Limited hepatitis B virus replication space in the chronically hepatitis C virus-infected liver.

We compared the kinetics and magnitude of hepatitis B virus (HBV) infection in hepatitis C virus (HCV)-naive and chronically HCV-infected chimpanzees in whose livers type I interferon-stimulated gene (ISG) expression is strongly induced. HBV infection was delayed and attenuated in the HCV-infected animals, and the number of HBV-infected hepatocytes was drastically reduced. These results suggest that establishment of HBV infection and its replication space is limited by the antiviral effects of type I interferon in the chronically HCV-infected liver.

Mutational analysis of the hepatitis C virus E1 glycoprotein in retroviral pseudoparticles and cell-culture-derived H77/JFH1 chimeric infectious virus particles.

Cell entry by enveloped viruses is mediated by viral glycoproteins, and generally involves a short hydrophobic peptide (fusion peptide) that inserts into the cellular membrane. An internal hydrophobic domain within E1 (aa262-290) of hepatitis C virus (HCV) may function as a fusion peptide. Retrovirus-based HCV-pseudotyped viruses (HCVpp; genotype 1a) containing Ala or Pro substitutions at conserved amino acid positions within this putative fusion peptide were generated. Mutation of conserved residues significantly reduced efficiency of HCVpp entry into Huh-7 cells. The majority of amino acid substitutions appeared to disrupt necessary interactions between E1 and E2. For some mutants, reductions in HCVpp-associated E1 were associated with the incorporation of a high molecular weight, hyperglycosylated E2 that displayed decreased CD81-binding. Other entry-deficient mutants displayed normal E1E2 incorporation into pseudoparticles and normal CD81-binding, and therefore might affect viral fusion. One mutant (S283P) consistently displayed two- to threefold higher infectivity than did wild-type. Three mutations that decreased HCVpp infectivity also reduced levels of HCVcc infectious virus production. However, the S283P mutation had a different effect in the two systems as it did not increase production of infectious HCVcc. This comprehensive mutational analysis of the putative HCV fusion peptide provides insight into the role of E1 in its interaction with E2 and in HCV entry.

Non-A, non-B hepatitis: ultrastructural evidence for two agents in experimentally infected chimpanzees.

Two different ultrastructural alterations were observed in liver cells of chimpanzees inoculated with plasma derived from two different patients with non-A, non-B hepatitis. During the acute phase of illness in one group of four chimpanzees, peculiar tubular structures, composed of two unit membranes with electron-opaque material in between, were observed in the cytoplasm of hepatocytes. In contrast, these structures were never detected in the liver cells of the second group of five chimpanzees that received the second inoculum, However, nuclear changes, usually associated with aggregates of 20- to 27-nanometer particles, were found in hepatocytes of the latter animals. Although these particles resembled viruses, they were not as uniform as small virus particles often appear. In five other chimpanzees inoculated with non-A, non-B hepatitis material not known to be related to the first two inocula, cytoplasmic structures were found in four, and nuclear structures were found in the remaining one. Thus, all 14 chimpanzees inoculated with transmissible non-A, non-B hepatitis agents could be classified as having either nuclear or cytoplasmic changes. These observations add support to epidemiologic data suggesting that there may be more than one agent of non-A, non-B hepatitis.

An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis.

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.

The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies.

The mechanisms by which hepatitis C virus (HCV) induces chronic infection in the vast majority of infected individuals are unknown. Sequences within the HCV E1 and E2 envelope genes were analyzed during the acute phase of hepatitis C in 12 patients with different clinical outcomes. Acute resolving hepatitis was associated with relative evolutionary stasis of the heterogeneous viral population (quasispecies), whereas progressing hepatitis correlated with genetic evolution of HCV. Consistent with the hypothesis of selective pressure by the host immune system, the sequence changes occurred almost exclusively within the hypervariable region 1 of the E2 gene and were temporally correlated with antibody seroconversion. These data indicate that the evolutionary dynamics of the HCV quasispecies during the acute phase of hepatitis C predict whether the infection will resolve or become chronic.

In vitro antigen-induced antibody responses to hepatitis B surface antigen in man. Kinetic and cellular requirements.

In this report we define the parameters of the human immune response after immunization with hepatitis B vaccine. 2 wk after booster immunization, there is significant spontaneous secretion of antibody to hepatitis B surface antigen (anti-HBs IgG), which is not further augmented by stimulation with antigen or pokeweed mitogen (PWM). By 4 wk there is little spontaneous secretion of specific antibody, and low doses of antigen or PWM produce significant increases in the amount of anti-HBs IgG produced. By 8 wk the peripheral blood mononuclear cells are refractory to stimulation by antigen, but anti-HBs IgG is produced in response to PWM. 0.5 yr or more after the last immunization, some individuals will manifest an antigen-induced specific antibody response. This induction of anti-HBs IgG by hepatitis B surface antigen (HBsAg) is monocyte- and T cell-dependent. Note that there is a dichotomy in the T cell response to HBsAg. The specific antibody response is clearly T cell dependent, but no in vitro T cell proliferative response to HBsAG could be demonstrated in the immunized individuals. Although the precise reason for the absent proliferative response to HBsAg despite well-established humoral immunity has not been determined, there was no evidence to suggest nonspecific suppression by HBsAg or the presence of an adherent suppressor cell population. The ability to evaluate antigen-induced, antigen-specific responses to HBsAg will be useful in defining the unique interaction between the human immune response and this clinically important viral agent.

Characterization of hepatitis E-specific cell-mediated immune response using IFN-gamma ELISPOT assay.

In developing countries, hepatitis E (HEV) and hepatitis A (HAV) are the major causes of acute viral hepatitis with similar feco-oral modes of transmission. In contrast to the high seroprevalence of hepatitis A infection, a low seroprevalence of HEV among children in endemic areas has been reported. These data suggest the possibility that silent HEV infection is undiagnosed by the current available methods. Many of the serological tests used for HEV diagnosis have poor specificity and are unable to differentiate among different genotypes of HEV. Moreover, the RT-PCR used for HEV isolation is only valid for a brief period during the acute stage of infection. Cell-mediated immune (CMI) responses are highly sensitive, and long lasting after sub-clinical infections as shown in HCV and HIV. Our objective was to develop a quantitative assay for cell-mediated immune (CMI) responses in HEV infection as a surrogate marker for HEV exposure in silent infection. Quantitative assessment of the CMI responses in HEV will also help us to evaluate the role of CMI in HEV morbidity. In this study, an HEV-specific interferon-gamma (IFN-gamma) ELISPOT assay was optimized to analyze HEV-specific CMI responses. We used peripheral blood mononuclear cells (PBMC) and sera from experimentally infected chimpanzees and from seroconverted and control human subjects to validate the assay. The HEV-specific IFN-gamma ELISPOT responses correlated strongly and significantly with anti-HEV ELISA positive/negative results (rho=0.73, p=0.02). Moreover, fine specificities of HEV-specific T cell responses could be identified using overlapping HEV ORF2 peptides.

Hepatitis C virus: historical perspective and current concepts.

Over the past 30 years, hepatitis C has emerged from shadowy enigma to important public health problem. The existence of the etiological agent of this disease was first appreciated two decades ago but significant progress in its understanding had to await its molecular characterization within the past 5 years. The virus is a member of the family Flaviviridae and is the cause of approximately 20% of clinical viral hepatitis in the United States. While the control of the transmission of hepatitis C virus in blood and blood products has been nothing less than spectacular, the control of community-acquired hepatitis C will be a major challenge to the scientific and medical communities.

Quasispecies in viral persistence and pathogenesis of hepatitis C virus.

Hepatitis C virus (HCV) is an important cause of chronic liver disease worldwide. This RNA virus circulates as a quasispecies and its genetic heterogeneity has been implicated in the lack of protective immunity against HCV and in its persistence following infection. HCV might escape from immune surveillance by developing mutations in proteins that are subject to immune pressure.

Recombinant vaccines for hepatitis E.

Hepatitis E virus causes epidemics of acute hepatitis in many developing countries. It infrequently causes disease in developed countries, but avirulent strains might circulate. Some evidence suggests that hepatitis E might be a zoonosis. There is probably only a single serotype. A candidate vaccine consisting of baculovirus-expressed recombinant capsid protein protected macaques from hepatitis E--it passed phase I clinical trials and is currently scheduled for phase II/III clinical trials.

Prevention of perinatal acquisition of hepatitis B virus carriage using vaccine: preliminary report of a randomized, double-blind placebo-controlled and comparative trial.

Hepatitis B is a serious disease of global significance. In developing countries, hepatitis B virus (HBV) infection and its sequelae rank among the public health problems of highest priority. Infants born to mothers who are chronic carriers of HBV are at particularly high risk of acquiring infection and becoming chronic HBV carriers. The efficacy of hepatitis B vaccine alone in preventing the transmission of HBV to infants born to HBV carrier mothers was determined in a double-blind placebo-controlled trial. Infants received plasma-derived vaccine at birth, 1 month, and 6 months of age. Of 180 infants born to hepatitis B surface antigen (HBsAg)-positive mothers, equal numbers received National Institute of Allergy and Infectious Disease (NIAID) vaccine, Beijing Institute of Vaccine and Serum (BIVS) vaccine, and placebo. The cumulative seroconversion to the vaccines at 1 year of age was 95% and 75%, respectively. Vaccine efficacy as measured by the prevention of HBsAg-positive events was 88% for the NIAID vaccine and 51% for the BIVS vaccine. Vaccine efficacy was similar among infants born to hepatitis Be antigen-positive mothers. Because of the low efficacy of the BIVS vaccine, an additional group of 28 infants was given vaccine and hepatitis B immune globulin at birth. The resulting efficacy was 83%. The results of this trial indicate that hepatitis B vaccine alone can substantially reduce perinatally acquired HBV infection and the resulting chronic carrier state.

Evidence for a bidirectional promoter complex within the X gene of woodchuck hepatitis virus.

The genetic organization of hepadnaviruses is unusual in that all cis-acting regulatory sequences are located within genes. Thus, in the mammalian hepadnavirus genome, the presurface, surface, and X transcript promoters reside within the polymerase gene while the pregenome transcript promoter is located within the X gene. In this study we have identified two additional promoters within the woodchuck hepatitis virus (WHV) X gene that stimulate production of transcripts in vitro. First, we cloned regions of the WHV X gene into a promoterless expression vector (pGL2) to examine their ability to promote expression of firefly luciferase and mapped a previously unidentified promoter to positions 1475-1625 of the WHV8 genome. Deletion analysis revealed that the essential domain of this promoter, termed the ORF5/deltaX transcript promoter, mapped to nucleotides 1525-1625. Analysis revealed that this transcript initiated at nucleotide 1572 in both human (HuH-7) and woodchuck (WLC-3) hepatoma cell lines. Consistent with this finding, DNA footprinting analysis revealed protection of nucleotides 1567-1578 on the positive strand of the WHV8 genome. The function of this transcript in vivo is unclear, however, it may be used to produce a truncated form of the X protein that initiates at an AUG codon at position 1743-1745 on the WHV8 genome. Next, a second promoter was identified at positions 1625-1975 that was responsible for production of an antisense transcript. The activity of this promoter was comparable to that of the previously characterized surface transcript promoter of WHV in the absence of an enhancer. The antisense transcript promoter resides immediately upstream of open reading frame (ORF) 6, a previously identified ORF on the strand opposite of the known WHV protein-encoding sequences, that is thought to represent a vestigial gene. Analysis indicates that the antisense transcript had multiple start sites: nucleotides 1683 and 1762 on the WHV8 genome when assayed in HuH-7 cells, and nucleotide 1786 when assayed in WLC-3 cells. These data are consistent with footprinting analysis of supercoiled WHV DNA that revealed that the regions encompassing nucleotides 1696-1685, 1781-1766, and 1801-1787 on the negative sense DNA strand were protected from nuclease degradation. It is possible that such a transcript was once used in protein expression in an ancestral virus and may now be used for genetic control of WHV replication and/or gene expression. Overall, these data are consistent with the presence of a bidirectional promoter complex within the WHV X gene.

Radioimmunoassay and characterization of woodchuck hepatitis virus core antigen and antibody.

Solid-phase radioimmunoassays for woodchuck hepatitis virus core antigen (WHcAg) and antibody (anti-WHc) were developed. WHcAg in woodchuck liver homogenates was characterized by ultracentrifugation in CsCl gradients; both heavy (1.35 g/cm3) and light (1.31 g/cm3) cores were obtained from the liver of an animal during acute WHV infection, which is consistent with observations in hepatitis B virus infection in man. Endpoint titers of anti-WHc were higher in chronic WHV carriers than in animals recovered from acute infections. Both IgM and IgG anti-WHc antibodies were produced by infected woodchucks. A survey of colony woodchucks demonstrated that 88/89 animals having one or more markers of past or ongoing WHV infection were positive for anti-WHc. Thus, serum anti-WHc appears to be a sensitive marker of WHV infection.

Heterogeneity of the woodchuck hepatitis virus genome in a chronically infected woodchuck.

The nucleotide sequence of an isolate of woodchuck hepatitis virus (WHV) from the serum of a woodchuck trapped in New York state (WHVNY) was compared with the sequences of previously published isolates. The nucleotide sequence of WHVNY was closest to that of an isolate originating from New Jersey: the two genomes shared a 15 nucleotide in-frame deletion in the region where the presurface and polymerase genes overlap (nucleotides 3260-3274) and differed by 54 point mutations (1.6% of genome). Amino acid differences ranged from 0.4% in the surface gene to 5.7% in the X gene. Three isolates from woodchucks that originated in Pennsylvania and Maryland did not contain the deletion and differed from WHVNY by 102 to 106 point mutations (3.0% to 3.2% of nucleotides). Amino acid changes ranged from 0.5% in the core gene to 5.7% in the X-gene. Thus, WHVNY differed little from previous isolates. Next, the genomes from 102 independent clones of WHVNY were compared to ascertain the extent of sequence variation among WHV genomes in a chronically infected animal. A total of 98 clones had genomes of unit length while 2 clones had genomes shorter than unit length and 2 clones had genomes longer than unit length. The clones not of unit length possessed deletions or inverted duplications of sequence. The rate of mutation in the viral genes was 2.65 mutations per 10,000 nucleotides in the precore domain, 1.27 per 10,000 in the X-gene, 0.98 per 10,000 in the presurface gene, and 3.77 per 10,000 at the 5' end of the core gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-directed ELISA identifies a highly antigenic region of the simian immunodeficiency virus transmembrane glycoprotein.

The transmembrane glycoprotein (gp32) of the simian immunodeficiency virus (SIV) contains a highly antigenic region that includes amino acid residues 606-628. A synthetic peptide representing this region was highly immunoreactive with sera from SIV-infected primates in a site-directed enzyme-linked immunosorbent assay (ELISA). This reactivity extended across four primate species from three genera and identified infection with at least two distinct isolates of SIV. This site-directed ELISA represents a simple, accessible method with broad specificity for screening large numbers of primates for antibodies against SIV.

Hepatitis B vaccines. On the threshold.

Experimental hepatitis B vaccines have recently been developed and are undergoing preliminary evaluation. These vaccines are unique in that they are prepared from viral antigen purified from the plasmas of human chronic carriers because hepatitis B virus has not been cultivated in vitro. They appear to be safe and antigenic, but care must be exercised in their evaluation because of the potential risks inherent in all vaccines.

In vivo transfection by hepatitis A virus synthetic RNA.

Marmosets injected intrahepatically with nucleic acids (cDNA and RNA transcripts) representing the full-length genome of the wild-type HM-175 strain of hepatitis A virus experienced acute hepatitis and seroconversion to hepatitis A virus capsid proteins. The hepatitis was comparable in severity to that caused by infection with the wild-type virus. The viral cDNA and the hepatitis A virus recovered from the feces of an injected animal contained the same marker mutation. Therefore, intermediate cell culture steps can be omitted and the virulence of a hepatitis A virus encoded by a cDNA clone can be evaluated by direct transfection of marmosets.

The epidemiology of hepatitis B antigen and antibody among Panamanian Cuna Indians.

Previous studies of hepatitis B antigen (HBsAg) and antibody to it (anti-HBs) showed widely differing exposures between Panamanian Indian tribes. Cuna Indians living on islands appeared infrequently exposed to HBsAg; we found no one antigenemic and low age specific anti-HBs rates. In contrast, mainland dwelling Guaymi and Chocó Indians had a high prevalence of anti-HBs. We have now measured HBsAg by counterelectrophoresis and anti-HBs by radioimmunoassay in two Cuna Indian groups who live in the Darien forest. The prevalence of HBsAg among Darien Cuna was low, 3 positive of 239 tested, but 106 (44%) had anti-HBs. Darien Cuna thus evidenced greater exposure to HBsAg than island Cuna (8% had anti-HBs) and had an anti-HBs prevalence similar to the neighboring Chocó Indians (42%). The Guaymi Indians of western Panama had a lower frequency of anti-HBs (29%) than either Chocó or mainland Cuna but their frequency of chronic antigenemia was significantly greater. These data suggest that while exposure may be a function of village habitat, chronic antigenemia may reflect differences in host responses.

Epidemiology of acute hepatitis in the Stann Creek District of Belize, Central America.

Hepatitis is common in the Stann Creek District of southern Belize. To determine the etiologies, incidence, and potential risk factors for acute jaundice, we conducted active surveillance for cases. Cases of jaundice diagnosed by a physician within the previous 6 weeks were enrolled. Evaluation included a questionnaire and laboratory tests for hepatitis A, B, C, D, and E, a blood film for malaria, and a serologic test for syphilis. Etiologies of jaundice among 62 evaluable patients included acute hepatitis A, 6 (9.7%), acute hepatitis B, 49 (79.0%), hepatitis non-A-E, 2 (3.2%), and malaria, 5 (8.1%). There were no cases of acute hepatitis E. One patient each with antibody to hepatitis C and D were detected. The annualized incidence of hepatitis A was 0.26 per 1,000. All cases of hepatitis A were in children 4-16 years of age. The annualized incidence of hepatitis B, 2.17 per 1,000, was highest in adults aged 15-44 years (4.4 per 1,000) and was higher in men (36 cases; 3.09 per 1,000) than women (13 cases; 1.19 per 1,000). Four (31%) of the women with hepatitis B were pregnant. The annualized incidence was significantly higher in Mestizo (6.18 per 1000) and Maya (6.79 per 1,000) than Garifuna (0.38 per 1,000) or Creole (0.36 per 1,000). Persons with hepatitis B were significantly more likely to be born outside of Belize (82%), had been in Belize < 5 years (73%), and lived and worked in rural areas (96%) than was the general population. Of those > or = 14 years of age with hepatitis B, only 36% were married. Few persons admitted to transfusions, tattoos, IV drug use, multiple sexual partners, visiting prostitutes, or sexually transmitted diseases. Only 1 of 49 had a reactive test for syphilis. Six patients were hospitalized (including 3 with acute hepatitis B and one with hepatitis A), and none to our knowledge died. Acute hepatitis B is the most common cause of viral hepatitis in the Stann Creek District, but the modes of transmission remain obscure. Infants, women attending prenatal clinics, and new workers are potential targets for immunization with hepatitis B vaccine.

Prevalence of antibodies to hepatitis E in two rural Egyptian communities.

A population-based serosurvey in two rural Egyptian communities was used to assess age-specific prevalence of antibody to hepatitis E virus (anti-HEV). One community is in the Nile Delta (11,182 inhabitants; 3,997 participants) and the other in Upper Egypt (10,970 inhabitants; 6,029 participants). Samples were tested for anti-HEV with a commercial enzyme-linked immunoassay (ELISA) based on antigens derived from open reading frame (ORF)2 and ORF3. Although there was a clear difference in sensitivity among the lots of the commercial test used, it was still possible to determine the seroprevalence. The seroprevalence of anti-HEV exceeded 60% in the first decade of life, peaked at 76% in the second decade and remained above 60% until the eighth decade. Prevalence of this magnitude is among the highest reported in the world, with an age-specific pattern more similar to hyperendemic hepatitis A virus transmission than generally described. Lot-to-lot variation in the sensitivity of the commercial ELISA kit highlights a problem when comparing seroepidemiologic studies of different populations.