Transmission of non-A, non-B hepatitis agent to chimpanzees from patients of epidemic hepatitis

Two chimpanzees were inoculated intravenously with acute-phase sera obtained from two patients with epidemic hepatitis. They developed histopathologically confirmed hepatitis. Electron microscopic examination of the liver showed peculiar cytoplasmic tubular structures in the hepatocytes. These ultrastructural findings were similar to those described for the livers of chimpanzees inoculated with the F strain of non-A, non-B hepatitis agent derived from a posttransfusion hepatitis case. The chimpanzee that had recovered from hepatitis caused by the F strain of non-A, non-B hepatitis agent was re-challenged with the serum from one of the patients. The chimpanzee developed neither clinical signs nor histological changes of hepatitis. These results suggested that non-A, non-B hepatitis agent was involved not only in post-transfusion hepatitis but also in epidemic hepatitis.     

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.     

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.     

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.     

Core antigen and antibody in woodchucks after infection with woodchuck hepatitis virus

The woodchuck hepatitis virus is a naturally occurring hepatitis B-like virus that infects the eastern woodchuck. Direct immunofluorescence staining for woodchuck hepatitis virus core antigen in liver biopsies demonstrated the presence of this antigen in 14 of 17 chronically infected woodchucks, and in 8 of 10 woodchucks undergoing acute infections. Fluorescent localization of woodchuck hepatitis virus core antigen was typically cytoplasmic, and this was confirmed further by electron microscopy. Experimental infection with woodchuck hepatitis virus was achieved in four of four woodchucks inoculated with serum from chronic carrier woodchucks. All infected animals developed a self-limited disease characterized by seroconversion to antibodies against the major viral antigens (core and surface antigens); naturally acquired acute infection demonstrated a similar course. A chimpanzee seronegative for all markers of hepatitis B virus developed a subclinical infection after inoculation with woodchuck hepatitis virus.     

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%.     

A molecularly cloned hepatitis B virus produced in vitro is infectious in a chimpanzee.

The infectivity of hepatitis B virus (HBV) produced in vitro by HepG2 cells transfected with HBV DNA (HepG2T14) has been assayed in a chimpanzee. Following inoculation, the chimpanzee underwent a typical course of type B hepatitis infection, characterized by elevation of serum aminotransferases and by histological identification of hepatic damage. Hepatitis B surface antigen and core-related antigen appeared in the serum at weeks 5 and 7, respectively, after infection. HBV DNA was detected in serum samples, and replicative forms of the HBV genome were identified in liver biopsies. Subtype identification of hepatitis B surface antigen and restriction enzyme analysis of HBV DNA in both the inoculum and the serum of the infected chimpanzee confirmed that the hepatitis B infection observed in this animal was caused by viral particles produced by HepG2T14 cells. These findings indicate that, although HepG2 cells do not seem to be susceptible to infection by HBV in vitro, they can produce biologically active infectious virions after transfection with cloned HBV DNA.     

The clinical chemistry of chimpanzees. I. Determination of aminotransferase baseline values for hepatitis studies

Viral hepatitis in chimpanzees produces negligible symptomatology, and serum aminotransferase changes may be minimal. To maximize the predictive value of these determinations, which are the only serum indicators available for non-A non-B (NANB) hepatitis infection, normal ranges for aspartate and alanine aminotransferases (AST, ALT) were examined and categorized according to age and sex. Males were found to have higher values than females, and adults higher values than juveniles. The kinetic method used and the values obtained are described. Differences in methodologies and reporting units are discussed.     

Transmission of human TT virus of genotype 1a to chimpanzees with fecal supernatant or serum from patients with acute TTV infection

Fecal supernatant or serum containing TT virus (TTV) of genotype 1a (10(5) copies/ml) from patients with acute TTV infection was inoculated intravenously into two naive chimpanzees. Serum samples were obtained weekly and tested for TTV DNA by genotype 1-specific polymerase chain reaction. TTV DNA was detected in chimpanzee 228 at weeks 5-15 after inoculation with 0.5 ml of serum, and in chimpanzee 234 at weeks 7-19 after inoculation with 1 ml of fecal supernatant. The TTV DNA titer peaked at weeks 12 and 13 in chimpanzee 228 and at weeks 14-16 in chimpanzee 234. Mild biochemical and histological changes in biopsied liver samples were observed in both chimpanzees in association with the reduction in TTV titer. TTV DNA was transient in chimpanzee 228, but in chimpanzee 234 it reappeared at week 21 and persisted through week 30. These results indicate that TTV in feces is infectious and suggest that TTV has hepatitis-inducing capacity.     

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.     

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.     

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.     

Detection of core antibody in hepatitis B infection.

Hepatitis B core antigen (HBcAg) is found on the decoated Dane particle and on a morphologically similar particle detected mainly in the nucleus of hepatocytes of patients with hepatitis B. HBcAg prepared from the liver of a chimpanzee infected with hepatitis B virus was used to test human serum for core antibody (anti-HBc) by complement fixation. Anti-HBc was found in serum collected from patients with hepatitis B in both the acute and convalescent stages, from carriers of hepatitis B surface antigen (HBsAg) and from patients with chronic liver or renal disease who were carriers of HBsAg. It was not found in patients with hepatitis A or infectious mononucleosis, or in healthy persons who were not carriers of HBsAg.     

Randomised controlled trial of interferon alfa (lymphoblastoid interferon) in chronic non-A non-B hepatitis.

OBJECTIVE--To determine the effect of low dose interferon alfa (human lymphoblastoid interferon) on aminotransferase activities in chronic non-A non-B hepatitis. DESIGN--Prospective randomised controlled parallel group study of active treatment versus no treatment carried out over 16 weeks and preceded by baseline measurements at weeks 8 and 4 and time zero. SETTING--HEPATOLOGY outpatient clinics in secondary referral centres. PATIENTS--Fourteen adults with histologically proved chronic hepatitis and persistently raised aminotransferase activities for six months or more. INTERVENTIONS--Seven patients randomised to receive interferon alfa 5 megaunits (MU) daily for one week, reducing to 5 MU thrice weekly for seven weeks, then 3 MU thrice weekly for eight weeks. Controls not treated. END POINT--Control of hepatic enzyme activity in chronic non-A non-B hepatitis. MEASUREMENTS AND MAIN RESULTS--Serum aspartate aminotransferase activity remained raised in controls (mean increase in study period 23.4 U/l) but fell rapidly to normal in the treated group (mean decrease 106.4 U/l). In four cases values were normal by eight weeks and in five cases by 16 weeks. Only minor side effects were recorded (fever, myalgia), which became less common as treatment progressed. CONCLUSIONS--Continuous low dose interferon alfa reduces aspartate aminotransferase activity to normal in most patients with chronic non-A non-B hepatitis and may prevent progression to cirrhosis.     

Chimeric Mouse Model for the Infection of Hepatitis B and C Viruses

While the chimpanzee remains the only animal that closely models human hepatitis C virus (HCV) infection, transgenic and immunodeficient mice in which human liver can be engrafted serve as a partial solution to the need for a small animal model for HCV infection. The established system that was based on mice carrying a transgene for urokinase-type plasminogen activator (uPA) gene under the control of the human albumin promoter has proved to be useful for studies of virus infectivity and for testing antiviral drug agents. However, the current Alb-uPA transgenic model with a humanized liver has practical limitations due to the inability to maintain non-engrafted mice as dizygotes for the transgene, poor engraftment of hemizygotes, high neonatal and experimental death rates of dizygous mice and a very short time window for hepatocyte engraftment. To improve the model, we crossed transgenic mice carrying the uPA gene driven by the major urinary protein promoter onto a SCID/Beige background (MUP-uPA SCID/Bg). These transgenic mice are healthy relative to Alb-uPA mice and provide a long window from about age 4 to 12 months for engraftment with human hepatocytes and infection with hepatitis C or hepatitis B (HBV) viruses. We have demonstrated engraftment of human hepatocytes by immunohistochemistry staining for human albumin (30-80% engraftment) and observed a correlation between the number of human hepatocytes inoculated and the level of the concentration of human albumin in the serum. We have shown that these mice support the replication of both HBV and all six major HCV genotypes. Using HBV and HCV inocula that had been previously tittered in chimpanzees, we showed that the mice had approximately the same sensitivity for infection as chimpanzees. These mice should be useful for isolating non-cell culture adapted viruses as well as testing of antiviral drugs, antibody neutralization studies and examination of phenotypic changes in viral mutants.     

Characterization of viral genomes in the liver and serum of chimpanzee long-term hepatitis B virus carriers: a possible role for supercoiled HBV-DNA in persistent HBV infection

In chimpanzee hepatitis B virus (HBV) carriers, the molecular mechanism for viral persistence has been examined by analyzing the properties of viral DNA molecules in liver and serum. Two extrachromosomal HBV-DNA molecules migrating on Southern blots at 4.0 kb and 2.3 kb were observed in chimpanzee liver DNA. There was no evidence for integration of HBV sequences into the host genome. The HBV-DNA molecule which migrated at 4.0 kb position represents a full-length "nicked," relaxed circular form, and the DNA molecules migrating at 2.3 kb position represents a supercoiled form of the HBV genome. Evidence for supercoiled HBV-DNA in serum was obtained by production of the relaxed circular intermediate upon digestion of Dane particle DNA with specific nucleases S1 and Bal 31. A possible role of these two extrachromosomal HBV-DNA molecules in the biology of hepatitis B virus infection and the mechanism for viral persistence are discussed.     

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.     

Trichinella spiralis: inhibition of sheep hemagglutinins in mice

One hundred and twenty-four mice were injected intraperitoneally with sheep red blood cells. The mice had been previously either orally inoculated with T. spiralis (16 mice), or injected intraperitoneally during 7 consecutive days with normal saline (12 mice), normal mouse serum (6 mice), or infected mouse serum (6 mice), normal rabbit serum (6 mice), sera from lightly (36 mice) or heavily infected rabbits (36 mice), and rabbit anti-lymphocyte serum (6 mice). The homologous serum clearly demonstrated an immunosuppressive effect on the production of sheep hemagglutinins; however, it was impossible to conclude that heterologous serum has such an activity since the normal rabbit serum used as control demonstrated the same activity. The inhibition of hemagglutinin production has also been observed in mice infected with T. spiralis. The presence of a suppressive agent released by the parasite or antigenic competition is discussed as the possible mediator of immunological unresponsiveness.     

Immunoenzyme determination of IgA specific anti-HAV in the serological diagnosis of hepatitis A

A capture enzyme immuno assay for the detection in the serum of specific IgA antibodies to HAV is described. A total of 203 sera from patients and controls were tested. IgA anti-HAV were present only in sera from patients with recent hepatitis A. 23 patients were followed prospectively and IgA anti-HAV were at detectable levels for at least six months after the onset. The detection of IgA anti-HAV is proposed as an important test to differentiate hepatitis A with persistent hypertransaminasemia from non-A, non-B patients.