These recommendations provide a data-supported approach. They are based on the following: (1) formal review and analysis of the recently published world literature on the topic; (2) guideline policies covered by the American Association for the Study of Liver Diseases/European Association for the Study of the Liver (AASLD/EASL) Policy on the Joint Development and Use of Practice Guidelines; and (3) the experience of the authors in the specified topic. Intended for use by physicians, these recommendations suggest preferred approaches to the diagnostic, therapeutic, and preventive aspects of care. They are intended to be flexible, in contrast to standards of care, which are inflexible policies to be followed in every case. Specific recommendations are based on relevant published information. To more fully characterize the available evidence supporting the recommendations, the AASLD/EASL Practice Guidelines Subcommittee has adopted the classification used by the Grading of Recommendation Assessment, Development, and Evaluation (GRADE) workgroup, with minor modifications (Table 1). The classifications and recommendations are based on three categories: the source of evidence in levels I through III; the quality of evidence designated by high (A), moderate (B), or low quality (C); and the strength of recommendations classified as strong (1) or weak (2). Literature review and analysis The literature databases and search strategies are outlined below. The resulting literature database was available to all members of the writing group (i.e., the authors). They selected references within their field of expertise and experience and graded the references according to the GRADE system [1]. The selection of references for the guideline was based on a validation of the appropriateness of the study design for the stated purpose, a relevant number of patients under study, and confidence in the participating centers and authors. References on original data were preferred and those that were found unsatisfactory in any of these respects were excluded from further evaluation. There may be limitations in this approach when recommendations are needed on rare problems or problems on which scant original data are available. In such cases, it may be necessary to rely on less-qualified references with a low grading. As a result of the important changes in the treatment of complications of cirrhosis (renal failure, infections, and variceal bleeding [VB]), studies performed more than 30 years ago have generally not been considered for these guidelines.

The X gene of the mammalian hepadnaviruses is believed to encode a protein of 17 kDa which has been shown to transactivate a wide range of viral and cellular promoters. The necessity for X gene expression during the viral life cycle in vivo has recently been suggested (H.-S. Chen, S. Kaneko, R. Girones, R. W. Anderson, W. E. Hornbuckle, B. C. Tennant, P. J. Cote, J. L. Gerin, R. H. Purcell, and R. H. Miller, J. Virol. 67:1218-1226, 1993). We have independently constructed two variants of woodchuck hepatitis virus (WHV) with mutations in the X coding region. Transient transfection of two different hepatoma cell lines showed that these WHV X gene mutants were competent for virus replication in vitro. To determine whether X expression was required for viral replication in vivo, we injected mutant and wild-type genomes into the livers of susceptible woodchucks. While the wild-type WHV genomes were infectious in all animals examined, the mutant genomes did not initiate a WHV infection in woodchucks. These results indicate that the X gene of the hepadnaviruses plays a major role in viral replication in vivo.

All known DNA polymerases require primers for the initiation of DNA synthesis. While cellular polymerases and reverse transcriptases use free hydroxyl groups of RNA or DNA, the DNA polymerases of certain animal viruses and bacteriophages depend upon hydroxyl groups of amino acid residues within proteins as primers for DNA synthesis. Recently, the reverse transcriptase of a hepadnavirus has been shown to prime RNA-directed DNA synthesis from an internal site of the polypeptide (G. H. Wang and C. Seeger, Cell 71:663-670, 1992). In this report we demonstrate that a tyrosine residue of the polymerase polypeptide is the site of a phosphodiester linkage with the first nucleotide of minus-strand DNA. This tyrosine residue is located within an aminoterminal domain of the polymerase polypeptide and is indispensable for the priming of reverse transcription. Our results demonstrate that the hepatitis B virus reverse transcriptase can initiate DNA synthesis without the requirement for tRNA as a primer.

Since the introduction of antiviral compounds such as lamivudine and famciclovir in the treatment schedules of patients with chronic hepatitis B virus (HBV) infection, the accumulation of a variety of mutations in the HBV polymerase gene has been observed. The selection of these mutations is generally considered the cause of viral nonresponsiveness and treatment failure. Therefore, the detection of these mutations is of clinical importance. Previously genotyped HBV strains isolated from treated and untreated patients were amplified with primers specific for the HBV polymerase region from amino acids 465 to 562. Amplified products were cloned into plasmid vectors. The clones were used as reference strains. A set of 38 highly specific oligonucleotide probes covering three different codon positions, L528M, M552V/I, and V/L/M555I, were selected. These probes were applied as 19 different lines on a membrane strip. The strips were then hybridized with PCR fragments from the reference panel, revealing the amino acids at the three codon positions simultaneously for each clone. PCR products generated from two patients infected with HBV genotypes A and C, respectively, and treated with nucleoside analogs were analyzed on these strips. A gradual increase in genetic HBV polymerase complexity was observed in follow-up samples compared to that in pretreatment samples. Additional analysis of HBV polymerase DNA fragments in recombinant plasmid clones demonstrated the existence of (i) clones with double mutations, (ii) clones with single mutations at either codon 528, 552, or 555, and (iii) the simultaneous occurrence of two or more viral populations within one sample. This line probe assay detected the complex quasispecies nature of HBV and provided some insight into the dynamics of resistance mutations.

No abstract