Hepatitis B virus(HBV) is a double-strand hepadnavirus.HBV genomics have unique properties:(1) Viral polymerase/reverse transcriptase(RT) is lacking in proofreading activity,leading to higher mutation occurrence than other DNA viruses.(2) Overlapping open reading frames in viral genomics may produce the possibility of mutation of two viral proteins mutation as a result of one-site genetic change.(3) A stable covalently closed circular DNA(cccDNA) form exists as an important intermediate in the life cycle.(4) Viral gene fragments may be integrated into host cellular genomic DNA.(5) Viral proteins could be a trans-regulator for certain host cellular protein expression.The virological characteristics of HBV are closely related with the chronicity and disease course of hepatitis B.Multi-site detection of HBV RT-gene mutations is very important for monitoring drug resistance and rational administration of anti-HBV therapy in clinic.Analysis of viral genomic variation and detection of cccDNA are highly valuable in better understanding virological pathogenesis and evaluate therapeutic efficacy.Methodological innovation and improvement is the basis for the study of virological characteristics of HBV.

Nucleoside analogs (NA) are currently the major anti-hepatitis B virus (HBV) agents in clinic. However,long-term use of NA may initiate a state of drug resistance due to HBV mutation. The genotyping and phenotyping are two basic methods for analysis of HBV drug resistance. Genotyping is done mainly by DNA sequencing and reverse hybridization line probe to detect the well-known drug-resistant mutations. Phenotyping is an essential tool used to identify "novel" and complex resistant mutations,and it is based on the comparison of HBV replication capacities of variants and wild-type counterparts by cell-transfer of individual viral genomes followed by quantitation of HBV replication intermediates in the presence of antivirals in different concentrations. Clinical choice of HBV resistance detection assays relies on their sensitivity,specificity and cost-effectiveness. In our recent studies,some novel HBV resistant features have been found,including multidrug-resistant strain with mutations due to the use of both entecavir and adefovir,association of HBV genotype/subgenotype with the resistant mutational patterns,transmission of the resistant HBV to cause acute hepatitis,replication compensation of rt229 variants,etc. These findings are important for better understanding the clinical features of HBV drug-resistant mutations and strengthening the management of HBV drug resistance in clinic.

Objective To compare the efficiency of detecting YMDD mutations by direct DNA sequencing and real time fluerescence PCR, and to explore the significance of the mutations of drug-resistance gene other than YMDD mutation. Methods 92 serum samples from 89 patients with chronic hepatitis B were collected and all the samples were detected by real-time PCR for YMDD mutation. HBV DNA was extracted from serum and was amplified by nest PCR to achieve HBV P gene RT region sequence. The PCR products were sequenced at both directions, and the sequencing results were contrasted through NTI program. The other 11 known drug-resistance mutation sites at the HBV RT region were also analyzed. Results Among the 37 samples with no YMDD mutation detected by real-time PCR, 33 samples were with M204M (without mutation), 1 sample with M204I and 3 samples with M204V by direct sequencing. Mutation and wild-type standard sequences were all coexisted in the 4 positive samples. There were 7 samples detected with ADV resistant mutation, accounting for 18.9% (7/37). Among the 55 samples with YMDD mutation detected by real-time PCR, 52 samples were detected by DNA sequencing, the accordance rate was 94.5% (52/55); 5 samples with ADV or ETV resistant mutation were detected, accounting for 9.1% (5/55). Conclusions Direct DNA sequencing is a high sensitive, repeatable method to detect drug-resistance mutation at RT region of HBV P gene. The result is well consistent with that attained by real-time PCR. Direct DNA sequencing can also detect various drug-resistance mutations as well as YMDD mutation, which is helpful to generally understand the nucleoside analogue resistant mutation and adopt more reasonable therapy projects against HBV.

Objective To investigate forkhead/winged helix transcription factor (FoxP3) expression in peripheral blood and liver-infiltrating lymphocytes (LIL) of patients with hepatitis B. Methods Flow cytometry, immunohistochemistry and real-time RT-PCR were employed for Fox P3 analysis of blood or live tissue obtained from patients with acute hepatitis B (AHB), chronic hepatitis B (CHB) and chronic severe hepatitis B (CSHB), as well as health controls. Results Fox P3 mRNA and protein expressions were specifically identified in CD4+CD25+ regulatory T cells. The level of Fox P3 mRNA in CD4+ T cells was identical with that in CD4+CD25+ regulatory T cell in peripheral blood. In CSHB patients a significant increase of Fox P3 mRNA expression was found in peripheral blood. The mean relative FoxP3 mRNA levels of CD4+ T cells in CSHB patients, AHB patients, CHB patients, and healthy controls were 0.199?0.174, 0.053?0.017, 0.059?0.053, and 0.056?0.021, respectively (CSHB group vs. other groups, P all

Objective To construct the yeast expression vector of a new gene transactivated by hepatitis B virus X protein (XTP11), and to explore the feasibility of cloning the hepatocyte proteins interacting with XTP11 protein by yeast two hybrid system. Methods The XTP11 gene was amplified by polymerase chain reaction (PCR) with specific primers, and the amplified fragment was subcloned into the Nco I/BamH I sites (5′ ends) of yeast expression vector pGBKT7,which was named as pGBKT7(-)-XTP11 encoding fusion proteins of full length of XTP11 and DNA binding domain of yeast protein GAL4. The pGBKT7 (-)-XTP11 plasmid was transformed in the yeast cells AH109 and the expression of XTP11 protein in yeast cells was detected by Western blotting assay. Then the yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp and SD/-Trp-His-Ade) containing X-?-gal, and their autonomous activation was tested. Results The yeast expression vector of XTP11 gene was constructed and the fusion protein in yeast cells was examined. The yeast cells transformed with pGBKT7-XTP11 plasmid could grew well on both of the media, and turned blue on medium containing X-?-gal for the production X-?-galactosidase. This phenomenon suggested that XTP11 protein acted to substitute the functional yeast protein GAL4 activation domain (AD) to activate transcription of reporter genes (ADE2, HIS3, MEL1 and LacZ). Conclusion The XTP11 protein fused to the GAL4 DNA-binding domain functioned as a transcriptional activation domain in yeast, and the transcriptional activation function of XTP11 protein in yeast might restrain researchers to use yeast two hybrid system to clone hepatocyte proteins interacting with XTP11 protein.

Objective To analyze the variation in CTL epitope of hepatitis B virus (HBV) popular in China. Methods Amino acid sequences of 13 cytotoxic T lymphocyte (CTL) epitopes previously reported abroad were taken as reference sequences, and comparison was made draw between these reference sequences and both the 45 CTL-epitopic sequences obtained from the Genbank and also 5 CTL-epitopic sequences from native patients with chronic hepatitis B, using Vector NTI software. Results Sequence variations were observed frequently within several CTL eptitopes of HBV genotype B and C compared with the reference sequences. In the 13 analyzed CTL epitopes, 4 showed variation accounting over 70% in genotype B, and 2 have variance over 70% in occurrence in genotype C. Among these, C18-27 (FLPSDFFPSV), HBcAg-derived CTL epitope which was the most frequently studied previously, is showed in variation more frequently in HBV genotype B and C popular in China, reaching 71% and 97%, respectively. Conlusion Some CTL epitopic sequences of HBV genotype B and C popular in China have their own charactiristic difference from previously reported ones, which should be considered in the study of CTL response of Chinese subjects.

Objective To screen the down-regulated genes in HepG2 cells transfected by pcDNA3.1(-)-IFN-? to further investigate the biological mechanism of IFN-?. Methods pcDNA3.1(-)-IFN-? was transfected into HepG2 cells as treatment group and the pcDNA3.1(-) transfected cells as the control to perform suppression subtractive hybridization (SSH). The subtractive library for down-regulated genes was obtained when pcDNA3.1(-) transfected HepG2 as tester and pcDNA3.1(-)-IFN-? transfected cells as driver. A housekeeping gene, G3PDH, was used to estimate the subtractive efficiency. Genes with lower expressions in IFN-? transfected HepG2 cells were obtained from the library after being randomly sequenced and analyzed. Among the obtained genes, regulation of IFN-? on heat shock 90kD (Hsp90) mRNA was further investigated by RT-PCR. Results G3PDH was subtracted efficiently indicating that the subtractive library was constructed successfully. 50 positive clones were randomly isolated for PCR identification. Results showed that most of the plasmids in the clones contained 200-1 000bp inserts. The down-regulated genes obtained were as follows: eukaryotic translation elongation factor 1 beta, ferritin, RAD23 homolog B, signal sequence receptor 2 (SSR2), tissue factor pathway inhibitor, heat shock 90kD protein 1, and adenosylmethionine decarboxylase 1. RT-PCR showed that hsp90 mRNA had a reduced expression in pcDNA3.1 (-)-IFN-? transfected HepG2 cells. Conclusion The down-regulated cDNA subtractive library in HepG2 cells after pcDNA3.1 (-)-IFN-? transfection was constructed successfully. IFN-? could down-regulate the hsp90 mRNA expression.

Objective To investigate the expression of ERBB3 protein,which encoded by ERBB3 gene,in gastric cancer(intestinal type and diffuse type)as well as the normal tissue,and to elucidate the possible role of ERBB3 gene in the progress of gastric cancer.Methods 65 samples of gastric cancer tissue(of which 43 samples were intestinal type and 22 were diffuse type,classified according to the Lauren patho-classification)and 65 samples of normal tissue(control group)were investigated immunohistochemically.The expression of ERBB3 in both control group and gastric cancer group was determined.Comparisons were made according to different pathological types and TNM clinical stages within and between groups.Results 7 of 65 samples(10.8%)in gastric cancer group were found to over-express the ERBB3 protein,while only 1 of 65 samples(1.6%)in control group was found to over-express the ERBB3 protein.Statistical differences(P

Objective To serially examine virus carrying condition of clinical specimens collected from SARS patients, in order to evaluate the dynamic changes of virus in the bady and its clinical significance. Methods A total of 494 samples, including plasma, urine, feces, sputum and throat swab, obtained from 84 cases of clinically diagnesed SARS patients at different time-points were examined for the study. Nested RT-PCR was employed for the detection of SARS-CoV using 2 pairs of primers targeting P and N region of the viral genome. The amplified viral genomic fragments were confirmed by DNA sequence analysis. Results Specific bands for SARS-CoV were amplified from various specimens of some SARS patients. Highest positive rate was found in sputum compared with the others. However, simultaneous examination of more than two specimens increased detectable rate of the virus. Moreover, dynamic examination revealed that the highest positive rate occurred in the 2nd week after the onset of the disease and next to the highest during the 3rd to 4th week. However, dynamic detectable rates for different specimens were not identical. Conclusion Nested RT-PCR is a practicable method for the detection of SARS-CoV and helpful for the early diagnosis or confirmation of the disease. Dynamic examinations for SARS-CoV by combined employment of the N and P primers and by involving multiple specimens may significantly increase the positive detectable rate, therefore would be helpful in diagnosis and eratuation of therapeutic effect of SARS.

Objective To observe dynamic responsive regularity of specific IgM and IgG antibodies in SARS patients. Methods 145 specimens of plasma from 25 cases of clinically diagnosed SARS patients were examined in the study. ELISA was employed to detect IgM and IgG antibodies against SARS coronaviral antigens. Nested RT-PCR was used to qualitatively determine SARS coronavirus (SARS-CoV). Results 49.0% (71/145) and 54.5% (79/145) of the samples were positive for IgM and IgG antibodies, respectively. Both antibodies were found to be detected in 84.0% of the patients. The antibodies were found to be detectable from the 2nd to 4th week after the onset of disease in most patients, and there was a fendeney of sising in positive rate until the 5th week after the onset. Thereafter, the detectable rate of IgM antibody began to decline, while that of IgG antibody remained to rise. Positive rate for serum SARS-CoV was 15.8% (18/114) for all samples or 40.0% (10/25) of patients. Most virus-positive samples were those which were collected within 4 weeks after disease onset. Conclusions Anti-SARS-CoV IgM/IgG antibody and detection of virus in plasma could serve as practical diagnostic indicators for SARS. In most cases, when the serum was pasitive for antibody the serum virus positive rate would soon declined. However, concomitant existence of antibody and viral sequence in plasma was observed in a few patients.