Jisu Li

Replication capacities of natural and artificial precore stop codon mutants of hepatitis B virus: Relevance of pregenome encapsidation signal

The emergence of hepatitis B virus variants unable to express HBe protein during late stage of viral infection may represent an important mechanism of viral persistence. The molecular mechanisms responsible for the elimination of HBe expression are nonsense or frameshift mutations or initiation codon mutations in part of its coding sequence, the precore region. So far only 2 of the 29 precore amino acid codons have been found mutated to stop codons in nature, although a total of 10 codons are convertible to stop codons by single nucleotide changes. Since the HBe-coding sequence is largely overlapped by the pregenome encapsidation signal (epsilon signal), a recently found cis-acting element required for the packaging of pregenomic RNA, the absence of other potential nonsense mutants could result from their impairment of the epsilon signal. Seven such potential stop codon mutants were constructed and tested for replication capacities by transfection into a hepatoma cell line. Five mutants were replication competent, but at levels lower than that of a prevalent natural stop codon mutant. The remaining two mutants were completely defective in DNA replication, which clearly explained why these two mutants are not found in nature. Northern blot analysis revealed wild-type levels of RNA transcription by these two mutants but complete lack of packaged pregenomic RNA. Additional studies lent further support to the importance of the epsilon signal in pregenome encapsidation and suggested relaxed sequence requirements for the computer-predicted hexanucleotide bulge region as compared to the hexanucleotide loop of the signal.

In vitro replication competence of a cloned hepatitis B virus variant with a nonsense mutation in the distal pre-C region

Hepatitis B virus (HBV) variants with a nonsense mutation in the distal pre-C region have been detected in patients positive for anti-HBe, and the complete nucleotide sequence of one cloned pre-C variant has been determined. Transfection of this HBV variant clone into the human hepatoma cell line HepG2 resulted in the appearance of major HBV transcripts, replicative forms of viral DNA evidenced by both molecular hybridization and endogenous DNA polymerase assay, as well as the expression and secretion of HBsAg and HBcAg particles. Western blotting revealed only the 21-kDa HBcAg but not the 17-kDa HBeAg. These results demonstrate the replication capacity of the HBV variant with a nonfunctional pre-C region despite its inability to express HBeAg.

Evidence of two major genotypes of hepatitis C virus in France and close relatedness of the predominant one with the prototype virus.

Hepatitis C virus (HCV) cDNA sequence in the nonstructural region NS3 was amplified from the serum of 66% French non-A, non-B hepatitis patients by the nested polymerase chain reaction. A 407 base-pair sequence was determined from four such cases, which revealed the presence of two different virus genotypes F1 (three cases) and F2 (one case) with 19-20% sequence divergence. F1 showed close homology (97.5%) to the prototype US isolate, but only limited (79%) homology to the reported Japanese isolates. In contrast, F2 had 91.6% homology to the Japanese isolate, but only 81% homology to the prototype US virus. Hybridization of the amplified products from 50 French samples with labeled F1 and F2 fragments suggested the F1-related strain(s) as the major hepatitis C virus genotype. Further studies involving a greater variety of samples will confirm whether the F1-related strain is the predominant hepatitis C virus strain circulating in France. Such data will have important implications for the PCR detection of HCV infection and production of HCV vaccines.

PCR detection of HCV RNA among French non-A, non-B hepatitis patients

Hepatitis C virus (HCV) cDNA was amplified from serum of 26/40 French chronic non-A, non-B hepatitis patients by the nested polymerase chain reaction. Compared with anti-C100, viral cDNA represents a more reliable marker of active HCV replication.

Pre-core mutation associated with lack of hepatitis B e antigenaemia in Moroccan asymptomatic carriers of the virus.

Recently, ongoing chronic liver disease with persistent viraemia has been described in hepatitis B virus (HBV) carriers despite the presence of anti-HBe. This has been attributed to infection with pre-C-region-mutated HBV variants. To investigate the possible existence and the prevalence of HBV variants in Morocco and the correlation between HBV DNA detection by polymerase chain reaction and pre-S1 antigenaemia, we tested twenty blood donors, HBsAg chronic carriers for more than one year. The diagnosis of such HBeAg-negative HBV variants was determined by a previously described rapid detection method using selective oligonucleotide hybridization. Probes M0, M1 and M2 correspond, respectively, to a non-mutated distal pre-C sequence, a one-point-mutated sequence with a TAG stop codon at pre-C codon 28 and a two-point-mutated sequence with codon 28 TAG and codon 29 GAC. All the 5 HBeAg-positive samples hybridized with the M0 wild-type probe only. Among the anti-HBe-positive samples, one hybridized with the M0 probe only, whereas another hybridized with none of the oligoprobes. The 13 remaining HBeAg-negative cases hybridized with the M1, M2 or combined M0, M1 and M2 probes. Seven of the 13 HBeAg-negative samples hybridized with more than one probe. DNA sequencing confirmed mixed distal pre-C sequence changes in samples hybridizing with more than one probe. These data demonstrate the existence, in patients, of HBV variants containing an inactive pre-C region and hence the incapacity to synthesize pre-C-region-derived HBeAg.

PreS1 antigen/antibody patterns following interferon therapy in acute and chronic hepatitis B

The relation between preS1 antigen/antibody system and different phases of hepatitis B virus infection were studied in 425 serum samples from 50 hepatitis B patients before, during and after antiviral therapy using interferon alone or in combination with corticosteroid withdrawal. A typical profile of self-limited acute hepatitis B was characterized by hepatitis B virus-DNA clearance using polymerase chain reaction and preS antigens using monoclonal radioimmunoassays and by antibody responses to the middle and the large HBs proteins (gp33/gp36 and p39/gp42) using immunoblotting quantitative analysis. After interferon therapy in patients with protracted hepatitis B, complete eradication of the virus was observed in 70% of patients, and antibody response directed to middle HBs and large HBs proteins could be induced. Conversely, this antibody response was never detected in follow-up studies of chronic active hepatitis B patients who responded well to antiviral therapy and lost HBs, preS2 and preS1 antigens. Most interesting, in 50% of patients with HBeAg-positive chronic active hepatitis B who received combination therapy and in 67% of patients with anti-HBe-positive chronic active hepatitis B given interferon alone, the elevated serum preS1Ag/HBsAg ratio persisted after treatment was discontinued and even increased until the end of the follow-up when hepatitis B virus DNA was undetectable in serum by the conventional hybridization technique. This rebound of preS1 antigen expression following antiviral therapy in patients with chronic active hepatitis B may indicate virus persistence, suggesting the possibility of relapse through wild-type hepatitis B virus or the emergence of hepatitis B virus mutants.

Rapid screening for bacterial colonies harbouring tandem hepatitis B virus sequences by an oligonucleotide probe

Transfection of the hepatitis B virus (HBV) genome requires the cloning of tandem HBV sequences into a plasmid vector, which is usually screened for by restriction enzyme digestion of plasmid minipreparations from at least a dozen bacterial colonies. We describe a simple alternative screening method based on in situ hybridization of bacterial colonies with a [32P]-labelled synthetic oligonucleotide which spans the head-to-tail junction site of two tandem HBV molecules. The accurate detection by the oligoprobe is confirmed by enzymatic digestion.

Limites du diagnostic immunosérologique et moléculaire de l'hépatite C

Hepatitis C is the most common cause of post-transfusion hepatitis, as well as of the viral chronic liver disease in the western world. However since it is even more often asymptomatic than HBV, this is not truly recognized. The detection of hepatitis C can only rely on serological and virological methods and require their extensive use in screening programs. Following the molecular identification characterisation of HCV, it became possible to detect virus specific antibodies. The first generation Elisas were limited in their scope and have been replaced by second and third generation tests with better sensitivity and specificity. These assays detect antibodies to several sets of HCV protein including the C22 core, the C33 and C100, which correspond to the non structural regions (NS3 and NS4 respectively). More recently, NS5 proteins have also been added and synthetic peptides have replaced some of the recombinant proteins used initially. In spite of improved sensitivity and specificity, last generation Elisas still require confirmation by supplemental assays which can be of different types (immunoblot or combined Elisas) and include sets of structural and non structural recombinant proteins or peptides. New tests are needed to improve sensitivity and proficiency of this mandatory confirmation procedure. It is unclear at this stage whether the dogma inherited from HIV to request two sets of reactive antibodies will be also warranted by experience in HCV infection. The biggest limitation of present HCV tests is the delayed appearance of anti-HCV following primary infection. Even more worrisome is the fact that 10% of chronic infection with liver disease still remain seronegative, despite circulating HCV RNA in serum and/or liver as well as expressing HCV antigen demonstrable in liver tissue by immunostaining. Such a proportion is even more common in settings with immune deficiencies including organ transplantation and HIV infection. DNA amplification methods, such as PCR or others, must be used in order to demonstrate HCV RNA in combination with reverse transcription steps. This new powerful technology must be however applied under stringent quality control procedures and cannot be yet considered for screening or routine diagnosis although it can detect viremia as early as a week after exposure and help to monitor interferon treatment. During acute hepatitis, the delay in the appearance of anti-HCV hampers acute phase diagnosis. The early detection of HCV RNA in peripheral blood, confirms the diagnosis and opens up therapeutic possibilities. In chronic hepatitis, the diagnosis of seronegative forms may only be resolved by PCR. Moreover, the presence of HCV RNA in peripheral blood represents the only marker of on going viral replication and coincides with the severity of liver damage. During treatment with interferon, the follow up of HCV RNA sequences makes it possible to monitor its efficacy. The search for HCV RNA sequences directly in liver tissue shows that HCV may replicate in the liver in the absence of viremia. The presence of HCV RNA in the liver and the serum of liver transplanted patients is essential for the etiological diagnosis and management of hepatitis and bone marrow failure occurring after transplantation. Epidemiological study using PCR is a major tool in documenting vertical transmission between mother and child. Finally, PCR is important for the analysis of the HCV genome. Thus, in France there are at least three main strains, one close to the US prototype, the other close to the Japanese strain, possibly responsible for a more severe illness, and a third one distinct from the previous two. Two major HCV genotypes, F1 and F2, corresponding to HCV type I and II (USA prototype and Japanese) with prevalence of 45% and 55% respectively, were found in France. F1 infected patients were younger and more often male than F2 group. Nine of 28 patients in F1 genotype infected group had history of drug abuse but none i

Sequence analysis of PCR amplified hepatitis C virus cDNA from French non-A, non-B hepatitis patients

Using nested PCR and hybridization techniques, it was found that the predominant HCV strain in France is related to the U.S. strain rather than to Japanese isolates.

Base Pairing in the Pregenome Encapsidation Signal of HBV: A Clue for the Prevalence of Naturally Occurring HBeAg-minus Precore Mutations

Emergence of hepatitis B virus (HBV) mutants defective in hepatitis B e antigen (HBeAg) expression is usually a consequence of nonsense or frameshift mutations owing to a single nucleotide change or insertion/deletion in the precore region. In two rare HBe-minus HBV mutants, a nonsense mutation at precore codon 28 and a frameshift mutation at codon 29 were respectively associated with additional nucleotide substitution(s). Were these additional changes accidentally associated with the HBe-abolishing mutations or did they play a role in viral replication? Construction of artificial mutants followed by transfection experiments revealed the importance of these sequence changes for the efficient packaging of pregenomic RNA. These results can be best explained by the presence of a base-paired region of the viral pregenome encapsidation signal overlapping HBe-coding sequences, which can tolerate primary sequence changes.