DNA sequences homologous to hepatitis C virus (HCV) in the extrachromosomal circular DNA in peripheral blood mononuclear cells of HCV-negative subjects / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

OBJECTIVE@#This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of HCV-negative subjects. We presumed that these experimentally proven 5'-noncoding region (5'-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA.@*METHODS@#Home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed.@*RESULTS@#The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known HCV genotypes. In contrast, BLASTn alignment searches of published HCV 5'-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5'-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with HCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5'-NCR revealed a broad diversity of individual patterns of methylation.@*CONCLUSIONS@#The experimental results confirm our assumption that parts of the HCV 5'-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.

Cleavage of in vitro transcripts of hepatitis B virus C gene by 10-23 DNA enzyme / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the cleavage activities of 10-23 DNA enzymes targeting at HBV C gene mRNA in vitro.</p><p><b>METHODS</b>10-23 DNA enzymes named DrzBC-7, DrzBC-8 and DrzBC-9 specific to HBV C gene ORF A1816UG were designed and synthesized. HBV C gene mRNA was obtained by in vitro transcription method. Cleavage activities were observed in vitro. The influence of MgCl2 concentration on RNA cleaving activity was examined with DrzBC-9. Values of kinetic parameters including Km, Kcat and Kcat/Km were calculated accordingly.</p><p><b>RESULT</b>Targeted substrate mRNA with the size of 300 nt was obtained by transcription in vitro. Under the certain cleavage conditions, DrzBC-7, DrzBC-8 and DrzBC-9 all efficiently cleaved target mRNA at specific sites in vitro. Cleavage products of 109 nt and 191 nt were obtained. No cleavage occurred without MgCl2. The most efficient cleavage was obtained at 150 mmol x L(-1) MgCl2. The efficiency of cleavage did not increase when the MgCl2 concentration was more than 200 mmol x L(-1). The kinetic parameters, Km, Kcat and Kcat/Km for DrzBC-9 were 1.4x10(-9) mol x L(-1), 1.6 min(-1) and 1.1x10(9) mol x L(-1) x min(-1), respectively.</p><p><b>CONCLUSION</b>10-23 DNA enzymes targeting at HBV C gene mRNA possess the specific cleavage activities in vitro.</p>

Character of HBV (hepatitis B virus) polymerase gene rtM204V/I and rtL180M mutation in patients with lamivudine resistance / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVES</b>To investigate the relationship between HBV (hepatitis B virus) polymerase gene 180 and 204 sites mutation and lamivudine resistance.</p><p><b>METHODS</b>One hundred forty-one patients with lamivudine resistance after lamivudine treatment and 60 chronic hepatitis B patients without lamivudine treatment were enrolled in this study. The serum HBV DNA mutation was analyzed by sequence detection via polymerase chain reaction (PCR). The sequences of the same patient were analyzed before and after lamivudine treatment.</p><p><b>RESULTS</b>One hundred and nine lamivudine resistance patients had HBV YMDD (tyrosine-methionine-aspartate-aspartate) mutation. Among them, 45 patients had rtL180M/M204V mutation (41.28%), 28 patients had rtL180M/M204I mutation (25.70%) and 36 patients had rtM204I mutation (33.02%). There were 6 patients with rtL180M mutation in 32 lamivudine resistance patients. Sixty chronic hepatitis patients without lamivudine treatment had no mutations.</p><p><b>CONCLUSIONS</b>HBV mutations, which play an important role in lamivudine resistance usually locate at polymerase gene 204 site; 180 site mutation was also observed in these patients. Evaluation of the anti-virus therapy by surveillance of the two sites mutations is of importance.</p>

Detection and analysis of the new kind of G743C and G743A point mutation of HBV P gene in hepatitis B virus infected patients resistant to lamivudine / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the point mutation in hepatitis B virus polymerase (HBV P) gene in HBV-infected patients resistant to lamivudine.</p><p><b>METHODS</b>HBV P gene was amplified by PCR and the products was sequenced to analyze the YMDD mutation. Then the variants were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with the following restriction enzymes: Fok I, Ssp I, Alw441 and were separated by 8.0% polyacrylamide gel electrophoresis.</p><p><b>RESULTS</b>Comparing with the sequences of standard HBV genome, there were 16 patients with G743C mutation and 1 patient with G743A mutation, and the codon ATG turned to ATC and ATA, YMDD motif changed into YIDD. But this kind of YIDD mutation was not proved by PCR-RFLP assay in the 17 patients.</p><p><b>CONCLUSIONS</b>The G743C and G743A mutations in HBV P gene, resulting in YMDD motif changed into YIDD, are detected only by direct sequencing, not by PCR-RFLP. The new kind of G743C and G743A point mutations in HBV P gene is important for the detection of HBV P gene YMDD mutation.</p>

Influence of HGV super-infected with HIV or HCV on the virus replication / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To realize human immunodeficiency virus(HIV) and hepatitis C virus(HCV) super-infected with hepatitis G virus(HGV or GBV/C) and to probe into the mechanism of these virus infection in the body.</p><p><b>METHODS</b>HIV and HCV load were tested by the quantitated RT-PCR in the HIV or HCV infected plasma samples respectively and the HGV RNA was detected in all of the samples. Then some of the HGV positive were sequenced.</p><p><b>RESULTS</b>123 of 317 HIV patients were positive for HGV, the positive rate was 38.8%. Among the 91 HCV patients, 19 were positive for HGV. The positive rate is 20.9% which was less than that of HIV patients. HIV load of the patients super-infected with HGV was less than that of those without HGV[(1.8+/-0.6)x10 copies/ml compared with (1.9+/-1.1)x10(2)copies/ml]; while HGV and HCV super-infection did not influence the HCV RNA load significantly [(1.5+/-0.6)x10(4) copies/ml compared with (5.4+/-1.8)x10(4)copies/ml]. The HGV sequences from HIV or HCV patients were compared and showed no difference markedly.</p><p><b>CONCLUSION</b>The rate of the HIV and HGV super-infection is higher than that of HCV. HGV may inhibit HIV reproduction in the body while superinfection.</p>

DNAzymes in vitro inhibit the expression of hepatitis B virus genes / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To evaluate the inhibition effects of DNAzymes specific to Hepatitis B Virus(HBV) s gene and e gene on the expressions of Hepatitis B surface antigen(HBsAg) and Hepatitis B e antigen(HBeAg).</p><p><b>METHODS</b>DNAzymes DrzBS and DrzBC specific to HBV s gene ORF A157UG and e gene ORF A1816UG, respectively, were designed and synthesized. The inhibition effects of DrzBS or DrzBC on the expressions of HBV s and e genes were observed in 2.2.15 cells.</p><p><b>RESULTS</b>The expression of HBV s or e genes was dramatically depressed after 2.2.15 cells treated by DrzBS or DrzBC. The concentration for effective inhibition was within 0.1-2.5 micromol/L and the inhibition showed a dose dependence within that concentration range. The maximum inhibition was 94.2% and 91.8% for DrzBS and DrzBC, respectively. The inhibition was maintained for 72 hours. The efficiency of inhibiting HbsAg, HbeAg in 2.2.15 cells by DrzBS, DrzBC was higher than that by antisense oligonucleotides for the same target genes. The concentrations for effective inhibition of the DNAzymes were at least 10-fold lower compared with antisense oligonucleotides. Neither inhibition on the replication of HBV DNA nor toxicity to 2.2.15 cells was observed.</p><p><b>CONCLUSION</b>DrzBS and DrzBC can highly block the expressions of HBV s gene and e gene in 2.2.15 HBV cell model and are proved a specific and effective anti-HBV gene therapeutic means.</p>