HBeAg and hepatitis B virus DNA as outcome predictors during therapy with peginterferon alfa-2a for HBeAg-positive chronic hepatitis B.

UNLABELLED: The aims of this study were to evaluate the usefulness of quantitative hepatitis B e antigen (HBeAg) values for predicting HBeAg seroconversion in patients treated with peginterferon alfa-2a and to assess the dynamic changes in quantitative HBeAg during therapy, compared with conventional measures of serum hepatitis B virus DNA. Data were analyzed from a large, randomized, multinational phase III registration trial involving 271 HBV-infected HBeAg-positive patients who received peginterferon alfa-2a plus oral placebo for 48 weeks. HBeAg levels were measured serially during therapy using a microparticle enzyme immunoassay validated with in-house reference standards obtained from the Paul Ehrlich Institute (PEIU/mL). In patients who achieved HBeAg seroconversion, levels of HBeAg consistently decreased during treatment and remained at their lowest level during the 24 weeks of posttreatment follow-up. After 24 weeks of treatment, 4% of patients with the highest levels of HBeAg (>or=100 PEIU/mL) achieved HBeAg seroconversion, yielding a negative predictive value of 96%, which was greater than that obtained for levels of HBV DNA (86%). Late responders to peginterferon alfa-2a could also be differentiated from nonresponders by continued decrease in HBeAg values, which were not evident by changes in HBV DNA. CONCLUSION: These analyses suggest quantitative HBeAg is a useful adjunctive measurement for predicting HBeAg seroconversion in patients treated with peginterferon when considering both sensitivity and specificity compared with serum HBV DNA.

Peginterferon Alfa-2a, lamivudine, and the combination for HBeAg-positive chronic hepatitis B.

BACKGROUND: Current treatments for chronic hepatitis B are suboptimal. In the search for improved therapies, we compared the efficacy and safety of pegylated interferon alfa plus lamivudine, pegylated interferon alfa without lamivudine, and lamivudine alone for the treatment of hepatitis B e antigen (HBeAg)-positive chronic hepatitis B. METHODS: A total of 814 patients with HBeAg-positive chronic hepatitis B received either peginterferon alfa-2a (180 microg once weekly) plus oral placebo, peginterferon alfa-2a plus lamivudine (100 mg daily), or lamivudine alone. The majority of patients in the study were Asian (87 percent). Most patients were infected with hepatitis B virus (HBV) genotype B or C. Patients were treated for 48 weeks and followed for an additional 24 weeks. RESULTS: After 24 weeks of follow-up, significantly more patients who received peginterferon alfa-2a monotherapy or peginterferon alfa-2a plus lamivudine than those who received lamivudine monotherapy had HBeAg seroconversion (32 percent vs. 19 percent [P<0.001] and 27 percent vs. 19 percent [P=0.02], respectively) or HBV DNA levels below 100,000 copies per milliliter (32 percent vs. 22 percent [P=0.01] and 34 percent vs. 22 percent [P=0.003], respectively). Sixteen patients receiving peginterferon alfa-2a (alone or in combination) had hepatitis B surface antigen (HBsAg) seroconversion, as compared with 0 in the group receiving lamivudine alone (P=0.001). The most common adverse events were those known to occur with therapies based on interferon alfa. Serious adverse events occurred in 4 percent, 6 percent, and 2 percent of patients receiving peginterferon alfa-2a monotherapy, combination therapy, and lamivudine monotherapy, respectively. Two patients receiving lamivudine monotherapy had irreversible liver failure after the cessation of treatment--one underwent liver transplantation, and the other died. CONCLUSIONS: In patients with HBeAg-positive chronic hepatitis B, peginterferon alfa-2a offers superior efficacy over lamivudine, on the basis of HBeAg seroconversion, HBV DNA suppression, and HBsAg seroconversion.

[Study of the relapse of patients with chronic hepatitis B undergoing first and repeated recombinant interferon-alpha therapy during long-term follow up].

OBJECTIVE: To investigate the relapse of patients with chronic hepatitis B (CHB) undergoing first and repeated recombinant interferon-alpha (rIFN-alpha) therapy during long-term follow up. METHOD: Five hundred and twenty three patients with chronic hepatitis B including 403 hepatitis B e-antigen (HBeAg) positive patients and 120 HBeAg negative ones were treated with 5MU recombinant interferon-alpha 1b (rIFN-alpha1b) subcutaneously thrice weekly for 6 - 25 (median 10) months. For each patient, serum alanine aminotransferase (ALT) was measured biochemically and serum HBV DNA level was detected by fluorescent-quantitative PCR, HBeAg with enzyme immunoassay every 1 - 3 month during therapy and every 3 - 6 month during the follow up period. Some of the individuals who relapsed during the follow-up period were treated with interferon-alpha repeatedly. RESULTS: Ratios of early response to interferon-alpha were similar in HBeAg positive patients (55.8%, 225/403), and HBeAg negative patients (64.2%, 77/120) at the end of naive treatment (chi(2) = 2.633, P = 0.105). 39.4% (119/302) of early responders relapsed during 39 +/- 22-month follow up, and relapse rates in HBeAg negative group (55.8%, 43/77) were higher than those in HBeAg positive group (33.8%, 76/225) at the end of follow up (chi(2) = 19.335, P = 0.000). Divided the follow-up period into six fragments as 1 - 12 months, 13 - 24 months, 25 - 36 months, 37 - 48 months, 48 - 60 months and > or = 61 months, we found that the differences of relapse incidence were significant (chi(2) = 73.518, df = 5, P = 0.000), and accumulative relapse rates were significant too (chi(2) = 32.167, df = 5, P = 0.000) in all follow-up periods. Constituent ratios of HBeAg in relapsed patients of every follow-up period were similar. 57 relapsed individuals (25 in HBeAg positive group and 32 in HBeAg negative group) were retreated with interferon-alpha, and complete response were achieved in all cases at the end of repeated therapy. The relapse rates in HBeAg positive group (52.0%, 13/25) were higher than in HBeAg negative group (21.9%, 7/32) during the follow-up period after the end of retreatment (chi(2) = 5.592, P = 0.018). CONCLUSION: Rates of early response to interferon-alpha therapy were similar in HBeAg positive and HBeAg negative patients at the end of nave treatment, and relapse rates in HBeAg negative group were higher than in HBeAg positive group during long term follow-up. Combined response was achieved in all relapse cases received repeated interferon-alpha therapy at the end of retreatment. The relapse rates in HBeAg positive group were higher than in HBeAg negative group during the follow-up period after repeated therapy.

[Viral breakthrough and neutralizing anti-interferon antibody production in chronic hepatitis B patients treated with recombinant interferon-alpha].

OBJECTIVE: 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). METHOD: 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. RESULTS: 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. CONCLUSION: 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.

[Retreatment with interferon of relapsed chronic hepatitis C patients].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[The factors associated with viral relapse after interferon treatment in chronic hepatitis C patients].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: The viral relapse of chronic hepatitis C patients after IFN treatment was significantly associated with the genotypes of the HCV.

Preliminary identification and analysis of point mutations correlated with response to interferon-alpha in hepatitis B virus post-transcriptional regulatory elements.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Predictive factors for sustained response to interferon treatment in patients with chronic hepatitis C: a randomized, open, and multi-center controlled trial.

BACKGROUND: This study was undertaken to investigate the predictive factors of sustained viral response in roferon-A or pegasys treated chronic hepatitis C patients after logistic regression analysis of the factors that might be associated with the therapeutic effects of interferon (IFN). METHODS: All patients enrolled into this randomized, open and multi-center controlled trial were divided into two groups randomly and treated with pegasys and roferon-A for 24 weeks, then followed up for another 24 weeks. Before treatment, hepatitis C virus (HCV) genotype was determined, and HCV-RNA in serum was detected before and at the end of treatment and follow-up. HCV-RNA turning negative was considered the major index for evaluating the therapeutic effect. The clinical characteristics including gender, age, infection route of HCV, treatment with IFN, platelet count, AST/ALT ratio and treatment drugs were analyzed by logistic regression. RESULTS: Intention to treat (ITT) and per-protocol (PP) population groups have 208 and 197 patients respectively. In the PP group, after treatment for 24 weeks, the response rates of female patients aged less than 50 years, infected through non-transfusion, relapsed after IFN treatment, and presented with a AST/ALT ratio/=1, virus load equal or more than 8 x 10(5) IU/ml, and genotype 1 infection, and treated finally with roferon-A. But, at the end of follow-up, the patients with a AST/ALT ratio>/=1 and virus load more than 8 x 10(5) IU/ml had a higher rate of sustained response than did those with a AST/ALT ratio

[A comparison of clinical and virological characteristics of 1686 cases of HBeAg-negative and HBeAg-positive chronic hepatitis B].

OBJECTIVE: To investigate the clinical characteristics of HBeAg-negative and HBeAg-positive chronic hepatitis B (CHB). METHODS: A total of 1686 hospitalized CHB cases were analyzed retrospectively. The serum ALT values, HBV DNA levels and hepatic inflammation and fibrosis were analyzed by their serum HBeAg status. RESULTS: Among the 1686 cases, 628 (37.3%) were HBeAg-negative and 1058 (62.7%) were HBeAg-positive. Compared with HBeAg-positive group, HBeAg-negative group had a lower serum ALT and HBV DNA levels. However, hepatic necroinflammation grading and fibrosis staging in HBeAg-negative group were more advanced than that of HBeAg-positive group. Irrespective to serum HBeAg status, patients with serum HBV DNA less than 10(5)copies/ml, had a lower hepatic necroinflammation activity. CONCLUSIONS: HBeAg-positive CHB is still the predominant form of CHB in Chinese patients. Compared with patients with low HBV replication, patients with active HBV replication had a higher hepatic necroinflammation activity. The liver histological grading and staging in HBeAg-negative CHB patients were more advanced than that in HBeAg-positive patients.

[Influences of the genotypes of HBV and HBeAg regarding their response to PEG-IFN in chronic hepatitis B patients].

OBJECTIVE: 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. METHODS: PCR-RFLP and S gene sequencing were conducted in 42 CHB patients. RESULTS: 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). CONCLUSION: Our results indicate that HBV genotype and HBeAg, especially the later, are main factors for predicting PEG-IFN therapy response in CHB patients.

Impact of virus genotype on interferon treatment of patients with chronic hepatitis C: a multicenter controlled study.

BACKGROUND: Some factors have been reported to be associated with a greater likelihood of sustained viral response (SVR) in the interferon (IFN) treatment of chronic hepatitis C. The factors include HCV genotype, HCV RNA level in serum, state of liver disease, baseline body weight, age, sex, and race. The aim of this trial was to investigate the influence of HCV genotype on the IFN treatment of patients with chronic hepatitis C. METHODS: The genotypes of HCV virus were determined in the patients with chronic hepatitis C from several hospitals of China enrolled into the randomized, opened and controlled trial of Peg-IFN alpha-2a (pegasys) treatment, controlled with IFN-alpha-2a (roferon-A). The serum ALT levels and HCV RNA concentrations of the patients were detected before and at the end of treatment and during the 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. RESULTS: The HCV genotypes of 202 patients were determined. Of these patients, 158(78.22%) were infected with genotype 1 HCV and 44(21.78%) with genotype non-1. The viral response at the end of treatment (ETVR) and sustained viral response (SVR) rates were 53.80% and 25.32% respectively in patients with genotype 1 HCV, but they were 61.36% and 43.18% in patients with genotype non-1. The difference of SVR between patients with genotype 1 HCV and those with genotype non-1 was significant (P=0.021). After being grouped by the used drugs, the ETVR rates of patients infected with genotype 1 and non-1 HCV were 76.83% and 80.95% in the patients treated with pegasys (P=0.686); but their SVR rates were 35.37% and 66.67% (P=0.01). The viral relapse rate of genotype 1 HCV (55.56%) was significantly higher than that of genotype non-1 HCV (23.53%) (P=0.02). In roferon-A group, the ETVR and SVR rates of patients with genotype 1 HCV were 28.95% and 14.47% respectively, which were lower but not more significant than those of patients with genotype non-1 HCV (43.48% and 21.74%). Moreover, the viral relapse rate of genotype 1 HCV (72.73%) was higher but not more significant than that of genotype non-1 HCV (50.00%) (P=0.21). CONCLUSION: HCV genotype could affect the efficacies, mainly sustained responses, of IFN treatment in patients with chronic hepatitis C, and the effects of IFN are related to drugs and therapeutic course.

[Expression of precore and core proteins of hepatitis B virus in human hepatoma cell line].

To further investigate the functions of proteins encoded by C open reading frame of hepatitis B virus (HBV) in HBV infectious cycle, cell lines expressing these proteins were established. The DNA fragments encoding 25 000, 22 000 precore-core and 21 000 core protein respectively were amplified from plasmid pCP10 which contained HBV genome, and inserted into a eukaryotic expression vector pcDNA-3, and then transfected into hepatoma cell line HepG2. The transformants were selected with neomycin, and three isolated colonies expressing precore or core proteins were obtained stably, which were demonstrated by immunohistochemistry and Western blotting analysis.

Preliminary identification and analysis of different points related with response to interferon-alpha in HBV post-transcriptional regulatory element.

OBJECTIVE: To screen the differential points related with response to interferon-alpha in HBV post-transcriptional regulatory element (HPRE). METHODS: HPRE sequence of 31 Chinese patients with HBV infection were detected by direct sequencing of PCR products. Differential points in HPRE between Chinese patients and White patients were compared. RESULTS: The T to C differential point at nt 1 504 and C to T(G) at nt 1 508 were found respectively in HPRE of 21 and 19 patients with chronic hepatitis B, the C to T differential point at nt 1 509 were found in 17 cases. These differential points were not found in HPRE of the White patients. The relationship between the HPRE differential points and the levels of HBV DNA was not observed. CONCLUSION: There were differential points in the HPRE between Chinese and White HBV patients, which might be correlated with response to interferon-alpha.

[Cloning and sequence analysis of a novel TT virus varian].

OBJECTIVE: To clone the DNA of a TT virus (TTV) variant isolated from a patient with elevated alanine transaminase (ALT) of unknown etiology, and conduct sequence analysis. METHODS: The long fragment of TTV DNA was amplified by nested PCR and then cloned into pGEM-T vector. A clone named 56-B containing 3.2 kb TTV DNA was selected for sequence analysis besides homology analysis with other 5 TTV variants retrieved from GenBank, and phylogenetic analysis was carried out by maximum likelihood method. RESULTS: The nucleotide identities of 56-B with the other 5 TTV strains TA278, JA10, US35, SANBAN and TUS01 were 42.4%, 48.2%, 47.9%, 49.8 % and 61.7 % respectively, and the corresponding amino acid identities were even lower. Phylogenetic analysis showed that 56-B was far from other TTV strains in genetic distance that ranged from 0.344 to 0.458. However, the sequences in the 5'- and 3'-end were still much conservative. CONCLUSION: The isolated 56-B showed high heterogeneity in genetic background and was therefore quite distinct from other TTV strains as a novel TTV variant that represents a new TTV genotype.

[Cloning and sequence analysis of the genome of a strain of duck hepatitis B virus].

OBJECTIVE: To clone and analyze the genome of a duck hepatitis B virus (DHBV) strain isolated from the ducks found in the local area. METHODS: The complete genome of DHBV was amplified by PCR prior to cloning into T vector and sequencing, with also homology and phylogenetic analyses. RESULT: Genome comparison of an isolated DHBV strain and 16 DHBV strains in GenBank revealed a homology of 89.4% to 99.3% at the nucleotide level, and the amino acid identity of the 3 open reading frames showed that the P region harbored more obvious variations than the other regions. CONCLUSION: The isolated DHBV strain might belong to a subtype of the virus found in the western countries.

[Application of multiplex PCR in genotyping of hepatitis B virus prevailing in Guangdong Province of China].

OBJECTIVE: To establish a convenient method for the genotyping of hepatitis B virus (HBV) using multiplex PCR. METHOD: Based on the alignment of 114 complete nucleotide sequences of HBV DNA belonging to different genotypes, acquired from the GenBank, genotype-specific sequences were identified according to which 6 pairs of primers were designed corresponding to each genotype. Subsequent genotyping of HBV was performed using these primers that were added, either alone or in conjunction with others, into a multiplex PCR reaction tube, and HBV genotype was determined according to the length of amplified DNA. RESULT: The genotyping result of multiplex PCR was consistent with that produced by PCR- restriction fragment length polymorphism as established by Lindh. We found in this study that among the HBV carriers in the vicinities Guangzhou of City, about 45% belonged to B genotype, 38.75% to C genotype and 16.75% to D genotype. CONCLUSION: This multiplex PCR method is simple, convenient and more differential.

[Effects of partial deletion in the core promoter of hepatitis B virus genome on viral antigen expressions].

OBJECTIVE: To study the effects of 20/21 bp partial deletion mutation (from nt 1 748 or nt 1 747 to nt 1 767) in the core promoter (CP) region of hepatitis B virus (HBV) genome complicated by precore stop condon mutation at nt 1 896 on the expression of the viral antigens. METHODS: Eukaryotic expression vector containing full-length HBV genome with the above mutations was constructed. After transfection of the recombinant HBV plasmids into HepG2 cells, the expression of the viral antigens was examined with enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. RESULTS: As shown by ELISA and Western blotting analysis, the amount of extracellular secretion of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) along with intracellular hepatitis B core antigen (HBcAg) in the cells transfected with vectors containing HBV genomes with partial deletion in the CP region was markedly reduced compared with that produced by wild-type HBV. CONCLUSION: The mutations in question causes marked reduction in viral antigen production by HBV in comparison that by wild-type HBV.

[Construction and biological activity assessment of core mutants of hepatitis B virus adr subtype].

OBJECTIVE: To construct the core mutant (L97 and V60) plasmids of hepatitis B virus (HBV) and assess their biological activity. METHODS: Site-directed mutagenesis was performed to induce specific core point mutations in HBV adr suhtype 1.2 copy genome plasmid p3.8 II. The plasmids were transfected into HepG2 cells via liposome, and intracellular HBV DNA was analyzed by Southern hybridization, while extracellular HBV antigens (HBsAg and HBeAg) in the culture supernatant were assayed by enzyme-linked immunosorbent assay. RESULTS: Sequence analysis of the constructed mutant plasmids p3.8 L97 and p3.8 V60 demonstrated mutations at 2 189 A C and 2 086 C HBV adr subtype genome core mutant (L97 and V60) plasmids we have constructed possess biological activity.G respectively. These mutant plasmids exhibited HBV DNA replication activity and gene expression in host cells. CONCLUSION: HBV adr subtype genome core mutant (L97 and V60) plasmids we have constructed possess biological activity.

Detection of the YMDD motif mutation of P gene of hepatitis B virus using polymerase chain reaction-restriction fragment length polymorphism.

OBJECTIVE: To establish a convenient approach to implement polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for detecting of gene mutation. METHODS: Serum samples were collected from l0 patients with chronic hepatitis B who had received oral lamivudine for at least l year, and 3 methods including mismatched PCR-RFLP, direct sequence analysis of the PCR product and cloning prior to secondary sequence analysis were employed respectively to examine the YMDD motif mutation of P gene of hepatitis B virus (HBV). RESULTS: PCR-RFLP revealed wild-type HBV infection in 7 cases mixed infection in 1 case and HBV mutant infection in 2 cases. With direct sequence analysis of the PCR product, 9 cases were found with Wild-type HBV infection and 1 with HBV mutant infection. However, 2 non-wild-type HBVV-infected cases as approved by PCR-RFLP, opposite to the results by direct sequence analysis, were both shown by sequence analysis after cloning to suffer mixed infection. CONCLUSION: Sequence analysis following cloning is the best way to detect YMDD motif mutation in HBV, but in terms of economy and practicability, mismatched PCR-RFLP is of greater significance in mass screening of YMDD motif mutant HBV than the other 2 approaches.

Hot-spot mutations in HBV pre-C region in HBeAg-negative patients with severe hepatitis B.

OBJECTIVE: To investigate the association of hot-spot mutations in hepatitis B virus (HBV) pre-C region with the occurrence and outcome of severe hepatitis B. METHODS: A total of 68 patients with severe hepatitis B negative for hepatits B e antigen (HBeAg) were enrolled in this study, including 6 cases of acute, 38 cases of subacute and 24 chronic severe hepatitis B, with another 44 HBeAg-positive patients with chronic hepatitis B serving as control. Mismatch PCR and restriction fragment length polymorphism analysis were employed to examine the mutations of T1862 and A1896 in this 2 groups of patients. RESULTS: The mutation rates at A1896 and T1862 were 66.7% (4/6) and 0 (0/6) respectively in acute severe hepatitis B cases, 42.1% (16/38) and 15.8% (6/38) in subacute severe hepatitis, 25.0% (6/24) and 16.7% (4/24) in chronic severe hepatitis, and 45.5% (20/24) and 2.3% (1/44) in chronic hepatitis cases. There were significant differences in terms of T1862 mutation between patients with severe hepatitis and chronic hepatitis (P<0.01). CONCLUSION: T1862 mutation is closely related to the exacerbation of chronic hepatitis, while the role of A1896 mutation in this process requires further investigation.