Replication of hepatitis B virus in primary duck hepatocytes transfected with linear viral DNA

AIM: To explore the expression and replication of hepatitis B virus (HBV) DNA in primary duck hepatocytes (PDHs).METHODS: Complete HBV genome was transfected into PDHs by electroporation (transfected group, 1.19×1012copies of linear HBV DNA/1×107 PDHs). After 1-5 d of transfection, HBsAg and HBeAg in the supernatant and lysate of PDHs were measured with the IMX System.Meanwhile, replicative intermediates of HBV DNA were analyzed by Southern blotting and Dot blotting. PDHs electroporated were used as control group.RESULTS: HBsAg in the hepatocyte lysates of transfected group was 15.24 (1 d), 14.55 (3 d) and 5.13 (5 d; P/N values, positive≥2.1) respectively. HBeAg was negative (＜2.1). Both HBsAg and HBeAg were negative in the supernatant of transfected group. Dot blotting revealed that HBV DNA was strongly positive in the transfected group and negative in the control group. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (rc DNA), covalently closed circular (ccc DNA), and single-stranded (ss DNA) HBV DNA replicative intermediates in the transfected group, there was no integrated HBV DNA in the cellular genome. These parameters were negative in control group.CONCLUSION: Expression and replication of HBV genes can occur in hepatocytes from non-mammalian species.HBV replication has no critical species-specificity, and yet hepatic-specific regulating factors in hepatocytes may be essential for viral replication.

The Effect of Gankang Suppository on Duck Hepatitis B Virus, Serum Biochemistry and Liver Histology in Ducklings

To examine the effect of Gankang Suppository on duck hepatitis B virus （DHBV）, the serum biochemistry and hepatic histology in an animal model of DHBV infection, a model of DHBV infection was established by infecting 1-day-old Yingtaogu ducklings with DHBV-positive serum. The successful model was confirmed by PCR assay and 48 ducklings infected with DHBV were randomly divided into 3 groups： a Gankang Suppository treatment group, an acyclovir （ACV） group and a DHBV model group （control）, with each group having 16 animals. All the animals were given the medicines for 4 weeks in a row. The serum of the animals was taken 14 and 28 days after the medica- tion and 7 days after drug discontinuation. Real-time PCR was performed to detect the copy numbers of DHBV DNA in the serum. ALT and AST were dynamically monitored. The ducklings were sacrificed on the 7th day after the discontinuation of the treatment and livers were harvested and examined for inflammation and degeneration of liver cells by using HE staining. The results showed that on day 14, 28 after the treatment and day 7 after the withdrawal, the logarithmic values （log） of DHBV DNA copy numbers in ducklings of Gankang Suppository treatment group were significantly lower than that before the treatment （P=0.0092, P=0.0070, P=0.0080, respectively）. Compared with DHBV model control group, the ALT level was significantly decreased （P=0.0020, P=0.0019, respectively） on day 28 after the treatment and on day 7 after the withdrawal. The AST level was also reduced on day 14 after the treatment （P=0.0298）. Compared with the ACV control group, the level of ALT was lower on day 7 after the withdrawal （P=0.0016）. Histologically, the hepatocyte swelling, vacuolous degeneration and acidophilic degeneration in Gankang Suppository treatment group were alleviated 7 days after the withdrawal as compared with model control group （P=0.0282, P=0.0084, P=0.0195, respectively）. It is concluded that Gankang Suppository can effectively suppress DHBV replication, reduce the levels of serum ALT and AST and improve hepatic histology.

Molecular Characterization of Duck Hepatitis B Virus Isolated from Hubei Brown Ducks

The objective of this study was to characterize the genome structure of duck hepatitis B virus （DHBV） isolated from Hubei brown ducks. The natural carrier rate of DHBV in adult ducks from Hubei area was investigated and the DHBV DNA-positive serum screened out. The complete genome of a DHBV strain was amplified by polymerase chain reaction （PCR） and cloned into T vector and sequenced. The results showed that the carrier rate of DHBV in Hubei brown ducks was 10 % This strain （GenBank accession number DQ276978） had a genome of 3024 nucleotides with three overlapping open reading frames encoding the surface, core and polymerase proteins respectively. Comparison of the strain with 17 DHBV strains registered in GenBank revealed a homology from 89.3 % to 93.5 % at the nucleotide level. The sequences of the structural and functional domains of these proteins were highly conserved. The strain was found to share more signature amino acids in the polymerase genes with the ＂Chinese＂ DHBV strains than those of the ＂Western＂ country strains. This finding was also corroborated by a phylogenetic tree analysis. Therefore, the DQ276978 might belong to a subtype of the Chinese DHBV strains.

DUCK HEPATITIS B VIRUS DNA WITHIN HEPATIC MULTICENTRIC CANCER AND/OR METASTATIC CANCER

Duck hepatitis B vims (DHBV) DNA was detected in different tumorous nodules of ducks with hepatic multicentric cancer or intrahepatic metastasis by Southern blot technique. Among 7 ducks with hepatocellular carcinoma of multiple tumor nodules, the hybridization pattern of Integrated DHBV DNA In different tumorous nodules was identical in 3 cases and different in 2 cases. One case showed a similar hybridization pattern in two tumorous nodules and other one was negative tor DHBV DNA. Integrated DHBV DNA was also identified in a metastatic lung cancer of ducks with hepatocellular carcinoma. The hybridization pattern of metastasis of lungs was as the some as that in primary hepatocellular carcinoma. The same discrete hybridization bands In the different tumorous nodules indicate that these nodules might arise from one transformed cell. The different hybridization patterns In various tumorous nodules show that these tumorous nodules might arise from various transformed cells. The results suggest that the hybridization pattern of different nodules of hepatocellular carcinoma with viral DNA probe could make a cell clone origin marker of tumor nodule to differentiate hepatic multlcentric cancer from Intrahepatic metastatic cancer.

Therapeutic effect of Styela plicata on duck hepatitis B virus in vivo

Objective:To evaluate the antiviral activity of the alcohol extract of Styela plicata on DHBV (duck hepatitis B virus) in vivo. Methods: Guangzhou-Sheldrake ducklings congenitally infected with DHBV were assigned to receive the alcohol extract of Styela plicata or lamivudine for 30 consecutive days. The DHBV DNA of sera was detected by RT-PCR. and the histological analysis of duckling liver was evaluated. Results:Thirty days after therapy,histological analysis of duckling liver showed that the ducklings receiving the alcohol extract of Styela plicata or lamivudine exhibited catabatic status in the degree of liver cell degeneration and inflammation compared with the ducklings receiving normal diet. DHBV DNA of sera from alcohol extract of Styela plicata-treated ducklings and lamivudine-treated ducklings all produced significantly lower levels compared with ducklings receiving normal diet (P<0. 01 ). Although these treatment groups all exhibited a rebound phenomenon 10 d after withdrawal of medication, they still exhibited a significant lower level of serum DHBV DNA compared with the control group responded to normal diet (P<0. 05, P<0. 01). Conclusion:Styela plicata may be an effective antiviral medicine in treating chronic hepatitis B. The data of this experiment will be valuable in studying the therapeutic role and the potential therapeutic mechanism of Styela plicata.

Detection of the covalently closed circular DNA of duck hepatitis B virus by Taq-Man fluorescent quantitative PCR assay

To develop a fluorescent quantitative PCR assay based on Taq-Man chemistry to detect the covalenfly closed circular DNA （eccDNA） of duck hepatitis B virus （DHBV）, a pair of primers was designed from both sides of the nick in the minus strand of DHBV and a Taq-Man probes between the primers, modified with 6-Fam at 5＇ end and Tamra at its 3＇ end was designed to detect the PCR products during PCR cycles. The DHBV DNA fragment was cloned into vector PUCm-T, and the recombinant plasmid was purified and subsequently qualified as the HBV DNA standard. The experimental conditions and reagents used in PCR assay for amplification were sophisticatedly optimized in order to yield a perfect amplification efficacy and reduce the possibility to produce non-specific amplification. It was demonstrated that the detect limit of assay was 10^3 copies/ml, and a linear standard curve was obtained between 10^5 -10^9 copies/ml [ C1 =-2.8361 ln（x） ＋ 41.45, r =-0.9985]. The coefficient of variation was 0.2%-3.14% and 2.22%-4.43% for intra- and inter-assay respectively. After a dynamic survey on the contents of DHBV DNA in serum of ducks, it was found that its peak value appeared at the second week of birth in ducks. It is evident that this method of Taq-Man fluorescent quantitative PCR assay appears to be simple, sensitive and specific.

Is hepatitis B-virucidal validation of biocides possible with the use of surrogates?

The hepatitis B virus(HBV)is considered to be a major public health problem worldwide,and a significant number of reports on nosocomial outbreaks of HBV infections have been reported.Prevention of indirect HBV transmission by contaminated objects is only possible through the use of infection-control principles,including the use of chemical biocides,which are proven to render the virus non-infectious.The virucidal activity of biocides against HBV cannot be predicted;therefore,validation of the virucidal action of disinfectants against HBV is essential.However,feasible HBV infectivity assays have not yet been established.Thus,surrogate models have been proposed for testing the efficacy of biocides against HBV.Most of these assays do not correlate with HBV infectivity.Currently,the most promising and feasible assay is the use of the taxonomically related duck hepatitis B virus(DHBV),which belongs to the same Hepadnaviridae virus family.This paper reviews the application of DHBV,which can be propagated in vitro in primary duck embryonic hepatocytes,for the testing of biocides and describes why this model can be used as reliable method to evaluate disinfectants for efficacy against HBV.The susceptibility levels of important biocides,which are often used as ingredients for commercially available disinfectants,are also described.

Immunogenicity and protective efficacy of DHBV DNA vaccines expressing envelope and capsid fusion proteins in ducks delivered by attenuated Salmonella typhimurium

Duck hepatitis B virus(DHBV) shares many basic characteristics with hepatitis B virus(HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks. Attenuated Salmonella typhimurium(SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response. Based on this strategy, novel DNA vaccines(SL7207-p VAX1-LC and SL7207-p VAX1-SC) were generated. Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy. The relative transcript levels of the CD4 and CD8 T genes and the antibody levels(Ig Y) in ducks receiving the vaccines were higher than those in single gene delivered groups. Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.

氧化苦参碱在鸭原代肝细胞中抗鸭乙肝病毒(DHBV)作用的研究

通过建立鸭原代肝细胞-DHBV感染模型研究氧化苦参碱抗DHBV的作用。分别在DHBV感染前、感染同时以及感染后给药，利用打点杂交、Southern印迹核酸杂交和荧光定量PCR方法分别检测培养细胞上清及细胞内病毒核酸，观察氧化苦参碱在病毒感染的各个环节所起的抗病毒作用。实验结果显示：1mg／mL氧化苦参碱处理细胞后，鸭原代肝细胞培养上清及细胞内的DHBV核酸明显低于病毒感染对照组，病毒抑制率达91．6％；在病毒感染同时加药对病毒的抑制率可达98．5％；感染后持续用药能使不同培养天数的鸭肝细胞内的DHBV核酸降低60．5％～96．6％；氧化苦参碱与DHBV共孵育后，可以使病毒感染力下降69．6％。结果说明氧化苦参碱可以在DHBV感染鸭原代肝细胞的多个环节，包括病毒吸附、进入细胞及细胞内复制等方面发挥抗病毒作用。

特异光敏效应对血细胞悬液中DHBV的灭活作用

目的 观察设计的靶向光敏剂所产生的特异光化学效应对血细胞悬液中病毒的灭活作用 ,以探索新型的血液病毒灭活技术。方法 以光化学效应为基本原理 ,利用反义核酸技术设计合成与DHBV DNA特异结合的TFO P ,将其作为特异的靶向光敏剂 ,在UVA的协同下将其作用于血细胞悬液中的DHBV。通过电泳转移印迹试验、原代鸭肝细胞感染试验 ,观察TFO P与DHBV DNA的结合效应和在UVA协同下灭活DHBV的效果。结果 所设计的TFO P能与不同株DHBV DNA样本产生不同程度的特异结合 ,在剂量为 0 .1nmol/ml的TFO P和照射强度为 180 0 μW/cm2 的UVA作用下 ,血细胞悬液中DHBV的活性下降 1.9～ 5 .4Log。 结论 TFO P所产生的光化学效应能显著灭活血细胞悬液中的DHBV。

水芹水醇提取物在雏鸭体内对DHBV-DNA的抑制效果

目的观察水芹(Oenanthe Javanica,OJ)水醇提取物在鸭体内对鸭乙型肝炎病毒(DHBV)-DNA 的抑制效果.方法将 DHBV 感染雏鸭随机分为高、中、低三个剂量组,分别为8,5,3g/kg 组进行药物治疗.每组5～6只,ig,2次/d×10 d;设病毒对照组(DHBV),以生理盐水(NS)代替药物,ig,2次/d×10 d.阳性药用阿昔洛韦(ACV)粉剂250 mg,ig,100mg/kg,2次/d×10 d.在感染7d 后,即用药前(TO),用药后5d(T5),用药10 d(T10)和停药后3 d(P3),自鸭腿胫静脉分别取血,分离血清,检查 DHBV-DNA 水平和肝功能.治疗结束经颈静脉放血处死动物,分别切取雏鸭肝脏,作肝病理检查.结果三批实验结果表明,水芹水醇提取物中,高剂量组对感染雏鸭无毒性,给药5,10 d 能显著降低 DHBV 感染鸭血清DHBV-DNA 水平,即 d5的测定值由1.04分别下降至0.52和0.44,抑制率分别为50.0%和50.6%;d10由0.85分别下降0.42和0.36,抑制率分别为57.7%和57.6%,与病毒对照组比较,均有显著性差异(P<0.05和 P<0.01).低剂量组对鸭血清 DHBV-DNA 也有一定疗效,并显示明显的量效关系.肝功能改善明显,给药10 d,中剂量雏鸭血中 ALT,AST,SB 含量下降率分别为44.1%,44.2%,33.3%.高剂量的下降率分别为61.0%,58.3%,64.4%.低剂量虽对肝功能有改善,但无统计显著性.肝组织病理观察证实,中、高剂量对雏鸭肝脏均有保护作用,肝细胞变性坏死均较轻,肝小叶结构基本完整.结论 OJ 在雏鸭体内有抗 DHBV 的作用,保肝效果显著.

PCR及Dot-blot法对先天感染DHBV筛选的比较

目的比较PCR及Dot-blot法对先天感染DHBV筛选的效果。方法分别利用PCR法及Dot-blot法对先天感染DHBV筛选,并进行比较。结果与Dot-blot法比较,PCR法有简单、快捷、敏感度高等优点,且可避免Dot-blot带来的放射性污染。但PCR法所需费用高,易因交叉污染而导致假阳性结果。结论PCR与Dot-blot法筛选先天感染鸭乙肝的方面各有其优缺点。由于PCR法更为敏感、快捷,很有必要对其加以改进和完善。

DHBV诱发鸭急性肝坏死动物模型的建立

本文首次报道了应用鸭乙型肝炎病毒(DHBV)加大肠杆菌内毒素诱导方法,建立了鸭急性肝坏死动物模型。结果发现:先肝内注射大肠杆菌内毒素,24h后再静脉内激发性注射DHBV者,易造成一叶或二叶肝坏死。而第一次肝内注射内毒素,24h后静脉内注入生理盐水,或二次均注入生理盐水者均未发现肝坏死病理变化。若第一次肝内注入DHBV,第二次静脉内注入内毒素,病理变化则明显轻于第一种方法,仅出现轻微散在斑点状坏死。

三螺旋结构寡核苷酸序列对DHBV基因的连接及封锁抑制作用研究

目的 筛选与鸭乙肝病毒 (DHBV) DNAX C区目标基因特异连接的三螺旋结构寡核苷酸(TFO) ,探讨TFO对DHBV DNA的特异连接及抑制作用。方法 将人工合成的TFO与目标基因片断和含(DHBV)DNA的重组细胞反应 ,用电泳转移印迹、PCR和鸭肝原代细胞培养等技术观察TFO对相应基因的连接以及对细胞内DHBV DNA复制的抑制效应。结果 在所设计的多条TFO中 ,( 3 )TFO和 ( 5 )TFO在 3～ 4h内能与细胞内外目标基因双链直接连接 ,导致DHBV DNA复制一定程度的抑制 ;在DHBV DNA阳性的原代鸭肝细胞培养液中连续加入终浓度为 4μmol L的TFO ,大多数细胞组的培养液中DHBV DNA的检测在 10～12d开始呈现阴性。结论 TFO能直接与病毒的DNA连接 ,导致目标基因的复制抑制 ,有着潜在的特异抗病毒价值。

上海地区麻鸭血中DHBV及其形态学的研究

本文应用电镜等方法对上海地区麻鸭中鸭乙型肝炎病毒(DHBV)及其形态学进行了研究。104只麻鸭血清标本DHBV阳性率为68.5%,其中1日～1月龄组为93.3%,6～12月龄组为72.7%,1～2年龄组为56.6%,表明DHBV阳性率随鸭龄增长而不断下降。鸭血清中除存在DHBV的40～45nm完全颗粒与40～60nm空心颗粒外,还发现一种20～30nm的小球形颗粒。鸭肝组织超薄切片的电镜观察,在肝细胞浆内质网中找到DHBV颗粒,同时在胞浆与核内均找到35～37nm裸露的核心颗粒。

一株鸭乙型肝炎病毒DHBV全基因的克隆和序列分析

目的从本地樱桃谷鸭血清中克隆鸭乙型肝炎病毒(DHBV)DNA,并进行序列分析.方法用PCR扩增DHBV全基因,连接至T载体上,挑选克隆进行测序分析,与GenBank中16株DHBV全基因组进行同源性比较,并进行分子进化树分析.结果24-18与16株DHBV基因组比较,核苷酸同源性介于89.4%99.3%之间,各开放阅读框氨基酸同源性比较显示差异显著地方位于P区.结论24-18属于DHBV西方基因型中的一个亚型.

鸭肝组织中DHBV cccDNA的实时荧光定量PCR方法的建立

目的 建立基于DHBV病毒正、负链缺口区设计引物、沉淀蛋白结合的DNA、酶切消化残留线性DNA的DHBVcccDNA荧光定量PCR方法.方法 根据DHBV cccDNA和rcDNA结构的不同,将引物设计在DHBV DNA负链缺口的两侧.采用十二烷基硫酸钾蛋白质沉淀法分离肝组织中cccDNA和rcDNA.并对提取的DNA进行紫外定量,按照每2UPSAD消化500ng cccDNA,计算PSAD的用量,酶切消化残存的线性DNA.以pBR322/2DHBV Core重组质粒为PCR标准品,通过优化反应体系和扩增条件,建立检测DHBV cccDNA的SYBR Green I实时荧光定量PCR方法,并进行方法学考察和实际应用.结果 优化的PCR扩增产物电泳后可见239 bp的DNA片段,与目标片段长度相同.标准曲线回归方程Y=-4.085 7X＋48.805,R2=-0.997 6.方法的灵敏度为103 copies/ml,线性范围可达103～108 copies/ml.方法特异性强,未检出DHV,HBV及E coli DNA.对已感染DHBV的鸭肝组织检测DHBV cccDNA,进行定量,含量从0.36～1733.08 copies/diploid genome.其中分布最广的是10～99 copies/dioploid genome.DHBV cccDNA占总DHBV DNA的比例平均仅为3.86％,范围从0.01％～13.3s％.结论 根据DHBV cccDNA与rcDNA的结构和理化性质的差异,设计能够特异性扩增DHBV cccDNA的荧光定量PCR方法,方法灵敏度高、特异度强,可广泛应用于DHBV和HBV抗病毒治疗策略的相关研究中.

扶正利湿活血复方对鸭乙肝模型血清DHBV DNA的影响

目的:观察扶正利湿活血中药复方对鸭乙型肝炎病毒(DHBV)的抑制作用。方法:PCR筛选先天感染DHBV的1日龄广东麻鸭,随机分为病毒对照组、拉米夫定组、扶正利湿活血复方高、低剂量组,每组7只,各组分别灌胃给药30天。在灌胃前、灌胃第10、20、30天及停药第5天分别采血,辛酸钠法提取血清DHBV DNA,采用荧光定量PCR法检测血清DHBV DNA的含量。结果:扶正利湿活血复方高剂量组在给药后的第10、20、30天时,鸭血清病毒载量均明显下降,(与D0比较,P<0.05;各时间点与病毒对照组比较,P<0.01);在停药5天后,与D0组内比较,血清病毒载量仍然显著降低(P<0.01),但与病毒对照组比较则无显著下降(P>0.05);扶正利湿活血复方低剂量组在给药后的第10、20、30天时,鸭血清病毒载量均明显下降,(与D0比较,P<0.01;各时间点与病毒对照组比较,P<0.01);在停药5天后,组内与D0比较及组间与病毒对照组比较均明显下降(P<0.05)。结论:扶正利湿活血复方可抑制DHBV DNA的复制,低剂量长期效应更为明显。

广东麻鸭DHBV全基因克隆及新读码框的发现

选取3份DHBV阳性广东麻鸭血清提取DHBVDNA,设计一对扩增DHBV全基因序列引物Q1和Q2,扩增并克隆DHBV全基因序列,测序并运用相关计算机软件及方法分析获得的全基因序列。结果表明,广东麻鸭DH-BV全长为3027bp。提交GenBank后获得的收录号分别为:AY433937、AY521226、AY521227(来自1号血清);AY392760、AY536371(分别来自2、3号血清)。AY521227的PreS/SORF出现了单碱基突变,在PreC/CORF之前发现一个新的ORF,暂命名为HORF,HORF也同时存在于GenBank中储存的另外8个DHBV全基因序列中。系统发育分析表明,广东麻鸭DHBV在分化程度上是目前储存于GenBank中的DHBV全序列中最高的。成功克隆了广东麻鸭DHBV全基因序列,序列分析为进一步研究广东麻鸭DHBV提供了有益的信息。

茵芍散体内抗DHBV实验研究

目的观察茵芍散体内以抗鸭乙肝病毒(DHBV)的作用。方法用PCR法筛选鸭乙肝病毒(DHBV)阳性血清,经静脉注射感染3日龄麻鸭,建立DHBV实验模型。将DHBV阳性麻鸭40只随机分为茵芍散高、中、低剂量组,拉米夫定组和模型对照组,常规饲养的同时分别给予不同剂量的药物干预。用荧光定量聚合酶链反应(FQ-PCR)检测血清DHBV DNA含量,ALT和AST水平,光学显微镜下观察肝组织病理改变。结果茵芍散各剂量组用药后血清DHBV DNA含量显著降低(P<0.01),其疗效与剂量和治疗时间密切相关。停药7 d后,茵芍散低剂量组和阳性对照组血清DHBV DNA有反跳现象,而茵芍散中、高剂量组血清DHBV DNA没有反跳现象。结论茵芍散有抑制DHBV DNA及降低血清转氨酶、改善肝脏炎症的作用。