Amplification of Full-Length Hepatitis B Virus Genomes from Samples from Patients with Low Levels of Viremia: Frequency and Functional Consequences of PCR-Introduced Mutations

Günther, Stephan; Sommer, Gunhild; Breunig, Franziska von; Iwanska, Alicja; Kalinina, Tatyana; Sterneck, Martina; Will, Hans

doi:10.1128/jcm.36.2.531-538.1998

Cited by 82 publications

(29 citation statements)

References 39 publications

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“…Recombinant proteins containing these same substitutions were readily detected by IMx HBsAg with a sensitivity equivalent to the wildtype antigen. This suggests that an alternative mechanism is involved in the explanation of these results [Carman et al, 1997], such as sensitivity endpoint differences in the initial screening assays coupled with a possible generation of artifacts due to PCR amplification [Gunther et al, 1998].…”

Section: Discussionmentioning

confidence: 99%

Immunoassay detection of hepatitis B surface antigen mutants

1999

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The increasing use of hepatitis B vaccination has had an overwhelming positive impact on the prevention of hepatitis B viral infection. Mutations in the hepatitis B surface antigen (HBsAg) gene occur as a result of vaccine escape mutants, anti-hepatitis B surface antigen immunotherapy, or in chronic hepatitis B viral infection. These mutants may present a challenge to immunoassay detection. Evaluation of the immunodetection of various HBsAg mutants has been sporadic, as the occurrence of these mutants is not common, and sufficient volume of serum samples is difficult to obtain. To investigate mutant detection, recombinant antigens were constructed to reflect mutations described in the literature occurring throughout the S gene. A limited number of serum samples exhibiting discordant immunoassay reactivity were also used to construct recombinant antigens. The evaluation of 25 HBsAg mutants across nine commercial assays of differing formats is described. Mutations affecting immunoassay performance were characterized as occurring mainly in loop 2 of the "a" determinant of HBsAg. It was determined that reagent epitope recognition was more significant for mutant detection than assay format.

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Section: Discussionmentioning

confidence: 99%

Immunoassay detection of hepatitis B surface antigen mutants

1999

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“…A method described previously was used to amplify the complete genomes of HBV DNA isolated from the patients. Briefly, the primers HBVP1:5 0 CCG GAA AGC TTG AGC TCT TCT TTT TCA CCT CTG CCT AAT CA 3 0 and HBVP2: 5 0 CCG GAA AGC TTG AGC TCT TCA AAA AGT TGC ATG GTG CTG G 3 0 having Sap1 recognition site flanks were used in a long PCR reaction including high fidelity taq polymerase [Gunther et al, 1998]. The sensitivity of long PCR was 1 Â 10 3 copy/ml in our hands.…”

Section: Hbv Dna Complete Genome Amplificationmentioning

confidence: 99%

Complete genome sequence and phylogenetic analysis of hepatitis B virus isolated from turkish patients with chronic HBV infection

Bozdayı

Turkyilmaz

İdilman

et al. 2005

Journal of Medical Virology

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Hepatitis viruses are the leading causes of chronic liver disease resulting in chronic hepatitis, cirrhosis, and hepatocellular carcinoma in the world and also in Turkey. Although Turkey has an intermediate rate of hepatitis B virus (HBV) infection with a prevalence reported as 5%, a complete HBV genome sequence has not been published. In this study, the molecular characterization and phylogenetic analysis are described of 11 complete HBV genomes isolated from 11 naïve patients (5 male, 6 female; ages: 18--54 years old, median 35 years old) with chronic HBV infection. Of 11 patients, 7 and 4 were HBeAg positive/anti-HBe negative and HBeAg negative/anti-HBe positive, respectively. All patients had no co-infection with HCV, HDV, or HIV. HBV DNA was extracted from the sera of the patients. The complete genome was amplified by PCR and cloned into a TA vector. The PCR products were sequenced directly and the complete HBV genome sequences were determined. Ten HBV genomes were 3182 base pairs in length. There was a 183 bp deletion (between nucleotides 2987--3169) in pre-S region in one HBeAg positive patient. There were two pre-core stop codons (G1896A) in two HBeAg negative and three core promoter dual mutations (T1762/A1764) in one HBeAg positive and two HBeAg negative patients' HBV genomes. Phylogenetic analysis of all complete genomes yielded that all Turkish sequences were clustered in genotype D branch (ten in subgenotype D1 and one in subgenotype D2). The analysis of S gene amino acid sequences revealed that surface gene subtypes of one and ten HBV strains were subtype ayw3 and ayw2, respectively. This study indicates that Turkish patients with chronic hepatitis B infection show very little genotypic heterogeneity. Genotype D of HBV DNA and subtype ayw2 of surface gene represent almost the whole Turkish patient population infected with HBV.

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“…1A. Full-length HBV (3.2 kb) was amplified according to the method described by Gunther et al,20,21 with the sense primer P1 containing HindIIIISapI sites, and the antisense primer P2 containing SacIISapI sites. A long PCR product of approximately 2,800 base pairs was amplified by 40 cycles (hot-start; 94°C for 40 seconds, 55°C for 1 minute, 68°C for 3 minutes plus 2 minutes after each 10 cycles) with a forward primer A containing a NotI site and a reverse primer B containing a NcoI site.…”

Section: Sampb Preparation and Pcr Ampzijcationsmentioning

confidence: 99%

A new strategy for studyingin vitro the drug susceptibility of clinical isolates of human hepatitis B virus

Durantel¹,

Carrou�e-Durantel²,

Werle-Lapostolle³

et al. 2004

Hepatology

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Resistance of hepatitis B virus (HBV) to antivirals has become a major clinical problem. Our objective was to develop a new method for the cloning of naturally occurring HBV genomes and a phenotypic assay capable of assessing HBV drug susceptibility and DNA synthesis capacity in vitro. Viral DNA was extracted from sera and was amplified by polymerase chain reaction, and amplicons were cloned into vectors that enable, after cell transfection, the initiation of the intracellular HBV replication cycle. Single or multiple clones were used to transfect Huh7 cells. The viral DNA synthesis capacity and drug susceptibility were determined by measuring the level of intracellular DNA intermediate, synthesized in absence or presence of antiviral, using Southern blot analysis. We have developed, calibrated, then used this phenotypic assay to determine the drug susceptibility of HBV quasispecies isolated throughout the course of therapy from patients selected according to their mutation profile. A multiclonal and longitudinal analysis enabled us to measure the variation of drug susceptibility of different viral quasispecies by comparison of IC(50)/IC(90)s with standards. The presence of famciclovir- or lamivudine-induced mutations in the viral population caused a change in viral DNA synthesis capacity and drug susceptibility in vitro, demonstrating the clinical relevance of the assay. In conclusion, our phenotypic assay enables the in vitro characterization of DNA synthesis capacity and drug susceptibility of HBV quasispecies isolated from patients. This assay should allow a better monitoring of patients undergoing antiviral therapy, as well as the screening of novel drugs on emerging resistant strains.

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Amplification of Full-Length Hepatitis B Virus Genomes from Samples from Patients with Low Levels of Viremia: Frequency and Functional Consequences of PCR-Introduced Mutations

Cited by 82 publications

References 39 publications

Immunoassay detection of hepatitis B surface antigen mutants

Immunoassay detection of hepatitis B surface antigen mutants

Complete genome sequence and phylogenetic analysis of hepatitis B virus isolated from turkish patients with chronic HBV infection

A new strategy for studyingin vitro the drug susceptibility of clinical isolates of human hepatitis B virus

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