Association of IL-28B, TBX21 gene polymorphisms and predictors of virological response for chronic hepatitis C.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease. The outcomes of both spontaneous HCV clearance and response to therapy depend on both viral and host factors. To investigate the influence of polymorphisms of IL-28B rs12979860 and TBX21 rs17250932, rs4794067 as well as viral factors (HCV genotype, F protein) on the outcome of HCV infection, we genotyped 565 patients with chronic HCV infection, 191 patients spontaneously resolved from HCV infection, 359 healthy controls and 383 treatment-naïve CHC patients with pegylated interferon-α and ribavirin (PEG IFN-α/RBV). Results showed that TBX21 rs4794067 variant genotypes significantly correlated with increased risk of HCV chronic infection (dominant model: OR = 5.690, 95% CI = 2.024-16.000) and susceptibility (dominant model: OR = 5.658, 95% CI = 2.514-12.735). We also found that the rs12979860, rs2227982 and rs36084323 polymorphisms showed no significant associations with susceptibility or spontaneous clearance of HCV in the anti-F antibody subgroup; however, the anti-F antibody positive subgroup might show an increased risk of N-SVR (all P < 0.001). Our results demonstrate that variant factors in both the host and pathogen are commonly important for HCV clearance. In addition rs4794067 and F protein status may be strong predictive markers in the Chinese population.

PD-1/PD-L1 signal pathway participates in HCV F protein-induced T cell dysfunction in chronic HCV infection.

Programmed cell death-1/programmed cell death-1 ligand 1 (PD-1/PD-L1) inhibitory signal pathway has been verified to be involved in the establishment of persistent viral infections. Blockade of PD-1/PD-L1 engagement to reinvigorate T cell activity is supposed to be a potential therapeutic scheme. Studies have verified the participation of PD-1/PD-L1 in hepatitis C virus (HCV) core protein-regulated immune response. To determine the roles of PD-1/PD-L1 signal pathway in HCV F protein-induced immunoreaction in chronic HCV infection, variations in T cells were examined. The results showed that PD-1 expression on CD8(+) and CD4(+) T cells was increased with HCV F stimulation in both chronic HCV patients and healthy controls, and could be reduced partly by PD-1/PD-L1 blocking. Additionally, by PD-1/PD-L1 blocking, HCV F-induced inhibition of T cell proliferation and promotion of cellular apoptosis were partly or even totally recovered. Furthermore, levels of both Th1 and Th2 cytokines were elevated in the presence of anti-PD-L1 antibody. All these results indicated that PD-1/PD-L1 signal pathway also participates in HCV F protein-induced immunoregulation. PD-1/PD-L1 blocking plays important roles in the restoration of effective functionality of the impaired T cells in chronic HCV patients.

HCV F protein amplifies the predictions of IL-28B and CTLA-4 polymorphisms about the susceptibility and outcomes of HCV infection in Southeast China.

Cytotoxic T lymphocyte associated antigen-4(CTLA-4) is an inhibitory receptor with great value in the progression of hepatitis C virus (HCV) infection related diseases. To determine the potential associations of IL-28B rs12979860 and CTLA-4 rs231775, rs3087243 and rs5742909 polymorphisms with the generation of HCV F protein, susceptibility and outcomes of HCV infection, a total of 375 healthy controls, 219 HCV spontaneous recovered patients and 600 chronic HCV patients from Southeast China were recruited and genotyped in this study. And the relative mRNA levels of CTLA-4 in T cells were detected. Logistic regression analysis showed that rs231775 A allele was associated with significantly higher rate of spontaneous viral clearance in anti-HCV F antibody negative patients (adjusted OR=0.512, P=0.008), but allele A was related to higher mRNA level of CTLA-4 with the generation of HCV F protein. And rs5742909 T allele added up to the risk of HCV infection chronicity significantly in patients with the presence of HCV F protein (adjusted OR=2.698, P=0.003). Also, the rs5742909 CC genotype, along with the presence of HCV F protein, indicated a significantly higher CTLA-4 level than that in anti-HCV F antibody negative patients. The AG+AA genotype of rs3087243 significantly increased the susceptibility to HCV infection in subjects over 56 years old (adjusted OR=1.595, P=0.011). Genotype-genotype interaction between IL-28B rs12979860 and CTLA-4 rs3087243 was found to be significantly associated with increased susceptibility to HCV infection (adjusted OR=1.509, P=0.005). Haplotype analysis in CTLA-4 also showed significant association with the generation of HCV F protein. All these results indicated the importance of IL-28B and CTLA-4 polymorphisms and their associations with HCV F protein in the risk and chronicity of HCV infection in Chinese Han population in Southeast China.

Potential Role of Hepatitis C Virus Alternate Reading Frame Protein in Negative Regulation of T-Bet Gene Expression.

Hepatitis C virus (HCV) is a major cause of chronic liver disease and has led to cirrhosis or hepatocellular carcinoma in a majority of infected individuals. We have previously demonstrated that the HCV alternate reading frame protein (F protein) is related to Th1/Th2 bias in chronic hepatitis C (CHC) patients, and we aimed to explore the relative molecular mechanisms here. A total of 104 cases including CHC patients and healthy donors were enrolled. T-bet and GATA-3 expression levels were analyzed in peripheral blood mononuclear cells (PBMCs). The levels of signal transducer and activator of transcription-1/-6(STAT1/6) and phosphorylated STAT1/6(pSTAT1/6) in PBMCs were measured by Western blotting. Our results showed that the levels of T-bet in PBMCs, as well as the levels of gamma interferon (IFN-γ) in sera, were decreased in anti-F protein antibody seropositive patients compared with anti-F protein antibody seronegative patients, whereas the levels of GATA-3 did not show difference between the two groups. Moreover, the decreased pSTAT1 and increased pSTAT6 were observed in PBMCs by HCV core/F protein stimulation with constant STAT1/6 expression. Taken together, it suggested that T-bet may be involved in Th1/Th2 bias induced by HCV F protein, and the disruption of STAT phosphorylation may participate in this mediation.

Genetic variations of IL-28B and PD-1 are in association with the susceptibility and outcomes of HCV infection in Southeast China.

Programmed cell death-1 (PD-1) is an important co-inhibitory molecule involved in the progression of chronic viral infections. To investigate the associations of three single nucleotide polymorphisms (SNPs) (rs10204525, rs2227982 and rs36084323) in PD-1 and a previously well-inquired SNP rs12979860 in IL-28B with the outcomes of hepatitis C virus (HCV) infection in Southeast China, a total of 375 healthy controls, 219 spontaneous resolved HCV patients and 600 chronic HCV patients were enrolled in this study. The generation of HCV F protein and PD-1 expression on T cells was determined. Multivariate logistic regression analysis showed no association of rs12979860 CC genotype with spontaneous clearance of HCV infection in our subjects. The generation of HCV F protein was significantly related to HCV infection chronicity, but no significant relationship was found between HCV F protein and SNPs in PD-1. The rs10204525 TT genotype was associated with an increased risk of HCV infection chronicity in age ⩽56years subgroup (adjusted OR=0.390, P=3.8×10(-4)). The C allele of rs10204525 played protective roles in females infected with HCV (adjusted OR=0.608, P=0.008). A significant higher percentage of PD-1 expression on T cells was observed in rs10204525 TT genotype when compared to CC genotype (P=0.047). Moreover, a significant genotype-genotype interaction between IL-28B rs12979860 CC and PD-1 rs10204525 TC+CC was found to be associated with higher rates of spontaneous clearance (adjusted OR=0.689, P=0.032). The combined effect of rs12979860 and rs10204525 was of great value in predicting the outcomes of HCV infection. These analyses showed the importance of IL-28B and PD-1 polymorphisms and their interactions in the outcomes of HCV infection in Chinese Han population in Southeast China.

Prediction of antiviral efficacy in patients with chronic hepatitis C by changes in forkhead box protein 3 levels.

The aim of the present study was to investigate the distribution of CD4+CD25+ regulatory T cells (Tregs) in the peripheral blood of patients with chronic hepatitis C; in addition to identifying whether the distribution of CD4+CD25+ Tregs predicts the efficacy of antiviral therapy for HCV. The expression of CD4+CD25+ forkhead box protein (FOXP) 3+ Tregs within a CD4+ T cell population was detected in the peripheral blood obtained from patients with chronic hepatitis C and from healthy control subjects using flow cytometry. The hepatitis C virus (HCV)-RNA load was measured using quantitative-fluorescence polymerase chain reaction. CD4+CD25+FOXP3+ Tregs accounted for 14.24±1.33% of the CD4+ T cells in the peripheral blood of patients with chronic hepatitis C, which was higher than that of the healthy control subjects (5.62±1.21%; P<0.001). Furthermore, the frequency of CD4+CD25+FOXP3+ Tregs in CD4+ T cells of the peripheral blood positively correlated with the HCV-RNA load (r=0.73; P=0.032). Therefore, the results of the present study indicated that the expression of CD4+CD25+FOXP3+ Tregs increased in patients that were chronically infected with HCV and positively correlated with the HCV-RNA load.

Anti-tumor efficacy of a hepatocellular carcinoma vaccine based on dendritic cells combined with tumor-derived autophagosomes in murine models.

The majority of hepatocellular carcinoma (HCC) patients have a poor prognosis with current therapies, and new approaches are urgently needed. We have developed a novel therapeutic cancer vaccine platform based on tumor cell derived autophagosomes (DRibbles) for cancer immunotherapy. We here evaluated the effectiveness of DRibbles-pulsed dendritic cell (DC) immunization to induce anti-tumor immunity in BALB/c mouse HCC and humanized HCC mouse models generated by transplantation of human HCC cells (HepG2) into BALB/c-nu mice. DRibbles were enriched from H22 or BNL cells, BALB/c-derived HCC cell lines, by inducing autophagy and blocking protein degradation. DRibbles-pulsed DC immunization induced a specific T cell response against HCC and resulted in significant inhibition of tumor growth compared to mice treated with DCs alone. Anti- tumor efficacy of the DCs-DRibbles vaccine was also demonstrated in a humanized HCC mouse model. The results indicated that HCC/DRibbles-pulsed DCs immunotherapy might be useful for suppressing the growth of residual tumors after primary therapy of human HCC.

Oxymatrine inhibits hepatitis B infection with an advantage of overcoming drug-resistance.

Oxymatrine (OMTR) is an anti-hepatitis drug used in China. Its mechanism of action is unknown. Recently, we found that OMTR inhibits hepatitis B virus (HBV) via down-regulating the expression of heat-stress cognate 70 (Hsc70), a host protein required for HBV DNA replication. Goal of this study was to assess the effect of OMTR on clinical HBV drug-resistance. OMTR monotherapy (oral, 12 months) reduced blood HBV DNA by 96% and HBeAg by 70% in the chronic hepatitis B (CHB) patients resistant to lamivudine (n = 17), equal to its efficacy in the naïve CHB cohort (n = 20). Liver biopsy study showed that OMTR treatment caused a decrease of Hcs70 mRNA in liver cells, parallel with a reduction of intracellular HBV DNA. Combination of lamivudine with OMTR (n = 15) (oral, 12 months) showed an enhanced anti-HBV effect as compared to lamivudine monotherapy (n = 25). The incidence of drug resistance against lamivudine in the combination group was significantly lower than that in the lamivudine group (1/15 vs 7/25; p<0.01). The results were further confirmed in vitro. Treatment of HBV(+) HepH2215 cells with sub-optimal dose of OMTR for 8 months suppressed HBV replication without inducing drug resistance, whereas lamivudine monotherapy caused drug-resistant mutation in 3 months. Combination of OMTR with lamivudine prevented HBV from developing drug resistance.

Serum microRNA profiles serve as novel biomarkers for HBV infection and diagnosis of HBV-positive hepatocarcinoma.

Diagnosis of hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC), particularly HCC independent of cirrhosis etiology, presents a great challenge because of a lack of biomarkers. Here we test the hypothesis that expression profiles of microRNAs (miRNAs) in serum can serve as biomarkers for diagnosis of HBV infection and HBV-positive HCC. We recruited 513 subjects (210 controls and 135 HBV-, 48 hepatitis C virus (HCV)-, and 120 HCC-affected individuals) and employed a strategy of initial screening by Solexa sequencing followed by validation with TaqMan probe-based quantitative reverse transcription-PCR assay. First, because of a close link between chronic hepatitis B and HCC, we compared miRNA expression profiles in HBV serum with that in control serum and successfully obtained 13 miRNAs that were differentially expressed in HBV serum. This 13-miRNA-based biomarker accurately discriminated not only HBV cases from controls and HCV cases, but also HBV-positive HCC cases from control and HBV cases. Second, we directly compared miRNA expressions in HCC serum with those in controls and identified 6 miRNAs that were significantly upregulated in HCC samples. Interestingly, 2 of these miRNAs, miR-375 and miR-92a, were also identified by our first approach as HBV specific. When we employed 3 of these miRNAs (miR-25, miR-375, and let-7f) as biomarkers, we could clearly separate HCC cases from controls, and miR-375 alone had an ROC of 0.96 (specificity: 96%; sensitivity: 100%) in HCC prediction. In conclusion, our study demonstrates for the first time that serum miRNA profiles can serve as novel and noninvasive biomarkers for HBV infection and HBV-positive HCC diagnosis.

[Design and application of a set of universal PCR primers for genotyping of hepatitis E virus].

To improve the reliability and credibility of genotyping hepatitis E virus (HEV) and to explore the possibility of unifying standards of HEV genotyping by designing HEV universal primers for amplification of a long genomic fragment of different HEV genotypes. A set of universal primers (HEVuPrimer) was designed based on conserved regions determined by alignment analysis of 82 HEV strains with complete genome in GenBank. HEVuPrimer was compared with a set of previously used primers (MXJ primers) for their sequence-matching to different HEV strains and applied to amplify HEV genomic fragments from HEV reference strains with known different genotypes and clinical serum samples with anti-HEV-IgM by RT-nPCR. HEV genotyping based on the fragments amplified with HEVuPrimer was compared and validated with that based on HEV full genome and fragments obtained with MXJ primers. HEV genotyping by the phylogenetic analysis supplemented with the percent of nucleotide identity of the HEVuPrimer-determined fragments showed good correspondence with that based on HEV full-length genome. In addition, HEVuPrimer was much better than MXJ primers in matching sequences of HEV strains available from GenBank, and was able to amplify all the reference HEV strains with different genotypes. Among 124 samples with anti-HEV-IgM, 60 were positive for HEV RNA determined by a 644bp amplicon of RT-nPCR with the HEVuPrimenr. All the positive isolates belonged to HEV genotype 4 with nucleotide homology of 80.0%-99.9%, and could be further divided into 4 subgenotypes. Moreover, a novel subtype was identified with 6 HEV strains isolated very recently. The RT-nPCR using the HEVuPrimer and phylogenetic analysis of the amplified region provided strong evidences for its feasibility in HEV genetic classification. Our data have new implication for the consensus of genotype classification of HEV.

Berberine reduces insulin resistance through protein kinase C-dependent up-regulation of insulin receptor expression.

Natural product berberine (BBR) has been reported to have hypoglycemic and insulin-sensitizing activities; however, its mechanism remains unclear. This study was designed to investigate the molecular mechanism of BBR against insulin resistance. Here, we identify insulin receptor (InsR) as a target of BBR to increase insulin sensitivity. In cultured human liver cells, BBR increased InsR messenger RNA (mRNA) and protein expression in a dose- and time-dependent manner. Berberine increased InsR expression in the L6 rat skeletal muscle cells as well. Berberine-enhanced InsR expression improved cellular glucose consumption only in the presence of insulin. Silencing InsR gene with small interfering RNA or blocking the phosphoinositol-3-kinase diminished this effect. Berberine induced InsR gene expression through a protein kinase C (PKC)-dependent activation of its promoter. Inhibition of PKC abolished BBR-caused InsR promoter activation and InsR mRNA transcription. In animal models, treatment of type 2 diabetes mellitus rats with BBR lowered fasting blood glucose and fasting serum insulin, increased insulin sensitivity, and elevated InsR mRNA as well as PKC activity in the liver. In addition, BBR lowered blood glucose in KK-Ay type 2 but not in NOD/LtJ type 1 diabetes mellitus mice that were insulin deficient. Our results suggest that BBR is a unique natural medicine against insulin resistance in type 2 diabetes mellitus and metabolic syndrome.

[The relationship between alpha-IFN anti-virus treatment and HLA-DRB1*11 gene mononucleotide polymorphism].

OBJECTIVE: To investigate the relationship between the distribution of mononucleotide polymorphism of five regulation regions of alpha-IFN among HLA-DRB1*11 gene episodes and the therapeutic effects of alpha-IFN treatment in chronic hepatitis B patients. METHODS: One hundred seven chronic hepatitis patients from Nanjing Second Hospital who were treated by alpha-IFN for 12 months and then followed at least six months without the treatment were randomly selected for this regressive analysis. They were grouped into a continuous responsive group and a non-continuous responsive group. Hepatitis B virus X interacting protein gene locus was searched in NCBI. Single nucleotide polymorphism (SNP) gene locus was detected based on a pooling sequencing method. Primer and TaqMan-MGB probes referring to different mononucleotide loci were designed respectively to detect SNP in five regulation regions of alpha-IFN. Then gene sequencing differences between the two groups were analyzed. RESULTS: Among the 107 cases there were 30 cases (28.0%) in the continuous responsive group and 77 cases (71.9%) in the non-continuous responsive group. CT occupation rate in five regulation regions of IFN reached 18.0% in the continuous responsive group and 23.8% in the non-continuous responsive group. AG occupation rate reached 10.8% in the former group and 15.8% in the latter group. The differences in CT and AG between the two groups were significant. CONCLUSIONS: The distribution of mononucleotide polymorphism of five regulation regions of alpha-IFN among HLA-DRB1*11 gene episodes affects the IFN anti-virus treatment. Detecting the gene distribution of mononucleotide in five regulation regions of alpha-IFN helps in predicting the therapeutic effects of alpha-IFN.

Double-antigen sandwich time-resolved immunofluorometric assay for the detection of anti-hepatitis C virus total antibodies with improved specificity and sensitivity.

Current anti-hepatitis C virus (HCV) antibody screening immunoassays are routinely based on an indirect format. Although their use for anti-HCV antibody detection has achieved a very high specificity and sensitivity, false-positive results are still a problem especially among populations with a low prevalence of HCV infection. One strategy to obviate this problem is to adapt the assay from an indirect format to a double-antigen sandwich one to further improve its specificity. In this study, a double-antigen sandwich time-resolved immunofluorometric assay (DAS-TRIFMA) has been developed to detect total anti-HCV antibodies based on biotin-streptavidin interaction. For comparison, 1025 samples were analysed by the DAS-TRIFMA and three indirect anti-HCV antibody detection methods. For samples with discordant results, PCR-ELISA and Inno-LIA were employed as supplementary assays to analyse the presence of HCV antibodies. With regard to the 1025 clinical samples, the overall concordance between the DAS-TRIFMA and the three indirect methods was 99.41, 98.93 and 98.93 % for Ortho ELISA 3.0, WAT ELISA and I-TRIFMA, respectively. The specificity/sensitivity of the DAS-TRIFMA, Ortho HCV ELISA 3.0, WAT HCV ELISA and I-TRIFMA were 100/99.09, 99.34/98.18, 99.23/97.27 and 99.01 %/98.18 %, respectively. The DAS-TRIFMA was able to detect HCV antibodies at a concentration about 1/10 of that detectable by indirect methods. From the obtained results and their comparison, it is concluded that the DAS-TRIFMA is a more specific and reliable method for screening anti-HCV antibodies, and weakly positive S/Co values by the DAS-TRIFMA were more predictive of HCV infection than those by indirect methods.

Antiviral therapeutic efficacy of foscarnet in hepatitis B virus infection.

Foscarnet (PFA), a viral DNA polymerase inhibitor, is a clinical agent for herpes viruses. The goal of the study was to evaluate the therapeutic efficacy of PFA in hepatitis B virus (HBV) infection. Intravenous infusion of PFA (1 g/day) for 4 weeks significantly reduced serum HBeAg (p<0.01) and HBV DNA copies (p<0.05) in 31 patients who were diagnosed with active chronic HBV infection (CHB) and had not received antiviral treatment previously. Alanine aminotransaminase (ALT), aspartate aminotransaminase (AST) and gamma glutamyl transpeptidase (gamma-GT) of the patients declined (p<0.001, 0.001 and 0.01, respectively). Kidney function (blood creatinine and urea nitrogen) remained unchanged. Another 21 lamivudine-resistant CHB patients with mutations at the tyrosine-methionine-aspartate-aspartate motif (YMDD) displayed a response to PFA similar to that mentioned above, with reductions in HBeAg (p<0.05), HBV DNA (p<0.01) and liver enzymes (ALT and AST, p<0.001; gamma-GT, p<0.05). Moreover, PFA reduced serum HBeAg (p<0.01), HBV DNA (P<0.05), AST (p<0.05) and ALT (p<0.02) in a cohort of 13 severe CHB patients with advanced liver damage. PFA was also evaluated in vitro and in vivo. PFA inhibited HBV DNA replication in HBV-transfected human HepG2 cells (2.2.15 cells) with reduced amount of HBV RC-DNA and DS-DNA. In the duck HBV-infected ducklings, PFA reduced viral DNA and duck HBsAg in the serum (p<0.01 for both).

Hepatitis gene chip in detecting HBV DNA, HCV RNA in serum and liver tissue samples of hepatitis patients.

OBJECTIVE: To study the preparation of diagnostic gene chip for detecting hepatitis B virus (HBV) and hepatitis C virus (HCV) and its accuracy in detecting HBV DNA and HCV RNA in serum and liver tissues. METHODS: The probes, which depend on the conservative gene fragment of hepatitis virus, was designed, synthesized and spotted on the modified glass. The probes and some other control probes were assembled on the diagnostic microarray of hepatitis virus. The gene of hepatitis virus, purified from blood or tissue, was labeled with fluorescence and hybridized to the microarray. The hybridized microarry was scanned with microarray scanner and the diagnostic result was analyzed from the scanning data. Fourty patients with hepatitis B virus and 40 healthy people or 40 patients with hepatitis C virus were subjected to detection of HBV DNA and HCV RNA with the hepatitis virus gene chip by the double-blind method. Paraffin liver specimens obtained from 99 cases of posthepatitic cirrhosis were used to detect HBV DNA. The liver tissues and serum from 15 cases of chronic hepatitis B were used to detect HBV DNA. Simultaneously, HBsAg and HBcAg were detected in the serum by fluorescence microparticle quantitation, HBV DNA and HCV RNA in the serum by PCR, and HBcAg in liver tissues by immunocytochemistry or HBV DNA by in situ molecular hybridization. RESULTS: Chip detection of serum specimens showed that 30 patients were HBV DNA positive and 10 HBV DNA negative in the 40 patients with HBV positive, 25 patients were HCV RNA positive and 15 patients were HCV RNA negative in the 40 patients with HCV positive, and all were HBV and HCV negative in the 40 healthy people. In 15 patients with HBV marker positive who were subjected to liver biopsy, 15 patients were detected HBV DNA positive in serum by gene chip, 15 patients HBcAg positive in liver tissues by immunocytochemistry, 14 patients HBV DNA positive in liver tissues by in situ molecular hybridization, and 14 patients HBV DNA positive in liver tissues by gene chip. Paraffin liver tissues specimens from the 99 patients with posthepatitis B cirrhosis showed that 67 patients were detected HBcAg positive by immunocytochemistry, 53 patients HBV DNA positive by in situ molecular hybridization, and 46 patients HBV DNA positive by gene chip. In the 46 patients, 40 patients were detected HBV DNA and HBcAg positive by in situ molecular hybridization and immunocytochemistry, 6 patients only HBcAg positive, and 33 patients HBcAg negative. CONCLUSIONS: The designed diagnostic gene chip can be used to simultaneously detect serum HBV DNA and HCV RNA, but the positive rate of HCV RNA diagnosed by this chip is lower. The gene chip can detect HBV DNA in serum and in liver tissue.

[Detection and identification of emergence of HBV YMDD motif mutation during lamivudine treatment by hybridization to an oligonucleotide array].

OBJECTIVE: Gene chip technology was set up to quickly and accurately detect and identify the HBV P gene YMDD motif mutation during the chronic hepatitis treated with lamivudine. METHODS: DNA microarrays were prepared by spotting fluorescence labeled probes of target genes onto specially lattice glass slides with robotics. The serum samples from 30 patients with hepatitis B after 68-week treatment with lamivudine were detected double-blind by gene chips and by nucleotide sequences assay technique to identify the rate of emergence of HBV P gene YMDD motif mutation. RESULTS: Twenty-one patients were found HBV P gene YMDD motif mutation by the gene chips including 11 cases with YVDD and 10 cases with YIDD motif mutation. By direct sequencing of the PCR products, 11 cases were found to have YVDD with adenine741 changed into cytidine resulted in methionine552 changed into valine in which 6 cases with adenine669 changed into cytidine and leucine changed into methionine, 10 cases had YIDD motif mutation with guanosine743 altered thymidine methionine552 changed into isoleucine, including 3 cases with thymidine281 changed into cytidine and leucine565 altered proline. CONCLUSION: The gene chip can be used to test HBV YVDD,YIDD motif mutation compared with nucleotide sequences assay technique, the accuracy rate was 100%.