Isolation of novel virus-like sequences associated with human hepatitis.

Two viruses, GB virus A (GBV-A) and GB virus B (GBV-B), were recently identified in the GB hepatitis agent. Human sera containing antibodies that recognize GBV-A and/or GBV-B recombinant proteins were subjected to polymerase chain reaction studies with degenerate oligonucleotides capable of amplifying a segment of the putative helicase genes from GBV-A, GBV-B or hepatitis C virus. Novel sequences related to members of the Flaviviridae were identified in sera from 12 individuals including 4 individuals with hepatitis. The limited nucleotide sequence identity between GBV-A, GBV-B and HCV sequences suggests that a novel virus, tentatively named GB virus C, may be responsible for some cases of non-A, non-B, non-C, non-D, non-E hepatitis.

Lack of protective immunity against reinfection with hepatitis C virus.

Some individuals infected with hepatitis C virus (HCV) experience multiple episodes of acute hepatitis. It is unclear whether these episodes are due to reinfection with HCV or to reactivation of the original virus infection. Markers of viral replication and host immunity were studied in five chimpanzees sequentially inoculated over a period of 3 years with different HCV strains of proven infectivity. Each rechallenge of a convalescent chimpanzee with the same or a different HCV strain resulted in the reappearance of viremia, which was due to infection with the subsequent challenge virus. The evidence indicates that HCV infection does not elicit protective immunity against reinfection with homologous or heterologous strains, which raises concerns for the development of effective vaccines against HCV.

GB hepatitis agent in cadaver organ donors and their recipients.

BACKGROUND: The cloning of yet another hepatitis virus, GB virus-C (GBV-C), has provided the opportunity to study the prevalence, and clinical and laboratory characteristics, associated with GBV-C infection among cadaver organ donors and recipients of organs from infected donors. METHODS: Stored sera from a cohort of cadaver organ donors from eight organ procurement organizations, representing different geographic regions of the United States previously screened for hepatitis C virus (HCV) infection, were tested for GBV-C RNA by polymerase chain reaction using degenerate primers derived from the NS3 helicase and 5'-untranslated regions of the GBV-C genome. Pre- and posttransplantation clinical data, and prevalence of GBV-C RNA among recipients of organs from GBV-C RNA-positive and -negative donors, were studied at one of the organ procurement organizations. RESULTS: Twenty-one of 76 (27.6%) anti-HCV ELISA1-positive donors tested positive for GBV-C RNA compared with 6 of 82 (7.3%) ELISA1-negative donors (P=0.001). The prevalence of GBV-C RNA, extrapolated to all cadaver organ donors, was 8.3% (95% confidence interval [CI]: 5.6-11.1%) and was higher than the prevalence of HCV RNA (2.4%). Among ELISA1-positive donors, GBV-C RNA was present in 13 of 35 (37%) donors with HCV RNA, compared with 8 of 41 (20%) donors without HCV RNA (odds ratio [OR]=2.44, P=0.09). Blood alcohol level of more than 100 mg/dl (OR=9.43, P=0.05) and a positive anti-HCV ELISA2 (OR=4.58, P=0.001) were significantly associated with GBV-C infection. In addition, there was a trend toward an association between history of drug abuse (OR=5.23, P=0.06) and younger age (OR=0.97/year, P=0.06) with GBV-C infection. Organs from four GBVC-positive donors and 47 GBV-C-negative donors procured by the New England Organ Bank (Newton, MA) were transplanted into 6 and 79 recipients, respectively. Among recipients of organs from GBV-C RNA. positive donors, the posttransplantation prevalence of GBV-C RNA (25%) was not significantly higher than among recipients of organs from GBV-C RNA-negative donors (23%). Among recipients in whom both pre- and posttransplantation sera were available, one of three (33%) recipients of kidneys from GBV-C RNA-positive donors acquired GBV-C RNA after transplantation, compared with 4 of 40 (10%) recipients of kidneys from GBV-C RNA-negative donors. After a median follow up of 6 years, the posttransplantation prevalence of liver disease, and graft and patient survival, were not significantly different between recipients of organs from GBV-C RNA-positive and -negative donors. CONCLUSIONS: Although GBV-C could be transmitted by organ transplantation, the results of this study preclude definitive conclusions. Further studies are required to determine the risk of transmission of GBV-C by organ transplantation and its role in posttransplantation liver disease.

Radioimmunoassay for detection of hepatitis B e antigen and its antibody. Results of clinical evaluation.

The performance of a solid phase radioimmunoassay (ABBOTT-HBe) for the detection of hepatitis B e antigen (HBeAg) and its antibody anti-HBe was evaluated in clinical studies. The reagents and procedure were found to be reproducible by seven investigators, and lab-to-lab variations were minimal. In HBsAg positive sera, ABBOTT-HBe detected HBeAg or anti-HBe in 90% of the specimens compared to 50% tested by immunodiffusion. During the early acute stage of viral infection, serum HBeAg coincided with the rise and decline of HBsAg and seroconversion to anti-HBe occurred most often prior to loss of HBsAg. Patients with clinical evidence of chronic liver disease showed persistence of HBsAg and HBeAg. Vertical transmission studies of hepatitis B virus infection from mother to newborns, showed that mothers whose sera were positive for both HBsAg and HBeAg resulted in a greater incidence of transmission of hepatitis B virus to their offspring than mothers whose sera were HBsAg positive but HBeAg negative.

Interpretation of various serological profiles of hepatitis B virus infection.

Serial serum specimens from 149 patients with clinically diagnosed hepatitis were tested for five hepatitis B serological markers: hepatitis B surface antigen and its antibody (anti-HBs); hepatitis B e-antigen and its antibody (anti-HBe); and antibody to hepatitis B core antigen (anti-HBc). The times of appearance, disappearance, and persistence of these markers were used to differentiate various serological profiles obtained from the study. Four distinctive profiles were found to be associated with acute hepatitis B followed by recovery, and three with chronic hepatitis. These serologic profiles were assessed as diagnostic and prognostic guides for clinical management of the disease.

Contrasting roles of rivers and wells as sources of drinking water on attack and fatality rates in a hepatitis E epidemic in Somalia.

In early 1988, an increased incidence of acute hepatitis was observed in villages along the Shebeli River in the Lower Shebeli region of Somalia. This was followed by a large epidemic that lasted until late 1989. In a survey of 142 villages with a population of 245,312 individuals, 11,413 icteric cases were recorded, of which 346 died, corresponding to an attack rate and a case fatality rate of 4.6% and 3.0%, respectively. The etiologic role of hepatitis E virus (HEV) in this epidemic was proven by demonstrating anti-HEV in 128 of 145 sampled cases as a sign of recent infection with HEV. In three villages, where a special study protocol was implemented, the attack rate was found to increase significantly with age from 5% in the group 1-4 years of age to 13% in the group 5-15 years of age and to 20% for persons older than 15 years of age. Among cases 20-39 years of age, the female-to-male ratio was 1.5:1, which was a significant predominance of females. As in other hepatitis E outbreaks, there was a high fatality rate in pregnant females, estimated to be 13.8%. The epidemic peaked with the rise in the level of the river during rainfall, suggesting that the disease was waterborne. The attack rate was higher (6.0%) in villages supplied with river water, while fewer cases were recorded in those relying on wells or ponds for their water supply, 1.7% and 1.2%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A biotin/avidin solid-phase sandwich enzyme immunoassay for the antibody to hepatitis B surface antigen (anti-HBs).

A biotin/avidin solid-phase enzyme immunoassay for the detection and quantitation of the antibody to hepatitis B surface antigen (anti-HBs) is described. The assay utilizes hepatitis B surface antigen (HBsAg) as a solid-phase 'capture' reagent and a mixture of biotinylated HBsAg and avidin-conjugated horseradish peroxidase as a probe 'detector' reagent. The assay was compared to a commercial radioimmune assay for anti-HBs detection. The two assays were found to measure the same molecules and to correlate well regarding anti-HBs titers.

The utilization of biotin-antibiotin interaction for the detection of antibody to hepatitis B surface antigen (anti-HBs).

A biotin-antibiotin solid-phase enzyme-linked immunoassay for the detection and quantitation of the antibody to hepatitis B surface antigen (anti-HBs) is described. The assay utilizes hepatitis B surface antigen as a solid-phase 'capture' reagent and a mixture of biotinylated HBsAg and antibiotin-conjugated horseradish peroxidase as a detector reagent. The assay was compared to a commercial enzyme immunoassay (AUSAB EIA) which used the biotin-avidin system for anti-HBs detection. The two assays were found to measure the same molecules and to correlate well regarding anti-HBs titers.

Re-examination and further characterization of a monoclonal antibody to hepatitis B e antigen (anti-HBe).

A purification procedure for serum hepatitis B e antigen (HBeAg) was developed to immunize mice for monoclonal anti-HBe production. Two monoclonal anti-HBe secreting hybridomas were identified. Immunoglobulin G (IgG2a) was isolated from each hybridoma and labeled with either 125I or horseradish peroxidase. Each label was used as a probe in solid phase immunoassays for HBeAg and anti-HBe detection. Both monoclonal antibodies recognized the beta epitope on HBeAg, but one consistently performed better as a probe. When this monoclonal probe was compared to commercially available polyclonal assays, it showed equivalent sensitivity and specificity.

An enzyme immunoassay for hepatitis B e-antigen and antibody.

A solid-phase enzyme-linked immunoassay for the detection of hepatitis B e-antigen (HBeAg) and antibody (anti-HBe) was developed and compared with rheophoresis and radioimmunoassay (RIA). The enzyme-immunoassay (EIA) was similar to RIA in sensitivity and was approximately 1000-fold more sensitive than rheophoresis for HBeAg, and approximately 6000-fold more sensitive than rheophoresis for anti-HBe.

Preparation and characterization of monoclonal antibodies to rotavirus.

By utilizing a strain of cultivable simian rotavirus (SA-11) as an immunizing antigen, we prepared 4 clones of mouse-mouse hybridoma, namely C127, C139, C172, and C214 which secreted monoclonal antibodies against the immunogen itself, SA-11 and also against other group A strains such as Wa and S2. Western blot analyses revealed that all of these antibodies are directed against VP6, a 42 kDa major inner capsid protein of group A rotavirus. Competitive experiments suggested that C127, C172 and C214 recognized three distinct epitopes on VP6, while C139 appeared to react with an epitope at or near the same epitope recognized by C172. We developed a two-step ELISA with excellent sensitivity and specificity for rotavirus detection by utilizing C127 and/or C214 as a capture antibody and rabbit anti-rotavirus conjugated with horseradish peroxidase as a probe. Also, when both monoclonal C127 capture antibody and polyclonal rabbit anti-rotavirus-HRP were incubated with rotavirus simultaneously in a one-step assay, equivalent sensitivity and specificity were observed. The data show that these generated anti-rotavirus antibodies can be utilized effectively as reagents for the detection of human rotaviruses in stool specimens.

Rapid detection of GB virus C RNA by reverse transcription-polymerase chain reaction (RT-PCR) using primers derived from the 5'nontranslated region.

A simple reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of GB virus C (GBV-C) RNA in serum or plasma is described. In this method, total nucleic acid, extracted from a small volume of human plasma, is reverse transcribed using random hexamers. An aliquot of cDNA is then utilized in PCR employing GBV-C specific primers designed to highly conserved regions of the 5'nontranslated region (NTR). For additional sensitivity, a second round of nested amplification is performed. Reactions are analyzed on an agarose gel and samples showing an ethidium bromide stained band of the appropriate size in the first and second amplification, or in the second amplification only, are designated to be positive. This protocol allows for the rapid and sensitive detection of GBV-C infection in human plasma or serum.

Rapid detection of Hepatitis E virus RNA by reverse transcription-polymerase chain reaction using universal oligonucleotide primers.

A rapid reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of Hepatitis E virus (HEV) RNA in serum is described. Total nucleic acids are extracted from a small volume of human serum and reverse transcribed using random hexamers. An aliquot of cDNA is then utilized in nested PCR employing degenerate HEV consensus primers. These primers are designed to sequences conserved between the Burma, Mexico, and US HEV strains, generating amplicons within each of the three open reading frames. Reactions are analyzed by agarose gel electrophoresis and samples showing an ethidium bromide stained band of the appropriate size in the first and second amplification, or in the second amplification only, are designated as positive. This protocol allows for the rapid and sensitive detection of HEV infection in human serum.

ELISA for detection of antibody to the E2 protein of GB virus C.

An enzyme linked immunosorbent assay (ELISA) for detection of antibodies to mammalian cell-expressed E2 protein of GB virus C (GBV-C E2) is described. Antibodies to GBV-C E2 are captured on a solid phase coated with affinity purified E2 protein. Bound antibody is detected in an indirect assay format using horseradish peroxidase (HRPO) labeled goat anti-human IgG as the secondary antibody. Following a color development step, absorbance at 492 nm is measured. A population of 100 volunteer blood donors was tested to assess the specificity of this assay. Individuals reactive for antibody to GBV-C E2 can be considered to have been exposed to GB virus C.

Examination of the buoyant density of hepatitis C virus by the polymerase chain reaction.

Sucrose and cesium chloride density gradients were used to fractionate hepatitis C virus (HCV) infectious chimpanzee plasma. The fractionated plasma was then evaluated for HCV RNA sequences using cDNA synthesis and the polymerase chain reaction (cDNA/PCR). cDNA/PCR detectable HCV RNA was identified repeatedly in two regions. One region was at the top of the gradients with a buoyant density of < or = 1.03 g/cm3, the other at a density of approximately 1.18-1.21 g/cm3.

Detection of hepatitis C viral RNA by the polymerase chain reaction in serum of patients with post-transfusion non-A, non-B hepatitis.

Serial serum samples from cardiac patients with a history of chronic or resolved post-transfusion non-A, non-B hepatitis were analyzed by a combination of cDNA synthesis and the polymerase chain reaction (cDNA/PCR) to amplify HCV RNA. Analysis of sera drawn after the acute hepatitis episode from 8 of the patients who had an acute, resolving HCV infection showed no detectable levels of HCV RNA when primers from the NS3 region were used. Evaluation of these sera with primers from the 5'-untranslated (5'-UT) region revealed that one patient was positive for HCV RNA. Further analysis of serial serum samples available from two of these patients indicated that a resolved infection was associated with a disappearance of detectable HCV RNA after a peak level during the acute phase of the disease. In contrast, post-acute samples from 4 of 6 patients with symptomatic acute HCV infection evolving to chronicity were positive for HCV RNA using primers from the NS3 region, however, upon retesting with primers from the 5'-UT region, all 6 patients were found to be positive. Analysis of serial serum samples from 2 of these patients showed the persistence of HCV RNA in 70% of the samples. These two patients were subsequently treated with interferon alpha-2b. One patient resolved his disease and normalized his aminotransferase level during treatment and thereafter, while the other relapsed upon cessation of treatment. In these two patients, normalization of ALT levels was consistent with the absence of HCV RNA while relapse of disease was confirmed by the reappearance of detectable levels of HCV RNA. These results indicate the utility of HCV RNA as a marker for persisting HCV viremia and in differentiating patients with ongoing active HCV infection from those with an acute resolving disease.

Identification of conserved nucleotide sequences within the GB virus C 5'-untranslated region: design of PCR primers for detection of viral RNA.

Recently, the discovery of a new human RNA virus, GB virus C (GBV-C), was reported. GBV-C was isolated from the serum of a West African individual using degenerate oligonucleotide PCR primers designed from a consensus sequence of the NS3 helicase genes of hepatitis C virus (HCV), GBV-A, and GBV-B. Seven other individuals were shown to be infected with GBV-C via RT-PCR using these primers. Subsequently, degenerate PCR primers based upon a consensus sequence of the eight original isolates were designed. These primers were shown to be superior to the original set. However, since they were derived from a region of the viral genome exhibiting up to 17% nucleotide sequence divergence, mismatch between the primers and template may result in an underestimation of the true GBV-C prevalence. To overcome this potential problem, we obtained the sequences at the 5'-untranslated region (UTR) of the GBV-C genome from 35 infected individuals and identified regions of high sequence conservation among the isolates. We describe the design and testing of PCR primers derived from conserved sequences within the 5'-UTR of the GBV-C genome. These primers were shown to be as effective as the helicase-derived primers in detecting GBV-C RNA in human sera.

Optimized PCR assay for the detection of TT virus.

A polymerase chain reaction (PCR)-based procedure for the detection of TT virus DNA is described. In this method. total nucleic acid extracted from a small volume of serum or plasma is utilized as a template in PCR employing TT virus specific primers designed to highly conserved regions of the virus genome. Additional sensitivity is obtained by carrying out a second round of amplification. Reactions are analyzed by agarose gel electrophoresis, and samples having an ethidium bromide stainable fragment of the appropriate size in the first and/or second amplification are designated as positive. This protocol allows for the rapid and sensitive detection of TT virus in human plasma or serum.

Solid-phase enzyme-linked immunosorbent assay for hepatitis E virus IgG and IgM antibodies utilizing recombinant antigens and synthetic peptides.

Four recombinant antigens representing two distinct antigenic domains from two different strains of hepatitis E virus (HEV), were used individually to develop four ELISAs designed to detect antibodies to HEV. Both IgG and IgM class antibodies to HEV were detected in 7 of 8 pedigreed serum/plasma from known outbreaks of HEV in Mexico, Burma, Somalia and Pakistan. In addition, specific HEV-antibodies were detected in cynomolgus macaques following inoculation with various HEV strains. Anti-HEV was also detected in 8 of 386 (2.1%) randomly selected American blood donors. Supplemental tests utilizing both synthetic peptides and specific blocking assays provided additional serologic data confirming the presence of anti-HEV. Similar prevalence studies on a limited number of available sera from other geographical regions (Alaska, Japan, Germany, New Zealand, Thailand and Mexico) confirmed the presence of anti-HEV in at least 1.1 to 7.6% of the specimens.

Biophysical characterization of GB virus C from human plasma.

Viral characterization studies were carried out on GB virus C (GBV-C) RNA positive plasma from normal human donors and from donors co-infected with GBV-C and hepatitis C virus (HCV). GBV-C RNA was detected by reverse-transcriptase polymerase chain reaction (RT-PCR) and probe hybridization in a single tube assay. Sequential filtration of GBV-C positive plasma indicated that GBV-C RNA is associated with a particle 50-100 nm in diameter. The peak of GBV-C RNA in sucrose gradients was observed at a buoyant density of 1.05-1.13 g/ml. GBV-C RNA titer was reduced following treatment with chloroform or with five detergents indicating that GBV-C has a lipid-containing envelope. Sucrose density gradients and self-forming cesium chloride gradients of detergent-treated GBV-C showed a shift in the RNA peak to heavier buoyant density only when RNase inhibitor (RNasin) and high detergent concentrations were present. The treated material was non-filterable and the RNA had a density of > 1.5 gm/ml.