Classical drug resistance mutations of HepG2.2.15 cells persistently induced by appropriate concentrations of adefovir / 解放军医学杂志

Objective To observe whether the classic drug-resistant mutations can be induced in various concentrations of adefovir (ADV)-treated HepG2.2.15 cells persistently and explore the mechanism for emergence of drug resistance.Methods HepG2.2.15 cells were cultured continually in 12-well plates with medium containing 0,0.01,0.1,1.0μmol/L concentration of ADV,and passaged every 3 days up to the 110th generations.The intracellular and supernatant HBV DNA was extracted every 10 generations.Intracellular HBV cccDNA was amplified by plasmid-safe ATP dependent DNase (PSAD) digestion in combination with rolling circle amplification and gap-spanning semi-nested PCR assay.And the RT region of supernatant HBV DNA was amplified by one-tube nested PCR assay.Then the classic drug-resistant mutations of the RT region of intracellular cccDNA and supernatant HBV DNA were analyzed using direct PCR sequencing combined with clonal sequencing (more than 20 clones/sample).Results HBV DNA stably replicated in ADV-untreated cells (control group).The intracellular total DNA and cccDNA levels,supernatant HBV DNA level decreased continuously with the prolonged ADV culture duration in 0.01 μmol/L and 0.1μmol/L ADV group.Drug resistant mutations were not detected up to the 110th generation in 0.01 μmol/L ADV group;while rtA181V+N236T mutations were detected at the110th generation in 0.1μmol/L ADV group.The 1.0μmol/L ADV group was ceased of culture at the 15th generation due to inhibited cell growth.Conclusion HBV cccDNA exists in HepG2.2.15 cells,and the classical drug-resistant mutations of rtA181V+N236T could be induced by proper concentration of ADV.

Phosphodiesterase type 5 inhibitors for lower urinary tract symptoms induced by benign prostatic hyperplasia: an update / 中华男科学杂志

Medication has become the first-line option for the management of lower urinary tract symptoms induced by benign prostatic hyperplasia (LUTS/BPH) for its advantages in controlling the symptoms, inhibiting BPH progression, and reducing serious complications and surgical risks. Recent years have witnessed remarkable achievement in the studies of phosphodiesterase type 5 inhibitors (PDE5-Is) in the treatment of LUTS/BPH. PDE5-Is can effectively alleviate LUTS/BPH, with even better efficacy when combined with al-ARAs.

Establishment of a method to detect duck hepatitis B virus covalently closed circular DNA based on rolling circle amplification / 病毒学报

Rolling circle amplification (RCA) is a newly developed experimental technique that can specific ally amplify circular DNA. Since 2008, RCA has been extensively used in hepatitis B virus (HBV) research, such as the amplification of the full-length sequence of the HBV genome, and the analysis of the drug-resistant mutations of HBV covalently closed circular DNA (cccDNA), amongst others. To create an easy assay for the analysis of duck hepatitis B virus (DHBV) cccDNA, this study established an RCA-based method. DHBV cccDNA was amplified from the DHBV DNA samples of duck liver with four pairs of sulfur-modified primers, which were designed according to the highly conserved sequence of DHBV using sera DHBV DNA as the negative control. DHBV cccDNA was detected in the obtained RCA products by the sequencing of RCA amplicons that were amplified with primer pairs on both sides of the gap of DH BV relaxed circular DNA, rather than by digesting RCA products with a restriction enzyme. The liver and sera DHBV DNA samples of 39 ducks infected with DHBV were examined with the RCA-based DHBV cccDNA detection method, and the results showed that while DHBV cccDNA was detected from all 39 liver DHBV DNA samples, no DHBV cccDNA was found in any of the sera DHBV DNA samples. These results suggest that the method established in the study is highly specific and sensitive for the detection of DHBV cccDNA. The establishment of this RCA-based DHBV method for cccDNA detection lays the groundwork for using a DHBV model to study the role of cccDNA in the pathogenesis of hepatitis B and to evaluate the effect of anti-virus therapies.

Quantitative analyses of intrahepatic HBV cccDNA and serum HBsAg in 54 patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To quantitatively detect intrahepatic HBV covalently closed circular DNA (cccDNA) and serum HBsAg; and to analyze the relationship between the two parameters and with serum HBV DNA level.</p><p><b>METHODS</b>Intrahepatic cccDNA (copies/cell) was quantitated by plasmid-safe ATP-dependent Danes (PSAD) digestion in combination with rolling circle amplification and gap-spanning selective real-time PCR assay using formalin fixed paraffin-embedded liver biopsy samples. HBsAg was measured by chemiluminescence's reagent manufactured by Abbott Company using sera sampled at time-point of liver biopsy.</p><p><b>RESULTS</b>Intrahepatic cccDNA level was positively correlated with serum HBsAg level (r = 0.459, P < 0.001), but not correlated with serum HBV DNA level. Serum HBsAg level was positively correlated with serum HBV DNA level (r = 0.328, P = 0.015), and reversely correlated with HBV replicative efficiency defined as the ratio of serum HBV DNA to cccDNA (r = -0.373, P = 0.005).</p><p><b>CONCLUSION</b>In patients with chronic hepatitis B, intrahepatic cccDNA level is correlated with serum HBsAg level. The two parameters combined with serum HBV DNA may comprehensively reflect HBV replication activity and help evaluation of antiviral therapeutic efficacy.</p>

Characterization of hepatitis B virus genotypes/subgenotypes in 1,301 patients with chronic hepatitis B in North China / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>There is still a paucity of data on hepatitis B virus (HBV) subgenotype prevalence in North China based on sequencing of large-size samples. In addition, whether HBV genotypes impact drug-resistance-associated and HBV e antigen (HBeAg)-loss-associated mutations in patients with chronic hepatitis B (CHB) is still under investigation. This study aimed to disclose clinical prevalence of HBV genotypes/subgenotypes in North China and the clinical implications of HBV genotype classification in respect to HBeAg loss and drug-resistant occurrence.</p><p><b>METHODS</b>Sera were collected from 1301 nucleos(t)ide analog-experienced CHB patients. Viral DNA was extracted and used as template for HBV genome amplification by nested PCR. DNA sequencing was performed for the analysis of HBV genotypes/subgenotypes, drug-resistance-associated mutations in polymerase gene and HBeAg-loss-associated mutations in precore/basal core promoter (BCP) regions.</p><p><b>RESULTS</b>HBV/B, HBV/C, and HBV/D were detected in 190 (14.6%), 1096 (84.2%), and 15 (1.2%) patients, respectively. HBV/B2 (182/190), HBV/C2 (1069/1096), and HBV/D1 (12/15) were predominant subgenotypes within individual genotypes. By contrast, C2 prevalence is relatively lower in Beijing area (77.2%) than in other north areas (84.9% - 87.4%). HBV/C-infected patients had an older age and a lower serum albumin level but similar HBV DNA and alanine aminotransferase (ALT) levels compared to HBV/B-infected patients. HBV/C infection had a higher incidence of lamivudine-resistant mutations rtM204I/V (44.9% vs. 30.2%, P < 0.01) and BCP mutations A1762T+G1764A (65.8% vs. 40.0%, P < 0.01) compared with HBV/B infection.</p><p><b>CONCLUSIONS</b>C2 is the most prevalent HBV subgenotype followed by B2 in CHB patients in North China; and HBV genotype prevalence is influenced by immigrant population. HBV/C infection is likely to have longer disease duration and severer liver functional impairment and might be more susceptible to develop lamivudine resistance compared to HBV/B infection.</p>

Comparative analysis of drug resistant mutations in the reverse transcriptase domain of hepatitis B virus covalently closed circular DNA and the viral relax circle DNA / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish a method for detection of reverse transcriptase region of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), and to compare the pattern and frequency of drug-resistant mutations in the region between intrahepatic HBV cccDNA and serum HBV relax circle DNA (rcDNA).</p><p><b>METHODS</b>HBV DNA were extracted from liver biopsy tissues of 20 patients with chronic hepatitis B. The RT region of HBV cccDNA was amplified by rolling circle amplification (RCA) followed by polymerase chain reaction (PCR) mediated by a pair of primers spanning across the gap region of HBV genome. The RT region of serum HBV rcDNA from the same patient was amplified by nested-PCR. The PCR products were directly sequenced and analyzed by Vector NTI Suite 8.0 and chromaslite 201 software. x2 test was used for statistical significance analysis of drug-resistant mutation occurrences between the HBV cccDNA and rcDNA.</p><p><b>RESULTS</b>The RT regions of HBV cccDNA were successfully amplified from liver tissues of all enrolled patients using the RCA plus PCR assay. Simultaneously, HBV the RT regions of rcDNA were amplified from these patients serum samples. Sequence analysis showed that the drug-resistant mutations were significantly more frequently detected in HBV rcDNA (40%) than in HBV cccDNA (10%) (P<0.05). Different mutational patterns were observed between the HBV cccDNA and rcDNA in a few cases.</p><p><b>CONCLUSION</b>The RCA in combination with PCR is a practical method for the detection of drug-resistant mutation in the RT region of HBV cccDNA. Drug-resistant mutational patterns could be discrepant between HBV cccDNA and rcDNA.</p>

Epitope screening of influenza A (H3N2) by using phage display library / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To screen the influenza A (H3N2) mimotopes by using phage display library.</p><p><b>METHODS</b>Using influenza A (H3N2) monoclonal antibody as selective molecule, a 7 mer phage peptide library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing.</p><p><b>RESULTS</b>21 positive clones were chosen for DNA sequencing. From the experiment and sequencing comparison results, one epitope was comfirmed as mimotope of influenza A (H3N2).</p><p><b>CONCLUSION</b>Influenza A (H3N2) mimotope was obtained by phage peptide library screening. The result provide a new approach for new Influenza virals vaccine development.</p>

Analysis and recombinant construction of HBV the reverse transcriptase gene with drug-resistant mutations from 40 patients with chronic hepatitis B / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyze genetic mutation associated with drug resistance in the reverse transcriptase (RT) domain of HBV from 40 patients with chronic hepatitis B, and to construct mutant RT gene recombinant vectors for drug-resistant phenotypic analysis.</p><p><b>METHODS</b>HBV DNA was extracted from sera of the 40 patients receiving anti-HBV nucleot (5) ide analogue. The complete RT domain-encoding gene was amplified by nested PCR, and then cloned into pGEM-T-easy vector. Three to Five clones were randomly selected for DNA sequencing. Data were analyzed by UNASTAR software. The pTriEx-HBV (C) 1.1 expression vectors were constructed by replacing the 1250-hp Xho I/Nco I fragments containing complete RT domain from individual patients samples.</p><p><b>RESULTS</b>All samples were detected with drug-resistant mutations associated with lamivudine, adefovir, and entacavir singly or in combination. Ninety-six mutant RT genes were cloned into pGEM-T-easy vector, from which 40 major mutant RT genes were replaced into pTriEx-HBV (C) 1.1 expression vectors. The construction was confinned to be successful by verifying mutation existence using DNA sequencing, and detectable HBsAg and HBeAg in the cell supernatant after transfecting recombinant expression vectors into Huh7 cells.</p><p><b>CONCLUSION</b>The analysis of drug-resistant mutation and the construction of mutant-recombinant expression vectors were successfully implemented using the samples frum clinical patients. The work lays a foundation for drug-resistant phenotypic analysis of HBV mutants.</p>

Altered expression profiles of microRNAs in a stable hepatitis B virus-expressing cell line / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>MicroRNAs (miRNAs) are highly conserved small non-coding RNAs of 18 - 25 nucleotides (nt) that mediate post-transcriptional gene regulation. Hepatitis B virus (HBV) can cause either acute or chronic hepatitis B, and is a high risk factor for liver cirrhosis and hepatocellular carcinoma. Some mammalian viruses have been shown to modulate the expression of host cellular miRNAs. However, interactions between the HBV and the host cellular miRNAs are largely unknown.</p><p><b>METHODS</b>miRNA microarray and Northern blotting analysis were used to compare the expression profile of cellular miRNAs of a stable HBV-expressing cell line HepG2.2.15 and its parent cell line HepG2. mRNA microarray assay and the miRanda program were used to predict the miRNA targets. A flow cytometric assay was further used to investigate the expression of human leukocyte antigen (HLA)-A.</p><p><b>RESULTS</b>Eighteen miRNAs were differentially expressed between the two cell lines. Among them, eleven were up-regulated and seven were down-regulated in HepG2.2.15 cells. Northern blotting analysis confirmed that the expression of miR-181a, miR-181b, miR-200b and miR-146a were up-regulated and the expression of miR-15a was down-regulated, which was in consistent with the results of the microarray analysis. Furthermore, some putative miRNA targets were predicted and verified to be linked with mRNA expression. The 3'-UTR of HLA-A gene had one partially complementary site for miR-181a and miR-181a might down-regulate the expression of HLA-A.</p><p><b>CONCLUSION</b>HBV replication modulates the expression of host cellular miRNAs, which may play a role in the pathogenesis of HBV-related liver diseases.</p>

Quantitative detection of hepatitis B virus covalently closed circular DNA in sera of chronic hepatitis B patients with a newly established assay / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To quantitatively detect hepatitis B virus covalently closed circular DNA (HBV cccDNA) in sera of chronic hepatitis B patients with a newly established assay.</p><p><b>METHODS</b>Primers and probe were designed in highly conservative region of HBV DNA. DNA was extracted from 175 sera samples of chronic hepatitis B patients, and was treated with plasmid-Safe-ATP-dependent Dnase(PSAD) to eliminate the relaxed circular DNA (rcDNA). The products were amplified by real-time PCR with primers spanning.</p><p><b>RESULTS</b>The detection rate of serum HBV cccDNA was found to correlate directly with serum HBV DNA loading. HBeAg positive chronic hepatitis B patients had higher serum HBV cccDNA levels than HBeAg negative chronic hepatitis B patients.</p><p><b>CONCLUSION</b>The method is good because of the high specificity. It can be used for detection of HBV cccDNA. DNA;</p>

A five-year analysis of HBV mutations in a multidrug-resistant patient with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate HBV mutations in reverse transcriptase (RT) gene and precore/basal core promoter (PC/BCP) regions in a chronic hepatitis B patient and to analyze the link between the mutations and drug resistance or HBeAg sero-conversion.</p><p><b>METHODS</b>Eighteen serum samples were collected from a chronic hepatitis B patient during his 14 hospitalizations from June 2002 to September 2007. HBV DNA was extracted and nested PCR was employed for amplification of target gene fragments. Direct sequencing of PCR products was performed followed by analysis with NTI software. The significance of the results was analyzed in combination with the clinical data of the patient.</p><p><b>RESULTS</b>Several mutations were identified in succession, including LAM-resistant mutations M204I/V and L180M+M204V, ETV-resistant mutation S202G, and HBeAg nonsense mutation G1896A. The results were in accordance with the disease progression of the patient.</p><p><b>CONCLUSION</b>Sequencing of HBV RT and PC/BCP regions is valuable for comprehensively checking the viral mutations and thus it is helpful in the surveillance of patients in clinics as a way for adopting reasonable antiviral therapy.</p>

Multiple-site analysis of HBV drug-resistant mutations in 340 patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To analyze HBV drug-resistant mutations against nucleos(t)ide analogues at 12 reported sites in 340 patients with chronic hepatitis B.</p><p><b>METHODS</b>Serum HBV DNA was extracted and a nested PCR assay was employed for the reverse transcriptase (RT) gene amplification. Direct sequencing of PCR product was performed. The significance of detected mutations was analyzed in view of clinical data of the patients.</p><p><b>RESULTS</b>Drug-resistant mutations were detected in 68 patients taking lamivudine (LAM), 10 taking adefovir (ADV), 8 taking entecavir, and 1 taking telbivudine (LdT). M204V and M204I were the most common LAM-resistant mutations. The former usually emerged with L180M while the latter often emerged alone. N236T +/- A181 substitution was the most frequently seen ADV-resistant mutation. ETV-resistant mutations occurred on the basis of LAM-resistant mutations and T184 change was the most common form. LdT-resistance was observed as M204I. Interestingly, these drug-resistant mutations were detected in a few patients who had not been treated with nucleos(t)ide analogues.</p><p><b>CONCLUSION</b>Detection of HBV drug-resistant mutations at multiple sites of the viral RT gene is valuable for discovering and verifying drug resistance and thus is very helpful in planning anti-HBV therapy.</p>

Screening and identification of proteins interacting with HCV NS4A via yeast double hybridization in leukocytes and gene cloning of the interacting protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To screen proteins interacting with HCV NS4A protein in leukocytes by yeast-double hybridization.</p><p><b>METHODS</b>The bait plasmid pGBKT7-NS4A was transformed into yeast AH109 was transformed, and the expressing of the fusion protein was identified by SDS-page. The transformed yeast was mated with yeast Y187 containing leukocytes cDNA library plasmid in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for selecting two times and screening. After extracting and sequencing of plasmid from blue colonies, analysis was conducted by bioinformatics. And, the gene encoding the interesting protein was cloned, and back-cross was performed.</p><p><b>RESULTS</b>Forty-five colonies were sequenced, among them, 29 colonies were human calcium modulating cyclophilin ligand (CAML). The gene encoding CAML was cloned, and the interaction between NS4A and CAML was ensured.</p><p><b>CONCLUSION</b>Seven kinds of proteins interacting with NS4A in leukocytes were successfully screened and the results brought some new clues for studying the pathogenesis of HCV.</p>

Transcriptional inhibitory effect of hepatitis B virus X protein on the expression of p53 tumor suppression gene / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To investigate the transcriptional inhibitory role of hepatitis B virus X protein on the expression of p53 tumor suppression gene.</p><p><b>METHODS</b>The promoter sequence of the p53 tumor suppression gene was identified and amplified by bioinformatics and polymerase chain reaction (PCR). The recombinant reporter gene expression vector pCAT3-p53p was constructed and transfected into the hepatoblastoma cell line HepG2 and cotransfected with pcDNA3.1 (-)-X by Fugene 6 transfection reagents. The chloramphenicol acetyl transferase (CAT) activity was detected by enzyme-linked immunosorbent assay (ELISA). The expression of p53 mRNA was further detected by RT-PCR with or without HBV X protein.</p><p><b>RESULTS</b>The reporter vector pCAT3-p53p has been successfully constructed and identified and the p53 promoter could cis-activate the transcription of the CAT gene. The relative expression level of CAT gene in HepG2 cells cotransfected with pCAT3-p53p and pcDNA3.1 (-)-X was lower than the control, and the inhibitory rate was approximately 78%, which indicate that HBV X protein could transcriptionally inhibit the activity of p53 promoter. After transfected with pcDNA3.1 (-)-X, the expression of p53 mRNA was lower than the control.</p><p><b>CONCLUSION</b>HBV X protein could transcriptionally inhibit the expression of p53 tumor suppression gene, which might be a possible molecular mechanism responsible for the development of HBV-associated hepatocellular carcinoma.</p>

Follow up study on viruses associated with SARS among the SARS patients / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To study the existence status of the SARS-CoV, retrovirus, and the poliovirus in the bodies of the patients with SARS and the possible relationship between the three viruses and SARS.</p><p><b>METHODS</b>The clinical specimens of the nasopharyngeal swabs, sputum (or saliva), urine, fecal specimens were collected on three consecutive days from 8 patients with SARS 2 years after the recovery from SARS. SARS-CoV, reovirus and poliovirus RNA was detected by using reverse transcription (RT)-PCR; IgG antibody to the poliovirus type 1 and 3 and the antibody to SARS-CoV were determined using enzyme linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>All the specimens were negative for SARS-CoV and reovirus by RT-PCR, but the fecal specimens from 4 persons were positive for poliovirus. The sequences of these poliovirus were highly homologous to that of human poliovirus type 1 strain sabin 1 genome at nucleotide level, but back mutations have occurred in the primary attenuating mutation sites at nucleotide position 480 (G --> A) in the 5' UTR and the nucleotide position 2795 (A --> G). No SARS-CoV, reovirus, and poliovirus were found in the normal controls. Three serum specimens were positive for the antibody to SARS-CoV. The IgG antibody to poliovirus were detected in 4 SARS patients and 23 healthy persons. No positive results for antibody to SARS-CoV were detected in the 25 healthy persons.</p><p><b>CONCLUSION</b>The positive rate of the poliovirus antibody in the serum of SARS patients 2 years after recovery was significantly different from that of the normal controls, and the positive rate of poliovirus in the fecal specimens was still very high, and more importantly back mutations have occurred in the attenuating mutation sites at nucleotide position which plays an important role in the poliomyelitis.</p>

Sequential analyses of circulating HBV specific T helper cell response in chronic hepatitis B patients receiving antiviral treatment / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To investigate the frequency of circulating HBV specific T helper cell and evaluate its association with serum levels of HBV DNA before and during lamivudine treatment in patients with chronic hepatitis B.</p><p><b>METHODS</b>The frequency of circulating HBV specific T helper cells in response to HBcAg in 25 chronic HBV-infected patients was determined by Elispot assay; serum HBV DNA was quantitated by real-time PCR.</p><p><b>RESULTS</b>The frequency of HBV specific T helper cell before antiviral treatment (47.30 +/- 25.50 SFCs /1 x 10(6) PBMC) was significantly higher than that at the third month of therapy (23.10 +/- 18.45 SFCs /1 x 10(6) PBMC, P < 0.05). All 8 patients observed dynamically had decreased frequency of HBV specific T helper cell at the third month of therapy; six patients with serum HBV DNA level reduced had higher frequency of HBV specific T helper cell before treatment than 2 patients without serum HBV DNA level decrease.</p><p><b>CONCLUSION</b>HBV specific T helper cell response at the time of hepatitis flare in chronic hepatitis B patients was significantly augmented compared to that at the time of catabasis.</p>

Study on the interaction between hepatitis virus C nonstructural protein 4A and calcium modulating cyclophipin ligand by in vivo coimmunoprecipitation / 中华传染病杂志

Objective To prove the interaction between hepatitis virus C(HCV)nonstruetural protein 4A(HCV NS4A)and calcium modulating cyclophilin tigand(CAML)with yeast-two hybrid- ization and coimmunoprecipitation.Methods The gene encoding CAML was cloned,and subcloned into the yeast expression vector pGADT7 and eucell expression vector pcDNA3.1/His-A.The back- cross test between HCV NS4A and CAML was performed in yeast cells.After that,the pCMV-Myc/ NS4A plasmid and pcDNA3.1/His-A-CAML plasmid were co transfected into 293 cells and,then, coimmunoprecipitation and Western blot were performed.Results The gene encoding CAML was cloned sucessfully,and then the gene was subcloned into yeast expression vectors,pGADT7.After the interaction between NS4A and CAML was ensured in yeast cells,the eukaryotic expression vec- tors of NS4A and CAML were constructed and their interaction was ensured again by Co-immunopre- cipitation.Conclusions The interaction between HCV NS4A and CAML is proved.CAML is one of the proteins involved in Ca~(2+)signaling,which suggests that the interaction of HCV NS4A and CAML may be a new clue of the chronic mechanism of HCV infection.Future studies will be required to de- fine the physiologic significance of this interaction.