Recombinant adenovirus-mediated expression of 1.2-copy hepatitis B virus DNA in three hepatocytes / 南方医科大学学报

<p><b>OBJECTIVE</b>To study hepatitis B virus (HBV) expression in 3 hepatocytes infected with recombinant adenovirus containing 1.2-copy HBV DNA.a</p><p><b>METHODS</b>A chicken hepatoma cell line and two human hepatocytes were infected by the recombinant adenovirus containing 1.2-copy HBV DNA at 25 pfu/cell. HBV-specific mRNA was detected by RT-PCR 3 days after the infection, and HBsAg and HBeAg were detected by ELISA and HBV DNA by real-time PCR daily after the infection.</p><p><b>RESULTS</b>HBV mRNA expression was detected in all the 3 cells after recombinant adenovirus infection, and the quantities of HBV DNA and HBV antigens in the culture supernatant increased with the passage of time.a</p><p><b>CONCLUSION</b>Infection with the recombinant adenovirus containing 1.2-copy HBV DNA can mediate HBV infection in the 3 cells in vitro.</p>

Factors related to chronic hepatitis B relapse after interferon-alpha treatment: a follow-up study / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the related to relapse of chronic hepatitis B (CHB) after recombinant interferon-alpha (rIFN-alpha) treatment.</p><p><b>METHODS</b>This investigation involved 523 pathologically confirmed CHB patients including 403 HBeAg-positive and 120 HBeAg-negative patients, who were treated with 5 MU rIFN-alpha subcutaneously thrice a week for 6-25 months. For each patient, serum alanine aminotransferase (ALT) was measured biochemically, serum HBV DNA level detected with quantitative fluorescent PCR, and HBeAg level with enzyme immuoassay every 1-3 months during therapy and every 3-6 months during the follow-up period.</p><p><b>RESULTS</b>Early response to rIFN-alpha treatment was observed in 302 (57.7%) patients at the end of treatment, among whom 39.4% (119/302) suffered relapse during the follow-up for 39.2-/+21.5 months. Age, HBeAg status before treatment, and follow-up duration were the predictive factors for post-treatment relapse. The mean age of patients with CHB relapse was significantly higher than that of the sustained responders (P<0.001), and the relapse rates in HBeAg-negative group (55.8%, 43/77) were significantly higher than that in HBeAg-positive group (33.8%, 76/225) at the end of follow up (P<0.001). The relapse rate and accumulative relapse rates at each year during the follow-up (for 5 years as the longest) differed significantly (P<0.001, P=0.000), but the accumulative relapse rates differed little between the years after the initial 2 of the follow-up (P=0.670). The relapse was not related to the patient's gender, pretreatment serum ALT, HBV DNA, grade of liver inflammation, stage of liver fibrosis, or duration of treatment. In HBeAg-positive patients, however, the mean HBV DNA was significantly higher in relapse group than in sustained response group (P=0.017).</p><p><b>CONCLUSION</b>Age, pretreatment HBeAg status, and follow-up duration are independent predictive factors for post-treatment CHB relapse. In HBeAg positive patients, pretreatment serum HBV DNA is also one of the risk factors for relapse.</p>

Dynamic analysis of HBV cccDNA in HepG2 cells infected with Ad-1.2 HBV / 南方医科大学学报

<p><b>OBJECTIVE</b>o study the replication of hepatitis B virus (HBV) in HepG2 cells infected with Ad-1.2 HBV.</p><p><b>METHODS</b>HepG2 cells were transfected with adenovirus containing 1.2 copies of HBV DNA. The expression of HBV antigens were detected in the culture medium by means of enzyme-linked immunosorbent assay (ELISA), and the covalently closed circular DNA (cccDNA) in the cells was extracted with plasmid extraction kit and detected by real-time PCR with selective primer after treatment with mung bean nuclease.</p><p><b>RESULTS</b>HBsAg, HBeAg and HBV cccDNA were all detected in HepG2 cells after tranfection with Ad-1.2 HBV. HBV cccDNA was detected 1 day after the infection, reaching the peak level 4 days after infection.</p><p><b>CONCLUSION</b>Ad-1.2 HBV-infected cells can serve as the model for screening and evaluation of antiviral agents.</p>

Viral breakthrough and neutralizing anti-interferon antibody production in chronic hepatitis B patients treated with recombinant interferon-alpha / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the relationship of virological breakthrough and production of neutralizing anti-interferon antibody (NAb) in chronic hepatitis B patients treated with recombinant interferon-alpha (rIFN-alpha).</p><p><b>METHOD</b>Four hundred eighty-five patients with histological proven chronic hepatitis B were treated with 5 MU recombinant interferon-alpha 1b (rIFN-alpha1b) thrice weekly for 6-37 months (median 10). Serum HBV DNA, HBeAg and NAb levels of the patients were detected by fluorescent-quantitative PCR, enzymoimmunoassay and antiviral neutralizing biological assay respectively during the therapy.</p><p><b>RESULTS</b>Virological breakthrough occurred in 66 patients (13.6%), and NAb was found in 98 patients (20.2%) of the total 485 patients. The rate of NAb positivity was higher in patients with viral breakthrough than those without it (68.2%, 45/66, vs 12.6%, 53/419, chi(2)=109.06, P < 0.01), and viral breakthrough occurred more in patients with positive NAb than with negative NAb (45.9%, 45/98, vs 5.4%, 21/387, chi(2)=109.06, P < 0.01). The time of the viral breakthrough occurrence and the time of NAb production had a significant correlation (P < 0.01). The occurrence of viral breakthrough was also influenced by the age of patients (P < 0.05) and HBeAg status (P < 0.01) before they were treated.</p><p><b>CONCLUSION</b>Viral breakthrough occurred in 13.6% of our 485 chronic hepatitis B patients treated with recombinant interferon-alpha. Their viral breakthrough and production of NAb production had a significant correlation.</p>

Stable expression of HBV C gene mutants in immortalized human B-cell lines / 南方医科大学学报

<p><b>OBJECTIVE</b>To provide an cell model of immortalized lymphoblstoid B-cell lines for studying the biological characteristics of full-length hepatitis B virus (HBV) genome carrying the hot-spot mutations V60, G87, and L97.</p><p><b>METHODS</b>V60, G87, and L97 mutation points were introduced into HBV p3.8 II plasmid containing 1.2 copy of HBV genome by means of site-directed mutagenesis. The HBV genome was amplified by PCR from p3.8 II and p3.8 II-V60, G87, L97 plasmid, and the PCR product was inserted into EBO-plpp eukaryotic expression vector. The recombinant vectors and the EBO-plpp vector were transfected into immortalized human lymphoblasts with lipofectamine 2000 and selected with hygromycin. Steady expression of the target genes was determined by RT-PCR, Western blotting and microparticle enzyme immunoassay.</p><p><b>RESULTS</b>DNA sequence analysis indicated that the desired mutation was introduced into wild-type HBV DNA. HBsAg, HBeAg and HBcAg could be detected in EBO-HBV-transfected cell lysate or culture supernatant.</p><p><b>CONCLUSION</b>Transfectants that stably express HBV mutant antigen may provide a cell model to study the biological characteristics of HBV carrying hot-spot mutation in vitro.</p>

The factors associated with viral relapse after interferon treatment in chronic hepatitis C patients / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the relationship between hepatitis C virus (HCV) genotype, serum viral load and ALT levels, and the factors associated with the viral relapse after IFN treatment in patients with chronic hepatitis C.</p><p><b>METHODS</b>The HCV RNA levels were determined with Cobas Amplicor Monitor Test, version 2.0, and HCV genotypes were examined by means of PCR products of 5' NTR digested with restriction endonucleases. The patients with chronic hepatitis C were treated with PEG-IFN alpha -2a and Roferon-A for 24 weeks. Those with a viral response after 24 week treatment were followed for an additional 24 weeks. The association of clinical characteristics, such as sex, age, the way of the HCV infection, IFN treatment history and platelet counts, and the HCV genotype, virus load and medicine used for the viral relapse after IFN treatment were analyzed.</p><p><b>RESULTS</b>Of the 208 chronic hepatitis C patients, the ALT levels were not related to HCV RNA levels (r = 0.093, P > 0.05). No difference of ALT levels between HCV genotypes was found, and the HCV RNA load was also of no difference between HCV genotype 1 patients and non 1 patients. Of the 119 patients with viral response after 24 week treatment, 58 cases (48.7%) relapsed after another 24 week's follow-up. Relapse was not significantly related to the clinical characteristics, such as sex, age, mode of the infection, treatment history of IFN, AST/ALT ratio, platelet counts and the baseline viral load. Among patients with genotype 1 virus, the relapse rate was significantly higher than those patients with non-genotype 1 virus (54.5% vs 32.1%, P=0.039). The relapse rate after PEG-IFN alpha -2a treatment was lower than that of Roferon-A treatment (47.0% vs. 52.8%), but not significantly.</p><p><b>CONCLUSION</b>The viral relapse of chronic hepatitis C patients after IFN treatment was significantly associated with the genotypes of the HCV.</p>

Retreatment with interferon of relapsed chronic hepatitis C patients / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy and investigate the influencing factors of the interferon (IFN) retreatment for patients with chronic hepatitis C relapsed after a previous IFN treatment.</p><p><b>METHODS</b>A retrospective study was designed to analyze the retreatment with IFN of 60 relapsed chronic hepatitis C patients. All patients were from a randomized, opened and multi-center clinical trial about the efficacy and security of PEG-IFNalpha-2a compared to CIFNalpha-2a in the treatment of chronic hepatitis C in China. There were 35 patients treated with PEG-IFNalpha-2a and 25 with CIFNalpha-2a. The main parameter to evaluate the efficacy was sustained viral response (SVR) rate. The influence of viral concentration in serum, genotype and drug categories on the responses to IFN were analyzed.</p><p><b>RESULTS</b>For all the patients, the end of treatment virus response (ETVR) and SVR rates were 55.00% and 35.00% respectively. ETVR rate of PEG-IFNalpha-2a was significantly higher than that of CIFNalpha-2a (74.29% and 28.00% respectively, P < 0.01). SVR rate of PEG-IFNalpha-2a was also markedly higher than that of CIFNalpha-2a (45.71% and 20.00% respectively, P < 0.05). However, there was no significant difference between the high and low viral load groups. Among the patients with genotype 1, ETVR and SVR rates of PEG-IFNalpha-2a (75.00%, 45.83%) were significantly higher than those of CIFNalpha-2a (22.22%, 11.11%), (P < 0.01, P < 0.05 respectively), but in patients with genotype non-1, there were no such differences between the two groups.</p><p><b>CONCLUSION</b>Some relapsed patients were not responsive to the IFN retreatment. The efficacy of PEG-IFNalpha-2a was superior to CIFNalpha-2a. The conventional IFN was not suggested to be used in the relapsed cases with genotype 1. The viral load was not associated with the efficacy of IFN retreatment.</p>

Influences of the genotypes of HBV and HBeAg regarding their response to PEG-IFN in chronic hepatitis B patients / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the effects of genotypes of HBV and HBeAg on the response to PEG-interferon alpha (PEG-IFN) in chronic hepatitis B (CHB) patients.</p><p><b>METHODS</b>PCR-RFLP and S gene sequencing were conducted in 42 CHB patients.</p><p><b>RESULTS</b>The sustained response (SR) rates were 66.7% in genotype B and 27.3% in genotype C group. The P value was 0.039 by the Pearson Chi-square test, while it was 0.06 by the Fisher's exact test. The results suggested a trend that patients with genotype B HBV compared to genotype C had better SR to PEG-IFN therapy, although the difference was not significant. Results also showed that SR rate in patients with HBeAg-negative CHB (7/8 87.5%) was significantly higher than that in HBe+ CHB patients (8/21 38.1%, P < 0.05).</p><p><b>CONCLUSION</b>Our results indicate that HBV genotype and HBeAg, especially the later, are main factors for predicting PEG-IFN therapy response in CHB patients.</p>

Preliminary identification and analysis of point mutations correlated with response to interferon-alpha in hepatitis B virus post-transcriptional regulatory elements / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>It is still unclear whether viral genetic variability influences response to interferon (IFN)-alpha treatment. Recent reports suggest that IFN-alpha effects may be associated with hepatitis B virus (HBV) post-transcriptional regulation. This study was designed to explore the heterogeneity of HBV post-transcriptional regulatory elements (HPRE) and the relationship between the diversity of HPRE and the response to IFN-alpha treatment.</p><p><b>METHODS</b>The HPRE sequences from 31 Chinese patients infected with HBV were determined by directly sequencing of polymerase chain reaction (PCR) product, and comparing them to those from Caucasian patients. Subsequently, eukaryotic expression vectors containing HPRE at various points were constructed and transfected into HepG2 cells, which were then exposed to recombinant human cytokines.</p><p><b>RESULTS</b>The T to C point mutation at nt 1504 and the C to T (G) at nt 1508 in HPRE were found in 21 and 19 patients with chronic hepatitis B, respectively; the C to T point mutation at nt 1509 was found in 17 patients. These point mutations did not exist in the HPRE of the Caucasian patients. The activity of the CAT gene obviously increased in the case of T to C point mutation at nt 1504, but did not change in the case of the C to T (G) mutations at nt 1508 and 1509. The activity of the CAT gene at these point mutations of HPRE could be inhibited by IFN-alpha/gamma and tumor necrosis factor (TNF)-alpha except for the point mutations at nt 1508 of HPRE which may escape the suppression role of IFN-alpha on HPRE.</p><p><b>CONCLUSIONS</b>There are point mutations between the HPRE of Chinese and Caucasian HBV patients, which might be correlated with response to IFN-alpha. The variation of HPRE might affect the function of HPRE and influence the regulative function of IFN-alpha other than that of IFN-gamma or TNF-alpha on HPRE.</p>

Establishment of stable expression cell lines for HBsAg variants and analysis of antigenicity / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the mechanism of hepatitis B virus infected patients who is negative for HbsAg.</p><p><b>METHODS</b>DNA sequences of 46 patients were analyzed. In these patients, HBsAg was negative but HBV DNA was positive and six new HBsAg variants were identified. Four of the six variants were combined point mutants and two were insertion variants. These S genes were subcloned into eukaryotic expression vector EBO-plpp, and the recombinant eukaryotic expression plasmids were transfected into COS7 cells. Cell lines expressing mutant type HBsAg were obtained. The supernatants were detected by ELISA and RIA.</p><p><b>RESULTS</b>Only the two-amino acid-insertion variants could be detected and the others failed to react with polyclonal and monoclonal antibodies against HbsAg.</p><p><b>CONCLUSION</b>The results indicated that the point mutations and insertions may result in a conformational change of the S gene, which affect HBsAg antigenicity, suggesting a possible relationship between the variants and the negative conversion of HBsAg of the patients.</p>

The pegasys using and HCV genotype were the independent predictors of the response to antiviral therapy in chronic hepatitis C / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the predictors of IFN therapy in patients with chronic hepatitis C through making the multivariate logistic regression analysis.</p><p><b>METHODS</b>The patients in the opened, randomized and controlled trial were enrolled into two group, pegasys and Roferon-A group, and were given 24 weeks of pegasys (injection of 180 microg a week), and Roferon-A (injection three times of Roferon-A 3 MU a week) therapy, and followed 24 weeks. The HCV RNA content was determined at the time before, end of treatment and at the followed-up. The association of the response to the treatment with the clinical characteristics including age, gender, way of HCV infection, history of IFN treatment, planet count, AST/ALT ratio, HCV RNA level, HCV genotype and treatment drugs was made trough multivariate logistic regression analysis.</p><p><b>RESULTS</b>The PP population containing 197 cases was analyzed. After controlling for age, gender, way of HCV infection, history of IFN treatment, planet count, AST/ALT ratio, HCV RNA level and treatment, the HCV genotype was not predictor of the end of treatment viral response (ETVR) to IFN therapy (OR 0.604, 95% CI 0.271-1.349, P = 0.219), but was the independent predictor of sustained viral response (SVR) (OR 0.408, 95% CI 0.189-0.881, P = 0.023). After controlling for other characteristics, the treatment drug was the predictors of ETVR (OR 0.105, 95% CI 0.052-0.212, P < 0.001) and SVR (OR 0.255, 95% CI 0.123-0.529, P < 0.001).</p><p><b>CONCLUSION</b>The pegasys using and HCV genotype were the independent predictors of the response to antiviral therapy in chronic hepatitis C.</p>

The influence of HCV genotype on the IFN treatment of patients with chronic hepatitis C / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the influence of HCV genotype on the IFN treatment of patients with chronic hepatitis C.</p><p><b>METHODS</b>The genotypes of HCV virus were determined in the patients enrolled into the Randomized, opened and controlled trial of Peg-IFN alpha-2a (Pegasys) treatment, controlled with IFN-alpha-2a (Roferon-A), on chronic hepatitis C patients in China. The serum ALT levels and HCV RNA concentration of the patients were detected in the time of before treatment, the end of therapy and follow-up. The influence of HCV genotype on the IFN treatment of patients with chronic hepatitis C was analyzed in intention to treat (ITT) population.</p><p><b>RESULTS</b>The HCV genotypes of 202 cases were determined. 158 (78.2%) cases infected with genotype 1 HCV and 44 (21.8%) cases with genotype non-1. For overall patients, the viral response at the end of treatment (ETVR) and sustained viral response (SVR) rates were 53.8% and 25.3% respectively in patients with genotype 1 HCV, but in genotype non-1 patients those was 61.4% and 43.2%, and the difference of SVR between genotype 1 and non-1 was significant (P=0.021). After grouped by the used drugs, in the patients given Pegasys treatment, the ETVR rates of patients with genotype 1 and non-1 HCV infection were 76.8% and 81.0%, the difference was not significant (P=0.686), but the difference of SVR rates, which were 35.4% and 66.7%, of the patients was significant (P=0.01). The viral relapse rate of genotype 1 was 55.6%; it was significant higher than that of genotype non-1 (23.5%) (P=0.02). In Roferon-A group, the ETVR and SVR rates of patients with genotype 1 HCV were 29.0% and 14.5%, which were lower, but not significant, than those of patients with genotype non-1 (43.5% and 21.7%). The viral relapse rate of genotype 1 was 72.7% and higher, but not significant, than that of genotype non-1 also (50.0%) (P=0.21).</p><p><b>CONCLUSION</b>HCV genotype could affects the efficacies, mainly the sustained responses, of IFN treatment of patients with chronic hepatitis C, and the effects of IFN were related to the kinds of drugs and therapeutic course.</p>

Clinical significance of neutralizing anti-interferon antibodies in chronic hepatitis B patients treated with recombinant interferon-alpha / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the clinical significance of neutralizing anti-interferon-alpha antibodies (NA) in chronic hepatitis B patients treated with recombinant interferon-alpha(rIFN-alpha).</p><p><b>METHODS</b>One hundred and eighty-one patients (128 male and 53 female) with histological proven chronic hepatitis B were treated with 5 MU recombinant interferon-alpha 1b (rIFN-alpha 1b) subcutaneously thrice weekly for 6 to 37 (median 10) months. For each patient, Specific detection of serum HBV DNA level with fluorescent-quantitative PCR, HBeAg with enzymoimmunoassay and NA with an antiviral neutralizing biological assay were performed during therapy.</p><p><b>RESULTS</b>NA was found in 61 (33.7%) of 181 patients. At the end of treatment, complete-response was achieved in 17 (27.9%) of 61 patients with NA and in 54 (45.0%) of 120 patients without NA, respectively (chi2=4.979). For NA positive patients, the complete-response rate was significantly lower in those who had not achieved partial-response prior to or at the same time as NA occurred than in those who did (3.8%, 1/26, vs. 45.7%, 16/35, chi2 = 7.457). Moreover, it was lower in patients who either had 20pg/ml of serum HBV DNA or above or HBV DNA had being reduced by less than 60% recent 3 months, but higher in those who had less than 20pg/ml of HBV DNA and HBV DNA had being reduced by 60% or above (20.0%, 9/45, vs. 56.3%, 9/16, chi2 = 11.009).</p><p><b>CONCLUSION</b>NA may negate the antiviral effects of rIFN-alpha in chronic hepatitis B patients treated with rIFN-alpha, especially if they appear before partial-response or at the occasion at which serum HBV DNA level was not below 20pg/ml or HBV DNA had being reduced by less than 60% recent 3 months.</p>

Analysis of three lamivudine-resistant HBV mutants with the method of restriction enzyme digestion and its application / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the prevalence of three lamivudine-resistant HBV mutants in lamivudine-treated Chinese patients.</p><p><b>METHODS</b>Using three pairs of of HBV polymerase gene B and C domain fragments were amplified by PCR. The PCR products were then digested by restriction enzyme Nde I and Nla III. The digested products were analyzed by electrophoresis. With this method, the prevalence of the three lamivudine-resistant mutants in lamivudine-treated Chinese patients was investigated.</p><p><b>RESULTS</b>After Nde I digestion of p24 and p29 amplified product, HBV wild type could be easily separated from YMDD mutant. At the same time, YIDD could be separated from YVDD mutant after Nla III digestion of p24 and p29 amplified product. By this method, the authors found that these eleven patients were infected with lamivudine-resistant mutants. Six of them were infected with M5501 mutant; five were infected with M550V mutant (one of them had both M550V and L526M mutations).</p><p><b>CONCLUSION</b>The method of the present study was demonstrated to be an easy way to detect HBV lamivudine-resistant mutants and can be applied to clinical monitoring of lamivudine resistance.</p>

Hepatitis B virus genotypes and the heterogeneity of its polymerase gene / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the heterogeneity of polymerase gene (P gene) within hepatitis B virus (HBV) genotypes based on a systematic analysis of 202 HBV P genes, providing some useful references for further studies on the relationship among HBV genotypes, P gene mutations, replication and nucleoside analogues drug-resistance.</p><p><b>METHODS</b>202 HBV complete sequences containing P genes were obtained from GenBank and were analysed using computer softwares.</p><p><b>RESULTS</b>There were some genotype-related characteristics of HBV P genes. As reverse transcriptase domain was concerned, there were more amino acid divergences in genotype C and D compared with these in genotype A. There were also amino acid substitutions in the A-F conserved regions of the reverse transcriptase domain within and between HBV genotypes.</p><p><b>CONCLUSIONS</b>There are divergences of P genes and amino acids within and between HBV genotypes, which should be considered when amino acid changes are analyzed whether they are proposed to be drug-resistance mutations or the results from quasispecies-selected. Moreover, these divergences may affect the antiviral effect of nucleoside analogues on HBV with different genotypes.</p>

Non-relationship between HCV genotype,serum viral load,and ALT,AST levels in chronic hepatitis C patients / 中华检验医学杂志

0.05),the difference of ALT,AST level and AST/ALT ratio between high viral load (serum HCV RNA≥8.5?10~5 IU/ml) group and low viral load (serum HCV RNA