Search

In order to study HDV replication without the difficulties caused by the use of a multimeric construction and to obtain a selectable expression vector, a minimal amount of antigenomic HDV cDNA, sufficient to initiate RNA dependent replication was cloned into the plasmid pUTSV1. The first plasmid, pUTdelta1.7, contained 1.7 genomes of HDV cDNA. After transfection of pUTdelta1.7 into HuH7 cells, antigenomic HDV RNA was produced, processed and could enter into the replicative cycle of HDV. However, after transfection of an antigenomic ribozyme mutant (pUTdelta1.7(AGR)) constructed on the same model, plasmid DNA dependent production of genomic HDV RNA was observed, especially in COS7 cells. It seems that a promoter within vector sequences downstream from the HDV insert may initiate counter-clockwise transcription of the plasmid. The presence of two genomic ribozymes in the insert permits the excision of a genome length genomic HDV RNA from this counter-clockwise transcript. In order to allow quantitative analysis of HDV replication, this problem was eliminated by removing the second genomic ribozyme from the insert to give the vector pUTdelta1.5. This vector can be used conveniently for transfection experiments to explore HDV biology.

Gene amplification may benefit from the construction of primers that augments the speed at which cloning and protein expression proceeds. Such primers include EcoRI or HindIII linkers as well as an in phase initiation or termination codon. PCR was carried out directly from viral particles of human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV) without DNA purification and from RNA extracted from WHV infected liver. Amplified products were directly cloned in the pKK223-3 expression vector under the control of the tac promoter. The characterization of the recombinant clones expressing the nucleocapsid protein (C protein) was done by direct incubation of the filter with 125I-labelled anti-HBc and confirmed by radioimmunoassay and Western-blot analysis. This procedure allows easy selection of recombinant clones expressing a given protein and could be applied to many other genes.

Duck hepatitis B virus (DHBV) was found in the serum of 1-6% of Pekin ducklings originated from French commercial flocks. The viremia was followed in the serum of 5 ducklings over a span of 3 mth by monitoring the levels of DHBV DNA and the endogenous DNA polymerase (DNAp) activity. The DHBV DNA levels in serum were quantified either by the DNA dot hybridization technique including counting of retained radioactivity, or by successive dilutions of each serum sample followed by DNA hybridization. The counting of the retained radioactivity was plotted on a curve and its evolution compared with that of viral DNAp activity. DHBV DNA levels in serum, estimated by both methods paralleled those of the DNAp activity, which peaked at the 4th or 5th week posthatch to decrease and fluctuate thereafter. Occasional discordance between DHBV DNA levels and the endogenous DNAp activity was observed, which could be correlated with the degree of repair of the single stranded gap of serum DHBV DNA. Parallel follow up studies comparing quantitative estimations of serum viral DNA and of DNAp activity, as presented here, may provide some clues for the understanding of the mechanisms involved in the establishment of the HEPA DNA virus carrier state. Such comparative studies may also be crucial for optimal monitoring of antiviral drugs in both human clinical trials and animal experimental studies.

The morphological similarities between B and non-A, non-B hepatitis viruses prompted the search for common antigenic determinants between the two agents. Major antigenic cross-reactivity was demonstrated by immunodiffusion between hepatitis B e/3 and the most common circulating antigen previously reported in the serum of patients with non-A, non-B hepatitis. Since it is the equivalent of an e antigen, this antigen will be hereafter referred to as non-A, non-B/e antigen. Screening with hepatitis B e/3 antigen or anti-hepatitis B e/3 by immunodiffusion may be used as an easy and efficient way to test for non-A, non-B hepatitis in the absence of specific reagents since it detected up to 91% of non-A, non-B antigen and 86% of anti-non-A, non-B/e. Antigenic cross-reactivity may be used to look for additional viruses belonging to the hepatitis B group.

Woodchucks hepatitis virus (WHV)-associated antigens and antibodies were studied using current sensitive radio- or enzyme immunoassays (RIA, EIA). A significant cross-reactivity was observed between hepatitis B surface antigen (HBsAg) and woodchuck hepatitis surface antigen (WHsAg) using RIA or EIA (Abbott Laboratories, North Chicago, Ill., U.S.A.) although not with two other commercial EIA tested (Organon Technika, Oss, The Netherlands; Behringwerke AG, Marburg, F.R.G.). A weak but significant reactivity was also found when woodchuck sera positive for WHsAg or anti-WHs by immunodiffusion were tested for HBeAg and anti-HBe by RIA, suggesting the existence of a WHeAg-anti-WHe system in infected woodchucks. The specificity of this e-anti-e reactivity in the woodchuck was further confirmed by successful absorption experiments. WHsAg and WHeAg could be distinguished serologically by immunodiffusion and separated from each other by ultracentrifugation and ammonium sulphate precipitation. A WHeAg preparation was used to boost the presumed natural antibody activity of an immune woodchuck. The specific anti-HBe response detected by RIA during the immunization experiments demonstrated the existence of a soluble WHeAg cross-reacting with the human HBe-anti-HBe system. This was confirmed in immunodiffusion by a partial identity between the precipitin lines formed by the WHeAg-anti-Whe and HBeAg-anti-HBe reaction. Whether the WHe-Ag-anti-WHe system wil mimick HBeAg and anti-HBe in all their clinico-pathological correlations, deserves further study.

Three distinct antigen/antibody systems supposedly associated with an HBV-like virus of non-A, non-B hepatitis have been identified. Because of previously demonstrated cross-reactivity with HBe/3 and HBc antigens and other analogies the following terminology is tentatively used. 1. The previously reported serum antigen has been redesignated non-A, non-B e antigen, since it is equivalent to HBe/3 Ag and cross-reacts with it. Non-A, non-BeAg or Ab were detected in 51/62 post-transfusion and 11/56 sporadic acute non-A, non-B hepatitis cases, and in 12/14 cases affecting staff members. In non-A, non-B chronic persistent or active hepatitis and cryptogenic cirrhosis, the prevalence was similarly high: 14/18, 22/48 and 12/18 respectively. Ten out of 26 implicated blood donors were found positive for non-A, non-BeAg accounting for 7 out of 8 post-transfusion cases. A high prevalence of non-A, non-BeAg was also found in haemophiliacs (11/48) and haemodialysed patients (6/42), whereas anti-non-A, non-Be was respectively detected in 4/48 and 6/42 of these cases. 2. Using immunofluorescence, a second antigen termed non-A, non-BcAg has been identified in liver biopsies from 55/84 non-A, non-B chronic hepatitis or cryptogenic cirrhosis cases. All 8 positive biopsies examined by electron microscopy revealed clusters of 22--25 nm intranuclear particles identical to those described in chimpanzees. Anti-non-A, non-Bc detectable by counter-electrophoresis and indirect immunofluorescence was found in the serum of all patients of which biopsy was positive for non-A, non-BcAg. Anti-non-A, non-Bc was also detected in 5/5 non-A, non-BeAg positive cases of post-transfusion hepatitis, 2--6 weeks after onset end remained positive for the 6 month follow-up period. 3. A third antigen, tentatively designated non-A, non-BsAg, has been found less frequently than non-A, non-BeAg in serum. However, it was detectable in 3/18 and 2/12 washed ultracentrifugation pellets of sera positive for non-A, non-BeAg or anti-non-A, non-Be, respectively.