non-A, non-B Hepatitis Agent Research Articles

Evaluation of Regulatory T Cells in Chronic Hepatitis C Patients

Hepatitis C virus (HCV), defined as a non-A non-B hepatitis agent in 1989, is a hepatotropic, noncytopathic, positive-stranded ribonucleic acid (RNA) virus from the family Flaviviridae. HCV infection is an acute or chronic necroinflammatory (...)

The road not taken or how I learned to love the liver: A personal perspective on hepatitis history

The road not taken or how I learned to love the liver: A personal perspective on hepatitis history

Hepatitis C Virus NS5B Polymerase Exhibits Distinct Nucleotide Requirements for Initiation and Elongation

The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) essential for replication of the viral RNA genome. Purified NS5B has been reported to exhibit multiple activities in vitro. Using a synthetic heteropolymeric RNA template with dideoxycytidine at its 3'-end, we examined de novo initiation and primer extension in a system devoid of self-priming and terminal nucleotide transferase activities. Products predominantly of template size and its multiples were detected. High concentrations of nucleoside triphosphates (K(app)(m) approximately 100-400 mum) corresponding to the first three incorporated nucleotides were found to be required for efficient de novo RNA synthesis. In the presence of initiating di- or trinucleotides, however, the amount of NTP needed to achieve maximal activity dropped 10(3)- to 10(4)-fold, revealing a much reduced nucleotide requirement for elongation (K(app)(m) approximately 0.03-0.09 microm). Accordingly, single round extension from an exogenous primer following preincubation of the enzyme with template and primer could also be supported by <0.1 microm levels of NTP. De novo synthesis at high NTP concentrations was shown to be preferred over primer extension. On a dideoxycytidine-blocked synthetic RNA template derived from the 3'-end of the HCV(-)UTR, the addition of the corresponding initiating trinucleotide also dramatically reduced the NTP levels needed to achieve efficient RNA synthesis. Thus, distinct nucleotide requirements exist for initiation and elongation steps catalyzed by the HCV NS5B polymerase.

Molecular biology and clinical implication of hepatitis C virus.

Hepatitis C virus (HCV) was first described in 1989 as the putative viral agent of non-A non-B hepatitis. It is a member of the Flaviviridae family and has been recognized as the major causative agent of chronic liver disease, including chronic active hepatitis, cirrhosis and hepatocellular carcinoma. HCV is a positive RNA virus with a genome containing approximately 9500 nucleotides. It has an open reading frame that encodes a large polyprotein of about 3000 amino acids and is characterized by extensive genetic diversity. HCV has been classified into at least 6 major genotypes with many subtypes and circulates within an infected individual as a number of closely related but distinct variants known as quasispecies. This article reviews aspects of the molecular biology of HCV and their clinical implication.

Modulation of Hepatitis C Virus RNA-dependent RNA Polymerase Activity by Structure-based Site-directed Mutagenesis

The hepatitis C virus (HCV) encodes an RNA-dependent RNA polymerase (NS5B), which is indispensable for the viral genome replication. Although structural comparison among HCV NS5B, poliovirus 3D-pol, and human immunodeficiency virus-reverse transcriptase RNA-dependent polymerase reveals the canonical palm, fingers, and thumb domains, the crystal structure of HCV NS5B highlights the presence of a unique A1-loop, which extends from the fingers to the thumb domain (amino acids 12-46), providing many contact points for the proposed "closed" conformation of the enzyme. The polymerase also possesses a tunnel, which starts at the active site and terminates on the back surface of the enzyme. This tunnel of 19 A contains five basic amino acids, which may be engaged in NTP trafficking. In the present study, we exploited the crystal structure of the enzyme to elucidate the involvement of these two structural motifs in enzyme activity by site-directed mutagenesis. As predicted, the replacement of leucine 30 located in the Lambda 1-loop is detrimental to the NS5B activity. Heparin-Sepharose column chromatography and analytical ultracentrifugation experiments strongly suggest a local alteration in the structure of the Leu-30 mutant. An analysis of amino acid substitutions in Arg-222 and Lys-151 within the putative NTP tunnel indicates that Arg-222 was critical in delivering NTPs to the active site, whereas Lys-151 was dispensable. Interestingly, the substitution of lysine 151 for a glutamic acid resulted in an enzyme that was consistently more active in de novo synthesis as well as by "copy-back" mechanism of a self-primed substrate when compared with the wild type NS5B enzyme. Burst kinetic analyses indicate that the gain in function of K151E enzyme was primarily the result of the formation of more productive pre-initiation complexes that were used for the elongation reaction. In contrast to the recent observations, both the wild type and mutant enzymes were monomeric in solution, whereas molecules of higher order were apparent in the presence of RNA template.

Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome [Science 1989;244:359–362

Abstract: A random-primed complementary DNA library was constructed from plasme containing the uncharacterized non-A, non-B hepatitis (NANBH) agent and screend with serum from a patient diagnosed with NANBH. A complementary DNA clone was isolated that was shown to encode an antigen associated specifically with NANBH infections. This clone is not derived from host DNA but from an RNA molecule present in NANBH infections that consists of at least 10,000 nucleotides and this is positive-stranded with respect to the encoded NANBH antigen. These data indicate that this clone is derived from the genome of the NANBH agent and are consistent with the agent being similar to the togaviridae or flaviviridae. This molecular approach should be of great value in the isolation and characterization of other unidentified infectious agents.

RT-PCR for the assessment of genetically heterogenous populations of the hepatitis C virus.

AbstractSince the discovery of the Hepatitis C Virus (HCV) in 1989 (1), known to that point as the infectious agent of Non-A Non-B hepatitis (2), there has been an intensive focus on understanding the underlying biology of its disease process. Of significant interest has been the study of heterogenous populations of closely related, but genetically non-identical HCV virions, commonly termed quasispecies (3,4), and their relationship to the pathogenesis of HCV infection. Studies of HCV quasispecies have predominantly focused on two viral genetic loci, Hypervariable Region 1 (HVR 1) and the Interferon Sensitivity Determining Region (ISDR) (5) (Fig. 1). HVR 1 encodes an approx 27 amino-acid sequence at the N-terminal of envelope glycoprotein 2 (E2), and sequence variation at this locus is used to characterize HCV quasispecies. HVR 1 acts as an immune escape epitope that, through continuous alteration of its antigenicity, is believed to hinder humoral immune-mediated clearance of the virus. The ISDR encodes an approx 40 amino-acid sequence in the C-terminal half of the nonstructural 5A (NS5A) protein. Protein kinase inhibition and transcriptional activation activities have been ascribed to NS5A, both of which are known to be modulated by sequence variation within the ISDR (6,7). The current view is that although the role of the NS5A protein in HCV disease progression is important, pathologically relevant viral genomic sequence evolution may not be strictly confined to the ISDR alone. Sequence variation of other regions in NS5A, such as the V3 region (8), nuclear localization signals, and phosphorylation motifs may also play important roles (9). Structure of the HCV genome and relative location of PCR products discussed in this chapter. (A) HCV genome comprising 5′ and 3′ untranslated regions (bold lines), flanking an open reading frame (box). Nucleotide (upper, italics), and amino acid (lower, plain) scales are shown. (B) Enlarged envelope 2 (E2) and nonstructural 5A (NS5A) genes showing (bold) HVR1 and 2, and ISDR, respectively. (C) Location of PCR products (hatched areas) in relation to HVR and ISDR loci. The NS5A PCR precisely covers the NS5A gene. Diagram based on HCV-H-strain nucleotide and amino-acid sequence (5). KeywordsLuria BertaniInterferon Sensitivity Determine RegionQuasispecies PopulationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Discovery of non-A, non-B hepatitis and identification of its etiology

The discovery of hepatitis C was the direct result of the landmark discoveries of hepatitis B virus (HBV) and hepatitis A virus (HAV) and their serologies. Screening tests for HAV and HBV made it possible in the mid-1970s to examine cases of transfusion-associated hepatitis (TAH) and to demonstrate that only approximately 25% resulted from HBV and that none were related to HAV. Consequently, approximately 75% of TAH became classified as non-A, non-B hepatitis (NANBH). Subsequently, chimpanzee studies demonstrated that NANBH was a result of a transmissible agent. Although it has been difficult to convince clinicians that NANBH was a serious disease because the overt manifestations are generally mild, it gradually became apparent that the NANBH agent often resulted in chronic hepatitis and sometimes evolved into cirrhosis. The NANBH agent remained a virologic enigma for the next decade until researchers at the Chiron Corporation used an ambitious molecular approach on large volumes of high-titer infectious chimpanzee plasma from the Centers for Disease Control and Prevention (CDC). They extracted RNA, cloned it into an expression vector, and screened the expressed product with presumed immune sera. A single positive clone was found in the millions screened, and, within a year, the entire genome was sequenced and the agent was identified as a novel flavivirus—the hepatitis C virus (HCV). Retrospective analysis of pedigreed samples at the National Institute of Health (NIH) showed that 70% to 90% of NANBH cases were HCV related. The impact of HCV blood donor screening has been enormous. The single-antigen first-generation enzyme immunoassay (EIA-1) prevented 40,000 HCV infections within the first year, and the second-generation assay (EIA-2) has actually reduced new transfusion-related HCV infections to almost zero.

A Continuous Assay of Hepatitis C Virus Protease Based on Resonance Energy Transfer Depsipeptide Substrates

Hepatitis C virus (HCV) is the major causative agent of non-A non-B hepatitis, an important health problem with an estimated 50 million people infected worldwide. Among the possible targets for therapeutic intervention, the serine protease contained within the N-terminal region of nonstructural protein 3 (NS3 protease) is so far the best characterized.In vitrocharacterization of synthetic substrates based on all the natural cleavage sites (as well as a series of analogs) has consistently revealed poor kinetic parameters, making them unsuitable for sensitive high-throughput screening. To overcome these difficulties, we have recently developed depsipeptide substrates incorporating an ester bond between residues P1and P′1. Due to ready transesterification of the scissile bond to the acyl-enzyme intermediate, these substrates showed very highkcat/Kmvalues, enabling detection of activity with subnanomolar NS3 concentrations. We have used the same principle to synthesize internally quenched depsipeptide fluorogenic substrates based on resonance energy transfer between the donor/acceptor couple 5-[(2′-aminoethyl)amino]naphthalene sulfonic acid/4-[[4′-(dimethylamino)phenyl]azo]benzoic acid, and developed a continuous assay for NS3 activity. Substrate cleavage is linear with enzyme concentration: depending on the conditions chosen, we estimated a detection limit for NS3 between 1 nmand 250 pm. The suitability of the assay for evaluation of inhibitors was established using as competitor a tridecapeptide corresponding to the natural NS4A/4B cleavage site; this gave an IC50of 30 μm, well in agreement with the previously foundKmvalue (40 μm).

Prevalence of anti-HCV among Chinese patients with acute and chronic liver disease.

To assess whether the hepatitis C virus plays an important role in Chinese patients with acute and chronic liver disease, antibodies to HCV (anti-HCV) were measured by enzyme immunoassay in 67 patients with type A and B acute viral hepatitis, 165 patients with non-A, non-B (NANB) hepatitis, 438 patients with chronic hepatitis, 200 patients with postnecrotic liver cirrhosis, 72 patients with alcoholic liver disease, 55 patients with non-alcoholic fatty liver, 24 patients with toxic and drug-induced hepatitis, and 20 patients with other chronic liver diseases. Anti-HCV was not detected in sera from patients with type A and B acute viral hepatitis, toxic and drug-induced hepatitis, primary biliary cirrhosis, Wilson's disease, or lupoid hepatitis. The anti-HCV prevalence was found to be highest in patients with NANB hepatitis (59% in sporadic and 73.2% in transfusion-associated), 16.4% in non-alcoholic fatty liver, 5.6% in alcoholic liver disease, 6.8% in chronic hepatitis, and 16% in postnecrotic liver cirrhosis. In patients with chronic hepatitis, the anti-HCV prevalence was significantly higher in HBsAg-negative (15/34, 44.1%) than in HBsAg-positive cases (15/404, 3.7%; P less than 0.0001). The results indicate that HCV is a major agent of NANB hepatitis and plays an important role in HBsAg-negative chronic liver disease in Taiwan.

Antibodies against hepatitis C virus in hemodialysis patients in the central Italian region of Umbria: evaluation of some risk factors.

The epidemiology of non-A, non-B hepatitis (NANBH) is still incomplete. To define the prevalence of antibodies against the main causative agent of NANBH, the hepatitis C virus (HCV) and the role of some risk factors, we tested sera from 269 patients on chronic dialysis at the hemodialysis units in our region in central Italy. We utilized the recently developed serological assay. Twenty-nine hemodialysis patients (13.3%) and 3 peritoneal dialysis patients (4.8%) were anti-HCV positive. Of these, 13 (40.6%) had antibodies to hepatitis B core antigen (anti-HBc) indicating prior hepatitis B infection. The anti-HCV seropositive patients had been on dialysis longer than the seronegative ones; they had received more transfusions than the others but without a significant difference. The prevalence rate of anti-HCV was statistically significantly higher among hemodialysis patients utilizing the same dialysis equipment for the previous 12 months.

Hepatis C Virus: A Giant Leap Forward

A random–primed complementary DNA library was constructed from plasma containing the uncharacterized non–A, non–B hepatitis (NANBH) agent and screened with serum from a patient diagnosed with NANBH. A complementary DNA clone was isolated that was shown to encode an antigen associated specifically with NANBH infections. This clone is not derived from host DNA but from an RNA molecule present in NANBH infections that consists of at least 10,000 nucleotides and that is positive–stranded with respect to the encoded NANBH antigen. These data indicate that this clone is derived from the genome of the NANBH agent and are consistent with the agent being similar to the togaviridae or flaviviridae. This molecular approach should be of great value in the isolation and characterization of other unidentified infectious agents. A specific assay has been developed for a blood–borne non–A, non–B hepatitis (NANBH) virus in which a polypeptide synthesized in recombinant yeast clones of the hepatitis C virus (HCV) is used to capture circulating viral antibodies. HCV antibodies were detected in six of seven human sera that were shown previously to transmit NANBH to chimpanzees. Assays of ten blood transfusions in the United States that resulted in chronic NANBH revealed that there was at least one positive blood donor in nine of these cases and that all ten recipients seroconverted during their illnesses. About 80 percent of chronic, post–transfusion NANBH (PT–NANBH) patients from Italy and Japan had circulating HCV antibody; a much lower frequency (15 percent) was observed in acute, resolving infections. In addition, 58 percent of NANBH patients from the United States with no identifiable source of parenteral exposure to the virus were also positive for HCV antibody. These data indicate that HCV is a major cause of NANBH throughout the world.

Non-A, non-B hepatitis: visualization of virus-like particles from chimpanzee and human sera

Virus-like particles (VLP), morphologically similar to togavirus particles, were detected in the sera of chimpanzees and humans with chronic non-A, non-B (NANB) hepatitis. Particles resembling the virus were also found in the cytoplasm of hepatocytes obtained from NANB hepatitis infected chimpanzees. Furthermore, two chimpanzees inoculated with materials containing the VLP developed NANB hepatitis. These results strongly suggest that the VLP may be closely associated with the agent of NANB hepatitis.

Isolation of a cDNA cLone Derived from a Blood-Borne Non-A, Non-B Viral Hepatitis Genome

A random-primed complementary DNA library was constructed from plasma containing the uncharacterized non-A, non-B hepatitis (NANBH) agent and screened with serum from a patient diagnosed with NANBH. A complementary DNA clone was isolated that was shown to encode an antigen associated specifically with NANBH infections. This clone is not derived from host DNA but from an RNA molecule present in NANBH infections that consists of at least 10,000 nucleotides and that is positive-stranded with respect to the encoded NANBH antigen. These data indicate that this clone is derived from the genome of the NANBH agent and are consistent with the agent being similar to the togaviridae or flaviviridae. This molecular approach should be of great value in the isolation and characterization of other unidentified infectious agents.

Hepatitis delta (delta) cDNA clones: undetectable hybridization to nucleic acids from infectious non-A, non-B hepatitis materials and hepatitis B DNA.

Hepatitis Delta (delta) cDNA clones were hybridized to RNA extracted from livers of chimpanzees infected with the blood-borne Non-A, Non-B hepatitis (NANBH) agent(s) and to total nucleic acids extracted from chimpanzee plasma containing a high titer of these NANBH agent(s). Since no hybridization was observed, the data suggests that the hepatitis Delta viral genome is not closely related to the genome(s) of the NANB agent(s). Our studies, in which the Hepatitis B virus genomic DNA was hybridized to hepatitis Delta cDNA clones, also confirm and extend previous studies [Hoyer et al, 1983], which report a lack of detectable homology between the hepatitis Delta genome and HBV DNA.

Characterization of an antigen-antibody system associated with epidemic non-A,non-B hepatitis in west africa and experimental transmission of an infectious agent to primates

Summary An epidemic of non-A, non-B hepatitis (NANBH) was studied in Tortiya, Ivory Coast, over a period of 17 months. Highly sensitive serologic tests performed on serum samples from patients with this type of hepatitis excluded both HAV (hepatitis A virus) and HBV (hepatitis B virus) as aetiologic agents. The mode of spread of the epidemic, clinical features and frequent cases of fulminant hepatitis in pregnant women resembled that of other recently described water-borne outbreaks of non-A,non-B hepatitis. Oral-faecal transmission of this form of NANBH was shown by experimental infection of African monkeys, which developed antibody of the IgM class specifically reacting with the antigen present in stools of infected persons. Using the immunoenzymatic test employing monkey IgM on solid phase and enzyme-labelled IgG purified from sera of patients convalescing from NANBH, we identified the antigen associated with the possible causative NANBH agent. These data confirm the existence of a distinct human NANBH agent responsible for epidemic out-breaks of NANBH. This agent is different from those of the postransfusion type of NANBH and antigenically unrelated to HAV. The direct serologic test presented here may be useful for diagnosis of an epidemic form of NANBH.

DETECTION OF REVERSE TRANSCRIPTASE ACTIVITY IN ASSOCIATION WITH THE NON-A, NON-B HEPATITIS AGENT(S)

Particle-associated reverse transcriptase activity was detected in four human serum specimens and in two plasma-derived products, all of which had been shown to transmit non-A, non-B hepatitis (NANBH) to other human beings and/or chimpanzees. Reverse transcriptase activity was also detected in all twelve sera from patients with acute or chronic NANBH. In contrast, reverse transcriptase activity was found in only 2 of 49 serum specimens from healthy plasma donors and laboratory workers. Sucrose density gradient fractions of two of the infectious human sera (peak reverse transcriptase activity at 1·14 g/ml) transmitted NANBH to chimpanzees. Biochemical and enzymatic data indicate that the NANBH agent(s) is a retrovirus or is retrovirus-like.

Characterization of a precipitating antigen detected in the serum of patients with viral hepatitis

During a search for the aetiological agent of non-A non-B hepatitis, a precipitating antigen was detected in the sera of some patients during the acute phase of their illness. The antigen was detected by agar gel diffusion using antibody from convalescent sera obtained from patients with non-A non-B hepatitis, and from haemophiliac sera. The antigen was usually detected early in the patient's illness, disappearing as liver function tests returned to normal. In some patients specific antibody appeared during the convalescent phase of the disease. The antigen does not appear to be specific for non-A non-B hepatitis, as it could be detected with similar frequency in patients with hepatitis A or hepatitis B and some patients with other liver disorders. Biochemical and biophysical studies suggest that the antigen is probably an abnormal lipoprotein produced as a result of acute liver damage.

Chronic non-A, non-B hepatitis: ultrastructural and serologic studies.

Liver biopsies from five patients with chronic non-A, non-B (NANB) hepatitis were examined by electron microscopy for hepatocellular alterations. Circular fused membranes were observed within the cytoplasm of hepatocytes of four of the patients. Aggregates of intranuclear particles, measuring 22 +/- 2 nm in diameter, were also seen in two of the biopsies in which fused membranes were identified. Sera of all five patients formed precipitin lines detectable by counterimmunoelectrophoresis when reacted with serum from an individual convalescent from posttransfusion NANB hepatitis. Using this convalescent serum as an antibody source, complexes of 22 +/- 2 nm particles were identified in three of the chronic patient sera by immunoelectron microscopy. These observations suggest that at last one of the agents responsible for NANB hepatitis elicits both nuclear and cytoplasmic modifications. Furthermore, the 22 +/- 2 nm particles circulating in the sera of the chronic NANB patients may represent components related to NANB hepatitis agent.

Transfusion-transmitted viruses study: experimental evidence for two non-A, non-B, hepatitis agents.

Plasma, serum, and a concentrate of factor VIII, implicated in human cases of non-A, non-B (NANB) hepatitis, were inoculated into four chimpanzees. All four animals demonstrated significant elevations of alanine aminotransferase levels within five weeks. After recovery from these NANB hepatitis episodes, the chimpanzees were cross-challenged with different inocula. A second episode of NANB hepatitis occurred after challenge in three animals. Reproducibility of the results was established by reversing the sequence of inoculation in two of the animals. A known infectious concentrate of factor VIII failed to induce hepatitis in a previously infected chimpanzee although the animal remained susceptible to a third inoculum, an event suggesting that the first inoculum might contain the same NANB hepatitis agent as the concentrate of factor VIII, which was supported by results of a subsequent cross-challenge experiment. Reinfection did not occur in two chimpanzees injected with their initial challenge strain of NANB hepatitis virus, providing evidence that strain-specific immunity had been established. Thus, experimental evidence is provided for the presence of two NANB hepatitis agents, supporting clinical and epidemiologic studies that favor the existence of more than one etiologic agent.