Expression of hepatitis B virus 1.3-fold genome plasmid in an SV40 T-antigen-immortalized mouse hepatic cell line.

AIM: To investigate the expression of the hepatitis B virus (HBV) 1.3-fold genome plasmid (pHBV1.3) in an immortalized mouse hepatic cell line induced by SV40 T-antigen (SV40T) expression. METHODS: Mouse hepatic cells were isolated from mouse liver tissue fragments from 3-5 d old Kunming mice by the direct collagenase digestion method and cultured in vitro. The pRSV-T plasmid was transfected into mouse hepatic cells to establish an SV40LT-immortalized mouse hepatic cell line. The SV40LT-immortalized mouse hepatic cells were identified and transfected with the pHBV1.3 plasmid. The levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the supernatant were determined by an electrochemiluminescence immunoassay at 24, 48, 72 and 96 h after transfection. The expressions of HBsAg and hepatitis B c antigen (HBcAg) in the cells were investigated by indirect immunofluorescence analysis. The presence of HBV DNA replication intermediates in the transfected cells and viral particles in the supernatant of the transfected cell cultures was monitored using the Southern hybridization assay and transmission electronic microscopy, respectively. RESULTS: The pRSV-T plasmid was used to immortalize mouse hepatocytes and an SV40LT-immortalized mouse hepatic cell line was successfully established. SV40LT-immortalized mouse hepatic cells have the same morphology and growth characteristics as primary mouse hepatic cells can be subcultured and produce albumin and cytokeratin-18 in vitro. Immortalized mouse hepatic cells did not show the characteristics of tumor cells, as alpha-fetoprotein levels were comparable (0.58 ± 0.37 vs 0.61 ± 0.31, P = 0.37). SV40LT-immortalized mouse hepatic cells were then transfected with the pHBV1.3 plasmid, and it was found that the HBV genome replicated in SV40LT-immortalized mouse hepatic cells. The levels of HBsAg and HBeAg continuously increased in the supernatant after the transfection of pHBV1.3, and began to decrease 72 h after transfection. The expressions of HBsAg and HBcAg were observed in the pHBV1.3-transfected cells. HBV DNA replication intermediates were also observed at 72 h after transfection, including relaxed circular DNA, double-stranded DNA and single-stranded DNA. Furthermore, a few 42 nm Dane particles, as well as many 22 nm subviral particles with a spherical or filamentous shape, were detected in the supernatant. CONCLUSION: SV40T expression can immortalize mouse hepatic cells, and the pHBV1.3-transfected SV40T-immortalized mouse hepatic cell line can be a new in vitro cell model.

[Functional analysis of hepatitis B virus immune escape mutants with insertion mutations in the surface antigen].

OBJECTIVE: To evaluate the influence of insertion mutations occurring in the hydrophobic region, between amino acids 114 and 115, of the hepatitis B surface antigen (HBsAg) on viral antigenicity and replication. METHODS: Hepatitis B virus (HBV) DNA was obtained from patients with HBsAg-positive chronic hepatitis B (CHB) infection and subjected to sequence analysis and comparison to GenBank reference sequences for HBV genotype B (AB073826) and genotype C (AF286594). Insertion mutations detected in the HBsAg region were used to make recombinant expression plasmids via site-directed mutagenesis. After transfecting the recombinant HBsAg into Huh7 cells, the mutants' effects on viral antigenicity and replication were evaluated by chemiluminescence microparticle immunoassay (CMIA) and Southern blot hybridization, respectively. The viral antigenicity of each mutant was predicted by bioinformatic analysis, using the Jameson-Wolf method to predict the antigenic index, the Hopp-Woods method to predict hydrophilicity, the Emini method to predict the probability of a region lying of the protein's surface, and the Karplus-Schulz method to predict the flexibility of the protein backbone. RESULTS: Two CHB patients harbored HBV with insertion mutations in HBsAg: one with two (NT) and one with three (NTT) inserted amino acids between 114 and 115. The NTT recombinant HBsAg mutant showed no impact on viral replication and reacted weakly with anti-HBs in CMIA (P = 0.02). The antigen indices for the insertion of NTT were 1.00, -0.16, and 0.18, and insertion of the three amino acids affected the index values of five proximal amino acid sites (with an average increase of 0.13). The hydrophilic indices for the insertion of NTT were 0.2, -0.4, and -0.4, with no significant effect on the proximal amino acids. The insertion of the three amino acids changed both the surface probability (range: -0.55 to 2.97; affecting eight proximal amino acids) and the flexibility (range: -0.01 to 1.1; affecting five proximal amino acids). CONCLUSION: The NTT three amino acids insertion in the HBsAg open reading frame, between 114 and 115 of the wild-type sequence, detected in a CHB patient may play a role in HBV immune escape without influencing the viral replicative capacity.

[A retrospective follow-up study of prolonged nucleos(t)ide analogue and interferon combination therapy in HBeAg-positive chronic hepatitis B patients].

OBJECTIVE: To investigate the efficacy of a 96-week course of nucleos(t)ide analogue and interferon (IFN) combination therapy for achieving seroconversion at 24 weeks after completion in patients with chronic hepatitis B (CHB). METHODS: One-hundred-and-thirty-five CHB patients with positivity for hepatitis B e antigen (HBeAg) were recruited for study between January 2005 and December 2008. All patients were given a 96-week course of nucleos(t)ide analogue (lamivudine or adefovir dipivoxil) alone (monotherapy controls, n = 45) or in combination with IFN or Pegylated-IFN-alpha-2a (Peg-IFNa-2a) (n = 90). At treatment weeks 12, 24, 48, 72, and 96, and at 24 weeks after treatment completion, serum samples were collected from all patients for assessment of biochemical, virological and serological responses to treatment. The biochemical response was indicated by normalization of the alanine aminotransferase (ALT) level. The virologic response was indicated by a reduction in the hepatitis B virus (HBV) DNA level to less than 1000 copies/ml. The serological response was indicated by seroconversion of either HBeAg or hepatitis B surface antigen (HBsAg). Statistical analysis was performed with the Chi-squared test. RESULTS: Among the patients treated with nucleos(t)ide analogue and IFN combination therapy, 41.1% (37/90) achieved HBeAg seroconversion and 18.9% (17/90) achieved HBsAg seroconversion at the end of treatment. However, significantly less of the patients treated with nucleos(t)ide analogue monotherapy achieved HBeAg seroconversion and none achieved HBsAg seroconversion by end of treatment (33.3% and 0%, respectively; x2= 8.08, P less than 0.01 vs. the combination therapy group). Age stratification of the 17 HBsAg-seroconverted patients treated with combination therapy indicated that the HBsAg seroconversion rate was significantly higher in patients less than 30-years-old than those 30 and older (x2= 12.62 and 4.24, respectively, P less than 0.05). At post-treatment week 24, the 17 HBsAg-seroconverted patients treated with combination therapy showed HBsAg titers of less than 250 IU/ml; moreover, 11.8% (2/17) of these patients remained HBeAg-positive and 17.6% (3/17) showed abnormal ALT levels and elevated HBV DNA. CONCLUSION: Prolonged nucleos(t)ide analogue plus IFN combination therapy can significantly improve the rate of HBsAg seroconversion in HBeAg-positive CHB patients, and this treatment regimen is especially efficacious in patients under the age of 30.

[Clinical implication of expression of perforin/granzyme in peripheral blood lymphocyte in severe hepatitis B].

OBJECTIVE: To explore role of perforin/granzyme expression of peripheral blood lymphocyte in injury of hepatocyte in severe hepatitis B, and evaluate relationship between perforin/granzyme expression levels and hepatitis B virus (HBV)-DNA load. METHODS: Thirty eight patients of severe hepatitis B were enrolled in the study. Fasting venous blood was collected on following morning of admission. Twenty adult healthy subjects served as healthy control group. Perforin/granzyme expression of peripheral blood lymphocyte was detected by flow cytometry, and serum HBV-DNA load was detected by fluorescence quantitative polymerase chain reaction (PCR). RESULTS: Positive rate of perforin/granzyme in peripheral blood lymphocyte in severe hepatitis B was higher than that of the healthy control group [perforin: (43.42+/-19.28)% vs. (19.65+/-9.27)%, granzyme: (40.35+/-12.26)% vs. (22.28+/-9.35)%, both P<0.01]. There was a significant negative correlation between the perforin/granzyme expression of peripheral blood lymphocyte and serum HBV-DNA load (r(perforin) =-0.92, r(granzyme) =-0.96, both P<0.01), the higher serum HBV-DNA load, the lower perforin/granzyme expression in severe hepatitis B. CONCLUSION: Perforin/granzyme overexpression in peripheral blood lymphocyte is an important factor in injury of hepatocyte in patients with severe hepatitis B, and the expression may be involved in HBV-DNA cleanup.

Detection of circulating antigen with a MAb-based sandwich-ELISA and its comparison with specific IgM detection in sera of patients with hemorrhagic fever with renal syndrome.

A monoclonal antibody (1H2) against nucleocapsid protein of Hantavirus was developed. A sandwich enzyme-linked immunosorbent assay (ELISA) with the monoclonal antibody (MAb) was established and evaluated for detecting circulating antigen (CAg) in serum of patients with hemorrhagic fever with renal syndrome (HFRS). Results were compared to that of an immunoglobulin M (IgM)-detecting ELISA. Of 143 patients with HFRS, 106 were positive for CAg of Hantavirus and 128 positive for specific IgM. Among the 15 HFRS patients in whom specific IgM was not detected, 10 were positive for CAg. Of 100 controls including 40 hepatitis B cases, 40 measles cases, and 20 healthy persons all were negative for both CAg and specific IgM. Detection of Hantavirus CAg with a MAb-based sandwich ELISA (MBS-ELISA) established in the present study adds a new diagnostic tool for HFRS, and it increases the diagnostic rate to conventional specific IgM detection, especially for patient in the early stage of HFRS.

[Study on the cost-effectiveness, benefit and utility analysis on the infant inoculation hepatitis B vaccine in Shanghai].

OBJECTIVE: To study the cost-effectiveness, benefit and utility of infant hepatitis B vaccination in Shanghai from 1992 to 2001. METHODS: To calculate the cost of hepatitis B vaccination by cost analysis method. Both the numbers of persons with HBsAg positive and patient with hepatitis B, cirrhosis and liver cancer decreased as the index of direct effect. To study the sick-time and the cost of treating hepatitis B, cirrhosis and liver cancer patients, a face to face questionnaire was used and quasi method was adopted to understand the effect of cure and the course of hepatitis B. The cost benefit analysis method was also used to calculate the cost benefit of HBV vaccine. The disability adjusted life years (DALY) was regarded as an index of utility to measure the disease burden. RESULTS: Input of 501,129.49 Yuan might have the result of reducing one liver cancer patient, ten cirrhosis patients, one hundred chronic hepatitis B patients and one thousand HBsAg positive people. The cost of hepatitis B vaccination was 0.24 hundred million Yuan during the past ten years in Shanghai, which had obtained the total benefit value of 41.22 hundred million Yuan, with a cost benefit ratio of 1:172 Yuan. It was estimated that the total disease burden of hepatitis B, cirrhosis and liver cancer patients was 59,762.55 DALY in order to reduce one DALY loss cost of 402.50 Yuan. CONCLUSION: HBV vaccine inoculation in infants seemed to be a low-cost input and high-effect output strategy.