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.

Seroprevalence of GB virus C and persistence of RNA and antibody.

Exposure to GB virus C (GBV-C) was determined in several U.S. populations by both reverse-transcription-polymerase chain reaction (RT-PCR) and by an enzyme linked immunosorbent assay (ELISA) for antibodies to mammalian cell-expressed GBV-C envelope protein, E2 (GBV-C E2). Most individuals exposed to GBV-C were either RNA positive/ELISA negative or ELISA positive/RNA negative. Exposure, therefore, was measured as the sum of GBV-C RNA positive and GBV-C E2 antibody positive specimens, and was higher in commercial plasmapheresis donors (40.5%) than in volunteer blood donors (5.5%). In intravenous drug users (IVDUs), GBV-C exposure was 89.2%. Serial bleed specimens tested for GBV-C RNA indicate that some patients remain viremic for at least 3 years and fail to produce detectable antibodies to GBV-C E2. In other exposed individuals who tested negative for GBV-C RNA, antibodies to E2 appear to be similarly long-lived (greater than 3 years) with a fairly constant titer (ranging in reciprocal endpoint dilution from 336 to 21,504). Since the detection of GBV-C RNA and GBV-C E2 antibody are mutually exclusive in most exposed individuals, studies pertaining to incidence and prevalence of GBV-C infection require both antibody and nucleic acid detection.

GB virus C E2 glycoprotein: expression in CHO cells, purification and characterization.

A 315 amino acid recombinant segment of the GB virus C (GBV-C) E2 envelope glycoprotein (E2-315) was expressed and secreted from CHO cells. E2-315 was purified by affinity chromatography using a monoclonal antibody directed to a FLAG sequence genetically engineered onto the C terminus of the recombinant protein. The secreted protein had a molecular mass of 48-56 kDa and was shown to be N-glycosylated. Amino acid sequencing confirmed the expected N-terminal sequence. Purified E2-315 was used to develop an ELISA for detection of E2 antibodies in human sera. Antibodies to GBV-C E2 appeared to be directed toward conformational epitopes since human sera reactivity was detected in ELISA using native E2-315, but it was extremely weak or non-existent with denatured E2 protein. The use of an ELISA which can detect human GBV-C E2 antibodies will be important in further understanding of the clinical significance and epidemiology of GBV-C.

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

An ELISA was developed for detection of antibodies to GB virus C (GBV-C) using a recombinant E2 protein expressed in CHO cells. Seroconversion to anti-E2 positivity was noted among several persons infected with GBV-C RNA-positive blood through transfusion. Of 6 blood recipients infected by GBV-C RNA-positive donors, 4 (67%) became anti-E2 positive and cleared their viremia. Thus, anti-E2 seroconversion is associated with viral clearance. The prevalence of antibodies to E2 was relatively low (3.0%-8.1%) in volunteer blood donors but was higher in several other groups, including plasmapheresis donors (34.0%), intravenous drug users (85.2%), and West African subjects (13.3%), all of whom tested negative by GBV-C reverse-transcription polymerase chain reaction (RT-PCR). These data demonstrate that testing for anti-E2 should greatly extend the ability of RT-PCR to define the epidemiology and clinical significance of GBV-C.

Expression of the GB virus C E2 glycoprotein using the Semliki Forest virus vector system and its utility as a serologic marker.

A 336-amino-acid segment of the GB virus C second envelope protein (E2) has been produced in BHK-21 cells using the Semliki Forest virus vector system. Secretion of this protein was facilitated by deletion of a hydrophobic region at the C-terminus that may represent the membrane anchoring domain. The E2 protein recovered from the culture supernatant exhibited a molecular mass of approximately 52 kDa, with the increase in size relative to the polyprotein backbone being contributed by N-linked glycosylation. A radioimmunoprecipitation assay using GBV-C E2 was developed to test for the presence of antibodies against this protein in human sera. The prevalence of antibodies to E2 was high among injection drug users and other individuals at risk for acquiring parenterally transmitted agents. There was a much higher percentage of anti-E2 seropositivity in GBV-C RT-PCR negative compared to GBV-C RT-PCR positive samples from these populations. In addition, serial samples from patients transfused with blood containing GBV-C showed seroconversion to anti-E2 positivity and loss of GBV-C viremia as measured by RT-PCR within 11 months of transfusion in five of seven individuals. Thus, this system provided a rapid means to identify GBV-C E2 as a useful antigen for the study of GBV-C exposure.

Antibody to hepatitis C virus second envelope (HCV-E2) glycoprotein: a new marker of HCV infection closely associated with viremia.

The second envelope protein (E2) of the hepatitis C virus (HCV) was cloned and expressed in Chinese hamster ovary (CHO) cells. This E2 glycoprotein was purified using ion exchange and lectin chromatography and used to construct an enzyme immunoassay for HCV E2 antibodies. The assay was shown to have good specificity, and detection of E2 antibodies was positively correlated (97.3%) to the presence of HCV RNA in serum and plasma. A high concordance between HCV 2.0 and E2 EIA reactivities was also observed. E2 antibody was the first serological marker to appear in 3/5 HCV seroconversion panels. This work demonstrated that 42.4% of core and 15.4% of NS3 indeterminate specimens also contained antibodies to E2, suggesting that HCV infection had occurred in these individuals. The E2 antibody assay was used to evaluate HCV 2.0 EIA-positive, HCV 3.0 EIA-negative plasma donors with indeterminate reactivity on RIBA HCV 2.0 or MATRIX HCV 1.0. Several HCV 3.0-negative specimens were shown to contain E2 antibodies in addition to an original indeterminate serological marker, primarily core. It is concluded that anti-E2 is a useful marker for determining HCV infection, and that the presence of antibodies to two nonoverlapping viral gene products suggests true HCV exposure. New HCV 3.0 blood screening tests should detect HCV 2.0-positive donors who present with an indeterminate pattern by RIBA or MATRIX and who also carry E2 antibodies.

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.

Improved methods for detecting enteric viruses in oysters.

New and improved methods for concentrating enteroviruses, reoviruses, and adenoviruses from oysters have been developed and evaluated. Viruses are efficiently adsorbed to homogenized oyster meat by adjusting the homogenate to pH 5.0 and a conductivity of less than or equal to 2,000 mg of NaCl per liter. After low-speed centrifugation, the virus-free supernatant is discarded and the viruses are eluted from the sedimented oyster solids with pH 7.5 glycine-NaCl having a conductivity of 8,000 mg of NaCl per liter. The oyster solids are removed by low-speed centrifugation and filtration, and the viruses in the filtered supernatant are concentrated to a small volume by either ultrafiltration or acid precipitation at pH 4.5. The concentrate is treated with antibiotics and inoculated into cell cultures for virus isolation and quantitation. When these methods were tested with oysters experimentally contaminated with polioviruses, reoviruses, and adenoviruses, recovery efficiencies averaged about 46%. With the exception of virus assay and quantitation, these methods are simple and inexpensive enough to be done in typical shellfish microbiology laboratories.