Detection of an antigen-antibody system in serum associated with human non-A, non-B hepatitis.

An antigen was detected by counterelectrophoresis in serum samples from six of seven chimpanzees during the acute phase of experimentally induced non-A, non-B hepatitis using antiserum from a chimpanzee convalescent from human non-A, non-B hepatitis. This antigen could not be detected in 35 preinoculation serum samples from these chimpanzees, or in 94 weekly bleedings from three chimpanzees with hepatitis A and three chimpanzees with hepatitis B. The antigen was detected in serum samples obtained from three humans with chronic non-A, non-B hepatitis whose blood had transmitted non-A, non-B hepatitis to other humans (including a nurse by accidental needlestick) and to chimpanzees by experimental inoculation. In addition, the antigen was detected in serum obtained retrospectively from 11 to 31 former blood donors whose blood had transmitted posttransfusion non-A, non-B hepatitis several years previously to recipients of a single unit of their blood. Antibody to this antigen was detected in convalescent serum samples from all seven chimpanzees studied, in convalescent serum from the nurse infected by accidental needlestick, and in serum from a hemodialysis patient convalescent from non-A, non-B hepatitis.     

Precipitin system detected in sera from patients with non-A,non-B hepatitis

A precipitin system detected by immunodiffusion was identified using sera from patients with non-A, non-B hepatitis. This system resembled those described by other authors as serological tests for non-A, non-B antigens and antibodies. However, testing of sera from a large number of patients with acute and chronic liver diseases revealed that this precipitin system was not specific for non-A, non-B hepatitis. Further characterization of the precipitin system demonstrated that it did not represent a true antigen-antibody reaction. Precipitin lines detected by immunodiffusion during the course of acute and chronic non-A, non-B hepatitis may not represent reactions specific for this disease.     

An enzyme-linked immunosorbent assay for an antigen related to non-A, non-B hepatitis and its antibody: partial characterization of the antigen and chimpanzee transmission

An enzyme-linked immunosorbent assay (ELISA) was developed based on sera from patients convalescent from non-A, non-B hepatitis and haemophilia A patients who had been frequently treated with commercial blood products. Using this ELISA, an antigen was detected which appears to be related to non-A, non-B hepatitis. The antigen is provisionally designated as DS-antigen (DS-Ag). The serum of another patient with haemophilia A, which was strongly positive for the DS-Ag, caused a typical case of non-A, non-B hepatitis in a chimpanzee. DS-Ag could be detected in the serum of the chimpanzee during the acute phase of the infection. The ELISA for DS-Ag reacted with neither hepatitis A or B virus antigens, nor Epstein-Barr virus or cytomegalovirus. The assay was provisionally evaluated using sera from different groups of patients. Out of 17 patients with posttransfusion hepatitis non-A, non-B, 11 were found positive in the ELISA for DS-Ag (65%). As expected, a relatively high prevalence of DS-Ag (9%) was found in patients with haemophilia, who are regularly treated with blood-clotting factor-concentrates. Antibodies to DS-Ag were found in 48% of these patients. The DS-Ag was found in 8 of 1400 (0.6%) volunteer blood donors, and antibody to DS-Ag in 3% of the sera. Remarkably, a high incidence (41%) of antibodies to DS-Ag was found in prostitutes, suggesting that this antigen may be transmitted by a sexual route. The DS-Ag was pelleted by ultracentrifugation for four hours at 100,000g and was found to have a buoyant density of 1.32 g/cm3 in a CsCl gradient.     

Post-transfusion hepatitis: antigen/antibody systems correlated with non-A, non-B hepatitis

In seven patients post-transfusion hepatitis (PTH) was due to non-A, non-B virus(es) (38.9% of all PTH, while 61.1% were due to hepatitis B virus (HBV). No clinical or biochemical differences were observed in non-A, non-B PTH when compared with PTH due to HBV, while incubation period was very ample, from 15 days to nine months (generally 45 days to two months). An antigen/antibody system was shared by five of our patients (their sera showed precipitin lines when assayed by immunodiffusion with known sources of antigen or antibody), while in one patient an antigen/antibody system was detected when onset serum was assayed with self-recovery serum but not when assayed with known sources of antigen and antibodies, nor with sera of the other five patients. Antigen was detected during the first weeks of illness, antibody at recovery, for both systems. The results suggest that there may be at least two antigen/antibody systems correlated to non-A, non-B hepatitis not necessarily linked to incubation period.     

AN6520 Ag: an antigen purified from liver with non-A, non-B hepatitis

An extract prepared from the liver of patient with chronic non-A, non-B (NANB) hepatitis was found to produce a precipitin line in immunodiffusion with a serum from a multiply transfused patient and those from patients convalescent from NANB hepatitis. The antigen was purified by gel filtration and density gradient centrifugation. The antigen had a buoyant density of 1.16-1.20 g/cm3 in cesium chloride, a sedimentation coefficient (S20,w) of 51.5, and a molecular weight of larger than 1.5 X 10(6) daltons. Electron microscopic examination revealed particles 29-34 nm in diameter (average 31.5 nm), which could be agglutinated by the specific antiserum. We developed a reverse passive hemagglutination (R-PHA) and a passive hemagglutination (PHA) technique for detection of the new antigen and antibody, respectively, and applied these to human sera. Antibody to the antigen was detected in 19/28 (67.9%) convalescent sera of NANB hepatitis. This prevalence was significantly higher than those found in convalescent sera of type A hepatitis patients (2/17 = 11.8%), type B hepatitis patients (2/15 = 13.3%), and normal blood donors (9/129 = 7.0%) (p less than 0.01); and the prevalence in hepatitis A and B patients did not differ significantly from that of normal donors. Furthermore, most (66.7%) of the cases of NANB hepatitis endemic in Shimizu City, Japan, showed clear seroconversion with respect to this antibody. These results suggest that the new antigen/antibody system is associated with NANB hepatitis.     

Non-A,non-b hepatitis virus: identification of a core antigen-antibody system that cross reacts with hepatitis b core antigen and antibody

Using immunodiffusion, a major cross-reactivity had been previously demonstrated between hepatitis B(HBe/3 Ag) and the antigen reported in the serum of non-A, non-B hepatitis patients, therefore redesignated NANBe Ag. By direct immunofluorescence a new antigen associated with, but distinct from, NANBe Ag has now been identified in the liver of 14/26 patients with NANB chronic active hepatitis. The homologous antibody was detected in the serum of these 14 patients. Behaving like HBc Ag and cross reacting with it by immunofluorescence, the new antigen was termed NANBc Ag. Anti NANBc also became detectable in serial acute phase and convalescence sera from 5/5 NANB Ag-positive posttransfusion hepatitis cases. Further characterization of NANBe and NANBc antigens achieved by fractionation of a NANB virus-infected liver showed NANBc Ag to be expressed on 22–25 nm HB core-like particles containing DNA polymerase activity. Cross-reactivity between NANBc and HBc antigens was confirmed by immunodiffusion. Liver-derived NANBe Ag identical to serum NANBe Ag exhibited the same physical properties as HBe/3 Ag and could be similarly released by disruption of the non-A, non-B virus cores. These results indicate that hepatitis B and NANB virions not only share the same structure and DNA polymerase activity but are also antigenically related and belong to the same new class of DNA viruses.     

An antigen/antibody system specific for an epidemic non-A, non-B hepatitis in patients of a cardiovascular surgical unit

The antigen/antibody systems specific for non-A, non-B hepatitis (NANB) were studied using urine samples as the antigen source and sera for the antibody source. Two immunologically distinct systems, SO-antigen/anti-SO and MI-antigen/anti-MI were discovered. This paper deals chiefly with the characterization of the SO-antigen, which was associated with an epidemic-type non-A, non-B hepatitis found during September 1979 to February 1980 (first outbreak) and October 1980 to January 1981 (second outbreak) in the Cardiovascular Surgical Unit of Tohoku University Hospital. All patients who developed non-A, non-B hepatitis during the first and second epidemic periods had SO-antigen in their urine (24 out of 24). After the epidemic, however, the detection rate of SO-antigen gradually decreased among patients in the same unit, although posttransfusion non-A, non-B hepatitis continued to be found. The final detection of SO-antigen occurred at or just after the elevation of alanine aminotransferase (ALT) levels during the episode of hepatitis and persisted in most cases throughout the elevated period. Anti-SO antibody was detected relatively late (eight months after blood transfusion, in most cases) and apparently persisted longer than five years. The immunological and physicochemical properties of SO-antigen were also studied. It appeared to be neither a plasma protein nor a liver tissue component when the cross-reactivity of SO-antigen was examined by the immunodiffusion method. Absorption with insolubilized human serum and liver tissues failed to affect the anti-SO antibody activity. The molecular weight of SO-antigen was estimated to be 250,000, the sedimentation coefficient to be 11.0 S, and the buoyant density in CsCl to be 1.215 g/cm3. Electron microscopy showed that the SO-antigen corresponded with uniform particles with a mean diameter of 11 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of SO-antigen revealed only a single protein band corresponding to molecular weight 38,000.     

Diagnostic value of anti-HBc IgM in high HBV prevalence areas

The diagnostic value of an anti-mu-capture immunoassay for the detection of IgM antibody against hepatitis B core antigen (anti-HBc) was evaluated. Strongly positive results were obtained from the acute phase sera of the 25 acute hepatitis B patients who were hepatitis B surface antigen (HBsAg) positive and of the 18 confirmed acute hepatitis B patients who had already cleared HBsAg when symptoms developed. Negative results were obtained in 5 hepatitis A patients, 20 non-A, non-B acute hepatitis patients serologically susceptible to HBV, 22 patients with chronic hepatitis B liver disease, 15 asymptomatic HBsAg carriers, and 10 healthy patients immune from past HBV infection. Fourteen of the acute hepatitis patients remained HBsAg positive for a follow-up period of at least 6 months, and 12 of these were found consistently anti-HBc IgM negative. These were considered as chronic HBsAg carriers with a superimposed form of acute liver injury. These data show that this assay can differentiate between acute from chronic (HBsAg positive) and recent from old (HBsAg negative) hepatitis B virus infection. Thus, it should be very useful in the complex diagnostic situations encountered commonly in areas with high prevalence of HBV infections.     

Etiology and outcome of acute viral hepatitis in Korean adults

One hundred and sixteen Korean adults with biopsy-proven acute viral hepatitis were studied to determine the etiology and the outcome of the disease using paired sera obtained during acute and convalescent phases. The prevalence of acute viral hepatitis A, B, D and non-A non-B were 3.4%, 60.3%, 0.9% and 35.3%, respectively: hepatitis B virus infection was the most common cause and the hepatitis D virus superinfection was almost negligible. Only eleven (26.8%) of 41 patients with AVH NANB were negative for all serological markers of HBV. The rest (73.2%) were positive for at least one HBV marker: HBsAg was positive in 31.7%. Therefore, the presence of HBV serologic markers in the sera does not exclude the diagnosis of AVH NANB in Korea. In patients with acute viral hepatitis B, 27% remained positive for HBsAg. Chronic hepatitis developed in 12.8% and 17% patients with acute hepatitis B and non-A non-B, respectively. Progression to chronic hepatitis in patients with acute viral hepatitis B and non-A non-B occurred more commonly, although statistically not significant, in male sex and in patients who did not have clinical jaundice during the acute phase and who showed bridging necrosis in their liver biopsies. Age did not influence the progression to chronic hepatitis.     

Non-a, non-b hepatitis: identification of hepatitis-B-like virus particles in serum and liver

Hepatitis B virus-like particles including: small spheres and filaments 15--25 nm in diameter together with a 35--40 nm Dane particle-like virion have been identified in sera of patients with non-A, non-B hepatitis. In a coded serological study, such particles were detected transiently in 3/4 acute, and persistently in 7/8 chronic cases of non-A, non-B hepatitis with non-A, non-B antigenemia. Only 2/12 similar cases without non-A, non-B antigens (Ag) in serum had detectable particles but neither patients with drugs, or type A hepatitis, nor cases of obstructive jaundice. The particles did not express hepatitis B surface (HBs) or non-A, non-B Ag at their surface but were associated, in three patients, with significant endogenous DNA polymerase activity. Furthermore, particles similar to hepatitis B cores (BHc) and also associated with DNA polymerase activity were demonstrated by sucrose gradient ultracentrifugation of a liver homogenate obtained from a patient who had died of non-A, non-B hepatitis. The non-A, non-B hepatitis virion described here appears, therefore, as a hepatitis B-like virus. The exact kinship between these two agents is currently being investigated.     

Determination of immunoglobulin M antibodies against hepatitis B core antigen and hepatitis A virus by reorienting sucrose gradient high-speed centrifugation for diagnosis of acute viral-hepatitis

Immunoglobulin M (IgM) antibodies against hepatitis B core antigen (anti-HBc) and hepatitis A virus (anti-HAV) were determined in 41 cases of acute viral hepatitis. In sera positive for anti-HBc or anti-HAV, IgM was separated from IgG by reorienting sucrose gradient high-speed centrifugation, and the IgG- and IgM-containing serum fractions were tested for the presence of specific antibody by radioimmunoassay. At the onset of illness, 4 of the 41 cases were classified as hepatitis A, 31 were hepatitis B, and 6 were non-A, non-B hepatitis, based on the results of these tests and of assays for hepatitis B surface antigen and antibody and hepatitis B e antigen and antibody. Fourteen of these 41 patients (34%) required IgM anti-HBc or IgM anti-HAV testing or both for appropriate classification. IgM anti-HBc persisted for at least 7 weeks after onset but no longer than 17 weeks in all patients tested with transient hepatitis B surface antigen-positive acute hepatitis. IgM anti-HAV persisted up to but not longer than 62 days in the patients with hepatitis A. Therefore, IgM anti-HBc and IgM anti-HAV determinants are valuable tools for the differential diagnosis of acute A, B, and non-A, non-B hepatitis.     

Toga virus-like particles in acute liver failure attributed to sporadic non-A, non-B hepatitis and recurrence after liver transplantation.

Toga virus-like particles (typically 60-70 nm: enveloped with small surface spikes) were detected in the native hepatectomy specimens in 7 of 18 patients grafted for acute liver failure attributed to sporadic non-A, non-B hepatitis and in 2 patients grafted for fulminant hepatitis attributed to anti-epileptic drug hepatotoxicity. These particles were not detected in the hepatectomies from 12 other patients grafted for other causes of acute liver failure, 12 for various chronic liver diseases, and 2 histologically normal livers. Acute hepatic failure, characterized histologically by severe haemorrhagic necrosis, developed 7 days after grafting in 5 patients, all in the non-A, non-B group with toga virus-like particles in native liver. Similar virus-like particles were detected in all grafts and were in greater abundance than in the native livers. The agent may be novel because pre- and post-grafting sera were negative for antibodies against representative panels of arboviruses and in first and second generation antibody tests for hepatitis C virus.     

Detection of an antigen (AN6520), possibly related to non-A, non-B hepatitis, by monoclonal antibodies. I

The antibody to AN6520 antigen, which was isolated from the liver of a patient with non-A, non-B hepatitis (NANBH), has been detected frequently in convalescent sera from patients with NANBH by the passive hemagglutination (PHA) test. In a further study, we established hybridoma cells secreting antibodies against AN6520 antigen and obtained ascitic fluids with PHA titers ranging from 1:10(5) to 1:10(7). In immunodiffusion with AN6520 antigen, all monoclonal antibodies were found to form an identical precipitin line. These lines were also identical to those formed by rabbit antiserum against AN6520 antigen and by convalescent sera from patients with NANBH. With one of the monoclonal antibodies, 1-F12, solid-phase radioimmunoassay (SP-RIA) for detecting AN6520 antigen was developed as well as blocking RIA for anti-AN6520 antibody detection. The antigen assay was 50 times more sensitive than the reverse passive hemagglutination (R-PHA) test, with a sensitivity threshold of the 1 ng/ml of antigen solution; the antibody assay was 10 times more sensitive than PHA. The results with this blocking RIA were mostly in agreement with the data obtained by PHA. Furthermore, the antigen in human sera, which had never been detected by R-PHA test, could be detected by SP-RIA.     

Spumaviruses isolated from sources containing agents of non-A, non-B (NANB) hepatitis do not cause NANB hepatitis

Serum and liver tissue containing infective non-A, non-B hepatitis virus were shown to contain a retrovirus-like agent that replicated when inoculated into chimpanzee liver cell cultures in vitro. The virus appeared to assemble its core particles in association with tubular structures reminiscent of those characteristically seen in non-A, non-B hepatitis virus-infected chimpanzee liver in vivo, and produced syncytial cytopathic effects in a number of continuous and a primary mammalian liver cells. The agents were neutralized by acute and convalescent sera from human and chimpanzee cases of non-A, non-B hepatitis, as well as by antisera against simian spumavirus type 7, but not type 6. Aluminum chloride failed to abolish viral infectivity. There was no evidence of virus replication or hepatitis in chimpanzees inoculated with a seventh passage of one of the isolates. Thus the data suggest that the isolates are not causally related to non-A, non-B hepatitis, as was previously postulated.     

Hepatitis with isolated serum antibody to hepatitis B core antigen. A variant of non-A, non-B hepatitis?

Antibody to hepatitis B core antigen (anti-HBc) has previously been recognized to be a sensitive marker of hepatitis B virus (HBV) infection. In addition, anti-HBc has recently been suggested to be a surrogate marker for non-A, non-B hepatitis agents in donated blood. The authors studied prospectively the HBV antigen and antibody status in four patients with chronic hepatitis and persistent presence of isolated anti-HBc in their sera. The serologic and histopathologic findings of these four patients were compared with those of three groups of patients having chronic hepatitis with or without HBV markers. A low concentration of serum HBV DNA was detected in only one of the four patients with hepatitis with isolated anti-HBc and in another patient with previous HBV infection. HBV antigens and HBV DNA were not detected in the sera and liver biopsies from the remaining patients with hepatitis with isolated anti-HBc and other patients with hepatitis with or without serologic markers of previous hepatitis A or HBV infection. In contrast, all patients with chronic HBV-associated hepatitis had detectable HBV DNA, hepatitis B surface antigen (HBsAg), and hepatitis B e antigen (HBeAg) in their sera and/or liver biopsies. These findings suggest that chronic hepatitis associated with isolated anti-HBc is a heterogenous pathologic entity. The condition of some of these patients may represent a variant of non-A, non-B hepatitis, whereas the remaining patients are chronic hepatitis B carriers with low serum concentrations of HBV.     

Autoantibodies against liver cell membrane antigens detected by enzyme-linked immunosorbent assay in acute and chronic liver disease

An enzyme-linked immunosorbent assay was developed to detect serum antibody binding to liver membrane antigen derived from human hepatoma cell line SK-Hep-1. When we tested sera from 214 patients with this assay, IgM antibodies were detected in 100% of patients with acute type A, but not with type B or non-A, non-B hepatitis. IgM antibodies were also found in highest frequency (76%) and titer in patients with autoimmune chronic active hepatitis (CAH) among chronic liver disease groups. IgG antibodies occurred in over 50% of patients with acute type A hepatitis, type B chronic active liver disease (CALD), and autoimmune CAH. IgA antibodies were present in 43% of the patients with alcoholic liver disease, but were also seen in other patient groups. When freshly isolated rat hepatocytes were used as target cells, prevalences and titers similar to those obtained with SK-Hep-1 were found. The levels of serum membrane binding antibody were significantly reduced by the addition of human liver-specific membrane lipoprotein in all patient groups. In particular, IgM antibodies became negative in over 50% of patients with CALD (both type B and non-A, non-B) and autoimmune CAH, whereas in acute hepatitis over 50% lost their positivity for IgG antibody. These results indicate that circulating liver membrane binding autoantibodies are heterogeneous, occurring in hepatitis virus-induced acute and chronic liver disease as well as in autoimmune CAH.     

Case report: Role of hepatitis E virus in the etiology of community-acquired non-A,non-B hepatitis in greece

The aim of this study was to determine the frequency of hepatitis E virus (HEV) infection in a population of Greek adults with community-acquired (sporadic) non-A, non-B hepatitis found to be seronegative for antibodies to hepatitis C virus (anti-HCV). All patients admitted to the Liver Unit of Western Attica General Hospital and diagnosed as having acute community-acquired non-A, non-B hepatitis between February, 1986, and May, 1990, were enrolled in follow up studies (n = 66). Nineteen patients with HCV infection and 11 patients with acute non-A, non-B, non-C hepatitis that progressed to chronicity were excluded. Convalescent sera were tested for antibody to HEV (anti-HEV) by a fluorescent antibody blocking assay in 33 of 36 eligible patients. One of the 33 (3%) patients was found to be positive for anti-HEV. Anti-HEV testing of all 20 available serum specimens from this patient showed evidence of anti-HEV seroconversion at the fourth week after the onset of hepatitis. The patient had not travelled abroad or within Greece or had not had apparent contact with people from foreign countries for the previous 3 months. These data show that HEV infection is not a major cause of community-acquired non-A, non-B hepatitis in Greece. However, the reported case of HEV hepatitis suggests that HEV may retain a low endemicity in Greece. More extensive seroprevalence studies are needed for an accurate estimation of the extent of HEV infection in the southeastern European countries. © 1994 Wiiey-Liss, Inc.     

Acute sporadic hepatitis E in children living in Cairo, Egypt.

Seventy-three pediatric patients with acute hepatitis and 19 control patients without liver disease living in Cairo, Egypt, were evaluated with a newly developed Western blot assay for IgM antibody to hepatitis E virus (IgM anti-HEV). The mean age of acute hepatitis patients was 6.4 years (range, 1-13 years); 56% were male. Among the 73 acute cases, hepatitis A was diagnosed in 30 (41%), possible acute hepatitis B in three (4%), hepatitis E in nine (12%), and by exclusion, non-A, non-B hepatitis in 29 (40%). Two additional acute cases were positive for both IgM anti-HAV and IgM anti-HEV. None of the 19 control subjects had IgM anti-HEV. Parenteral risk factors were associated with cases of non-A, non-B hepatitis but were not associated with acute hepatitis E. Contact with a family member with jaundice was associated with acute hepatitis A. In contrast to prior epidemics of enterically-transmitted non-A, non-B hepatitis, HEV was found to be a common cause of acute hepatitis in a pediatric population. This study provides additional evidence that HEV may be a frequent cause of acute sporadic hepatitis among children living in some developing countries.     

Excretion of hepatitis A virus in the stools of hospitalized hepatitis patients

A study was carried out to determine whether hepatitis A virus (HAV) can be detected in the stools of patients hospitalized for HAV infection. Acute phase samples of whole blood and stool, as well as completed questionnaires, were obtained from 31 patients hospitalized at any of 13 hospitals in the Phoenix metropolitan area. Blood specimens were tested for hepatitis B surface antigen (HBsAg), IgG antibody to HAV (IgG anti-HAV), and IgM antibody to HAV (IgM anti-HAV). Stools were tested for HAV by radioimmunoassay. Five patients (16.1%) had acute hepatitis B, five (16.1%) had acute non-A/non-B hepatitis, and 21 (67.7%) had acute hepatitis A. Of these 21 patients with acute hepatitis A, 11 (52.4%) were found to have HAV in their stools. These results confirm the potential for infectivity of stools of patients hospitalized for hepatitis A and emphasizes the need for caution when dealing with such stools.     

Studies on transmission of human non-A, non-B hepatitis to marmosets

Two sera obtained from four healthy blood donors, which caused non-A, non-B post-transfusion hepatitis in two recipients, were experimentally inoculated into nine marmosets. Three of seven marmosets developed acute hepatitis characterized by the elevation of serum concentrations of glutamic pyruvic transaminase (GPT) and/or isocitric dehydrogenase (ICD) 8-11 weeks after inoculation. Four of seven showed histopathological changes of acute hepatitis in liver biopsy specimens during the biochemically acute phase. In electron microscopic examination, attached membrane-like structures, which consisted of two-unit membranes of two neighboring endoplasmic reticula with electron-dense material between them, were noted in cytoplasm of hepatocytes during the acute phase of hepatitis. Furthermore, acute-phase sera obtained from two animals were inoculated into four additional marmosets, and non-A, non-B hepatitis was successfully passaged in two of them. The results of this study indicate that certain species of marmoset monkeys are susceptible to human non-A, non-B hepatitis agents and provide a useful animal model for non-A, non-B hepatitis.