PBMC from 5 (36%). This suggests that, at least under circumstances of impaired immunity associated with HIV infection, HCV is lymphotropic in vivo. However, the clinical implications of these findings need to be further investigated.

Hepatitis C Virus Quasispecies in Patients Infected with HIV-1: Correlation with Extrahepatic Viral Replication

Hepatitis C virus (HCV) sequences recovered from serum, peripheral blood mononuclear cells (PBMCs), and various tissues from human immunodeficiency virus type 1 (HIV-1) positive patients were compared by single strand conformational polymorphism (SSCP) and sequencing. In five patients, paired serum and PBMCs samples were analyzed while in two other patients multiple autopsy tissues were studied. Sequences amplified from the NS5 and E2 regions were consistently identical in the same patient; however, three PBMCs samples and three different tissue samples (pancreas and adrenal gland in one patient and lymph node in the other patient) contained 5′ untranslated region (5′UTR) sequences that were different from circulating sequences. The presence of 5′ UTR sequences differing from circulating sequences correlated with the presence of HCV RNA negative strand, as the latter was detected by a Tth-based strand-specific assay in all but one of these samples. These two independent lines of evidence: viral sequence differences and the presence of RNA negative strand in the same tissues strongly argue for the genuine presence of extrahepatic HCV replication, at least in the setting of HIV-1 infection.

Ribozyme gene therapy for hepatitis C virus infection

Background: The development of antiviral drugs for hepatitis C virus (HCV) infection represents a substantial challenge. Similar to human immunodeficiency virus (HIV), HCV is highly prone to mutation. It is, therefore, expected that potential HCV therapeutics currently under development, such as protease inhibitors, will suffer from the same shortcomings of HIV therapeutic drugs; the emergence of drug resistant viral mutants. Ribozymes (Rz) are enzymatic RNA molecules that can be engineered to specifically target any given RNA molecule. A therapeutic Rz can be manufactured and administered as a drug, or a Rz gene can be delivered and expressed intracellularly by gene therapy. For HCV therapeutics, we favour the gene therapy approach as delivery and in vivo expression of Rz genes will result in a constant and continuous supply of multiple intracellular Rz, offering less opportunity for the development of drug-resistant viral variants. Objectives: To utilise direct intravenous injection of hepatotropic viral vectors to transfer Rz genes directly into the hepatocytes of HCV-infected patients, resulting in degradation of the HCV positive strand RNA genome, the viral mRNAs, and even the negative strand RNA replication intermediate. We plan to circumvent the emergence of drug-resistant viral mutants by targeting multiple, highly conserved HCV RNA sequences simultaneously with multiple Rz genes expressed from a single vector. Study design: Rzs targeting conserved regions of the HCV positive and negative RNAs were transcribed in vitro and used to cleave HCV target RNAs. The most effective Rzs identified were then incorporated into adeno associated viral (AAV) vectors and adenoviral (AV) vectors and tested for their ability to inhibit HCV core expression in a tissue culture model. Results: Several Rzs targeting highly conserved HCV sequences effectively degraded positive and negative strands of HCV RNA in vitro. Furthermore, substantial inhibition of HCV gene expression was observed in tissue culture using viral vectors to deliver and express Rz genes. Conclusions: Rz gene therapy has potential for the production of anti-viral drugs directed against HCV. Initial studies employing Rz gene therapy to produced anti-viral drugs against HCV have proved successful. Rz gene therapy may be a useful approach to overcome problems associated with anti-HCV drug design, such as the emergence of drug-resistant mutants.