Search

Erythrocytes bind circulating immune complexes (ICs) and facilitate IC clearance from the circulation. Chronic hepatitis C virus (HCV) infection is associated with IC-related disorders. In this study, we investigated the kinetics and mechanism of HCV and HCV-IC binding to and dissociation from erythrocytes. Cell culture-produced HCV was mixed with erythrocytes from healthy blood donors, and erythrocyte-associated virus particles were quantified. Purified complement proteins, complement-depleted serum, and complement receptor antibodies were used to investigate complement-mediated HCV-erythrocyte binding. Purified HCV-specific immunoglobulin G (IgG) from a chronic HCV-infected patient was used to study complement-mediated HCV-IC/erythrocyte binding. Binding of HCV to erythrocytes increased 200- to 1,000-fold after adding complement active human serum in the absence of antibody. Opsonization of free HCV occurred within 10 minutes, and peak binding to erythrocytes was observed at 20-30 minutes. Complement protein C1 was required for binding, whereas C2, C3, and C4 significantly enhanced binding. Complement receptor 1 (CR1, CD35) antibodies blocked the binding of HCV to erythrocytes isolated from chronically infected HCV patients and healthy blood donors. HCV-ICs significantly enhanced complement-mediated binding to erythrocytes compared to unbound HCV. Dissociation of complement-opsonized HCV from erythrocytes depended on the presence of Factor I. HCV released by Factor I bound preferentially to CD19 + B cells compared to other leukocytes. Conclusion: These results demonstrate that complement mediates the binding of free and IC-associated HCV to CR1 on erythrocytes and provide a mechanistic rationale for investigating the differential phenotypic expression of HCV-IC-related disease., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

Extrahepatic disease manifestations are common in chronic hepatitis C virus (HCV) infection. The mechanism of HCV-related lymphoproliferative disorders is not fully understood. Recent studies have found that HCV in peripheral blood mononuclear cells from chronically infected patients is mainly associated with cluster of differentiation 19-positive (CD19 + ) B cells. To further elucidate this preferential association of HCV with B cells, we used in vitro cultured virus and uninfected peripheral blood mononuclear cells from healthy blood donors to investigate the necessary serum components that activate the binding of HCV to B cells. First, we found that the active serum components were present not only in HCV carriers but also in HCV recovered patients and HCV-negative, healthy blood donors and that the serum components were heat-labile. Second, the preferential binding activity of HCV to B cells could be blocked by anti-complement C3 antibodies. In experiments with complement-depleted serum and purified complement proteins, we demonstrated that complement proteins C1, C2, and C3 were required to activate such binding activity. Complement protein C4 was partially involved in this process. Third, using antibodies against cell surface markers, we showed that the binding complex mainly involved CD21 (complement receptor 2), CD19, CD20, and CD81; CD35 (complement receptor 1) was involved but had lower binding activity. Fourth, both anti-CD21 and anti-CD35 antibodies could block the binding of patient-derived HCV to B cells. Fifth, complement also mediated HCV binding to Raji cells, a cultured B-cell line derived from Burkitt's lymphoma., Conclusion: In chronic HCV infection, the preferential association of HCV with B cells is mediated by the complement system, mainly through complement receptor 2 (CD21), in conjunction with the CD19 and CD81 complex. (Hepatology 2016;64:1900-1910)., Competing Interests: Potential conflict of interest: Nothing to report., (© 2016 by the American Association for the Study of Liver Diseases. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Unlabelled: The goal of this study was to determine whether an association exists between circulating microRNA (miRNA) levels and disease progression in chronic hepatitis C (CHC), whether plasma or extracellular vesicles (EVs) were optimal for miRNA measurement and their correlation with hepatic miRNA expression, and the mechanistic plausibility of this association. We studied 130 CHC patients prospectively followed over several decades. A comprehensive miRNA profile in plasma using microarray with 2578 probe sets showed 323 miRNAs differentially expressed between healthy individuals and CHC patients, but only six that distinguished patients with mild versus severe chronic hepatitis. Eventually, let-7a/7c/7d-5p and miR-122-5p were identified as candidate predictors of disease progression. Cross-sectional analyses at the time of initial liver biopsy showed that reduced levels of let-7a/7c/7d-5p (let-7s) in plasma were correlated with advanced histological hepatic fibrosis stage and other fibrotic markers, whereas miR-122-5p levels in plasma were positively correlated with inflammatory activity, but not fibrosis. Measuring let-7s levels in EVs was not superior to intact plasma for discriminating significant hepatic fibrosis. Longitudinal analyses in 60 patients with paired liver biopsies showed that let-7s levels in plasma markedly declined over time in parallel with fibrosis progression. However, circulating let-7s levels did not parallel those in the liver., Conclusion: Of all miRNAs screened, the let-7 family showed the best correlation with hepatic fibrosis in CHC. A single determination of let-7s levels in plasma did not have superior predictive value for significant hepatic fibrosis compared with that of fibrosis-4 index, but the rate of let-7s decline in paired longitudinal samples correlated well with fibrosis progression. Pathway analysis suggested that low levels of let-7 may influence hepatic fibrogenesis through activation of transforming growth factor β signaling in hepatic stellate cells. (Hepatology 2016;64:732-745)., (© 2016 by the American Association for the Study of Liver Diseases. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Unlabelled: Interleukin (IL)28B polymorphisms are associated with spontaneous clearance of hepatitis C virus (HCV) infection and response to therapy. Whether IL28B genotype affects fibrosis progression or clinical outcome is unclear. Our aim was to study the relationship between IL28B genotype and both histological and clinical outcomes in patients with chronic hepatitis C (CHC). Hepatic fibrosis was scored using the Ishak (0-6) scale; progression was defined as a 2-point increase in Ishak score between biopsies. Multiple logistic and Cox regressions were used to identify variables associated with fibrosis progression. In all, 1,483 patients were included in a baseline cross-sectional analysis, from which 276 were eligible for a paired biopsy analysis (median time between biopsies 4 years), and 400 for a clinical outcome analysis. At baseline biopsy, patients with IL28B CC genotype had significantly higher portal inflammation (2.4 versus 2.2) and alanine aminotransferase (ALT) levels (133 versus 105 U/L; P < 0.05 for all). In the paired biopsy analysis, there was no difference in the frequency of fibrosis progression between patients with IL28B CC and non-CC genotypes (17% versus 23%). In logistic regression, only higher baseline alkaline phosphatase, lower platelets, and greater hepatic steatosis were associated with fibrosis progression. Patients with IL28B CC were twice as likely to develop adverse clinical outcomes compared to non-CC (32% versus 16%; P = 0.007)., Conclusion: IL28B CC genotype was associated with greater hepatic necroinflammation, higher ALT, and worse clinical outcomes in CHC patients. This suggests that IL28B CC is associated with a state of enhanced immunity that, on the one hand, can promote viral clearance, but alternately can increase necroinflammation and hepatic decompensation without enhancing fibrosis progression., (© 2013 by the American Association for the Study of Liver Diseases.)

Unlabelled: Recent studies have found hepatitis C virus (HCV) RNA in peripheral blood mononuclear cells (PBMCs) of the majority of presumed recovered subjects. We investigated this unexpected finding using samples from patients whose HCV RNA and anti-HCV status had been serially confirmed. HCV RNA was detected in PBMCs from 66 of 67 chronic HCV carriers. Subpopulation analysis revealed that the viral load (log copies/10(6) cells) in B cells (4.14 ± 0.71) was higher than in total PBMCs (3.62 ± 0.71; P < 0.05), T cells (1.67 ± 0.88; P < 0.05), and non-B/T cells (2.48 ± 1.15; P < 0.05). HCV negative-strand RNA was not detected in PBMCs from any of 25 chronically infected patients. No residual viral RNA was detected in total PBMCs or plasma of 59 presumed recovered subjects (11 spontaneous and 48 treatment induced) using nested real-time polymerase chain reaction with a detection limit of 2 copies/μg RNA (from ≈ 1 × 10(6) cells). PBMCs from 2 healthy HCV-negative blood donors became HCV RNA positive, with B-cell predominance, when mixed in vitro with HCV RNA-positive plasma, thus passively mimicking cells from chronic HCV carriers. No residual HCV was detected in liver or other tissues from 2 spontaneously recovered chimpanzees., Conclusion: (1) HCV RNA was detected in PBMCs of most chronic HCV carriers and was predominant in the B-cell subpopulation; (2) HCV detected in PBMCs was in a nonreplicative form; (3) HCV passively adsorbed to PBMCs of healthy controls in vitro, becoming indistinguishable from PBMCs of chronic HCV carriers; and (4) residual HCV was not detected in plasma or PBMCs of any spontaneous or treatment-recovered subjects or in chimpanzee liver, suggesting that the classic pattern of recovery from HCV infection is generally equivalent to viral eradication., (Copyright © 2012 American Association for the Study of Liver Diseases.)

To investigate the quantitative characteristics of humoral immunity in patients with hepatitis C, we established an enzyme-linked immunosorbent spot (ELISpot) assay for detection of anti-hepatitis C virus (HCV)-secreting B cells. Receiver operating characteristic curve analysis demonstrated 100% specificity and 58% to 92% sensitivity for detecting B-cell responses to NS5b, NS3, E2, and core antigens. The median sum of anti-HCV-secreting B cells to all HCV antigens tested was significantly higher in 39 patients with chronic hepatitis C (47.3 spot forming cells [SFCs]/10(6) peripheral blood mononuclear cells [PBMCs]) than in 9 recovered subjects (15.3 SFCs/10(6) PBMCs; P = .05) or 11 uninfected controls (5.3 SFCs/10(6) PBMCs; P < .001); the significant difference (P = .018) in chronic versus recovered patients was in reactivity to nonstructural antigens NS3 and NS5b. Anti-HCV immunoglubulin M (IgM)-secreting B cells were also readily detected and persisted decades into HCV infection; there was no difference in IgM-positive cells between chronic and recovered patients. ELISpot reactivity to genotype 1-derived antigens was equivalent in patients of genotypes 1, 2, and 3. There was significant correlation between the numbers of anti-HCV IgG-secreting B cells and serum aminotransferase and to the level of circulating antibody. In conclusion, ELISpot assays can be adapted to study B-cell as well as T-cell responses to HCV. Measurement at the single-cell level suggests that humoral immunity plays a minor role in recovery from HCV infection and that B-cell immunity is strongest in those with persistent infection.

Active and/or passive immunoprophylaxis against hepatitis C virus (HCV) remain unachieved goals. Monoclonal antibodies might provide one approach to protection. We derived human monoclonal antibodies from the bone marrow of a patient with a well-controlled HCV infection of 22 years duration. Five distinct antibodies reactive with the E2 glycoprotein of the homologous 1a strain of HCV were recovered as antigen-binding fragments (FAbs). They demonstrated affinity constants as high as 2 nanomolar. "Neutralization of binding" titers paralleled the affinity constants. All five FAbs reacted with soluble E2 protein only in nonreducing gels, indicating that the relevant epitopes were conformational. The FAbs could be divided into two groups, based on competition analysis. Three of the FAbs neutralized the infectivity of pseudotyped virus particles (pp) bearing the envelope glycoproteins of the homologous HCV strain (genotype 1a). The three FAbs also neutralized genotype 1b pp and one also neutralized genotype 2a pp. In conclusion, one or more of these monoclonal antibodies may be useful in preventing infections by HCV belonging to genotype 1 or 2, the most medically important genotypes worldwide.

Hepatitis C virus (HCV) has a high propensity for persistence. To better define the immunologic determinants of HCV clearance and persistence, we examined the circulating HCV-specific T-cell frequency, repertoire, and cytokine phenotype ex vivo in 24 HCV seropositive subjects (12 chronic, 12 recovered), using 361 overlapping peptides in 36 antigenic pools that span the entire HCV core, NS3-NS5. Consistent with T-cell-mediated control of HCV, the overall HCV-specific type-1 T-cell response was significantly greater in average frequency (0.24% vs. 0.04% circulating lymphocytes, P =.001) and scope (14/36 vs. 4/36 pools, P =.002) among the recovered than the chronic subjects, and the T-cell response correlated inversely with HCV titer among the chronic subjects (R = -0.51, P =.049). Although highly antigenic regions were identified throughout the HCV genome, there was no apparent difference in the overall HCV-specific T-cell repertoire or type-1/type-2 cytokine profile relative to outcome. Notably, HCV persistence was associated with a reversible CD4-mediated suppression of HCV-specific CD8 T cells and with higher frequency of CD4(+)CD25(+) regulatory T cells (7.3% chronic vs. 2.5% recovered, P =.002) that could directly suppress HCV-specific type-1 CD8 T cells ex vivo. In conclusion, we found that HCV persistence is associated with a global quantitative and functional suppression of HCV-specific T cells but not differential antigenic hierarchy or cytokine phenotype relative to HCV clearance. The high frequency of CD4(+)CD25(+) regulatory T cells and their suppression of HCV-specific CD8 T cells ex vivo suggests a novel role for regulatory T cells in HCV persistence.

This study was performed to examine the immunologic basis for the apparent ethnic difference in clinical outcome of hepatitis C virus (HCV) infection between African Americans (AA) and Caucasian Americans (CA). To this end, we recruited 99 chronically HCV-infected and 31 spontaneously HCV-cleared subjects for clinical, virologic, and immunologic analysis. In particular, CD4-proliferative T-cell response to genotype 1-derived HCV antigens (core, NS3-NS5) was examined in 82 patients chronically infected with genotype 1 (54 AA, 28 CA) and in all HCV-cleared subjects (14 AA, 17 CA). HCV-specific Th1 response also was examined in 52 chronic and 13 recovered subjects. Our results showed that HCV clearance was associated with a vigorous HCV-specific Th1 response irrespective of ethnic origin. Although the HCV-specific CD4 T-cell response clearly was weaker during chronic infection, AA ethnicity in this setting was associated with a significantly greater CD4-proliferative T-cell response to HCV, particularly to the nonstructural antigens (22% AA vs. 0% CA, P =.007) as well as better clinical parameters of liver disease. Interestingly, most HCV-specific CD4 T-cell proliferative responses in AA patients were unaccompanied by concurrent interferon gamma (IFN-gamma) production, suggesting a dysregulated virus-specific, CD4 T-cell effector function during chronic HCV infection. In conclusion, our results suggest that host ethnicity does influence the clinical outcome and antiviral T-cell response during HCV infection. AA ethnicity is associated with a more robust antiviral CD4 T-cell response than CA ethnicity, although these T cells are limited in direct virus or disease control due to their dysfunctional nature.

A vaccine strategy directed to increase Th1 cellular immune responses, particularly to hepatitis C virus (HCV) nonstructural protein 3 (NS3), has considerable potential to overcome the infection with HCV. DNA vaccination can induce both humoral and cellular immune responses, but it became apparent that the cellular uptake of naked DNA injected into muscle was not very efficient, as much of the DNA is degraded by interstitial nucleases before it reaches the nucleus for transcription. In this paper, cationic liposomes composed of different cationic lipids, such as dimethyl-dioctadecylammonium bromide (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), or 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (DOEPC), were used to improve DNA immunization in mice, and their efficiencies were compared. It was found that cationic liposome-mediated DNA immunization induced stronger HCV NS3-specific immune responses than immunization with naked DNA alone. Cationic liposomes composed of DDAB and equimolar of a neutral lipid, egg yolk phosphatidylcholine (EPC), induced the strongest antigen-specific Th1 type immune responses among the cationic liposome investigated, whereas the liposomes composed of 2 cationic lipids, DDAB and DOEPC, induced an antigen-specific Th2 type immune response. All cationic liposomes used in this study triggered high-level, nonspecific IL-12 production in mice, a feature important for the development of maximum Th1 immune responses. In conclusion, the cationic liposome-mediated gene delivery is a viable HCV vaccine strategy that should be further tested in the chimpanzee model.

The SEN virus (SENV) is a recently identified single-stranded, circular DNA virus. A strong association between 2 SENV variants (SENV-D and SENV-H) and transfusion-associated non-A-to-E hepatitis has been reported. To clarify the effect of SENV infection on coexisting chronic hepatitis C and the effect of interferon alfa (IFN-alpha) therapy on SENV replication, SENV DNA was quantitated by polymerase chain reaction in serum samples from 186 patients with chronic hepatitis C. Thirty-nine of 186 (21%) patients with chronic hepatitis C were positive for SENV DNA. There were no differences in the clinical, virologic and histologic features between patients with and without SENV infection. Eighteen of 102 patients with chronic hepatitis C who received IFN-alpha were positive for SENV DNA. The sustained response rate for hepatitis C virus (HCV) clearance after IFN-alpha treatment did not differ significantly between patients with SENV (28%) and without SENV infection (39%). SENV DNA levels decreased during therapy in 15 of 16 patients, and 11 of the 16 patients (69%) had a sustained loss of SENV DNA in response to IFN-alpha. In coinfected patients, SENV responses to IFN-alpha were significantly better in those who failed to clear HCV RNA than in those who lost HCV RNA (P =.013). In conclusion, SENV infection was frequently found in patients with chronic hepatitis C. SENV infection had no apparent influence on the severity of HCV-related liver disease or the HCV response to IFN-alpha. SENV was sensitive to IFN-alpha therapy and the majority of patients had a sustained virologic response.

SEN virus (SEN-V) is a recently identified single-stranded, circular DNA virus. Two SEN-V variants (SENV-D and SENV-H) were assayed by polymerase chain reaction (PCR) to investigate their role in the causation of transfusion-associated non-A to E hepatitis. The incidence of SEN-V infection after transfusion was 30% (86 of 286) compared with 3% (3 of 97) among nontransfused controls (P < .001). Transfusion risk increased with the number of units transfused (P < .0001) and donor-recipient linkage for SEN-V was shown by sequence homology. The prevalence of SEN-V in 436 volunteer donors was 1.8%. Among patients with transfusion-associated non-A to E hepatitis, 11 of 12 (92%) were infected with SEN-V at the time of transfusion compared with 55 of 225 (24%) identically followed recipients who did not develop hepatitis (P < .001). No effect of SEN-V on the severity or persistence of coexistent hepatitis C virus (HCV) infection was observed. In 31 infected recipients, SEN-V persisted for greater than 1 year in 45% and for up to 12 years in 13%. SEN-V-specific RNA (a possible replicative intermediate) was recovered from liver tissue. In summary, SENV-D and -H were present in nearly 2% of US donors, and were unequivocally transmitted by transfusion and frequently persisted. The strong association of SEN-V with transfusion-associated non-A to E hepatitis compared with controls raises the possibility, but does not establish that SEN-V might be a causative agent of posttransfusion hepatitis. The vast majority of SEN-V-infected recipients did not develop hepatitis.

Persons with non-A, non-B hepatitis (cases) identified in 5 transfusion studies in the early 1970s have been followed ever since and compared for outcome with matched, transfused, non-hepatitis controls from the same studies. Previously, we reported no difference in all-cause mortality but slightly increased liver-related mortality between these cohorts after 18 years follow-up. We now present mortality and morbidity data after approximately 25 years of follow-up, restricted to the 3 studies with archived original sera. All-cause mortality was 67% among 222 hepatitis C-related cases and 65% among 377 controls (P = NS). Liver-related mortality was 4.1% and 1.3%, respectively (P =.05). Of 129 living persons with previously diagnosed transfusion-associated hepatitis (TAH), 90 (70%) had proven TAH-C, and 39 (30%), non-A-G hepatitis. Follow-up of the 90 TAH-C cases revealed viremia with chronic hepatitis in 38%, viremia without chronic hepatitis in 39%, anti-HCV without viremia in 17%, and no residual HCV markers in 7%. Thirty-five percent of 20 TAH-C patients biopsied for biochemically defined chronic hepatitis displayed cirrhosis, representing 17% of all those originally HCV-infected. Clinically evident liver disease was observed in 86% with cirrhosis but in only 23% with chronic hepatitis alone. Thirty percent of non-A, non-B hepatitis cases were unrelated to hepatitis viruses A,B,C, and G, suggesting another unidentified agent. In conclusion, all-cause mortality approximately 25 years after acute TAH-C is high but is no different between cases and controls. Liver-related mortality attributable to chronic hepatitis C, though low (<3%), is significantly higher among the cases. Among living patients originally HCV-infected, 23% have spontaneously lost HCV RNA.

This study was performed to compare the vigor and phenotype of virus-specific CD4(+) and CD8(+) T-cell responses in patients with different virologic and clinical outcomes after hepatitis C virus (HCV) infection. The results show that a vigorous and multispecific CD4(+) proliferative T-cell response is maintained indefinitely after recovery from HCV infection whereas it is weak and focused in persistently infected patients. In contrast, the HCV-specific CD8(+) T-cell response was quantitatively low in both groups despite the use of sensitive direct ex vivo intracellular interferon gamma (IFN-gamma) staining. Furthermore, although HCV-specific cytolytic CD8(+) memory T cells were undetectable ex vivo, they were readily expanded from the peripheral blood of chronically HCV-infected patients but not from recovered subjects after in vitro stimulation, suggesting that ongoing viremia is required to maintain the HCV-specific memory CD8(+) T-cell response. HCV-specific CD8(+) T cells displayed a type 1 cytokine profile characterized by production of IFN-gamma despite persistent HCV viremia. The paradoxical observation that HCV-specific CD4(+) T cells survive and CD8(+) T cells are lost after viral clearance while the opposite occurs when HCV persists suggests the existence of differential requirements for the maintenance of CD4(+) and CD8(+) T-cell memory during HCV infection. Furthermore, the relative rarity of circulating CD8(+) effector T cells in chronically infected patients may explain the chronic insidious nature of the liver inflammation and also why they fail to eliminate the virus.

The aim of this study was to analyze the clinical performance of a new enzyme immunoassay (EIA) for hepatitis C virus (HCV) core antigen in comparison with the reverse transcription polymerase chain reaction (RT-PCR). A total of 310 patients with acute or chronic hepatitis C, and 132 HCV-negative controls were studied. Chemiluminescence EIA with monoclonal anti-HCV core antigen was used, and qualitative and quantitative commercial RT-PCRs and an in-house nested RT-PCR were performed. Compared with nested RT-PCR, the core antigen assay showed 97% sensitivity and 100% specificity in 75 patients with chronic hepatitis C and 132 controls. HCV core antigen was positive in 16 (94%) of 17 patients with acute hepatitis C at initial consultation. In 3 persons prospectively followed, core antigen was detected in the first available (1-3 weeks) post-transfusion sample. In 167 anti-HCV-positive individuals, 129 (77%) were viremic; core antigen was detected in 126 (98%) compared with 129 (100%) for nested RT-PCR and 121 (94%) for the commercial RT-PCR. In 48 patients with chronic hepatitis C treated with interferon alfa, the concentration of core antigen before treatment was significantly (P <.002) lower in patients with sustained response than in nonresponders. All responders had a sustained loss of core antigen, whereas all nonresponders remained core antigen positive. The concentrations of HCV core antigen and HCV RNA correlated significantly (n = 48, r =.627, P <.001). In conclusion, the HCV core antigen assay is useful for the diagnosis of acute and chronic hepatitis C, and for predicting and monitoring the effect of interferon alfa treatment.

TT virus (TTV) has been proposed as the causative agent of non-A to E hepatitis. We studied the association between TTV viremia and biochemical evidence of hepatitis in blood donors and prospectively-followed patients. TTV was found in 7.5% of 402 donors and in 11.0% of 347 patients before transfusion. The rate of new TTV infections was 4.7% in 127 nontransfused, and 26.4% in 182 transfused patients (P <.0001). The risk of infection increased with the number of units transfused (P <.0001). The rate of new TTV infections in 13 patients with non-A to E hepatitis (23.2%) was almost identical to the rate in 124 patients who were transfused, but did not develop hepatitis (21.8%). Of 45 patients with acute hepatitis C, 40.0% were simultaneously infected with TTV. TTV did not worsen the biochemical severity (mean ALT: 537 in TTV+; 550 in TTV-) or persistence of hepatitis C. In non-A to E cases, the mean ALT was 182 in those TTV-positive and 302 in TTV-negatives. No consistent relationship between alanine transaminase level and TTV DNA level was observed in 4 patients with long-term, sequential samples. Of 21 viremic subjects, 67% cleared TTV within 5 years (38% in 1 year); 33% were viremic throughout follow-up extending to 22 years. We conclude that TTV is a very common, often persistent infection that is transmitted by transfusion and by undefined nosocomial routes. We found no association between TTV and non-A to E hepatitis and no effect of TTV on the severity or duration of coexistent hepatitis C. TTV may not be a primary hepatitis virus.

The second envelope protein (E2) of the hepatitis G virus (HGV) was expressed in Chinese hamster ovary (CHO) cells and showed a molecular weight of approximately 60 to 70 kd, with 15 to 25 kd of the size contributed by N-linked glycosylation. An enzyme-linked immunosorbent assay (ELISA) using HGV-E2 was developed to test for antibodies to this protein (anti-E2) in human sera. High sensitivity was achieved by developing monoclonal antibodies (mAbs) to HGV-E2, which were used as capture antibodies in the ELISA. Our studies revealed that 16% of healthy Spanish blood donors were exposed to HGV, indicating that additional routes of viral transmission besides parenteral exposure might exist. An even higher prevalence of exposure to HGV (52%-73%) was found in several groups at risk of parenteral exposure to infectious agents, i.e., intravenous drug users, transfusion history, hemophiliacs, and hepatitis C virus (HCV)-positive patients. Most anti-E2-positive patients were HGV-RNA-negative and vice versa, indicating an inverse correlation of these two viral markers. A panel of 16 posttransfusion patients followed for up to 16 years revealed that patients who develop an anti-E2 response become HGV-RNA-negative, while patients who do not develop anti-E2 are persistently infected. Immunity to HGV seems to be long-lasting, because circulating antibody to E2 could still be detected 14 years after seroconversion. Sequence comparisons showed that E2 is highly conserved among isolates collected worldwide, indicating that immune escape variants are not common in HGV infections. This reflects on a molecular level why HGV infections usually are cleared spontaneously by the host. However, possible mechanisms of HGV persistence, as found in some patients, remain to be elucidated.

Among 248 asymptomatic blood donors positive for antibody to hepatitis C virus (anti-HCV) enrolled in a long-term prospective study, 86% had chronic HCV infection and 14% appeared to have recovered as assessed by serial determinations of serum alanine aminotransferase (ALT) levels and HCV RNA by polymerase chain reaction. Established parenteral risk factors for HCV transmission were identified in 75% of donors. In addition, there was a strong independent association between HCV positivity and cocaine snorting, suggesting that shared snorting devices may be a covert route of parenteral transmission. Ear piercing in males was also significantly associated with transmission. There was no evidence for sexual spread. Although the majority of HCV carriers had both biochemical and histological evidence of chronic viral hepatitis, the extent of liver injury was generally mild. Among a larger population of 280 HCV RNA-positive donors, 17% had repeatedly normal ALT levels, 45% had levels that did not exceed twice, and only 22% had levels that exceeded five times the upper limit of the normal range. Among 81 patients who underwent liver biopsy, only 13% had evidence of severe hepatitis (8%) or cirrhosis (5%), despite a duration of infection that generally exceeded 15 years. No severe histological lesions were observed in blood donors with chronic HCV infection who had repeatedly normal ALT levels. In both donors and blood recipients, the frequency of severe morbidity or mortality related to HCV infection was less than 10% during the first two decades of infection. Further long-term studies are required to see if the progression to severe outcomes continues to accrue at this slow pace or whether it accelerates during subsequent decades.

We have evaluated the clinical and histopathological outcomes of patients who contracted chronic non A, non B hepatitis as a result of transfusions administered during heart surgery at the National Institutes of Health. Posttransfusion hepatitis developed in 65 of 1,070 (6.1%) patients and became chronic in 45 (69%) of those cases. Antibody to hepatitis C virus was detectable in 53 patients (82%) with posttransfusion non A, non B hepatitis. Thirty-three patients with chronic non A, non B hepatitis agreed to liver biopsy (group 1). In addition, six other patients with chronic posttransfusion non A, non B hepatitis were evaluated (group 2). These 39 patients were followed between 1 and 24 yr (mean = 9.7 yr). Cirrhosis developed in 8 patients (20%) between 1.5 and 16 yr after blood transfusion. Of the 33 patients in group 1, 11 (33%) died during follow-up. In two cases (6%), this was related to liver failure. At this writing, two additional patients (6%) have decompensated cirrhosis and one (3%) had debilitating fatigue. Twenty of 33 patients (61%) with histological evidence of chronic active hepatitis or cirrhosis are asymptomatic and have no clinical evidence of liver disease. Thus chronic non A, non B posttransfusion hepatitis appeared to be due to hepatitis C virus infection in most cases. It was associated with the development of cirrhosis in approximately 20% of cases and end-stage liver disease in 12% of patients followed prospectively. Most patients with histological evidence of cirrhosis or chronic active hepatitis, however, had minimal clinical evidence of liver disease within the time frame of this study.

Convalescent phase non-A, non-B (NANB) human and chimpanzee sera were utilized in a solid-phase radioimmunoassay (RIA) in an attempt to identify a specific NANB antigen in human plasma, plasma-derived pellets and NP-40 disrupted pellets proven to transmit NANB infection to chimpanzees. RIA reactivity was noted in 5 of 8 pedigreed NANB infectious plasmas, but did not appear to be virus-specific. The RIA reactive fraction: (i) had a density of 1.27 to 1.32 gm per cm3 in cesium chloride; (ii) showed immunoreactivity corresponding to normal IgM, but peak RIA activity coincided with an aberrant IgM having a peak sedimentation coefficient of 56 S, and (iii) eluted at higher pH (5.6 to 5.0) than normal IgM on chromatofocusing columns. The reactant shared properties with rheumatoid factor in that it was absorbed by aggregated IgG and was unstable to 2-mercaptoethanol, but it did not react in standard agglutination tests for rheumatoid factor. The reactant was detected in both the acute and chronic phase of NANB infections in chimpanzee and man. By reacting with IgG in presumptive NANB convalescent sera, this aberrant rheumatoid factor could simulate a NANB antigen and represent a cause of false-positive reactions in putative NANB assays.