中国河南株丁型肝炎病毒全基因组的cDNA克隆和序列分析

从我国河南－抗丁型肝炎病毒抗原（ａｎｔｉ－ＨＤＡｇ）及丁型肝炎病毒（ＨＤｖ）ＲＮＡ双阳性的ＨＢｓＡｇ携带者血清中提取ＲＮＡ,采用人工合成的引物进行逆转录和聚合酶链反应（ＰＣＲ）,获得了贯穿ＨＤＶ全基因组的６个相互重叠的ｃＤＮＡ片段。经双脱氧末端终止法进行核苷酸序列分析,得到了长度为１６７４ｂｐ的我国人河南株ＨＤＶｃＤＮＡ全序列。计算机分析表明,该株与我国台湾株（ＨＤＶＩＡ型）、美国－１株（ＨＤＶＩＢ型）、日本－１株（ＨＤＶⅡ型）和秘鲁－１株（ＨＤＶⅢ型）的核苷酸同源性分别为的９４．３％、８６．８％、７５．４％和６６．３％,氨基酸序列的同源性分别为８９．７％、８５．１％、７１．９％和６４．６％,并在核苷酸和推导的ＨＤＡｇ氨基酸序列中分别发现了５个和２个集中保守的区域。这些区域均与ＨＤＶ的某些重要功能密切相关。     

乙型肝炎病人尿细胞中HBV的实验研究

本文采用间接免疫荧光法（ＩＦ）,ＲＰＨＡ法,ＥＬＩＳＡ法及斑点杂交技术检测１０例无症状ＨＢ－ｓＡｇ携带者及８９例乙肝病人尿细胞中的ＨＢｓＡｇ、ＨＢｅＡｇ及ＨＢＶＤＮＡ,发现尿细胞中有ＨＢｓＡｇ、ＨＢｅＡｇ、ＨＢＶＤＮＡ存在。结果提示：乙肝无症状携带者及乙肝病人尿细胞中具有ＨＢｓＡｇ、ＨＢｅＡｇ、ＨＢＶＤＮＡ,因此更进一步证实尿液具有传染性。     

乙型肝炎病人重叠感染丙型肝炎的评价

应用ＥＬＩＳＡ和ＰＣＲ法检测５０２例乙肝病人血清,４０１例ＨＢｓＡｇ阳性血清中,有１１４例（２８．４％）抗－ＨＣＶ和ＨＣＶＲＮＡ双项阳性,２５例（６．２％）ＨＣＶＲＮＡ单项阳性;２１例（５．２％）抗－ＨＣＶ单项阳性。将ＨＢｓＡｇ乙肝病人分成ＨＢＶＤＮＡ,ＨＢｅＡｇ阳性组和ＨＢＶＤＮＡ,ＨＢｅＡｇ阴性组。前者抗－ＨＣＶ阳性率为１１．６％～２０．５％,ＨＣＶＲＮＡ阳性率为１６．２％～２０．５％。后者抗－ＨＣＶ阳性率为２０．２％～５５．６％,ＨＣＶＲＮＡ阳性率为２３％～６０．３％。结果说明长期携带ＨＢＶ者和慢性乙肝病人均可重叠ＨＣＶ感染。ＨＢＶＤＮＡ阳性组抗－ＨＣＶ和ＨＣＶＲＮＡ阳性率明显高于ＨＢＶＤＮＡ阳性组     

恩拉霉素抗乙型肝炎病毒的体外实验研究

以ＨｅｐＧ２．２．２．１５细胞株为模型,以其分泌的ＨＢｓＡｇ、ＨＢｅＡｇ、ＨＢＶＤＮＡ及细胞存活率为观察指标,综合评价天然多肽类抗生素恩拉霉素体外抗ＨＢＶ效果。结果表明恩拉霉素对ＨＢｓＡｇ和ＨＢｅＡｇ的５０％抑制浓度ＩＣ５０分别为２７μｇ／ｍＬ和３４μｇ／ｍＬ,治疗指数（ＴＩ）分别为５．９和４．６。Ｓｏｕｔｈｅｒｎ结果显示,５０μｇ／ｍＬ恩拉霉素对细胞内游离ＨＢＶＤＮＡ抑制率为５６．８％。     

用套式多聚酶链反应技术监测乙型肝炎病毒的母婴垂直传播

用套式多聚酶链反应（Ｎｅｓｔｅｄ－ＰＣＲ）技术对１６９对ＨＢｓＡｇ及ＨＢｓＡｇ／ＨＢｅＡｇ阳性孕妇及其新生儿外周血清进行了ＨＢＶ－ＤＮＡ检测。１０３对ＨＢｓＡｇ阳性孕妇及其新生儿外周血清中ＨＢＶ－ＤＮＡ阳性率分别为７２．８％和３３．０％;６６对ＨＢｓＡｇ和ＨＢｅＡｇ双阳性的孕妇及其新生儿外周血清中ＨＢＶ－ＤＮＡ阳性率分别为８６．４％和４３．９％。对５５例ＨＢｓＡｇ及ＨＢｓＡｇ／ＨＢｅＡｇ阳性产妇产后的初乳进行了ＨＢＶ－ＤＮＡ检测,结果ＨＢＶ－ＤＮＡ阳性率为３６．４％。结果表明ＨＢｓＡｇ和ＨＢｅＡｇ双阳性的孕妇及其新生儿外周血清ＨＢＶ－ＤＮＡ检出率较ＨＢｓＡｇ单阳性的孕妇及其新生儿要高,其初乳中ＨＢＶ－ＤＮＡ的检出率也高。还对１０５例注射了乙肝疫苗及高价乙肝特异性免疫球蛋白的６月龄婴儿的外周血清进行了ＨＢＶ－ＤＮＡ检测,结果有２３例阳性。     

用套多聚酶链反应技术监测乙型肝炎病毒的母婴垂直传播

用套式多聚酶链反应（Ｎｅｓｔｅｄ－ＰＣＲ）技术对１６９对ＨＢｓＡｇ及ＨＢｓＡｇ／ＨＢｅＡｇ阳性孕妇及其新生儿外周血清进行了ＨＢＶ－ＤＮＡ检测,１０３对ＨＢｓＡｇ阳性孕妇及其新生儿外周务中ＨＢＶ－ＤＮＡ阳性率分别为７２．８％和３３．０％;６６对ＨＢｓＡｇ和ＨＢｅＡｇ双阳性的孕妇及其新生儿外周血清中ＨＢＶ－ＤＮＡ阳性率分别为８６．４％和４３．９％,对５５例ＨＢｓＡｇ及ＨＢｓＡｇ／ＨＢｅＡｇ阳性产妇产后     

HBsAg携带者重叠感染HCV、HDV的血清学调查

对３６２名无症状高滴度ＨＢｓＡｇ携带者用ＲＩＡ法检测ＨＢｅＡｇ、Ａｎｔｉ－ＨＢｅ、Ａｎｔｉ－ＨＣＶ、Ａｎｔｉ－ＨＤＶ。结果表明,ＨＢｅＡｇ阳性率随ＨＢｓＡｇ滴度的增高而增加,且有显著性差异（Ｐ＜０．０５）。重叠感染以ＨＢＶ＋ＨＣＶ最高,占２７．６％;其次是ＨＢＶ＋ＨＤＶ,占９．１％;ＨＢＶ＋ＨＣＶ＋ＨＤＶ最少,占６．４％。但是,以上重叠感染率均与ＨＢｓＡｇ滴度及／或ＨＢｅＡｇ阳性率高低无关（Ｐ＞０．０５）。调查显示,无症状ＨＢｓＡｇ携带者中ＨＢＶ＋ＨＣＶ,或ＨＢＶ＋ＨＤＶ,或ＨＢＶ＋ＨＣＶ＋ＨＤＶ的重叠感染均可发生。     

乙型肝炎、肝硬变和肝癌中HBxAg的表达及其在肝癌发生中的作用

对３７９例良、恶性肝组织进行的免疫组织化学研究显示,３３％的慢性迁延性肝炎（６／１８）、７６％的慢性活动性肝炎（２６／３４）、９２％的肝硬变（５７／６２）和９７％的肝细胞性肝癌（ＨＣＣ）（５８／６０）中有ＨＢｘＡｇ表达,阳性率高于ＨＢｓＡｇ或ＨＢｃＡｇ。癌周肝中的ＨＢｘＡｇ阳性率显著高于非癌周肝。与其它２种ＨＢＶ抗原不同,ＨＢｘＡｇ表达在细胞类型上有较明显的选择性,在肝小多角细胞（ＳＰＬＣ）、小细胞性不典型增生（ＳＣＤ）及ＨＣＣ中较强。与ＩＧＦⅡ、ｃ－ｅｒｂＢ－２、ｃ－ｍｙｃ和ＥＧＦ－Ｒ表达进行的对照研究表明ＨＢｘＡｇ与ＩＧＦⅡ和ｃ－ｅｒｂＢ－２这２种ＨＣＣ发生相关基因的表达关系密切。ＰＣＮＡ染色结果显示ＨＢｘＡｇ阳性组织的细胞增殖活性显著高于ＨＢｘＡｇ阴性组织。我们的结果还表明ＨＢｘＡｇ表达与肝细胞不典型增生的发生和进展有关、提出ＨＢＶＸ基因可能通过其表达产物（ＨＢｘＡｇ）首先激活ＩＧＦⅡ、ｃ－ｅｒｂＢ－２基因,继而引起显著的ＳＰＬＣ增生和ＳＣＤ而参与ＨＣＣ发生的．     

硫代磷酸反义寡脱氧核苷酸抗HBV细胞的实验研究

针对ＨＢＶ的５个基因位点作为靶序列,设计合成硫代反义寡聚核苷酸（Ｓ－ａｓＯＤＮ）．应用ＥＬＩＳＡ方法、ＭＴＴ比色法、电子显微镜等手段,观察Ｓ－ａｓＯＤＮ对ＨｅｐＧ２２２１５细胞ＨＢｓＡｇ、ＨＢｅＡｇ抗原的表达,及细胞的毒性、细胞形态和超微结构的影响．结果显示不同靶位点的Ｓ－ａｓＯＤＮ对ＨＢｓＡｇ、ＨＢｅＡｇ表达都有显著的抑制作用,且表现为序列特异性、剂量相关性、联合协同性和一定的抗核酸酶性,在浓度为２０μｍｏｌ／Ｌ时,对细胞无明显杀伤作用,对细胞的超微结构无显著的改变．结果提示Ｓ－ａｓＯＤＮ有望发展为抗ＨＢＶ的有效药物,但靶序列的选择、透细胞膜性及联合用药配伍等仍值得进一步研究和解决．     

反义寡聚硫代磷酸脱氧核苷抑制体外细胞培养系统中乙肝抗原表达的研究

设计合成了两个分别互补于乙肝病毒２．１ｋｂ ｍＲＮＡ起始区（片段Ａ）和增强子区（片段Ｂ）的硫代磷酸的ＤＮＡ片段,在经克隆ＨＢＶ ＤＮＡ转染ＨｅｐＧ２细胞建立的ＨＢＶ短暂表达系统及稳定产生ＨＢＶ的２２１５细胞中研究二者对ＨＢｓＡｇ及ＨＢｅＡｇ表达的抑制作用。结果表明反义寡聚物能不同程序抑制乙肝抗原表达,并与剂量呈一定正相关。在ＨｅｐＧ２细胞ＨＢＶ短暂表达系统中,６μｍｏｌ／Ｌ浓度时,片段Ａ、Ｂ对ＨＢ     

乙肝病毒增强子对其基因表达的调控

本文构字线性化的含有从核心抗原启动子Ｃｐ起始的ＨＢＶ完整转录单元的基因组隆,以此为模型,通过增强子Ｉ和增强子ＩＩ的缺失或点突变分析,研究了ＥＮＩ和ＥＮＩＩ对ＨＢＶ基因组基因宾调控。结果用ＥＮＩＩ对Ｓ基因表达均有增强作用,且相互协同。     

Hepatitis B,Hepatitis C and HIV-1 Coinfection in Two Informal Urban Settlements in Nairobi,Kenya

Background

HIV-1 and Hepatitis B and C viruses coinfection is common in Sub-Saharan Africa due to similar routes of transmission and high levels of poverty. Most studies on HIV-1 and Hepatitis B and C viruses have occurred in hospital settings and blood transfusion units. Data on Hepatitis B and C viruses and HIV-1 coinfection in informal urban settlements in Kenya are scanty, yet they could partly explain the disproportionately high morbidity and mortality associated with HIV-1 infections in these slums.

Objectives

The objective of this study was to determine the prevalence of HIV and Hepatitis B and C dual infection in urban slums in Nairobi.

Methods

Blood samples were collected from residents of Viwandani and Korogocho between 2006 and 2007. A structured questionnaire was used to obtain socio-demographic data from participants. Samples were screened for Hepatitis B surface antigen (HBsAg), anti-HCV and anti-HIV-1. Statistical analysis was done using STATA.

Results

Samples were successfully collected from 418 (32%) men and 890 (68%) females. The HIV-1, HBV and HCV prevalence was 20.4%, 13.3% and 0.76% respectively at the time of the study. Of the 268 (20.4%) HIV-1 positive participants, 56 (4.26%) had HBV while 6 (0.46%) had HCV. Of the 1041 HIV-1 negative participants, 117 (8.9%) had HBV while 4 (0.31%) had HCV. Only two people (0.15%) were co-infected with all the three viruses together.

Discussion

The odds of getting hepatitis infection were higher in HIV-1 participants (for HBV OR 2.08,p<0.005 and for HCV OR 5.93, p<0.005). HIV prevalence rates were similar in both informal settlements. HIV infection was highest in age group 35-39 years and among the divorced/separated or widowed. Prevalence of all viruses was highest in those who did not have any formal education.

Conclusion

The HIV prevalence in these informal settlements suggests a higher rate than what is observed nationally. The prevalence rates of HBV are significantly higher in the HIV-1 positive and negative populations. HCV as well as triple HIV-1, HBV and HCV coinfection are uncommon in Korogocho and Viwandani. This clearly indicates the need for HIV-1 control programmes and hepatitis B virus vaccination to be promoted through public awareness as preventive strategy.     

High Hepatitis B Surface Antigen Levels Predict Insignificant Fibrosis in Hepatitis B e Antigen Positive Chronic Hepatitis B

Introduction

There is no data on the relationship between hepatitis B surface antigen (HBsAg) levels and liver fibrosis in hepatitis B e antigen (HBeAg)-positive patients with chronic hepatitis B (CHB).

Methods

Serum HBsAg and HBV DNA levels in HBeAg-positive CHB patients with liver biopsies were analyzed. The upper limit of normal (ULN) of alanine aminotransferase (ALT) was 30 and 19 U/L for men and women respectively. Histologic assessment was based on Ishak fibrosis staging for fibrosis and Knodell histologic activity index (HAI) for necroinflammation.

Results

140 patients (65% male, median age 32.7 years) were recruited. 56 (40%) had ALT ≤2×ULN. 72 (51.4%) and 42 (30%) had fibrosis score ≤1 and necroinflammation grading ≤4 respectively. Patients with fibrosis score ≤1, when compared to patients with fibrosis score >1, had significantly higher median HBsAg levels (50,320 and 7,820 IU/mL respectively, p<0.001). Among patients with ALT ≤2×ULN, serum HBsAg levels achieved an area under receiver operating characteristic curve of 0.869 in predicting fibrosis score ≤1. HBsAg levels did not accurately predict necroinflammation score. HBsAg ≥25,000 IU/mL was independently associated with fibrosis score ≤1 (p = 0.025, odds ratio 9.042).Using this cut-off HBsAg level in patients with ALT ≤2×ULN, positive and negative predictive values for predicting fibrosis score ≤1 were 92.7% and 60.0% respectively. HBV DNA levels had no association with liver histology.

Conclusion

Among HBeAg-positive patients with ALT ≤2×ULN, high serum HBsAg levels can accurately predict fibrosis score ≤1, and could potentially influence decisions concerning treatment commencement and reduce the need for liver biopsy.     

Effects of Entecavir on Hepatitis B Virus Covalently Closed Circular DNA in Hepatitis B e Antigen-Positive Patients with Hepatitis B

The aim of this study was to assess the effect of 48-week entecavir therapy on serum and intrahepatic hepatitis B virus, covalently closed circular DNA (HBV cccDNA) levels in hepatitis B e antigen (HBeAg)-positive patients. A total of 120 patients with HBeAg-positive chronic hepatitis were treated with entecavir for 48 weeks. Serum HBV markers, total HBV DNA, and HBV cccDNA levels were measured at baseline and week 48. Biopsies from 20 patients were available for both intrahepatic total HBV DNA and cccDNA testing at these timepoints. HBV cccDNA levels were decreased from a median level of 5.1×10⁶ copies/mL at baseline to a median level of 2.4×10³ copies/mL at week 48. Reduction magnitudes of HBV cccDNA in patients with normalized alanine aminotransferase levels and those undergoing HBeAg seroconversion were significantly greater than those in alanine aminotransferase-abnormal and HBeAg positive patients. Intrahepatic HBV cccDNA was decreased significantly after 48 weeks of treatment, but could not be eradicated. In conclusion, treatment of HBeAg-positive hepatitis B patients with entecavir for 48 weeks decreased serum and intrahepatic HBV cccDNA significantly, and the magnitude of HBV cccDNA reduction was related to total HBV DNA decrease, alanine aminotransferase normalization, and HBeAg seroconversion.