有偿献血者HIV-1、HCV、HBV合并感染调查

目的调查山西省农村某既往有偿献血地区人类免疫缺陷病毒（HIV）、丙型肝炎病毒（HCV）和乙型肝炎病毒（HBV）合并感染状况。方法对4个村年龄18～59岁的村民进行问卷调查和血样采集，检测项目包括HIV-1抗体、HCV抗体和HBV表面抗原（HBsAg）。结果人群HIV、HCV、HBV感染率分别为1．3％（40／3062），12．7％（389／3062），3．5％（103／2982）；40名HIV感染者中，85．0％伴HCV感染，2．5％伴HBV感染。多因素Logistic回归分析提示，既往有偿献血（浆）史是HIV、HCV及HIV／HCV合并感染的危险因素，而与HBsAg阳性呈负相关，过去5年外出打工史与HIV、HBsAg、HIV／HCV感染差异有统计学意义；未发现吸毒、终生性伴数、婚外性行为、商业性行为和各种性行为中安全套的使用与HIV、HCV、HBV感染及合并感染有关联。结论应重点关注HIV、HCV和HBV感染者的治疗、健康教育和行为干预。

Inhibition of hepatitis B virus surface antigen expression by small hairpin RNA in vitro

AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA)expression vector.METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method.RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control.Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively.CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).

Epigallocatechin gallate inhibits HBV DNA synthesis in a viral replication-inducible cell line

AIM:To analyze the antiviral mechanism of Epigallocatechin gallate(EGCG)against hepatitis B virus(HBV) replication.METHODS:In this research,the HBV-replicating cell line HepG2.117 was used to investigate the antiviral mechanism of EGCG.Cytotoxicity of EGCG was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Hepatitis B virus e antigen(HBeAg)and hepatitis B virus surface antigen(HBsAg)in the supernatant were detected by enzyme-linked immunosorbent assay.Precore mRNA and pregenomic RNA(pgRNA) levels were determined by semi-quantitative reverse transcription polymerase chain reaction(PCR)assay.The effect of EGCG on HBV core promoter activity was measured by dual luciferase reporter assay.HBV covalently closed circular DNA and replicative intermediates of DNA were quantified by real-time PCR assay.RESULTS:When HepG2.117 cells were grown in the presence of EGCG,the expression of HBeAg was suppressed,however,the expression of HBsAg was not affected.HBV precore mRNA level was also downregulated by EGCG,while the transcription of precore mRNA was not impaired.The synthesis of both HBV covalently closed circular DNA and replicative intermediates of DNA were reduced by EGCG treatment to a similar extent,however,HBV pgRNA transcripted from chromosome-integrated HBV genome was not affected by EGCG treatment,indicating that EGCG targets only replicative intermediates of DNA synthesis.CONCLUSION:In HepG2.117 cells,EGCG inhibits HBV replication by impairing HBV replicative intermediates of DNA synthesis and such inhibition results in reduced production of HBV covalently closed circular DNA.

Development of HBsAg-Binding Aptamers that bind HepG2.2.15 cells via HBV surface antigen

Hepatitis B virus surface antigen （HBsAg）, a specific antigen on the membrane of Hepatitis B virus （HBV）-infected cells, provides a perfect target for therapeutic drugs. The development of reagents with high affinity and specificity to the HBsAg is of great significance to the early-stage diagnosis and treatment of HBV infection. Herein, we report the selection of RNA aptamers that can specifically bind to HBsAg protein and HBsAg-positive hepatocytes. One high affinity aptamer, HBs-A22, was isolated from an initial 115 met library of -1.1 ×10^15 random-sequence RNA molecules using the SELEX procedure. The selected aptamer HBs-A22 bound specifically to hepatoma cell line HepG2.2.15 that expresses HBsAg but did not bind to HBsAg-devoid HepG2 cells. This is the first reported RNA aptamer which could bind to a HBV specific antigen. This newly isolated aptamer could be modified to deliver imaging, diagnostic, and therapeutic agents targeted at HBV-infected cells.

Effect of lamivudinein in BeAg-positive chronic hepatitis B: Discordant effect on HBeAg and HBV DNA according to pretreatment ALT level

AM: To clarify differences in antiviral effect of the drug in patients with different ALT levels, we examined the changes in HBV markers in patients with high or low ALT levels with or without lamivudine treatment. METHODS: Thirty-seven HBeAg-positive patients were studied. Ten patients with ALT levels higher than 200 IU/L (group 1) and 8 patients with ALT below 200 IU/L (group 2) were treated orally with 100 mg/d of lamivudine. As untreated control, 9 patients with ALT above 200 IU/L (group 3) and 10 patients with ALT below 200 IU/L (group 4) were examined. ALT level, HBeAg/HBeAb status, and HBV DNA level were examined monthly for 11.9±0.4 mo. RESULTS: The ALT level normalized in all 10 patients of group 1, 7/8 of group 2, 4/9 of group 3, and 1/10 of group 4 within 6 mo (groups 1 vs2, P= NS; groups 1 vs 3, P= 0.002; groups 1 vs4, P<0.0001). HBV DNA fell below the detection limit in all 10 patients of group 1, 7/8 of group 2, 0/9 of group 3, and 0/10 of group 4 within 6 mo (groups 1 vs 2, P - NS). HBeAg became seronegative in 7/10 patients of group 1, 1/8 of group 2, 3/9 of group 3, and 0/10 of group 4 within 12 mo (groups 1 vs2, P= 0.02; groups 1 vs 3, P= NS). CONCLUSION: Our data suggest that HBeAg-positive patients with higher ALT levels can be considered good candidates for lamivudine therapy, probably because lamivudine accelerates the natural seroconversion of HBeAg, accompanied by HBV DNA loss, in these patients.

Novel Evidence Suggests Hepatitis B Virus Surface Proteins Participate in Regulation of HBV Genome Replication

Naturally occurring mutations in surface proteins of Hepatitis B virus(HBV) usually result in altered hepatitis B surface antigen(HBsAg) secretion efficiency.In the present study,we reported two conserved residues,M75 and M103 with respect to HBsAg,mutations of which not only attenuated HBsAg secretion(M75 only),but also suppressed HBV genome replication without compromising the overlapping p-gene product.We also found M75 and M103 can initiate truncated surface protein(TSPs) synthesis upon over-expression of full-length surface proteins,which may possibly contribute to HBV genome replication.However,attempts to rescue replicationdefective HBV mutant by co-expression of TSPs initiated from M75 or M103 were unsuccessful,which indicated surface proteins rather than the putative TSPs were involved in regulation of HBV genome replication.

Significance of hepatitis B virus surface antigen, hepatitis C virus expression in hepatocellular carcinoma and pericarcinomatous tissues

AIM： To investigate the correlation between hepatitis B virus surface antigen （HBsAg）, hepatitis C virus （HCV） expression in hepatocellular carcinoma （HCC）, the HAI score of the noncancerous region of the liver and the serum Alpha fetoprotein （AFP） level. METHODS： The patterns of HBsAg and HCV in 100 cases of HCC and their surrounding liver tissues were studied on paraffin-embedded sections with immunohistochemistry, the histological status was determined by one pathologist and one surgeon simultaneously using the hepatitis activity index （HAIl score, and AFP was detected by radioimmunity. The study included 100 consecutive patients who underwent curative resection for HCC. Based on HBsAg and HCV expression, the patients were classified into 4 groups： patients positive for HBsAg （HBsAg group）, patients positive for HCV （HCV group）, patients negative for both HCV and HBsAg （NBNC group） and patients positive for both HBsAg and HCV （BC group）. RESULTS： The BC group had significantly higher HAI scores than the other three groups. （BC 〉 HCV 〉 HBsAg 〉 NBNC）. HBV and HCV virus infection was positively correlated with HAI （rs = 0.39, P = 0.00011. The positive rate of AFP （85.7%） and the value of AFP （541.2 ng/mL） in the group with HBV and HCV co-infection were the highest among the four groups. The positive rate （53.3%） of AFP and the value of AFP （ 53.3 ng/mL） in the group with none-infection of HBV and HCV were the lowest. HBV and HCV virus infection was positively correlated with AFP（rs = 0.38, P = 0.0001）. CONCLUSION： The AFP increase in patients with liver cancer was positively correlated with the infection of HBV and HCV. The-serum AFP elevation by the infection of HBV and HCV is one of mechanisms which lead to hepatocarcinogenesis, and the antivirus intervening treatment of hepatitis is significant for the prognosis of liver cancer. From our Spearman＇s rank correlation analysis, we can conclude that the severity of virally induced inflammation is correlated with HBsAg and HCV expression in HCC tissues and noncancerous tissues. Prior co-infection of HBV in HCV patients may be an adverse risk factor for intrahepatic inflammation.

Positive Rate of Different Hepatitis B Virus Serological Markers in Peking Union Medical College Hospital,a General Tertiary Hospital in Beijing

Objectives To investigate the positive rate of different hepatitis B virus （HBV） serological markers, and the demographic factors related to HBV infection. Methods We enrolled all patients tested for HBV serological markers, such as HBV surface antigen （HBsAg）, HBV surface antibody （HBsAb）, hepatitis B e antigen （HBeAg）, hepatitis B e antibody （HBeAb）, HBV core antibody （HBcAb）, and HBV-DNA from July 2008 to July 2009 in Peking Union Medical College Hospital. The positive rate of each HBV serological marker was calculated according to gender, age, and department, respectively. The positive rates of HBV-DNA among patients with positive HBsAg were also analyzed. Results Among 27 409 samples included, 2681 （9.8%） were HBsAg positive. When patients were divided into 9 age groups, the age-specific positive rate of HBsAg was 1.2%, 9.6%, 12.3%, 10.9%, 10.3%, 9.7%, 8.0%, 5.8%, and 4.3%, respectively. The positive rate of HBsAg in non-surgical department, surgical department, and health examination center was 16.2%, 5.8%, and 4.7%, respectively. The positive rate of HBsAg of males （13.3%） was higher than that of females （7.3%, P=0.000）. Among the 2681 HBsAg （＋） patients, 1230 （45.9%） had HBV-DNA test, of whom 564 （45.9%） were positive. Patients with HBsAg （＋）, HBeAg （＋）, and HBcAg （＋） result usually had high positive rate of HBV-DNA results （71.8%, P=0.000）. Conclusions Among this group of patients in our hospital, the positive rate of HBsAg was relatively high. Age group of 20-29, males, and patients in non-surgical departments were factors associated with high positive rate of HBsAg.

Quantification of HBsAg:Basic virology for clinical practice

Hepatitis B surface antigen (HBsAg) is produced and secreted through a complex mechanism that is still not fully understood. In clinical fields, HBsAg has long served as a qualitative diagnostic marker for hepatitis B virus infection. Notably, advances have been made in the development of quantitative HBsAg assays, which have allowed viral replication monitoring, and there is an opportunity to make maximal use of quantitative HBsAg to elucidate its role in clinical fields. Yet, it needs to be underscored that a further understanding of HBsAg, not only from clinical point of view but also from a virologic point of view, would enable us to deepen our insights, so that we could more widely expand and apply its utility. It is also important to be familiar with HBsAg variants and their clinical consequences in terms of immune escape mutants, issues resulting from overlap with corresponding mutation in the P gene, and detection problems for the HBsAg variants. In this article, we review current concepts and issues on the quantification of HBsAg titers with respect to their biologic nature, method principles, and clinically relevant topics.

A prophylactic approach for bone marrow transplantation from a hepatitis B surface antigen-positive donor

It has been accepted that bone marrow transplantation （BMT） is the only curative therapeutic option for certain hematologic malignancies. The southeast Asia region is an endemic area of hepatitis B virus （HBV） infection; thus, BMT using a hepatitis B surface antigen （HBsAg）- positive donor is occasionally unavoidable. Organ transplantation using a HBsAg-positive donor can lead to post-transplantation de novo HBV infection and severe HBV-related hepatitis if no effective prophylactic measures are taken prior to and after transplantation. In this report, a four-level approach was designed for a patient with chronic myeloid leukemia, beginning with a booster HBV vaccination before performing BMT with a HBsAg-positive donor. Prior to BMT, the HBV viral load of the donor was reduced to an undetectable level by anUviral therapy. After BMT, hepatitis B immunoglobulin was administered intramuscularly for 1 wk together with a long-term antiviral drug, lamivudine. One year after discontinuation of lamivudine, the patient is still free of HBV infection.

High affinity mouse-human chimeric Fab against Hepatitis B surface antigen

AIM： Passive immunotherapy using antibody against hepatitis B surface antigen （HBsAg） has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal （5S） against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody.METHODS： We cloned the VH and V, genes of this mouse antibody, and fused them with CH1 domain of human IgG1 and C, domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. coli.RESULTS： The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. This chimeric antibody fragment was further expressed in different strains of E. coli to increase the yield.CONCLUSION： We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a fulllength chimeric antibody for therapeutic uses.

“Anti-HBc alone” in human immunodefi ciency virus-positive and immuno-suppressed lymphoma patients

Hepatitis B virus (HBV) infection is endemic in various parts of the world. A proportion of patients have resolved prior exposure to HBV, as evidenced by the clearance of circulating hepatitis B surface antigen and the appearance of antibody to hepatitis B core antigen (anti-HBc), which could produce protective antibody to hepatitis B surface antigen (anti-HBs). With time, anti-HBs in some patients may become negative. Such patients are described as having occult HBV infection or "anti-HBc alone". In the context of immunodef icient patients, such as HIV patients or lymphoma patients undergoing immunosuppressive immunotherapy, the lack of protective anti-HBs may increase the risk of hepatitis B reactivation. Serum HBV DNA testing may be necessary in "anti-HBc alone" patients, to detect patients at a high risk of developing HBV infection allowing appropriate prophylactic management.

Impact of comorbidities on the severity of chronic hepatitis B at presentation

AIM:To evaluate the clinical relevance of each cofactor on clinical presentation of chronic hepatitis B.METHODS:Out of 1366 hepatitis B surface antigen(HBsAg) positive subjects consecutively observed in 79 Italian hospitals,53(4.3%) showed as the only cofactor hepatitis D virus(HDV) infection [hepatitis B virus(HBV)/HDV group],130(9.5%) hepatitis C virus(HCV)(group HBV/HCV),6(0.4%) human immunodeficiencyvirus(HIV)(group HBV/HIV),138(10.2%) alcohol abuse(group HBV/alcohol);109(8.0%) subjects had at least two cofactors and 924 were in the cofactor-free(CF) group.RESULTS:Compared with patients in group CF those in group HBV/alcohol were older and more frequently had cirrhosis(P < 0.001),those in group HBV/HDV were younger(P < 0.001),more frequently resided in the south of the country and had cirrhosis(P <0.001),those in group HBV/HCV were older(P < 0.001) and more frequently had cirrhosis(P < 0.001).These cofactors were all independent predictors of liver cirrhosis in HBsAg positive patients.Multivariate analysis showed that an older age [odds ratio(OR) 1.06,95% CI:1.05-1.08],alcohol abuse with more than 8 drinks daily(OR 2.89,95% CI:1.81-4.62) and anti-HDV positivity(OR 3.48,95% CI:2.16-5.58) are all independently associated with liver cirrhosis.This association was found also for anti-HCV positivity in univariate analysis,but it was no longer associated(OR 1.23,95% CI:0.84-1.80) at multivariate analysis.CONCLUSION:Older age,HDV infection and alcohol abuse are the major determinants of severe liver disease in chronic HBV infection,while HCV replication plays a lesser role in the severity of hepatic damage.

Effect of interferon-γ and tumor necrosis factor-α on hepatitis B virus following lamivudine treatment

AIM： To evaluate anti-hepatitis B virus （HBV） activity and cytotoxicity of interferon-α, （IFN-3,） and tumor ne- crosis factor-α （TNF-α） following lamivudine treatment of HepG2.2.15 cells. METHODS： HepG2.2.15 cells were treated with 2 pmol/L lamivudine for 16 d （lamivudine group）, cultured for 10 d, followed by 5 ng/ml TNF-α and 1000 U/mL IFN-γ, for 6 d （cytokine group）, or treated with 2 ~tmol/L lami- vudine for 10 d followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ, for 6 d （sequential group）, or cultured without additions for 16 d （control group）. Intracellular DNA was extracted from 3 ×10^ HepG2.2.15 cells from each group. The extracted DNA was further purified with mung bean nuclease to remove HBV relaxed circu- lar DNA that may have remained. Both HBV covalently closed circular DNA （cccDNA） and HBV DNA were exam- ined with real-time polymerase chain reaction. The titers of hepatitis B surface antigen （HBsAg） and hepatitis B e antigen （HBeAg） were quantified with enzyme-linkedimmunosorbent assay. Cell viability was measured with the cell counting kit-8 assay. RESULTS： Compared to lamivudine alone （22.63%±0.12%）, both sequential （51.50% ± 0.17%, P = 0.034） and cytokine treatment （49.66% ± 0.06%, P = 0.041） showed a stronger inhibition of HBV cccDNA; the dif- ference between the.sequential and cytokine groups was not statistically significant （51.50% ± 0.17% vs 49.66% ± 0.06%, P = 0.88）. The sequential group showed less inhibition of HBV DNA replication than the lamivudine group （67.47% ±0.02% vs 82.48% ± 0.05%, P = 0.014）; the difference between the sequen- tial and cytokine groups was not statistically significant （67.47% ± 0.02% vs 57.45% ± 0.07%, P = 0.071）. The levels of HBsAg and HBeAg were significantly de- creased in the sequential treatment group compared to the other groups [HBsAg： 3.48 ± 0.04 （control）, 3.09 ± 0.08 （lamivudine）, 2.55± 0.13 （cytokine）, 2.32 ± 0.08 （sequential）, P = 0.042 for each between-group comparison; HBeAg 3.48 ± 0.01 （control）, 3.08 ± 0.08 （lamivudine）, 2.57 ± 0.15 （cytokine）, 2.34 ± 0.12 （se- quential）, P = 0.048 for each between-group compari- son]. Cell viability in the cytokine group was reduced to 58.03% ± 8.03% compared with control cells （58.03% ± 8.03% vs 100%, P = 0.000）. Lamivudine pretreat- ment significantly reduced IFN-γ, ± TNF-αmediated toxicity of HepG2.2.15 cells [85.82% =1= 5.43% （sequen- tial） vs 58.03% ± 8.03% （cytokine）, P = 0.002]. CONCLUSION： Sequential treatment overcame the lower ability of lamivudine alone to inhibit cccDNA and precluded the aggressive cytotoxicity involving IFN-y and TNF-α by decreasing the viral load.

Treatment of chronic viral hepatitis with nitazoxanide and second generation thiazolides

Nitazoxanide,the first thiazolide,was originally developed for the treatment of Cryptosporidium parvum.More recently,antiviral activity of nitazoxanide against hepatitis B virus(HBV)and hepatitis C virus was recognized in in vitro systems.These basic studies led to phaseⅡclinical trials that demonstrated the safety and efficacy of nitazoxanide in combination with peginterferon,with or without ribavirin,in the treatment of chronic hepatitis C genotype 4.The sustained virologic response rate was 79%and 80%in two studies,which was higher than the response rate of 50%with the standard of care with peginterferon plus ribavirin.In very preliminary studies of patients with chronic hepatitis B,nitazoxanide suppressed serum HBV DNA and led to loss of hepatitis B e antigen in the majority of patients and hepatitis B surface antigen in approximately a quarter of patients.Randomized controlled studies of naive and nonresponder patients with chronic hepatitis C genotype 1 are underway,new second generation and controlled release thiazolides are being developed,and future studies of patients with chronic hepatitis B are planned.