To explore the relationship between HPRE mutations and noncytolytic anti-HBV infection, the objective eukaryotic expression vectors were constructed by molecular cloning and PCR-based site-directed mutagensis in vitro, and identification was performed using PCR and sequencing analysis. The results showed that eukaryotic expression vectors containing HPRE segment and mutating point were constructed successfully as confirmed by sequencing analysis. The activity of CAT gene obviously increased in the T to C mutation at nt 1504 of HPRE and no alteration in the C to T(G) at nt 1508. The mutation at nt 1508 of HPRE may escape the suppression role of IFN-?on HPRE. These results suggested that the mutation of HPRE might be affected the function of HPRE and influence the regulative function of IFN-? on HPRE, but not of 1FN-? nor of TNF-?.

Infection rate of HBV in the high-risk population, such as dentists and the patients undertaking hemodialysis, was not higher than those in general population. The occurrence of the subclinical infection state might be resulted from hypo-responsiveness of humoral and cellular immunity to HBsAg. Of 257 cases of liver biopsy, 44% of them were diagnosed as chronic hepatitis of various categories. No relationship was found between the expression patterns of viral antigens and the inflammatory activity in the liver. The infection state was quite stable. HBeAg/anti-HBe seroconversion was not a turning point from replicative to nonreplicative phase of the virus. The individuals with intrahepatic integrated HBV DNA might be the genome carriers with sero-negative HBsAg. The above results illustrate the characteristics of hyporesponsive HBV infection in hyperendemic area in China.

Objectives To evaluate the relationship between the expression of B7-1 in liver tissue and the effect of interferon-alpha(?-IFN) treatment in patients with chronic hepatitis B(CHB).Methods The expression of B7-1 in liver biopsy specimens from 68 CHB patients was studied with immunohistochemistry before ?-IFN treatment.Results B7-1 was expressed in 45(66 2%) liver tissues among 68 patients with CHB,but none in 5 normal controls.The total response ratio of ?-IFN in patients with B7-1 positive was 66 7%(30/45),which was significantly higher than 39 1%(9/23)in the patients with B7-1 negative (? 2=7 20,P

To study tissue distribution of TTV in experimentally infected Rhesus monkey and if the TT virus is hepatotropic. Total DNA was extracted from tissues of 5 experimentally infected Rhesus monkeys. TTV was detected by PCR,and dot hybridization was done with virus double DNA strand probe or single antisense strand probe. The double strand probe was hybridized with DNA of liver, bone marrow, and spleen, stomach, small intestine, colon. and sera. In the above tissues , the virus was also positive as shown by PCR. The single strand antisense probe was only hybridized with DNA of liver, bone marrow,and small intestine of all 5 monkeys, but not with that of other tissues. It suggested that TTV could infect many tissues of Rhesus monkey. Only the liver ,bone marrow, and small intestine presented the virus positive single strand, which might be a replicative intermediate of the virus. It suggests that TT virus replicates in liver , bone marrow, and small intestine,and it might be hepatotropic.

To elucidate whether the mutations in X region of hepatitis B virus (HBV) might be responsible for the different clinical profiles in cases positive for antibody to hepatitis B e antigen. The nucleotide sequences of X gene regions in serum HBV were examined in 14 asymptomatic carriers (AsC) and 14 chronic active hepatitis (CAH) patients with antibody to hepatitis e antigen. The results showed that 12 of 14 AsCs (85.7%) had insertions, deletions or point mutations in nucleotide sequence of X region resulting in truncation of the X protein by creating frame shift mutation or a new stop codon, whereas no patient with CAH had those X gene mutations( P

Objective To study the biological significance of the common mutant preC/C gene in clinical HBV in China. Methods Site directed mutagenesis based on the unique enzyme site elimination was used to construct eukaryocyte expression vector with mutant HBV/C gene(V60、G87、L97). Expression vectors with wild and mutant preC/C gene were transferred into HepG2 cell. Culture supernatant was detected by ELISA for HBeAg. Results Result of DNA sequencing showed that the constructed mutant HBV preC/C gene had only one specific site variation compared with the wild type sequence. Goal DNA fragment was detected positive in the HepG2 cells transferred with wild and mutant preC/C gene. A value of HBeAg in the supernatant of the cells harboring L97 variant was higher than that of the wild and other variant strains( P

Objective In order to further study the influence of a mutant on viral replication and transfection, a eukaryotic vector with mutation of 20/21 bp deletion (1748/ 1747 to nt 1767) in core promoter region and precore stop mutation (nt.1896) was constructed. Methods A linearized genome containing the entire HBV 3.5kb mRNA transcriptional units (P3.8Ⅰ vector) and initiating from the basic core promoter upstream sequences was used as a tool, the objective eukaryotic vectors were constructed by the molecular cloning and PCR based site directed mutagenesis in vitro. The capability of progeny virus production and transcription were examined with Southern blot and Northern blot analysis respectively, after transfection of the recombinant HBV plasmids into HepG2 cells by using liposome. Results The eukaryotic vectors were constructed successfully and their sequences were confirmed by clone sequencing. Both Southern and Northern blotting of DNA and RNA extracted from the transfected cells showed markedly reduced mutant activity to produce progeny virus, to transcript both 3.5kb precore/pregenome mRNA and 2.1kb preS/S mRNA. Conclusions The levels of replication and transcription are markedly reduced in the mutant compared with those in wild type HBV.

The aim of the present study was to investigate the inhibitory effect of galactosides modified lamivudine (LA) on hepatitis B virus (HBV) and examine the liver targeting ability of lamivudine modified by galactosides in vitro and in vivo (mice). Lamivudine nanoparicles modified by galactosides (LAP GSLN) were prepared and delivered into 2.2.15 cells. After 10 days, hepatitis virus B e antigen (HBeAg) expression in 2.2.15 cells was detected by ELISA, and immune fluorescence levels of HBV DNA in the medium were examined by quantitative polymerase chain reaction (PCR). The cytotoxicity of LAP GSLN on 2.2.15 cells was observed as well. In the in vivo experiment, ten male mice were randomly divided into 2 groups: lap GSLN group (i.v.injection of LAP GSLN) and LA group (i.v.injection of LA). Lamivudine levels in serum, hepatic, renal, pulmonary, and splenic tissues were detected by reversed phase high performance liquid chromatography (RP HPLC). On the 6th day, the expression of HBeAg was found inhibited by LPA GSLN. HBV DNA replication was also inhibited by LAP GSLN on the 4th day. Hepatic LAP GSLN concentrations in LAP GSLN group were 3.3 fold higher that of the LA group. The above results suggested galactosides modified lamivudine could effectively inhibit the antigen expression and DNA replication of HBV, and it showed a high liver targeting ability in vivo .

Objectives To evaluate the relationship between the expression of B7-1 in liver tissue and the effect of interferon- alpha(α- IFN) treatment in patients with chronic hepatitis B(CHB).Methods The expression of B7 -1in liver biopsy specimens from 68 CHB patients was studied with immunohistochemistry before α-IFN treatment.Results B7-1 was expressed in 45(66.2%) liver tissues among 68 patients with CHB,but none in 5 normal controls.The total response ratio of α- IFN in patients with B7-1 positive was 66.7%(30/45),which was significantly higherthan 39.1%(9/23)in the patients with B7-1 negative(x2 =7.20,P <0.01).B7-1 expression was closely corelat-ed with the histological activity grade(HAI) and serum alanine transaminase(ALT) level.Conclusions The level of B7-1 expression in liver tissue may be regarded as an effective parameter for predicting α-IFN response in patients with CHB.

Objective In order to study the biological significance of HBV/C gene variant in vitro.Methods Retroviral vector pXT1 was used to construct the HBV/C gene expression vector and the recombinant plasmid pXT1-HBV/C was used to transfect immortalized human peripheral blood B cell lines to express HBcAg steadily in the host cells.Results plasmid pXT1-HBV/C was detected positive by PCR as well as enzyme digestion with Bgl Ⅱ and Xho Ⅰ.Meanwhile.transfected cells was detected to contain HBV/C gene by PCR and HBcAg was expressed in 47.4%of the ceils by means of flow cytometry.Conclusion Retroviral expression vector with HBV/C gene can transfeet into eucaryotic cells effectively and express the goal gene steadily.The recombinant cells may be used in the systematic studies on the biological significance of HBV/C gene variant.