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GB virus B (GBV‐B) is a New World monkey‐associated flavivirus used to model acute hepatitis C virus (HCV) infection. Critical for evaluation of anti‐viral or vaccine approaches is an understanding of the effect of HCV on the liver at different stages of infection. In the absence of longitudinal human tissue samples at defined time points, we have characterised changes in tamarins. As early as two weeks post infection histological changes were noticeable, and these were established in all animals by six weeks. Despite high levels of liver‐associated viral RNA there was reversal of hepatic damage on clearance of peripheral virus though fibrosis was demonstrated in four tamarins. Notably, viral RNA burden in the liver dropped to near undetectable or background levels in all animals which underwent a second viral challenge, highlighting the efficacy of the immune response in removing foci of replication in the liver. These data add to the knowledge of GBV‐B infection in New World primates which can offer attractive systems for the testing of prophylactic and therapeutic treatments and the evaluation of their utility in preventing or reversing liver pathology.

Co-infection with HBV and HCV seems to be associated with more severe liver disease in retrospective and cross-sectional studies in adults, but no data are available when co-infection is acquired in youth. The long-term outcome of infection acquired in youth was assessed in patients co-infected with HBV and HCV and in patients with HBV infection only. Twenty-seven patients with HBV and HCV co-infection and 27 patients infected with HBV only were enrolled. Seventy-six per cent of the patients were treated with alpha-interferon for 1 year. After a median follow-up of 23 years, the annual progression rate of fibrosis was 0.07 in patients co-infected with HBV and HCV, and in those infected with HBV it was 0.07 and 0.11 (P < 0.004) for HBe and anti-HBe-positive patients, respectively. In co-infected patients, the development of cirrhosis was observed in 2 (7.4%) and of hepatocellular carcinoma (HCC) in 1 (3.7%), while in those with HBV, cirrhosis appeared in one patient (3.7%). Alcohol intake (OR = 9.5 +/- 1.2; 95% CI = 6.6-13.9; P < 0.0001) was independently associated with cirrhosis and HCC. alpha-interferon showed no efficacy during treatment, but the treated group showed higher HCV RNA clearance during post-treatment follow-up. Co-infection with HBV and HCV and single HBV infection acquired in youth showed a low rate of progression to liver fibrosis, no liver failure, and low development of HCC during a median follow-up of 23 years (range 17-40).

Acute exacerbations in HBeAg negative patients with chronic hepatitis B virus (HBV) infection are invariably associated with concurrent increases in the index of IgM class antibodies against the core protein (anti-HBc) of the virus. This study aimed to investigate whether this was related to the clearance of variants from the quasispecies pool and the appearance of new ones, with aminoacid substitutions in well recognized B-cell epitopes. In this study, 5 HBeAg negative patients (A to E) with 13 sequential serum samples (A1-A2, B1-B2-B3, C1-C2, D1-D2-D3, E1-E2-E3) were investigated after amplification of the entire core encoding region followed by cloning/sequencing studies. The sequences at different time points were compared with those from a single HBeAg positive patient with no apparent acute exacerbations. The results from sequence comparison showed that virus variants emerged in all (A2, B3, C2, D3, E2, and E3) but two (B2 and D2) subsequent sera with amino-acid substitutions affecting B-cell epitopes. It is concluded that the rise in the values of IgM anti-HBc may be attributed to the alteration of the antigenic epitopes leading to new antibody production in the majority of the cases. However, it appears that increases in IgM anti-HBc indexes in a few cases may relate to other possible mechanisms which are discussed.

Fludarabine is used widely for the treatment of chronic lymphocytic leukaemia, but not as yet implicated in the emergence of hepatitis B surface antigen (HBsAg) variants following hepatitis B virus (HBV) reactivation. Such a variant was detected in a 78-year-old female who was HBsAg(-)/anti-HBc(+)/anti-HBs(+)/anti-HBe(+), and with normal ALT levels, who developed HBV reactivation after fludarabine treatment. She had high HBV-DNA levels, and became positive for HBeAg, in the absence of detectable HBsAg. HBV-DNA was extracted from serum and the HBsAg encoding region of the genome was amplified by PCR, followed by cloning and sequencing. The HBV strain appeared to be subtype adw, but had higher nucleotide homology with ayw than adw isolates, supported further by phylogenetic tree analysis. Amino-acid sequence comparisons over the alpha determinant region revealed the following substitutions: C124N, G130R, and N146S. There were also unique substitutions outside the alpha determinant. All these mutations appeared to have a profound effect on the antigenicity of this region, which resulted in failure to detect HBsAg by commercially available diagnostic assays. It is concluded that a surface variant emerged in an HBsAg(-)/anti-HBs(+) patient with chronic lymphocytic leukaemia following fludarabine treatment, with an unprecedented number of amino-acid substitutions in the alpha determinant region of HBsAg, including a subtype switch.

To determine the pattern and significance of the HCV genetic heterogeneity before and during treatment with recombinant-2b or lymphoblastoid α-interferon, hypervariable region 1 (HVR-1) and NS5A quasispecies were characterised by cloning and sequencing in 12 HCV-1b-infected subjects. Patients were either responder-relapsers or non-responders to treatment. Extensive amino acid sequence analysis was applied to reveal the significance of HCV variation at key sites within HVR-1 and NS5A regions. Genetic complexity, genetic diversity, and the non-synonymous to synonymous substitution ratios of HVR-1 quasispecies decreased during treatment in responder-relapser patients only, and more markedly so following lymphoblastoid α-interferon. In non-responders, the HVR-1 quasispecies broadened. Amino acids G406 and Q409, which represent a major viral epitope, were highly conserved throughout treatment. Responder-relapser patients had a higher mutation frequency in NS5A than non-responders. Lymphoblastoid α-interferon promoted the selection of intermediate Interferon Sensitivity Determining Region (ISDR) sequences, whereas recombinant-2b α-interferon favoured maintenance or selection of conserved ISDR sequences. Variability upstream of the ISDR was associated with treatment response, but the amino acid substitutions conferring higher replicative ability to in vitro HCV replicons were absent in in vivo isolates. In conclusion, the pattern of HVR-1 quasispecies evolution correlates with the clinical response, and the conservation of specific amino acids may be useful for immune targeting in vivo. In responder-relapser patients, the initial HVR-1 evolution resembles that found in sustained responders. Variability within the entire NS5A, as opposed to a single region (ISDR), may have a role in influencing α-interferon treatment outcome. A differential effect of different α-interferon preparations on HCV quasispecies kinetics may exist. J. Med. Virol. 70:62–73, 2003. © 2003 Wiley-Liss, Inc.

The N-terminal end of the hepatitis C virus (HCV) envelope glycoprotein E2 contains a stretch of 27 amino acids that exhibit increased variability. This hypervariable region 1 (HVR-1), as it is normally referred to, is thought to contain epitopes that come under humoral immune attack. In the present study, 10 patients (5 children and 5 adults) with humoral immune defects and chronic HCV infection were investigated, to see how HVR-1 sequences behave over time in these patients who are unable to produce antibodies. Amplicons of this region showed little or no variation at all over time, indicating that quasispecies variation in this region is driven by the host's humoral immune response.

Hospital-acquired hepatitis B (HBV) and C virus (HCV) infections continue to occur despite increased awareness of this problem among the medical community. One hundred six patients were infected in a haematology oncology ward for children, over the time period 1996 to 2000. Serum samples from 45 such patients and 3 from infected medical personnel were used for nucleic acid amplification. HBV core, as well as HCV core and hypervariable region 1 (HVR1) nucleotide sequences, were analysed by phylogenetic tree analysis, in order to characterise the epidemiological pattern of viral transmission on the ward. Samples from 32 patients were positive for HBV-DNA or HCV-RNA by PCR. Ten patients were positive for both markers. Seventeen out of twenty-three HCV core gene sequences were found to be evolutionarily related and clustered separately from other local sequences in the phylogenetic tree, indicating nosocomial transmission. This was confirmed by analysis of HVR1 gene sequences. One nurse and one physician from the ward were HCV RNA positive, but their HCV sequences were not related evolutionarily to those of the patient cluster. Fifteen out of nineteen HBV core gene sequences were also clustered together and were positioned separately in the relevant tree. Epidemiological investigation excluded a common source infection and indicated that spread of infection was most likely due to inappropriate infection control measures on the ward. No obvious risk factors for transmission were identified during the retrospective survey in patients with related sequences, except use of multidose vials for saline and poor staff compliance with routine hand hygiene procedures. The preventive measures that were introduced reduced the incidence of infection significantly. No new cases of HBV infection and only three anti-HCV seroconversions occurred over a period of 19 months. The introduction and maintenance of strict prevention measures over a 2 year period, combined with HBV vaccination, reduced significantly the incidence of new HCV and HBV infections.

One hundred forty-two precore/core sequences were obtained from Gambian chronic hepatitis B virus (HBV) carriers and the predominant variants defined. The two point mutations, from A to T and G to A at nt positions 1762 and 1764 in the basic core promoter region, were found in only 7/99 (7%) of the samples where this region was sequenced. These mutations were found in both HBeAg-positive and -negative patients. The precore stop-codon mutation at nt position 1896 was found in 14/51 (27%) of HBeAg-negative samples, which is a lower prevalence rate in comparison with other parts of the world with high carrier rates. In HBeAg-positive patients the core amino acid sequences were conserved, but after seroconversion to anti-HBe significantly more changes were apparent. Several of the amino acid substitutions found have been described previously been in wild-type viruses of other genotypes.

T-cell responses to hepatitis B virus nucleocapsid antigens (HBcAg and HBeAg) play an important role in disease outcome in those infected with hepatitis B virus (HBV). The woodchuck is naturally infected in the wild with woodchuck hepatitis virus (WHV), which shows a high degree of genetic homology to HBV and produces a similar pattern of infection in its natural host. Twenty-three overlapping peptides were constructed to cover the entire WHV core region and used to identify immunodominant cellular epitopes in the nucleocapsid antigen using peripheral blood lymphocytes from 12 chronic WHV carrier and 4 uninfected control animals. A peripheral blood lymphocyte response was seen in all of the chronic WHV carrier animals to at least one peptide, and in 8 of the 12 chronic carrier animals a response was observed to 5 common peptides: peptide analogues of amino acids 16-30, 38-52, 50-69, 76-90 and 91-105. Peptide 91-105 produced maximal proliferation in 5 out of 12 infected animals. In addition, a difference in response was observed between wild and laboratory infected animals; the latter appeared to have a lower response to peptides than animals infected in the wild. This study provides evidence that the woodchuck has a population of peripheral blood cells which are sensitised to epitopes within the nucleocapsid protein and provides a basis on which to develop the use of the woodchuck as an immunological model of HBV infection for testing therapeutic means of enhancing this response.

Thirty-nine patients (62 sera) who, after interferon-alpha therapy for chronic hepatitis B virus (HBV) infection, were seronegative for HBeAg and HBV-DNA by dot blot hybridisation, were tested using the polymerase chain reaction (PCR) for residual viraemia. Overall, 59% of the HBsAg-positive sera and 43% of the HBsAg-negative sera were positive by PCR. All except one of the HBsAg-negative patients had seroconverted to anti-HBs. Between 13 and 18 months after therapy, 33% of the HBsAg-positive and 20% of the HBsAg-negative patients remained viraemic. Eighteen months after the end of treatment, no patient tested was positive. Twenty-three patients were tested sequentially over periods from 1 to 43 months: Thirteen lost HBV-DNA by PCR, three remained positive, five remained negative, and two patients relapsed. The merits and disadvantages of PCR for assessing interferon treatment of HBV carriers are discussed.

Antibody to the common “a” determinant of hepatitis B surface antigen (HBsAg) protects against infection with hepatitis B virus. A number of variant surface antigens with amino acid substitutions within the “a” determinant have been described in patients around the world. Both wild type and variant HBsAgs were expressed in the yeast Pichia pastoris and the antigens were semi-purified and quantitated. The effect on antigenicity of these changes was investigated in a quantitative fashion using four monoclonal antibodies known to bind to different epitopes within the common “a” determinant. The results suggest that amino acid substitution of T131I, K141E and G145R and insertion of 3 amino acids between residues 123 and 124 markedly affect the antigenic structure of HBsAg. These substitutions and insertions in the viral envelope may lead to evasion of the virus neutralizing antibody response and also to reduce efficiency of detection by immunoassays used for diagnosis and blood-bank screening. J. Med. Virol. 60:113–121, 2000. © 2000 Wiley-Liss, Inc.

The case of a young woman with GB virus C/hepatitis G virus (GBV-C/HGV) infection and with a severe exacerbation of chronic hepatitis of unknown etiology during pregnancy is described. In the offspring, severe neonatal hepatitis with subsequent mild chronic liver disease of at least 16-month duration was followed by the development of antibodies to the envelope protein (E2) of GBV-C/HGV, suggesting that the child was recovering from GBV-C/HGV infection. There was an improvement in clinical and biochemical parameters in the mother following delivery and alpha-interferon therapy was associated with a transient biochemical response.

We have successfully limited the level of hepatitis delta viraemia occurring after superinfection of hepadna-virus infected woodchucks by prior immunisation with the short form of the hepatitis delta virus antigen expressed by a recombinant baculovirus or vaccinia virus. This phenomenon occurred in the absence of detectable circulating antibody to hepatitis delta virus antigen and in the absence of evidence of priming of the humoral immune response and may reflect the induction of a cytotoxic T-cell response. The latter would control viraemia by rapid lysis of delta antigen expressing hepatocytes. It is suggested that the T-cell epitopes involved may be located on the carboxyl end of the delta protein (amino acids 77-195).

Four woodchucks chronically infected with hepatitis delta virus (HDV) were treated with cyclosporin-A (CyA) for 11 weeks. All animals had detectable HDAg in the liver and two of them were also positive for HDAg and HDV-RNA in serum. Reappearance of HDV in serum was noted in one of the two non-viraemic animals and increased viraernia in the two viraemic. HDV-RNA levels became elevated within a week of starting treatment and an inverse relationship between HDV-RNA and WHV-DNA became apparent during the treatment period. With discontinuation of treatment, HDV-RNA levels either returned to pretreatment levels or became negative. The remaining animal showed no return of viraemia during CyA treatment; HDV-RNA remained negative and WHV-DNA levels did not change. Liver biopsies revealed a slight increase in lobular activity during CyA treatment in the animals showing increased viraemia. These data are consistent with the hypothesis that the host immune response exerts a negative control on the level of HDV viraemia and that HDV influences HBV replication independently of the host immune response. In an animal that may have been clearing HDV, immunosuppression did not result in recurrence of viraemia.

Eighteen red-bellied tamarins (Saguinus labiatus), experimentally infected with hepatitis A virus (HAV), were followed for up to 1.5 years after initial challenge. Half of these animals developed protracted alanine aminotransferase (ALT) abnormalities, which lasted for between 23 and 55 weeks post-challenge. IgM anti-HAV was detected intermittently during the early phase of their relapsing hepatitis and never after return of ALT levels to normal. The possibility that these findings may be related to continued HAV replication, coinfection with another agent, or the result of an autoimmune phenomenon triggered by HAV are discussed.

In a large population of patients, chronic hepatitis delta virus (HDV) infection was usually associated with absence of hepatitis B virus (HBV) replication. However, acute HDV superinfection progressing to chronic HDV infection in two hepatitis B e antigen (HBeAg)-positive HBV carriers and coinfection in two other patients who progressed to chronic HBV (HBeAg-positive) and HDV infection was associated with continuing high-level HBV replication for several years. Thus HDV infection does not always inhibit HBV replication. The hypothesis that the different effects of HDV coinfection and superinfection on HBV replication may stem from variability in the capacity of the host to produce and respond to interferon is discussed.

An antigen/antibody system associated with one form of parenterally transmitted non-A, non-B (NANB) hepatitis has been identified by solid-phase radioimmunoassay (RIA). The antigen is found in 3% of normal subjects and in increased frequency in patients with haemophilia, renal homograft recipients, homosexual men, prostitutes, drug addicts, and patients with chronic hepatitis B virus (HBV) infection. The antigen was found in normal frequency in patients with acute hepatitis A and B, sporadic NANB hepatitis (nondrug addicts), autoimmune and alcohol-induced chronic liver disease, and primary biliary chirrhosis.

Two reactive sequences of the pre-S regions of hepatitis B surface antigen were synthesized chemically and used in micro-ELISAs for the assay of pre-S1 and pre-S2 antigens in serum from patients with acute and chronic hepatitis B. Pre-S1 antigen correlated well with the presence of HBV-DNA and was no longer detectable on cessation of viral replication, after natural recovery and after successful treatment with alpha-interferon. Pre-S2 proteins were also lost after treatment with alpha-interferon. The results show that the assay of pre-S1 and pre-S2 proteins in serum provides additional useful markers for assessing patients with acute and chronic hepatitis B infection and for monitoring the response to treatment with interferon.

In Kenya hepatitis B virus (HBV) infection and its sequelae are common. We followed up 49 hepatitis B surface antigen (HBsAg)- positive mothers and their newborn infants for 9 months to determine the importance of perinatal transmission in the African and to relate this to the HBe and HBV-DNA status of the mother. Our study shows that perinatal transmission is relatively unimportant in Kenya and that this may be a consequence of the low levels of circulating HBV-DNA in the maternal plasma. These results imply that vaccination without hyperimmune globulin may be adequate to control HBV infection in Kenya.

Non-A, non-B (NANB) viral hepatitis was successfully transmitted to two colony-born Tamarins following inoculation with antihaemophilic factor VIII concentrate or the "H" inoculum. Both animals showed histological and ultrastructural evidence of viral hepatitis, with raised alanine aminotransferase (ALT) levels from the second week after inoculation through to the end of follow-up, 5 months later.

Some studies suggest that there is antigenic and nucleic acid homology of one type of non-A, non-B hepatitis virus with hepatitis B viral (HBV) proteins and DNA. Using molecular hybridization under high and low stringency conditions with high specific activity 32P-HBV DNA as a probe, serum and liver samples from patients and nonhuman primates infected with non-A, non-B hepatitis were examined. Our results provide no evidence of significant homology between the DNA extracted from serum and liver of patients and nonhuman primates infected with one type of non-A, non-B hepatitis and HBV DNA.

Seventy-eight patients with congenital coagulation disorders were treated with hepatitis B vaccine either subcutaneously or intradermally. All the children (eight vaccinated subcutaneously and eight vaccinated intradermally) responded. Seventeen of 19 (90%) anti-HIV-negative adults vaccinated subcutaneously and 14/25 (56%) anti-HIV-negative adults vaccinated intradermally showed an immune response. At 24 months, the anti-HBs level was greater than 10 IU/l in all children vaccinated subcutaneously, 83% of children vaccinated intradermally, 77% of adults vaccinated subcutaneously, and 55% of adults vaccinated intradermally. Eight of 15 (53%) adult patients who were anti-HIV positive were also anti-HBc positive before vaccination and 6/8 (75%) failed to produce an amnestic response to vaccine. Subcutaneous vaccination with regular monitoring of anti-HBs levels and appropriate boosting is recommended.

Markers for acute hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis non-A, non-B (HNANB) infections were examined in the sera of 94 patients presenting with acute hepatitis in Kenya. Hepatitis B virus was responsible for 70% of cases, HNANB for 18%, and HAV for only 12%. The use of an IgM anti-HBc assay increased the rate of diagnosis of acute HBV infection, thereby reducing the proportion of cases designated as NANB.

The hepatitis D virus (HDV; previously called the "delta agent") is a defective organism which can replicate only in the presence of the hepatitis B virus (HBV). We tested the serum of 95 substance abusers, all of whom had sufficient evidence of chronic liver disease to warrant a liver biopsy, for hepatitis D virus antibody (anti-HDV). Anti-HDV was detected in five of eight hepatitis B surface antigen (HBsAg)-positive patients and 12 of 87 (14%) HBsAg-negative patients. Antibody to the hepatitis B core antigen (anti-HBc) was the sole hepatitis B marker in eight of the 12 (67%) anti-HDV-positive, HBsAg-negative patients but in only 14 of 75 (19%) anti-HDV-negative, HBsAg-negative patients (P less than 0.005). None of the anti-HDV-positive, HBsAg-negative patients had detectable IgM anti-HBc in the serum or hepatitis D antigen in liver tissue, and they had similar clinical features and liver biopsy diagnoses to HBsAg-negative patients without anti-HDV. We conclude that anti-HDV in HBsAg-negative substance abusers reflects infection with HDV and HBV in the distant past and does not indicate more severe liver disease than that seen in HBsAg-negative patients without anti-HDV.

Hepatitis A virus (HAV) shedding in the faeces, appearance of HAV-Ag (antigen) in the liver, and development of humoral immunity to HAV have been studied in experimentally infected tamarins. The appearance of liver damage measured by transaminase elevation and histology, in relation to the above variables, suggests that the virus is not cytopathic and the immune system contributes to the production of liver cell damage. Preliminary data suggest that HAV replication may occur in the mucosa of the small intestine and in the liver.

Chromatography with a solid-phase polyelectrolyte based on ethylene maleic anhydride polyelectrolyte (EMA PE) has been shown to remove a large proportion of hepatitis B surface antigen (HBsAg), including hepatitis B virus (HBV) particles, from contaminated cryoprecipitate. This process may reduce the risk of transmission of HBV infection when factor VIII concentrates are administered.

An immunoradiometric assay for hepatitis B surface antigen (HBsAg) that employs monoclonal antibodies directed against the common epitope(s) of HBsAg was used to analyse 3,694 samples of human serum. Further analysis of those sera identified as HBsAg-positive in this assay demonstrated that the findings with the monoclonal-antibody-based assay correlated with the presence of HBsAg as determined by Austria II. A small proportion of apparently false-positive reactions were observed, in that some sera, although reactive with the monoclonal antibodies, were not positive in conventional immunoassays using polyclonal antisera, nor were they neutralisable with polyclonal anti-HBs. The material purified by monoclonal immunoabsorbants from representative "true" and "false-positive" sera was run on polyacrylamide gels and examined under the electron microscope. The antigen in the apparently false-positive sera contained some polypeptides of similar size to those found in HBsAg, but no virus particles were seen by electron microscopy. The majority of patients with this monoclonal-antibody-reactive antigen gave either a history of hepatitis B virus (HBV) contact or had signs of liver disease.