A new avian hepadnavirus infecting snow geese (Anser caerulescens) produces a significant fraction of virions containing single-stranded DNA.

We describe the identification and functional analysis of an evolutionary distinct new avian hepadnavirus. Infection of snow geese (Anser caerulescens) with a duck hepatitis B virus (DHBV)-related virus, designated SGHBV, was demonstrated by detection of envelope proteins in sera with anti-DHBV preS and S antibodies. Comparative sequence analysis of the PCR-amplified SGHBV genomes revealed unique SGHBV sequence features compared with other avian hepadnaviruses. Unlike DHBV, SGHBV shows an open reading frame in an analogous position to orthohepadnavirus X genes. Four of five cloned genomes were competent in replication, gene expression, and virus particle secretion in chicken hepatoma cells. Primary duck hepatocytes were permissive for infection with SGHBV, suggesting a similar or identical host range. SGHBV was found to secrete a significant fraction of virion-like particles containing single-stranded viral DNA. This was observed both in cell culture medium of SGHBV DNA-transfected LMH cells and in viremic sera of several birds, suggesting that it is a stable trait of SGHBV. Taken together, SGHBV has several unique features that expand the knowledge of the functional and evolutionary diversity of hepadnaviruses and offers new experimental opportunities for studies on the life cycle of hepadnaviruses.     

Hepatitis B virus genomes that cannot synthesize pre-S2 proteins occur frequently and as dominant virus populations in chronic carriers in Italy.

The heterogeneity of hepatitis B virus (HBV) pre-S sequences coding for envelope proteins was tested by DNA amplification and direct sequencing of viral genomes from sera of 22 unselected chronic carriers resident in Southern Italy. The sequences of the dominant viral genome populations from 15 carriers were very similar to known published "wildtype" HBV genomes and showed no deletions. In contrast, in the HBV populations of six patients, deletions in the pre-S region, mainly clustered at the amino terminal end of the pre-S2 region, were found. Four of these mutant genome populations and those from another patient cannot express pre-S2 proteins due to deletions or a mutation of the translation initiation codon. Emergence of the pre-S mutant viruses either during the natural course of infection or after interferon treatment was found in follow-up sera of one and two patients, respectively. These data indicate a high prevalence of pre-S mutant viruses which cannot express pre-S2 and normal-size pre-S1 proteins. This has important implications for the usefulness of diagnostic pre-S protein assays and possibly for interferon treatment and the efficacy of new vaccines containing pre-S proteins.     

Monoclonal antibody recognizing pre-S(2) epitope of hepatitis B virus: characterization of pre-S(2) epitope and anti-pre-S(2) antibody

A hybrid cell line producing monoclonal antibodies recognizing an epitope encoded by the pre-(S)2 region of hepatitis B virus (HBV) genome was obtained by fusion of mouse myeloma cells with lymphocytes from mice immunized with HBV. The monoclonal antibody Mo-F124 secreted from the hybrid line reacted with the pre-S(2) epitope expressed on the surface of both viral and recombinant HBsAg particles--pre-S(2) and S gene product--localised on 34 kD glycoprotein of the viral envelope. The pre-S(2) epitope was sensitive to digestion with V8 protease from Staphylococcus aureus. The enzyme abolished reactivity with Mo-F124 and polymerized human serum albumin (pHSA) binding activity of recombinant particles. Mo-F124 antibody was used to develop highly sensitive radioimmunoassays for determination of pre-S(2) epitope and anti-pre-S(2) antibody in sera of hepatitis B patients. Detection of a pre-S(2) epitope by the monoclonal antibody-based assay in the early phase of acute HBV infection correlated well with the presence of markers of active viral replication (HBeAg, HBV DNA). The appearance of anti-pre-S(2) antibody, usually in the third month after onset of symptoms, was followed by elimination of circulating HBsAg and seroconversion to anti-HBs in all tested cases of uncomplicated acute hepatitis followed by recovery. Anti-pre-S(2) response was not observed in patients with chronic hepatitis B or acute HBV infection progressing to chronic disease. The observed correlation of anti-pre-S(2) response with recovery suggests that the pre-S(2) epitope may represent one of the epitopes inducing antibodies that neutralize the hepatitis B virus.     

Studies by electron microscopy on the assembly of duck hepatitis B virus in the liver

Liver specimens from 1-day-old ducklings infected in ovo with maternally transmitted duck hepatitis B virus (DHBV) were examined by electron microscopy. Complete and incomplete DHBV particles were located within hypertrophied cisternae of the endoplasmic reticulum of the hepatocytes. The complete viral particles found intracellularly have inner cores with a diameter ranging from 35 to 37.5 nm and an outer coat or envelope. The whole particle measures approximately 45-65 nm in diameter. Naked core particles were located in the nuclei, free in the cytoplasm, and also near or on the cisternal membrane of the endoplasmic reticulum on the cytoplasmic face. Duck hepatitis B virions appear to share morphological characteristics with the viral coat and core of human hepatitis B virus (HBV). Electron microscopy suggested that the core particles of DHBV migrate from the nucleus into the cytoplasm through the nuclear pores. The complete viral particles are probably formed by protrusion of the core particles through the endoplasmic reticulum and by simultaneous encapsulation with a coat derived from the endoplasmic reticulum.     

Decreased replication capacity of a duck hepatitis B virus mutant with altered distal pre-S region

We have observed in a previous study that insertion, deletion and partial frameshift mutation in the distal pre-S region did not abolish replication capacity of the duck hepatitis B virus (DHBV) (Li et al., 1989, J. Virol. 63, 4965-4968). To compare further the relative replication capacity between the pre-S mutant and wild type virus, ducts were infected with either the wild type DHBV strain or a pre-S mutant (FS-17) characterized by a total change of nine consecutive amino acid codons in the distal pre-S region. Compared with the wild type virus, FS-17 exhibited decreased replication capacity whether in separate or mixed infection. The decreased viral replication was correlated with delayed appearance of supercoiled DNA and viral RNA in the hepatocytes. Besides, FS-17 induced persistent viremia when inoculated into 1-day-old ducklings; hence the transient viremia which had been observed in the previous study was probably due to the time delay needed to generate compensatory deletion mutation.     

DNA vaccines expressing the duck hepatitis B virus surface proteins lead to reduced numbers of infected hepatocytes and protect ducks against the development of chronic infection in a virus dose-dependent manner

We tested the efficacy of DNA vaccines expressing the duck hepatitis B virus (DHBV) pre-surface (pre-S/S) and surface (S) proteins in modifying the outcome of infection in 14-day-old ducks. In two experiments, Pekin Aylesbury ducks were vaccinated on days 4 and 14 of age with plasmid DNA vaccines expressing either the DHBV pre-S/S or S proteins, or the control plasmid vector, pcDNA1.1Amp. All ducks were then challenged intravenously on day 14 of age with 5 x 10(7) or 5 x 10(8) DHBV genomes. Levels of initial DHBV infection were assessed using liver biopsy tissue collected at day 4 post-challenge (p.c.) followed and immunostained for DHBV surface antigen to determine the percentage of infected hepatocytes. All vector vaccinated ducks challenged with 5 x 10(7) and 5 x 10(8) DHBV genomes had an average of 3.21% and 20.1% of DHBV-positive hepatocytes respectively at day 4 p.c. and 16 out of 16 ducks developed chronic DHBV infection. In contrast, pre-S/S and S vaccinated ducks challenged with 5 x 10(7) DHBV genomes had reduced levels of initial infection with an average of 1.38% and 1.93% of DHBV-positive hepatocytes at day 4 p.c. respectively and 10 of 18 ducks were protected against chronic infection. The pre-S/S and the S DNA vaccinated ducks challenged with 5 x 10(8) DHBV genomes had an average of 31.5% and 9.2% of DHBV-positive hepatocytes on day 4 p.c. respectively and only 4 of the 18 vaccinated ducks were protected against chronic infection. There was no statistically significant difference in the efficacy of the DHBV pre-S/S or S DNA vaccines. In conclusion, vaccination of young ducks with DNA vaccines expressing the DHBV pre-S/S and S proteins induced rapid immune responses that reduced the extent of initial DHBV infection in the liver and prevented the development of chronic infection in a virus dose-dependent manner.     

Molecular cloning and sequence analysis of duck hepatitis B virus genomes of a new variant isolated from Shanghai ducks

The genomes of duck hepatitis B virus (DHBV) from a brown duck (S5) and a white duck (S31) kept independently in Shanghai, China, were cloned and the complete nucleotide sequence of each virion DNA (DHBV-S5 and DHBV-S31) was determined. DHBV-S5 and DHBV-S31 were both 3027 bp in length and 6 bp longer than the other two DHBVs analyzed previously, DHBV16 and DHBV3. The genomes of DHBV-S5 and DHBV-S31 encoded three long overlapping open reading frames designated as P, S, and C. A possible new open reading frame was found in a complementary strand of each viral genome, as 336 bp for DHBV-S5 and 306 bp for DHBV-S31, respectively. A pair of 3-bp insertions were found in the overlapping region of pre-S2 and P and so two amino acids were inserted in this region in DHBV-S5 and DHBV-S31. The nucleotide sequence variation between DHBV-S5 and DHBV-S31 (4.9%) was similar to that between DHBV16 and DHBV3 (5.6%), and less than the variations between either of these Shanghai clones and DHBV16 or DHBV3 (9.5-10.4%). The amino acid sequence was also conserved in the two Shanghai clones but showed group difference from DHBV16 or DHBV3. Thus these two independent Shanghai clones of DHBV showed geographical characteristics of genomic structure.     

The insertion domain of the duck hepatitis B virus core protein plays a role in nucleocapsid assembly

Synthesis of hepadnaviral DNA is dependent upon both the viral DNA polymerase and the viral core protein, the subunit of the nucleocapsids in which viral DNA synthesis takes place. In a study of natural isolates of duck hepatitis B virus (DHBV), we cloned full-length viral genomes from a puna teal. One of the clones failed to direct viral DNA replication in transfected cells, apparently as a result of a 3 nt inframe deletion of histidine 107 in the core protein. Histidine 107 is located in the center of a predicted helical region of the "insertion domain", a stretch of 45 amino acids which appears to be at the tip of a spike on the surface of the nucleocapsid. The mutation was introduced into a well-characterized strain of DHBV for further analysis. Core protein accumulated in cells transfected with the mutant DHBV but was partially degraded, suggesting that it was unstable. Assembled nucleocapsids were not detected by capsid gel electrophoresis. Interestingly, the mutant protein appeared to form chimeric nucleocapsids with wild-type core protein. The chimeric nucleocapsids supported viral DNA replication. These results suggest that the insertion domain of the spike may play a role either in assembly of stable nucleocapsids, possibly in formation of the dimer subunits, or in triggering nucleocapsid disintegration, required during initiation of new rounds of infection.     

Occurrence of pre-S1 antigen in viremic and nonviremic carriers of hepatitis B surface antigen

The proteins of viral envelope, encoded by the pre-S1 region of HBV-DNA, were measured quantitatively with enzyme immunoassay using monoclonal antibodies directed to pre-S1 epitope and correlated with the expression of pre-S2 region encoded epitope and other HBV markers. In acute HBV infection, both pre-S encoded proteins were detected in sera along with markers of viral replication and disappeared shortly before complete virus clearance while high HBsAg titers were still present. Pre-S1 antigen was present in most (95.5%) symptomatic and asymptomatic chronic HBsAg carriers. There was no correlation between the presence of pre-S1 and HBeAg or HBV-DNA in serum: 73% of sera with pre-S1 determinants were anti-HBe positive, and only 25.4% were positive for HBV-DNA. Most pre-S1 activity in sera of viremic carriers was detected in fractions of sucrose gradient containing subviral 22-nm particles, and much less in those containing infectious virions. In asymptomatic, nonviremic HBsAg carriers, pre-S1 was located only on subviral 22-nm forms. Pre-S1 positive particles had no accessible pre-S2 epitope, which is recognized specifically by monoclonal anti-pre-S2 (F124) antibody. These results show that the synthesis of the large protein of HBV envelope may occur also in the absence of active viral replication, and in these cases pre-S1 encoded sequences are on subviral particles of HBsAg. Therefore, pre-S1 is not a serologic marker of infectious virus. Disappearance of pre-S1 epitopes on HBsAg occurs only before complete clearance of the virus, and this may have potential prognostic relevance.     

Comparative sequence analysis of duck and human hepatitis B virus genomes

We have cloned and sequenced an infectious, functionally active genome of a duck hepatitis B virus (DHBV). It is 3,021 base pairs (bp) in length and shows little DNA sequence homology to the genome of human hepatitis B virus (HBV). However, the amino acid sequences of predicted viral gene products are similar between DHBV and HBV, and the genome organization present in DHBV reflects that of HBV. As in the mammalian virus the long minus strand of the DHBV genome encodes three long overlapping reading frames designated as P, S, and C. The fourth open reading frame, termed X, is absent in DHBV. A comparison with a sequence of a second DHBV isolate [Mandart et al, Journal of Virology 49:782-792, 1984] revealed a nucleotide sequence variation of 5.6% and confirmed the presented overall gene organization of DHBV.     

Encapsidation of truncated human hepatitis B virus genomes through trans-complementation of the core protein and polymerase

Mutational analyses and complementation tests were used to analyze the strategy of packaging and of replication of human hepatitis B virus (HBV). By creating new restriction enzyme sites and by varying the genome length of HBV mutants, we identified that the mutated genomes could be encapsidated through trans-complementation of the polymerase and/or core protein. This study demonstrates that the polymerase of HBV, similar to that of duck hepatitis B virus (DHBV), is synthesized de novo instead of through a core-polymerase fusion protein. The results also indicate that both the polymerase and the core protein can be supplied in trans during viral packaging, and that the complementation is not due to recombination between the cotransfected plasmids. Furthermore, HBV genome deleted down to 2.4 kb is still able to be encapsidated, as measured by the endogenous polymerase reaction. Taken together, these results provide a basis for using HBV as a vector to deliver foreign genes into hepatocytes and for defining the location of the packaging signal on the HBV genome.     

Interrupted replication of hepatitis B virus in liver tissue of HBsAg carriers with hepatocellular carcinoma

To search for events underlying reduction of peripheral viremia and integration of hepatitis B virus (HBV) DNA into the liver cell genome in long-term virus carriers with hepatocellular carcinoma, paired samples of liver and tumor tissue were analyzed by molecular hybridization and immunological methods. Most tumor tissues contained integrated viral DNA; in none was extrachromosomal HBV DNA detected. Integrated HBV DNS was also found in peritumor liver tissue in the majority of patients. However, liver of patients either with or without peripheral viremia also contained free HBV DNA and replicative intermediates. In three nonviremic patients with replicative HBV DNA in liver, viral core antigen expression was markedly reduced or absent, whereas viral envelope protein (surface antigen) expression was normal. In one case, replicative intermediates in liver were sensitive to DNase I digestion, indicating that viral DNA was not encapsidated in normal viral core particles. These results suggest that decreased or defective core antigen production can lead to reduced viremia associated with blocked virus assembly/secretion and accumulation of unencapsidated HBV DNA replicative intermediates in the liver cell. Accumulation of such HBV DNA molecular forms in the liver may lead to an increased propensity for HBV DNA to integrate into the host genome, which has been found with high frequency in hepatic neoplasms from patients infected with hepatitis B virus.     

乙型肝炎病毒前C区和BCP区突变株复制质粒的构建

目的　构建乙型肝炎病毒 (HBV)前C区和基本核心启动子区 (BCP)突变株的复制质粒。方法　以含 1 2倍拷贝HBVDNA全基因的质粒 (pHBV1 2 )为工具 ,采用分子克隆、PCR定点诱变、限制性酶切长度多态性和序列分析等技术构建目的质粒。转染肝癌细胞株Huh7后 ,测定培养上清HBsAg、HBVDNA来了解病毒抗原的表达和病毒复制。结果　成功构建了 10种HBV全基因前C区 /BCP区突变的表达质粒 ,转染肝癌细胞株 ,获得病毒的表达和病毒颗粒的分泌。其中 ,pUC HBVT176 2、A176 4双突变以及pUC HBVT175 3突变株复制效率较高 ,转染细胞培养上清的HBVDNA为3 16× 10 5拷贝 ml。野生型复制子培养上清HBVDNA含量稍低于BCP突变复制子。结论　获得 10株含有不同前C区和BCP区突变的HBV全基因复制质粒 ,为体外进一步研究上述变异的生物学意义提供基本模型。     

Myristylation of a duck hepatitis B virus envelope protein is essential for infectivity but not for virus assembly

In addition to the major surface (S) protein, the envelope of the duck hepatitis B virus (DHBV) contains a related presurface (preS) protein whose N-terminus bears a covalently attached myristate group. We have explored the functional significance of this modification by examining the replicative potential of a mutant viral genome whose myristylation signal has been inactivated. Following transfection into permissive hepatoma cells, the mutant expresses an unmyristylated preS protein of normal size, immunoreactivity and stability. Cytoplasmic cores containing viral DNA are synthesized, and Dane particles are assembled and exported into the medium. However, the mutant is noninfectious when inoculated into susceptible ducklings. We conclude that myristylation of preS proteins is essential for hepadnaviral infectivity but not for viral assembly; myristylation is most likely required for an early step of the life cycle involving the entry or uncoating of virus particles.     

Comparison of two defective hepatitis A virus strains adapted to cell cultures.

The replication of two defective hepatitis A virus strains in cell culture was examined. The w.t. HAS-15 strain growing in FRhK-4 cells produced infectious icosahedral virions 27 nm in size as well as round shaped particles with lipids attached to their surface. The morphogenesis of HAV was membrane-dependent and the detected particles were in various degree of maturation. The MBB 11/5 strain growing in PLC/PRF/5 cells produced mainly noninfectious empty procapsids without RNA genome. The translation of viral proteins was uninhibited in both strains. The reason for restricted replication competence of both strains seemed to be different. In HAS-15, highly efficient encapsidation of the progeny RNA positive-strand lowered the formation of replicative intermediate forms. In MBB 11/5, nearly exclusive empty procapsid production gave evidence for the failure of the VPg primer protein attachment to viral RNA. Changes in the efficacy of viral genome replication were a result of the adaptation of HAV to propagation in vitro.