OBJECTIVETo investigate the variants and quasispecies of reverse transcriptase region in polymerase gene of hepatitis B virus (HBV) during lamivudine treatment and their relationship with genotypes and viral loads.

METHODSHBV DNA of 117 chronic hepatitis B patients treated with lamivudine were amplified by using PCR. The PCR products including the YMDD motif were sequenced by DNA sequencer, of which, HBV DNA viral loads of 99 patients were determined by real-time PCR and 64 samples were sequenced by Pyrosequencing.

RESULTSIn HBV YMDD variant group and no variant group, the HBV genotypes were 79.6% and 86.7% of type C, 18.5% and 12.7% of type B, 1.9% of A/B recombinant type and 2.6% of type D, respectively. The viral loads (log 10) were 6.5699 and 6.6165, respectively. There was no significant difference in HBV genotypes and viral loads between these two groups. The rtL180M variant was found in association with the rtM204I/V variant, HBV variants and wild-type in YMDD motif all existed together in these two groups.

CONCLUSIONSHBV variants (quasispecies) in YMDD motif could be quantified by pyrosequencing, which would be a feasible measure during nucleoside or nucleotide analogue therapy against chronic HBV infection.

Antiviral Agents ; pharmacology ; Genotype ; Hepatitis B virus ; drug effects ; genetics ; Lamivudine ; pharmacology ; Polymerase Chain Reaction ; RNA-Directed DNA Polymerase ; genetics ; Sequence Analysis, DNA

OBJECTIVETo detect the mutations of rpoB gene in Mycobacterium tuberculosis by pyrosequencing and to evaluate the values on detection of rifampin resistance in clinical isolates.

METHODSUsing the new technology of pyrosequencing, the mutations in the rifampin resistance determining region (RRDR) of rpoB gene were analyzed. The results were compared with those obtained from methods of the absolute concentration and the minimum inhibitory concentration (MIC).

RESULTSAmong the 150 Mycobacterium tuberculosis clinical isolates, 84 were susceptible and 66 resistant to RIF. 54 of the 66 resistant isolates were multidrug-resistant (MDR) strains. Ser531Leu and His526Asp or Tyr, including twelve different genotypes and six codons, were the most common mutations. In the drug susceptibility testing, the accordance rates of the pyrosequencing and the absolute concentration method as well as MIC were 92.7% and 97.8% respectively.

CONCLUSIONNot only is the pyrosequencing technology a fast, sensitive and high throughput method in detecting rifampin resistance in Mycobacterium tuberculosis, but also a useful tool in the research of rifampin resistance mechanism.

Bacterial Proteins ; genetics ; DNA-Directed RNA Polymerases ; Drug Resistance, Bacterial ; genetics ; Humans ; Microbial Sensitivity Tests ; Mutation ; Mycobacterium tuberculosis ; drug effects ; genetics ; Phosphoric Acids ; Polymerase Chain Reaction ; Rifampin ; pharmacology

OBJECTIVETo study resistance evolution pathway of HIV-1 CRF_BC under drug selection pressure, and compare with B subtype.

METHODSBased on the reverse transcriptase region of CRF_ 97BC HIV-1 from 588 treatment-naive and 274 treatment patients, selection pressure based method was used to select resistance-associated mutations, and Bayesian network was used to construct the resistance evolutionary pathway under antiretroviral therapy. Meanwhile, it was constructed that the resistance evolutionary pathway for B subtype with the same regimens using the data from HIV resistance database, and made a comparison with CRF_07BC.

RESULTSThe major resistance mutations for CRF_07BC were identified including K103N, Q197K, V179D and Y188L. While for B subtype, the major resistance mutations include M184V, K103N,Y181C, T69N,G190A, K238T,Y188H and P225H. Much difference was observed between these two classes. However, the classical TMA1 (41L, 210W and 215Y) and TMA2 (67N, 70R and 219E/Q) pathways exist in both pathways. As different from B subtype, the predicted major drug resistance mutations for CRF_07BC did not contain TAM-related mutations, and nucleoside reverse transcriptase inhibitor-related mutations and non-nucleoside reverse transcriptase inhibitor-related mutations were mutually depending on each other.

CONCLUSIONHIV-1 CRF_07BC showed distinctive resistance evolutionary pathway, the mutations K103N,Q197K,V179D and Y188L were the major resistance mutations, and different resistance evolutionary pathways were observed between HIV-1 CRF_07BC and B subtype.

Anti-HIV Agents ; pharmacology ; Bayes Theorem ; Drug Resistance, Viral ; genetics ; Evolution, Molecular ; HIV-1 ; drug effects ; enzymology ; genetics ; Humans ; Mutation ; RNA-Directed DNA Polymerase ; genetics

OBJECTIVETo establish a simple and accurate method for rapid detection of lamivudine related mutations in hepatitis B virus (HBV) polymerase gene.

METHODSHBV polymerase gene fragments of covering B and C active region were amplified by nested polymerase chain reaction (nPCR) or nested mismatched PCR. The PCR products were digested with Nde I or Nia III and subjected to electrophoresis on agarose gel, respectively. The patterns of restriction fragment length polymorphism (RFLP) were distinguished. Using this method, thirty patients with chronic hepatitis B and treated with lamivudine for at least one year were analysed for the lamivudine related mutations in polymerase gene. Sixteen cases without lamivudine therapy were used as controls. Some of the patients were also analysed by clone sequencing.

RESULTSThe nested mismatched PCR-RFLP method was simple, accurate and rapid. The whole experiments could be finished in eleven hours. The least titers of HBV DNA which could be detected was 10.3 copies/ml. The wild or mutant strains judged by RFLP were identified by clone sequencing. Mutation in the tyrosine methionine aspartic aspartic acid (YMDD) motif of HBV polymerase gene was found in eight patients and mutations of YMDD motif associated with L526M were found in another three patients. However, there were no such mutations in the control cases.

CONCLUSIONThe nested PCR-RFLP is considered as a simple and accurate method for rapid detection of lamivudine related mutations in HBV polymerase gene. It is suitable for larger number of sample detection.

Antiviral Agents ; pharmacology ; DNA-Directed DNA Polymerase ; genetics ; Drug Resistance, Viral ; genetics ; Hepatitis B virus ; drug effects ; enzymology ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Lamivudine ; pharmacology ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length

NEDDylation has been shown to participate in the DNA damage pathway, but the substrates of neural precursor cell expressed developmentally downregulated 8 (NEDD8) and the roles of NEDDylation involved in the DNA damage response (DDR) are largely unknown. Translesion synthesis (TLS) is a damage-tolerance mechanism, in which RAD18/RAD6-mediated monoubiquitinated proliferating cell nuclear antigen (PCNA) promotes recruitment of polymerase η (polη) to bypass lesions. Here we identify PCNA as a substrate of NEDD8, and show that E3 ligase RAD18-catalyzed PCNA NEDDylation antagonizes its ubiquitination. In addition, NEDP1 acts as the deNEDDylase of PCNA, and NEDP1 deletion enhances PCNA NEDDylation but reduces its ubiquitination. In response to HO stimulation, NEDP1 disassociates from PCNA and RAD18-dependent PCNA NEDDylation increases markedly after its ubiquitination. Impairment of NEDDylation by Ubc12 knockout enhances PCNA ubiquitination and promotes PCNA-polη interaction, while up-regulation of NEDDylation by NEDD8 overexpression or NEDP1 deletion reduces the excessive accumulation of ubiquitinated PCNA, thus inhibits PCNA-polη interaction and blocks polη foci formation. Moreover, Ubc12 knockout decreases cell sensitivity to HO-induced oxidative stress, but NEDP1 deletion aggravates this sensitivity. Collectively, our study elucidates the important role of NEDDylation in the DDR as a modulator of PCNA monoubiquitination and polη recruitment.
DNA Damage ; drug effects ; DNA Repair ; genetics ; DNA Replication ; genetics ; DNA-Binding Proteins ; genetics ; DNA-Directed DNA Polymerase ; genetics ; Endopeptidases ; genetics ; Gene Knockout Techniques ; Humans ; Hydrogen Peroxide ; toxicity ; NEDD8 Protein ; genetics ; Oxidative Stress ; genetics ; Proliferating Cell Nuclear Antigen ; genetics ; Ubiquitin-Conjugating Enzymes ; genetics ; Ubiquitin-Protein Ligases ; genetics ; Ubiquitination ; genetics ; Ultraviolet Rays

Adult ; Aged ; Antiviral Agents ; pharmacology ; DNA-Directed DNA Polymerase ; genetics ; Drug Resistance, Viral ; genetics ; Female ; Genes, Viral ; Hepatitis B virus ; drug effects ; genetics ; Humans ; Lamivudine ; pharmacology ; Male ; Middle Aged ; Nucleotides ; pharmacology ; Young Adult

OBJECTIVETo elucidate the characters of rpoB mutation in rifampin-resistant clinical isolates of Mycobacterium tuberculosis.

METHODS286 bp DNA fragment of rpoB gene including 81 bp code region (rifampin resistance deteremination region, RRDR) was analyzed by PCR-single-strand conformation polymorphism(SSCP). The 286 bp DNA fragment of each strain which had been proved to have mutation by PCR-SSCP was then sequenced. 110 strains of M. tuberculosis, including 73 rifampin-resistant strains, 11 rifampin-susceptible drug-resistant strains and 26 drug-susceptible strains were studied.

RESULTS47 rifampin-resistant strains were detected to have mutations by PCR-SSCP method. 76.6% rifampin-resistant strains showed that rpoB gene was carrying single point mutation analyzed by direct sequencing technique, which mainly located at 531-Ser (61.1%) and 526-His (25.0%). The combined mutation rate was 23.4%. In addition, 2 rifampin-susceptible drug-resistant strains and 1 drug-susceptible strain were mutated, detected by PCR-SSCP method. Sequencing results showed that the mutations located at 511-Leu, 526-His and 535-Pro.

CONCLUSIONMutations in the 81 bp RRDR of rpoB were the main reasons of M. tuberculosis resistant to rifampin. 531-Ser and 526-His were the most common positions of mutations.

Antibiotics, Antitubercular ; pharmacology ; Bacterial Proteins ; genetics ; DNA Mutational Analysis ; DNA-Directed RNA Polymerases ; Drug Resistance, Bacterial ; Mycobacterium tuberculosis ; drug effects ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Rifampin ; pharmacology

BACKGROUNDThe most frequently used therapy for post-transplantation recurrence of hepatitis B virus (HBV) infection is lamivudine, but this drug is associated with a high resistance rate due to YMDD mutant. In preliminary reports, adefovir dipivoxil (ADV) has been shown to have activity against lamivudine-resistant strains of HBV. However, clinical experience in treatment of HBV infection after liver transplantation (LT) is still not entirely clear. This study was aimed to evaluate the prophylactic efficacy of ADV plus hepatitis B immunoglobulin (HBIG) in patients with YMDD mutant before LT.

METHODSFrom March 2004 to March 2006, 16 patients with chronic hepatitis B had lamivudine-resistant YMDD mutants detected prior to liver transplantation and received treatment with ADV plus additional intramuscular HBIG after LT as prophylaxis against graft reinfection. Tests for liver function, serum HBsAg, anti-HBs (HBIG), HBeAg, anti-HBc, anti-HBe, HBV-DNA, and creatinine were assessed pre- or post-liver transplantation.

RESULTSThe median follow-up of these patients post-liver transplantation was 19.4 months. Fifteen patients survived and one patient died of recurrence of hepatocellular carcinoma (HCC). There was significant difference (10.98% vs. 2.26%, P < 0.05) in YMDD mutant rate between the patients with HBV-DNA over 10(6) copies/ml and those with HBV-DNA less than 10(6) copies/ml. Fifteen patients (93.8%) had undetectable HBV-DNA at 4 weeks and 1 (6.3%) at 6 months after LT. No hepatitis B recurrence was detected by persistent testing of HBsAg, HBeAg, and HBV-DNA and no increase of serum creatinine level associated with ADV was observed in any of the patients.

CONCLUSIONADV combined with intramuscular HBIG can effectively prevent patients with pre-transplantation YMDD mutant from HBV recurrence after LT.

Adenine ; analogs & derivatives ; therapeutic use ; Adult ; Aged ; Antiviral Agents ; therapeutic use ; DNA-Directed DNA Polymerase ; genetics ; Drug Resistance, Viral ; Hepatitis B ; prevention & control ; Humans ; Lamivudine ; therapeutic use ; Liver Transplantation ; adverse effects ; Male ; Middle Aged ; Mutation ; Organophosphonates ; therapeutic use ; Recurrence

BACKGROUND/AIMS: Adefovir dipivoxil (ADV) is a nucleotide analogue that inhibits wild-type hepatitis B virus (HBV) and lamivudine (LMV)-resistant HBV mutants. The aim of this study was to elucidate the efficacy of ADV monotherapy and the incidence of genotypic resistance to ADV in patients with LMV-resistant chronic HBV infection. METHODS: This study involved 124 patients with chronic HBV infection who had received ADV monotherapy due to the presence of LMV-resistant HBV mutants. The efficacy of ADV was evaluated by the normalization of serum alanine aminotransferase (ALT) level and by the reduction of serum HBV DNA level (with cutoff levels of 2x10(4) IU/mL and 2x10(2) IU/mL). The cumulative rate of HBeAg loss or seroconversion was assessed in HBeAg-positive patients. The development of mutations in the reverse trancriptase region of HBV DNA polymerase was evaluated by direct sequencing analysis during ADV monotherapy. RESULTS: The mean serum HBV DNA level was 5.94 log10IU/mL. At 12 and 24 months after ADV monotherapy, the cumulative rates of serum ALT normalization were 69.4% and 75.5%, respectively, and those of serum HBV DNA reduction were 79.8% and 89.2% for a cutoff level of 2x10(4) IU/mL, and 44.2% and 59.0% for a cutoff of 2x10(2) IU/mL. The mean serum HBV DNA levels at 12 and 24 months were significantly lower than baseline, at 3.24 and 3.04 log10IU/mL, respectively (P<0.001). At 12 months after ADV treatment, the cumulative rates of HBeAg loss and seroconversion were 15.8% and 10.5%, respectively, and the rtN236T and rtA181T/V mutants in HBV DNA polymerase were identified in 25% and 64% of patients, respectively. CONCLUSIONS: Although ADV monotherapy is effective, it leads to a high rate of mutations of HBV DNA reverse transcriptase gene in patients with chronic HBV infections who have LMV-resistant HBV mutants.
Adenine/*analogs & derivatives/therapeutic use ; Adult ; Amino Acid Substitution ; Antiviral Agents/*therapeutic use ; DNA, Viral/analysis ; DNA-Directed DNA Polymerase/drug effects/genetics ; Drug Resistance, Viral ; Female ; Genotype ; Hepatitis B e Antigens/blood ; Hepatitis B virus/*drug effects ; Hepatitis B, Chronic/diagnosis/*drug therapy ; Humans ; Lamivudine/*therapeutic use ; Male ; Middle Aged ; Mutation ; Phosphonic Acids/*therapeutic use ; Sequence Analysis, DNA

OBJECTIVETo study the inhibitory effect of binding peptides on duck hepatitis B virus (DHBV) replication in duck hepatocytes.

METHODSSpecific binding peptides to duck hepatitis B virus polymerase (DHBVP) were screened by phage display technology (PDT), then were sequenced and synthesized. Binding peptides were added into primary culture of duck hepatocytes infected with DHBV in vitro. DHBV-DNA in the cytoplasm, cell nucleus and medium supernatant was assayed over time.

RESULTSSeven binding peptides were obtained after 3-round screening by PDT. Duck primary hepatocytes infected by DHBV were treated with above obtained binding peptides. The DHBV-DNA levels in medium supernatant and cytoplasm of duck hepatocytes treated with synthesized peptides (the 3rd and the 6th peptide) were significantly lower than those of control cells (P<0.05).

CONCLUSIONSpecific binding peptides to DHBVP could inhibit the replication of DHBV.

Animals ; Cells, Cultured ; Ducks ; Hepadnaviridae Infections ; virology ; Hepatitis B Virus, Duck ; drug effects ; genetics ; Hepatitis, Viral, Animal ; virology ; Hepatocytes ; virology ; Peptide Nucleic Acids ; pharmacology ; RNA-Directed DNA Polymerase ; metabolism ; Virus Replication ; drug effects