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Biomedical subjects

L Vitvitski

Publications and source records attributed to L Vitvitski.

At least 55 records · Page 3Linked to original sources
PubMed1992

New assays for quantitative determination of viral markers in management of chronic hepatitis B virus infection.

We performed a quantitative study of serum hepatitis B virus (HBV) markers, including new parameters such as pre-S1 antigen (Ag), pre-S2 Ag, and anti-HBx, in 88 chronic hepatitis B surface antigen (HBsAg) carriers. New IMx assays for HBsAg and immunoglobulin M (IgM) anti-HBc detection were also used. The population studied was composed of 65 chronic hepatitis cases (40 positive for hepatitis B antigen [HBeAg] and 25 positive for anti-HBe) and 23 anti-HBe-positive, asymptomatic HBsAg carriers. Serum HBsAg levels detected by IMx were higher in HBeAg-positive than in anti-HBe-positive HBsAg carriers (all patient subgroups included) and correlated with the serum HBV DNA level (P = 0.0001). Both pre-S1 and pre-S2 Ags were detected by enzyme immunoassays in almost all HBsAg carriers. Both pre-S1 and pre-S2 Ag titers correlated positively with the serum HBsAg concentration (P = 0.0001), but only the pre-S1 Ag titer correlated with the level of serum HBV DNA (P = 0.02). The detection of low levels of IgM anti-hepatitis B core (anti-HBc) antibodies by IMx was associated with the presence of liver disease (P = 0.05) but not with the level of viral replication. The prevalence of anti-HBx antibodies detected by the enzyme immunoassay was slightly, although not significantly, higher in patients with high levels of HBV DNA (greater than 100 pg/ml) than in patients without detectable HBV DNA (P = 0.16). In anti-HBe-positive chronic HBsAg carriers, the quantitative detection of serum HBV DNA, pre-S Ag titers, and IgM anti HBc allowed us to predict which patients suffered from chronic liver disease and/or supported viral replication (P < 0.05). In a follow-up study of eight patients undergoing antiviral therapy, the clearance of both pre-S1 Ag and HBV DNA was associated with a subsequent clearance of HBV. Therefore, the quantitative determination of HBV DNA, pre-S Ags, IgM anti-HBc may prove useful for the decision to use and the monitoring of antiviral therapy, especially in anti-HBe-positive HBsAg carriers.

Carrier State↗
PubMed1991

Two French genotypes of hepatitis C virus: homology of the predominant genotype with the prototype American strain.

A hepatitis C virus (HCV) cDNA covering part of the nonstructural region, NS3, was amplified from the serum of 50 out of 76 French non-A, non-B hepatitis patients by the nested polymerase chain reaction (PCR). Determination of a 407-bp sequence from four such cases revealed the presence of two different virus genotypes, F1 and F2, which exhibited 19-20% sequence divergence. F1 was represented by three of the four isolates and showed a sequence homology of about 97.5% to the prototype American HCV isolate, but of only 79% to a reported Japanese isolate. In contrast, F2 had 91.6% homology to the Japanese isolate, but only 81% homology to the prototype American HCV. PCR products from the 50 samples were hybridized with labeled F1 and F2 fragments under stringent conditions; results indicated the F1-related strain(s) as the major HCV genotype. Furthermore, a total of 1477 bp of sequence has been determined for one of the isolates belonging to the F1 category. These results will have implications for the PCR detection of HCV infection and production of HCV vaccines, especially for European countries.

Americas↗
PubMed1991

Generation of a 1.5-kb cDNA fragment of the hepatitis C virus genome by overlap extension.

Hepatitis C virus (HCV) is a recently identified RNA virus responsible for most of non-A, non-B hepatitis. Genetic analysis of HCV sequences and their encoded proteins has been hampered by the difficulty in obtaining cDNA fragments of sufficient lengths: construction of cDNA library requires technical expertise while amplification by the polymerase chain reaction (PCR) usually yields fragments of less than 400 base pairs. In this report we have generated a 1.5-kb HCV cDNA fragment by overlap extension of smaller PCR fragments and by ligation through restriction sites. Sequencing of the cloned fragment confirmed the absence of significant sequence alteration produced by this procedure. Overlap extension may represent an easy method for generating relatively large HCV cDNA clones for the expression of HCV-encoded proteins.

Base Sequence↗
PubMed1991

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.

DNA Replication↗
PubMed1991

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.

Base Sequence↗
PubMed1991

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.

Antisense Elements (Genetics)↗
PubMed1991

In vitro and in vivo replication capacity of the precore region defective hepatitis B virus variants.

Although the precore region defective hepatitis B virus variants have been implicated in chronic liver disease and fulminant hepatitis, our knowledge on the molecular biology of these variants is still limited. Using an in vitro transfection assay, we confirmed the replication competent but HBeAg-negative nature of the major variants containing a TAG stop codon in the distal precore region associated with one or two point mutations. Transfection of the two-point-mutated variant into a chimpanzee induced serological responses including anti-HBc and anti-HBs. Interestingly, anti-HBe response was found in the absence of HBeAg antigenemia, suggesting that anti-HBe can be stimulated by degraded HBc. Using the rabbit reticulocyte system the possible effect of the different precore region mutations on the expression of HBcAg from precore- and core-mRNAs was also studied.

Animals↗
PubMed1991

Detection of pre-S1 proteins in peripheral blood mononuclear cells from patients with HBV infection.

The presence of pre-S1 proteins in peripheral blood mononuclear cell (PBMC) samples from 115 patients with different forms of hepatitis B virus (HBV) infection was investigated by Western blot. Among 67 chronic HBsAg carriers, HBV antigens were detected in the PBMC in 80% for HBsAg, 27% for HBc/e Ag and 34% for pre-S1 proteins. The detection of pre-S1 proteins in PBMC was significantly associated with the presence of serum markers of HBV replication (HBV DNA and/or DNA polymerase). In the group of 48 consecutive patients negative for serum HBsAg, but positive for anti-HBc with or without anti-HBs, HBsAg and pre-S1 proteins could be detected in PBMC. This finding was more frequent among anti-HIV-positive patients (77 and 23% of the cases, respectively) than in the negative ones (23 and 4% of the cases, respectively). The detection of HBV DNA and polyadenylated RNA in some of the PBMC samples positive for HBV proteins suggests that these proteins may be expressed in PBMC, especially during intense HBV replication. In patients negative for serum HBsAg, PBMC may constitute a reservoir of HBV.

Blotting, Western↗
PubMed1990

Active hepatitis B virus replication in the presence of anti-HBe is associated with viral variants containing an inactive pre-C region.

Although rise of anti-HBe immunity in the course of hepatitis B virus (HBV) infection is generally followed by clearance of the infectious virions, ongoing chronic liver disease with circulating virions has been repeatedly observed in a significant number of anti-HBe patients, especially in Mediterranean countries. To investigate the possible role of HBV variants, we cloned HBV DNA from the serum of three such anti-HBe cases. Comparative restriction mapping of HBV clones suggested circulation of different HBV genomes in the three cases. DNA sequencing revealed an inactive pre-C region in all 11 HBV clones derived from the three cases, either as one or two point mutations in the 3' terminus generating an in-frame TAG stop codon, or a 1 nucleotide insertion in the 5' terminus resulting in frameshift mutation. Furthermore, for one clone the complete 3182 nucleotide sequence was determined and no significant mutation was found in the remainder of the genome. We conclude that chronic hepatitis cases positive for anti-HBe are associated with HBV variants containing an inactive pre-C region and hence cannot synthesize pre-C region-derived HBeAg. This finding may provide a molecular explanation for the continued viral replication despite presence of anti-HBe immunity.

Base Sequence↗
PubMed1990

Rapid detection and further characterization of infection with hepatitis B virus variants containing a stop codon in the distal pre-C region.

Recently, hepatitis B virus (HBV) replication in the absence of HBe antigenaemia has been attributed to HBV variants with a TAG stop codon in the distal pre-C region associated with one or two point mutations. We describe here a rapid detection method for the diagnosis of such HBeAg-negative HBV variants using selective oligonucleotide hybridization. The entire pre-C region was amplified by the polymerase chain reaction and hybridized under stringent conditions with non-mutated (M0), one (M1) and two (M2) point-mutated oligonucleotide probes. Of the 15 HBeAg-positive (group I) and 20 HBeAg-negative (group II) serum samples studied, 14 samples in group I and one sample in group II hybridized with M0 only and 18 samples in group II hybridized with M1 or M2, or both. The remaining two samples (from groups I and II, respectively) failed to hybridize with any of the three probes. DNA sequencing confirmed mixed distal pre-C sequences in samples hybridizing with more than one probe and also revealed novel mutations in the distal pre-C region of the two samples which failed to hybridize with any of the probes. The latter sample had a +2 frameshift and hence represented a new type of HBeAg-negative HBV variant. This method may therefore prove useful in the diagnosis of infections by HBeAg-negative HBV variants resulting from common mutations in the pre-C region, as well as for the identification of less common variants with novel mutations in the same region.

Base Sequence↗
PubMed1987

Prevalence and significance of hepatitis B virus antigens: expression in peripheral blood mononuclear cells in chronic active hepatitis.

One-hundred four chronic active hepatitis (CAH) patients were investigated for the expression of the hepatitis B virus (HBV) surface and core gene products (HBs Ag, HBc/HBe Ags) in peripheral blood mononuclear cells (PBMC). Two-thirds of 59 HBs antigenemic patients expressed HBs Ag in PBMC but 26% of cases positive for both anti-HBs and anti-HBc also expressed HBs Ag while none of the controls reacted. Among HBs antigenemic patients, only those who replicated HBV express the core gene products (HBc and/or HBe Ag) in PBMC, and high replicators did so more often than low replicators (P less than 0.05). The HBs Ag prevalence in PBMC, although slightly higher among HBe Ag/DNAp-positive cases could not be correlated with the intensity of HBV replication. In 16 cases (8 replicants and 8 nonreplicants) HBV DNA was detected by DNA hybridization spot test, while 8 controls devoid of HBV markers were negative. Both T and non-T cells reacted similarly for antigenic or genomic HBV markers. When the expression of HBV gene products in PBMC among 43 cases with HBs antigenemia was compared with that in the liver, a good correlation was found in 70% of cases for HBs Ag but in only 40% for HBc and HBe Ags. By contrast, among 38 cases lacking HBs Ag in the serum but positive for anti-HBc with or without anti-HBs, concordance between liver and PBMC expression of core gene products (69%) was better than for HBs antigenemic patients (40%). These data suggest that PBMC including T lymphocytes may represent the second-best HBV target and may mimic the steps of HBV cycle within hepatocytes.

Adult↗
PubMed1986

Evidence for the presence of duck hepatitis B virus in wild migrating ducks.

A virus closely related to duck hepatitis B virus (DHBV) was isolated from serum and liver samples of wild migratory ducks (mallards) caught in two separate wildlife reserve parks in France. In the first one (Dombes region) 12% of wild mallards were positive for DHBV, and in the second (River Somme) 3% of mallards were found positive. The DHBV isolated from the serum of wild mallards was also associated with an endogenous DNA polymerase activity capable in vitro of completing a partially double-stranded viral DNA into a fully double-stranded DNA of 3 kb. The various replicative DNA forms reported for DHBV were also detected in the liver of wild viraemic mallards. The DNA restriction enzyme pattern of the wild mallard strain differed from that of American and French strains of DHBV. The wild mallard strain DHBV was experimentally transmitted to mallard and Pekin ducklings and induced a chronic viraemia in both varieties of infected birds. This strain might be the common ancestor of all DHBV strains isolated from domestic ducks world-wide. The discovery of a DHBV-related virus in the natural wild population might be an important clue in the study of the different roles of environmental, host and viral factors in the pathogenesis of DHBV infection, and their possible oncogenic action in ducks.

Animal Population Groups↗
PubMed1985

Woodchuck hepatitis virus infection: serologic and histopathologic course and outcome.

Five out of seven American woodchucks inoculated with woodchuck hepatitis virus developed antigenemia after 2 to 13 weeks followed by an antibody response. One animal became a carrier, and another animal exhibited a primary antibody response. Clinical disease was not obvious and aminotransferase elevation could not be demonstrated. Liver biopsy showed mononuclear portal infiltration and little parenchymal cell necrosis.

Animals↗
PubMed1985

Comparative study of DHBV DNA levels and endogenous DNA polymerase activity in naturally infected ducklings in France.

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.

Animals↗
PubMed1984

Comparison of properties of woodchuck hepatitis virus and human hepatitis B virus endogenous DNA polymerases.

The principal properties of the DNA polymerases of woodchuck hepatitis virus and human hepatitis B virus were compared. The enzymes of both viruses exhibited optimal activities in the same range of pH, ionic strength, and MgCl2 concentration. Like human hepatitis B virus DNA polymerase, the woodchuck hepatitis virus DNA polymerase was strongly inhibited by phosphonoformic acid but not by phosphonoacetic acid and aphidicolin. Similar inhibition patterns for both enzymes were observed with arabinofuranosyl nucleotides (9-beta-D-arabinofuranosyladenine-5'-triphosphate, 1-beta-D-arabinofuranosylcytosine-5'-triphosphate, 1-beta-D-arabinofuranosylthymine-5'-triphosphate) and dideoxythymidine triphosphate, whereas no effect was obtained with corresponding nucleosides. The therapeutic significance of these results and the relevance of the woodchuck as an experimental animal model for the study of human hepatitis B virus infections are discussed.

Animals↗
PubMed1983

Use of the cross-reactivity with hepatitis B virus antigens and antibodies for the demonstration of a woodchuck hepatitis virus 'e' antigen-antibody system.

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.

Animals↗
PubMed1983

Serial transmission of hepatitis B like non-A, non-B hepatitis and associated markers to chimpanzees successfully immunized against HBV.

In order to demonstrate that the HBV like strain of NANB hepatitis bred true as non-B together with its associated markers, 2 chimpanzees with high titer anti-HBs (45 and 125 AUSAB RU respectively) immunized with the Pasteur HB vaccine received 1 ml IV of a NANB inoculum. Two neighbour captive animal served as controls. The inoculum was the serum of a leukemic patient in remission for over 3 years with NANB chronic active hepatitis which serum contained HBV like particles and was found positive for NANBe Ag and anti-NANBc whereas in the liver typical numerous "SHIMIZU" dense soft edge aggregated intranuclear structures were demonstrated by electron microscopy. After 4 weeks portal inflammation and hepatocyte necrosis with significant aminotransferase elevation lasting for over 10 weeks developed in the 2 infected chimpanzees. No change in anti-HBs titer was seen and HBs, HBc, HBe Ag and/or AB could neither be detected in serum by RIA nor in liver by immunofluorescence during the 6 month follow up period. By contrast NANBc Ag became clearly demonstrable by immunofluorescence in the liver nuclei together with anti-NANBc in the serum after the 6th week and both persisted for over 4 months. Double unit structures similar to those of NANB/F strain were demonstrable in the cytoplasm at the acute phase. None of these changes was seen in the two control animals. Inoculation of the acute phase serum in the 2 previous control animals was again followed by the same sequence of events. Hybridization studies with HBV DNA of liver biopsies of infected chimps were negative.

Animals↗