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The cellular localization of the precore/core and core proteins was studied by immunofluorescence following transfection of 143 thymidine kinase-negative (TK ) and Hep-G2 cells with expression constructs containing wild-type (hepatitis B e antigen [HBeAg]-positive) and precore mutant (HBeAg-negative) sequences. Precore/core constructs with the wild-type phenotype result in strong nuclear staining, while, in contrast, constructs expressing core antigen alone have strong cytoplasmic staining. These differences in the pattern of immunofluorescence staining may be caused by expression of the precore/core protein, some of which may be translocated into the nucleus, following removal of the signal peptide. In vitro translation experiments showed that the main protein products obtained in the presence of microsomal membranes were the precore/core protein and a truncated product representing the same protein without its signal peptide. Core protein expression from the precore mutant constructs was very much reduced, indicating that translational re-initiation was not very efficient. The significance of the precore/core protein being present in the nucleus is not clear, but suggests that it may be important in the replicative cycle of the virus. Finally, HBeAg produced by some of the constructs could not be detected because amino acid substitutions affected antibody-binding epitopes.

The precore stop-codon variant of hepatitis B virus (HBV) has been implicated in fulminant hepatitis. The precore/core regions of such variants from two sets of patients with interpartner transmission resulting in fulminant hepatitis in the contact, were sequenced to establish whether further sequence variations in the core region are specifically associated with the fulminant disease. In both sets of patients, there was sequence diversity of the precore/core region from the wild type, leading to numerous amino acid substitutions in the core region. Between the infecting source and the contact, there was only one amino acid change in one set of patients and none in the other. In addition, in the second set of patients, serum samples from four different time points were investigated. Sequence data showed no variation in each patient at the nucleotide level in the core region, even in the case of the source, who was followed for 3 years. In this same pair of subjects, the remainder of the genome was sequenced and was identical at the nucleotide level. Therefore, it appears that, at least in some cases of fulminant hepatitis caused by infection with the precore variant, the nucleotide sequence of the patient with fulminant hepatitis is identical to that observed in the asymptomatic source of infection. These data indicate that the severity and outcome of infection in such cases are unrelated to any additional variation in the entire HBV genome, and that the changed clinical picture is dependent on host factors, possibly the HLA environment.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of unique hepatitis B virus (HBV) variants has been investigated in two Chinese patients with chronic liver disease, whose sera were positive for HBV-DNA by dot blot hybridization or polymerase chain reaction (PCR) but hepatitis B surface antigen (HBsAg)–negative by conventional polyclonal antibody based immunoassays. PCR amplification of HBV-DNA followed by direct sequencing showed an insertion of six nucleotides, which introduced two additional amino acids between codons 122 and 123 in one patient (Isolate 1), whereas a nine nucleotide insertion in the other patient (Isolate 2) gave rise to three amino-acids between codons 123 and 124 immediately upstream from the ‘a’ determinant in the S gene. These insertions have not been described previously in any published sequences of the known subtypes and were absent from sequences of 30 HBsAg-positive Chinese patients from the same region. In the cases under study, the insertion is associated with four consecutive adenine molecules from nucleotides 516 to 519. It seems likely that this area is a hot spot for insertions in HBV. We found none of the previously described amino-acid deletions or substitutions in the pre-S1, pre-S2 and S genes, which are involved in unusual antigenic profiles. This finding suggests that genetic mutations in the S gene outside the ‘a’ determinant may be responsible for failure to detect HBsAg in some Chinese patients with chronic hepatitis caused by HBV infection. (HEPATOLOGY 1995;21:273–278.)

An in situ hybridization method using radiolabeled oligonucleotide probes was developed to study primary sites of hepatitis A virus replication in an experimental animal model of infection. Hepatitis A genomic sequences were demonstrated in hepatocytes of four marmosets with acute hepatitis A by use of antisense probes. In two of these animals, staining was also found when a sense probe was used, which is consistent with active replication in the hepatocytes. The specificity of the hybridization signal was confirmed by neutralization with “cold” (i.e., unlabeled) probes and by absence of hybridization with non-A hepatitis and reverse antisense probes. The hepatocyte appeared to be the only cell type showing staining. No hybridization was found in other organs, including the intestine (n = 4) and, in one animal, the kidney and spleen. (HEPATOLOGY 1992;16:642–648.)

The presence of hepatitis delta virus genomic RNA and hepatitis delta antigen was investigated in woodchuck liver and extrahepatic tissues by in situ hybridization using synthetic radiolabeled probes, Northern-blot analysis and immunohistochemical staining for hepatitis delta antigen. Hepatitis D virus RNA and hepatitis delta antigen were detected in the nuclei of infected hepatocytes but in none of the other tissues examined. Northern-blot analysis of total cell RNA confirmed these findings and revealed a series of hepatitis D virus transcripts, including full-length genomic RNA and dimers and trimers of hepatitis D virus RNA that may represent replicative intermediates. Use of single-stranded probes showed genome-size monomers and dimers to be both of genomic and antigenomic polarity, although dimers were found to be predominantly antigenomic. These findings document the strict hepatotropism of hepatitis D virus and support the rolling-circle model of genome replication for this unique, defective RNA virus.

To assess the role of immunization against hepatitis delta antigen in the prevention of hepatitis delta virus infection, woodchuck carriers of woodchuck hepatitis virus were immunized with a 64 amino acid portion of hepatitis delta antigen from its N-terminal region. The protein was expressed in Escherichia coli and contained a major immunogenic epitope. A significant anti-hepatitis delta response was observed that did not, however, protect the animals from hepatitis delta virus superinfection. Unexpectedly, the period of detectable viremia was longer in the immunized than in the control animals. We conclude that immunization with this recombinant hepatitis delta antigen does not afford protection against subsequent hepatitis delta virus exposure. (HEPATOLOGY 1990;12:1125–1128).

Hepatitis B virus DNA (HBV DNA) in serum was measured by a Spot hybridization technique in a consecutive series of 79 cases with chronic HBV infection from Taiwan. HBV DNA was found in 96.3% (52/54) of HBeAg-positive, 66% (2/3) with neither HBeAg or anti-HBe and in 63.6% (14/22) of anti-HBe positive patients. The levels of HBV DNA in the HBe-Ag-positive patients were significantly higher than in the anti-HBe positive patients (median, 944 vs. 58 pg per ml, p less than 0.001). The mean ages increased from 28.7 years for the cases with high levels of HBV DNA, to 34.7 years for those with low levels (p less than 0.01) and to 41.0 years in those without HBV DNA in serum (p less than 0.05 when compared with those with low level of HBV DNA). Ninety per cent of patients (27/30) with high levels of HBV DNA showed only minor hepatic inflammatory activity, as did 91% (10/11) of those without HBV DNA. In contrast, histologic signs of chronic active hepatitis or chronic lobular hepatitis were demonstrated in 76% of cases (29/38) with low levels of HBV DNA. These data are consistent with the hypothesis that liver damage occurs during the period of clearance of hepatocytes supporting HBV replication, and are inconsistent with the view that HBV may be directly cytopathic. Thus, the natural history of chronic HBV infection may be divided into three phases.(ABSTRACT TRUNCATED AT 250 WORDS)

A previous randomized controlled study has shown a 30% rate of HBe antigen/antibody seroconversion within 1 year of a month course of adenine arabinoside-5'-monophosphate; no seroconversion occurred in the control group. In this study of patients derived from the same population, 45 hepatitis B virus carriers with chronic liver disease were randomized to receive either a short (4-week) course of adenine arabinoside-5'-monophosphate, a long (7 to 8-week) course of adenine arabinoside-5'-monophosphate or a 12-week course of lymphoblastoid interferon. Long-lasting suppression of hepatitis B virus replication with disappearance of serum hepatitis B virus DNA and clearance of HBeAg occurred within 12 months of treatment in four patients who received the short course of adenine arabinoside-5'-monophosphate and in five who received interferon. Of the nine responders, four also lost HBsAg. A response to antiviral therapy was accompanied by clinical and biochemical evidence of improvement in liver disease. None of the patients who received a long course of adenine arabinoside-5'-monophosphate responded. Peripheral neuropathy and myalgia were the most serious adverse effect affecting three recipients of the short course of adenine arabinoside-5'-monophosphate and eight recipients of the long course. Thrice weekly administration of interferon was well-tolerated. Further studies to identify the characteristics of the "responder patients" and large-scale controlled trials of antiviral therapy in these subgroups are indicated.

Twenty-one pretreatment variables were assessed for their significance in response prediction using data from 114 patients given alpha-interferon for chronic hepatitis B virus infection. In those patients who had received a minimum of 90 million units per m2 total dose over 12 weeks, a negative anti-human immunodeficiency virus antibody status (p less than 0.001), chronic active hepatitis on liver biopsy (p less than 0.005), high AST level (p less than 0.001), low hepatitis B virus DNA level (p less than 0.001) and a history of acute hepatitis (p less than 0.005) were all associated with an increased likelihood of response on univariate analysis. On stepwise logistic regression analysis, hepatitis B virus DNA, AST and a history of acute hepatitis predicted response independently (p less than 0.05). The most reliable combination of predictive factors was a negative anti-human immunodeficiency virus antibody status, with either a positive history of acute icteric hepatitis and AST greater than 45 IU per liter or no history of acute icteric hepatitis and AST greater than 85 IU per liter, which predicted response in 77% with a specificity of 79% (p less than 0.001). The loss of HBsAg in addition to HBeAg and hepatitis B virus DNA was more likely to occur in patients with chronic infection of less than 2 years duration (p less than 0.001).