Lack of susceptibility of congenitally athymic nude mice to human non-A, non-B hepatitis

Nude mice were inoculated intravenously with chimpanzee serum containing a human non-A, non-b hepatitis agent. Control groups of nude mice were inoculated with normal saline or normal chimpanzee serum. During 77 days of observation, evidence of non-A, non-B hepatitis was not detected. Serum alanine aminotransferase levels remained within normal limits, and normal liver histology was seen in serially killed mice.     

Primate animal models and titered inocula for the study of human hepatitis A, hepatitis B, and non-A, non-B hepatitis

Although many primate species have been inoculated with the agents of human hepatitis A, B, and non-A, non-B, only a small number of species have been shown to be susceptible, and only the chimpanzee (Pan troglodytes) has been shown to be reproducibly susceptible to all three types of human hepatitis. Infectious inocula containing each agent have been identified in different laboratories and the end-point titers of infectivity determined, in most cases by inoculation of chimpanzees. These inocula and the nonhuman primate models have permitted investigators to study the inactivation of these agents and to evaluate passive and active immunization against the agents.     

Ultrastructural alterations in serial liver biopsy specimens from chimpanzees experimentally infected with a human non-A, non-B hepatitis agent

Four chimpanzees experimentally infected with an agent of human non-A, non-B hepatitis were studied to determine the sequence of ultrastructural alterations in hepatocytes during infection. Three of the four types of cytoplasmic alterations previously described in association with non-A, non-B hepatitis were observed in the hepatocytes. Sponge-like cytoplasmic inclusions (designated C-I) were detected at or near the time of peak serum aminotransferase elevations in two of the four chimpanzees. Undulating membranes (designated C-II) were observed in all four chimpanzees, at the time of the first elevation of serum aminotransferase levels. Cytoplasmic tubules (designated C-III) were first observed four, eight, and twelve weeks, respectively, after inoculation in three of the chimpanzees. Four weeks after the peak of serum aminotransferase elevations, cytoplasmic alterations could no longer be detected in hepatocytes of the four chimpanzees. Intranuclear inclusions consisting of 20-27 nm granules and vermicular particles were observed in hepatocytes from preinoculation liver biopsy specimens, as well as biopsies obtained during non-A, non-B hepatitis. The number of these particles was greatest near the time of peak elevation of serum aminotransferase levels, however. Tubulo-crystalline inclusions were noted as well in the endothelial cells from both preinoculated and infected chimpanzees. Cytoplasmic alterations in hepatocytes of chimpanzees experimentally infected with an agent of non-A, non-B hepatitis appear characteristic of infection with this agent. In contrast, intranuclear particles were not specifically related to the non-A, non-B hepatitis infection.     

A cDNA fragment of hepatitis C virus isolated from an implicated donor of post-transfusion non-A, non-B hepatitis in Japan.

Recently, a cDNA from the hepatitis C virus (HCV) RNA genome has been isolated in the USA from a chronically infected chimpanzee. In order to isolate HCV cDNA derived from human material, RNA was extracted from plasma of a Japanese blood donor implicated in post-transfusion non-A, non-B hepatitis and HCV cDNA was synthesized and amplified by the PCR method using HCV-specific oligonucleotide primers. The cDNA fragment, 583 nucleotides long, showed 79.8% homology at the nucleotide level and 92.2% homology at the amino acid level compared with the prototype HCV cDNA. These results provides further evidence to show that HCV is closely associated with the development of post transfusion non-A, non-B hepatitis.     

Non-A, non-B hepatitis-related hepatocellular carcinoma in a chimpanzee

Epidemiology has indicated the possible association of non-A, non-B hepatitis (NANBH) with hepatocellular carcinoma (HCC) in man, but there are no means for confirmation. Chimpanzees are recognized models for studying hepatitis B and NANBH, and may become carriers of both. The first case of HCC to be reported in chimpanzees was found after longitudinal study of a hepatitis B-free chimpanzee 7 years after inoculation with human plasma from a patient reported to have chronic NANBH.     

Blood transfusion and hepatitis: still a threat?

The incidence of post-transfusion hepatitis (PTH) in recipients of blood products is reviewed. PTH was observed in 10%-12% of recipients of blood products in the United States, 2%-4% in northern Europe and 15%-20% in southern Europe. All studies indicate that 80%-90% of all PTH cases are attributed to non-A/non-B. At least 40% of the patients with PTH non-A/non-B will develop chronic hepatitis or cirrhosis. No specific tests for the detection of the non-A/non-B agent(s) exist. However, several independent studies indicate that part of the donors carrying the infectious non-A/non-B agent have increased levels of alanine amino transferase (ALT). When donors are excluded with elevated ALT values, it is estimated that about 30% of the PTH non-A/non-B cases would be prevented. Some studies indicate that anti-hepatitis B core (anti-HBc) positive donors may carry an increased risk to transmit the non-A/non-B agent, but more recent studies do not confirm this. There is hope that a specific non-A/non-B test will be developed soon.     

Association of human hepatocellular membrane fusions with non-A, non-B hepatitis

Liver biopsies from patients with alcoholic hepatitis, chemical hepatitis, or viral hepatitis types A, B, or non-A, non-B were examined by electron microscopy. Circular, fused, cytoplasmic membranes were observed in hepatocytes of 17% of patients with hepatitis type B and 92% of patients with hepatitis type non-A, non-B. The membrane alterations were not observed in hepatocytes of patients with the other types of hepatitis. The greater frequency of altered cytoplasmic membranes in hepatocytes of patients with non-A, non-B hepatitis was shown to be statistically significant (p less than 0.05) when compared to that in patients with viral hepatitis type B.     

A cDNA clone closely associated with non-A, non-B hepatitis.

A lambda gt11 cDNA library was constructed from RNA purified from hepatitis B viral surface antigen-negative human plasma with high alanine aminotransferase activity. A cDNA clone, designated as C8-2, was isolated by immunoscreening with mixed sera from non-A, non-B hepatitis (NANBH) carrier and convalescent chimpanzees. The recombinant protein produced by C8-2 reacted specifically with sera of patients in the chronic phase of NANBH. The sequence of C8-2, 269 bp, did not hybridized with any human or chimpanzee genomic DNA, and had no homology with those of primates and viruses. The existence of this sequence in RNA of possibly infectious plasma was shown by RNA blot hybridization and by Southern blot analysis of products amplified by the polymerase chain reaction. These results strongly suggest that C8-2 is derived from the agent of this viral hepatitis.     

Lymphocyte and neutrophil dysfunction associated with hepatitis B virus and hepatitis non-A, non-B virus infection in the chimpanzee.

Chimpanzees were examined for the effect of viral hepatitis infections on specific and nonspecific immune response mechanisms. The data suggest that infection with either hepatitis B virus or hepatitis non-A, non-B virus may result in suppression of cellular immune response components. Mitogen-induced lymphocyte proliferation was lower in virus-infected chimpanzees than in naive animals. Neutrophils from virus infected animals exhibited decreased or altered chemiluminescence kinetics.     

Occurrence of non-A, non-B post-transfusion hepatitis in heart surgery. Prospective study on the value of the determination of serum alanine aminotransferase and detection of anti-HBc antibodies in blood donors

We studied a group of 64 patients undergoing cardiac surgery for the occurrence of post-transfusion hepatitis during a follow-up period of 5 months. They received blood units (packed red cells in saline-adenine-glucose medium and/or fresh frozen plasma exclusively) from 447 volunteer donors. Post-transfusion hepatitis was identified in 5 patients: 1 patient had cytomegalovirus hepatitis and the remaining 4 cases were defined, by exclusion, as non-A, non-B hepatitis (with prevalence and incidence rates of 80% and 6.25% respectively). We found no statistically significant differences between the numbers of transfused blood product units in patients who developed non-A, non-B hepatitis as compared to those who did not. Our analysis of the predictive effectiveness of alanine aminotransferase and anti-HBc antibodies screening in blood donors to prevent non-A, non-B post-transfusion hepatitis led to the following conclusions: we failed to confirm the association between anti-HBc in blood donors and enhanced risk of non-A, non-B hepatitis in recipients since no case developed among patients receiving blood products from anti-HBc positive donors. So, 20 donors (4.5%) would have been discarded without any reduction of the incidence of non-A, non-B hepatitis. we could not confirm nor exclude the possibility that screening donor blood for elevated alanine aminotransferase levels would have reduced the number of non-A, non-B hepatitis in recipients.     

Isolation of the novel agent from human stool samples that is associated with sporadic non-A, non-B hepatitis.

The agent(s) responsible for sporadic non-A, non-B hepatitis in humans was serially transmitted in rhesus monkeys by intravenous inoculation of the stool extract from a patient. A novel agent called HFV (hepatitis French [origin] virus) was present as 27- to 37-nm particles in the infectious stool extract. Hepatopathic lesions were noticed in infected monkeys during the acute phase of illness. The purified viral 27- to 37-nm particles consist of a double-stranded DNA of approximately 20 kb and are detected in infected monkey liver. Analysis of cell culture detects the approximately 20-kb-long viral DNA in stool samples from infected monkeys and sporadic enteric non-A, non-B hepatitis patients. Furthermore, the 27- to 37-nm viral particles were able to protect monkeys challenged with infectious stool extract. Our results indicate that 27- to 37-nm virus like particles are responsible for sporadic non-A, non-B hepatitis in rhesus monkeys.     

The clinical chemistry of chimpanzees: II. Gamma glutamyl transferase levels in hepatitis studies

Normal ranges for gamma glutamyl transferase (GGT) in chimpanzees were determined and categorized according to age and sex. Enzyme patterns presented for 36 cases of non-A, non-B (NANB) hepatitis and compared to others with hepatitis A and/or B show that the response of this enzyme to these viral agents in chimpanzees is comparable to that seen in human patients. The value of GGT determinations, in addition to aspartate aminotransferase and alanine aminotransferase for the differentiation of various types of viral hepatitis, is described.     

The territorial and age-related characteristics of the distribution of patients with non-A, non-B viral hepatitis infected via the fecal-oral mechanism in foci of elevated viral hepatitis morbidity in the Uzbek SSR

The study of patients from 10 foci of acute viral hepatitides for the presence of HBsAg (in the passive reverse hemagglutination test) and anti-hepatitis A virus IgM (in the radioimmunoassay) has shown high frequency and variability in the spread of hepatitis non-A, non-B, the prevalence of adults aged 20-29 years and children aged 2-4 years among persons involved into the epidemic process and the tendency towards an increase in the proportion of hepatitis non-A, non-B in the total number of cases of viral hepatitides in the republic.     

Incidence of hepatitis non-A,non-B compared with types A and B in hospital patients.

To establish the relative frequencies of types A, B and non-A, non-B hepatitis, stored samples of blood from all the cases of acute viral hepatitis seen over a period of 9 years in a general hospital for adults were classified according to their type by presently available serologic methods. The study included 456 episodes of hepatitis in 447 patients, distributed as follows: 114 episodes of hepatitis A (25%), 282 of hepatitis B (62%) and 60 of hepatitis non-A, non-B (13%). The episodes of non-A, non-B hepatitis were equally distributed between the sexes, suggesting a mode of transmission different from that of hepatitis A or B, which had male/female ratios of 2.4 and 3.1 respectively. The low proportion of hepatitis non-A, non-B may not reflect its real frequency, since it often escapes clinical recognition.     

Analyses of amino acid sequences in hypervariable region-1 of hepatitis C virus (HCV) in sera from chimpanzees infected three times with the same HCV strain

Background  To investigate whether or not the same strain of hepatitis C virus (HCV) can twice re-infect the same chimpanzee, we analyzed nucleic and amino acid sequences in HCV hypervariable region-1 (HVR1). Two chimpanzees were inoculated, three times each, with the same HCV strain during the 1983–1991. After each inoculation, chimpanzees developed acute hepatitis C, and then recovered.
Methods  Using sera, HVR1 cloning and antibody to HVR1 major clone measurement were performed.
Results  Clones from the first inoculum were divisible into major and minor types. Clones from the second and third inocula, as well as all post-inoculation sera, were essentially identical to the major type. Titers of antibody to HVR1 major clone were consistently low in pre- and post-inoculation sera.
Conclusions  Both chimpanzees were re-infected twice with the same strain of HCV. The sequences from the second and third infections were similar to the major sequences in the first inoculum.     

Mapping of linear B cell epitopes on open reading frames 2- and 3-encoded proteins of hepatitis E virus using synthetic peptides

Abstract Hepatitis E virus (HEV) is the causative agent of non-A, non-B hepatitis which is transmitted by the fecal-oral route and occurs principally in the form of large epidemics and outbreaks in developing countries. Two overlapping synthetic peptides corresponding to overlapping DNA sequences of the ORF 3 of HEV genome were found to be immunoreactive with sera from patients involved in two epidemics of enterically transmitted non-A, non-B hepatitis. The results suggested the existence of two distinct epitopes. The four synthetic peptides representing these two epitopes from Burma and Mexico strains of hepatitis E virus, were used to investigate anti-HEV reactivities. HEV antibodies were detected in 84–88% of HEV-infected individuals according to the peptide used. The results suggest that a peptide-based ELISA can provide an accurate tool for the diagnosis of acute hepatitis type E.     

Characterization of a vaccinia-derived recombinant HIV-1 gp160 candidate vaccine and its immunogenicity in chimpanzees.

The human immunodeficiency virus (HIV-1) envelope glycoprotein gp160 was produced in large-scale microcarrier cultures of Vero cells, using a system involving coinfection with two recombinant vaccinia viruses. The immunogenicity of this material was studied in conjunction with a number of different adjuvant formulations, and chimpanzees were then immunized with gp160 in conjunction with Al(OH)3, Al(OH)3 and sodium deoxycholate, and a lipid-based adjuvant. The Al(OH)3-gp160 vaccine formulation elicited very poor immune responses in two chimpanzees, and these animals were further immunized with gp160 in conjunction with a lipid-based adjuvant. Immunization with the latter formulation lead to induction of high-titer neutralizing antibodies, and, following challenge with HIV-1, one chimpanzee demonstrated no evidence of virus infection over a period of 3 years. The second chimpanzee, which had previously been infected with non-A, non-B hepatitis, and two animals immunized with gp160 with Al(OH)3 and deoxycholate were not protected against challenge.     

Prospective study of post-transfusion hepatitis after cardiac surgery in a British centre.

A series of 248 consecutive patients undergoing cardiac surgery were examined in a prospective study of post-transfusion hepatitis in a single British centre. Patients received a total of 1796 units of blood or blood products (mean blood transfusion 6.28 units per patient). During five to 30 days after operation 38 of the patients showed an increase in serum transaminase activities. There was no serological evidence for fresh infection by hepatitis A or B virus, cytomegalovirus, Epstein-Barr virus, or herpes virus in any of these patients. The increase in transaminase activities was unexplained and reached over 100 IU/l (normal less than 40 IU/l) in six patients. The incidence of acute short incubation post-transfusion non-A, non-B hepatitis was therefore thought to be 2.4%. These six patients had normal liver function six months after transfusion but a further two of the surviving 228 patients had raised serum transaminase activities at six months. In one of these, liver biopsy disclosed chronic persistent hepatitis; in the other, alcoholic liver disease was suspected. The incidence of significant chronic liver disease after blood transfusion possibly attributable to a non-A, non-B hepatitis agent was therefore only 0.4%.     

Prevalence of hepatitis C virus antibody among healthy blood donors and non-A, non-B hepatitis patients in Thailand

With the antigen expressed in yeast from a cDNA clone encoding a non-structural region of newly discovered hepatitic C virus (HCV) genome, the prevalence of HCV antibody in people in Thailand was investigated. Antibody was detected in 2.6% of healthy blood donors and in 2.8% of healthy pregnant women. These prevalence rates were higher than those reported previously from Japan, USA and European countries. Among community-acquired, sporadic cases of acute and chronic non-A, non-B hepatitis, however, only 5.7% and 15.4% were shown to possess the antibody, respectively. Among hepatocellular carcinoma patients who were negative for hepatitis B surface antigen in the sera, 11.1% had antibody to HCV. These seroepidemiological data suggest that HCV plays an important role as an etiological agent in Thailand; however, other agents must also be involved in etiologic agents of viral hepatitis and chronic liver disease.     

Protein F. A novel F(ab)-binding factor, present in normal liver, and largely released in the digestive tract during hepatitis

Significant percentages of patients suffering from non-A non-B hepatitis (43%) and B hepatitis (35%) were found to release an Ig-binding factor in their stools. This factor, which we called "protein F" was less frequently observed (20%) in patients suffering from other liver disorders, and was found in only 6.7% of healthy subjects (p less than 10(-7), less than 10(-4), and less than 0.03, respectively). The specificity of the detection test (a nonimmune ELISA-like assay) was confirmed by inhibition experiments. Binding was located on the F(ab) fragment of Ig, irrespectively of their isotype. Protein F was inactivated by pepsin, neuraminidase, and high concentrations of subtilisin, whereas it was resistant to trypsin and chymotrypsin. Molecular sieving by HPLC indicated an apparent molecular mass of 175 kDa. In preparative SDS-PAGE, the molecular mass was 85 kDa in favor of a dimer disrupted under dissociating conditions. Preparative IEF showed the isoelectric charge to lie between 3.9 and 4.1. Analysis of liver extracts from two patients suffering fron non-A non-B hepatitis, and from a transplant donor, revealed the presence of the factor in the three cases.