Animal models for the study of hepatitis B virus infection

Even with an effective vaccine, an estimated 240 million people are chronically infected with hepatitis B virus （HBV） worldwide. Current antiviral therapies, including interferon and nucleot（s）ide analogues, rarely cure chronic hepatitis B. Animal models are very crucial for understanding the pathogenesis of chronic hepatitis B and developing new therapeutic drugs or strategies. HBV can only infect humans and chimpanzees, with the use of chimpanzees in HBV research strongly restricted. Thus, most advances in HBV research have been gained using mouse models with HBV replication or infection or models with HBV-related hepadnaviral infection. This review summarizes the animal models currently available for the study of HBV infection.

Advances in Animal Models of Hepatitis B Virus Infection

Hepatitis B virus (HBV) infection seriously affects human health. Stable and reliable animal models of HBV infection bear significance in studying pathogenesis of this health condition and development of intervention measures. HBV exhibits high specificity for hosts, and chimpanzee is long used as sole animal model of HBV infection. However, use of chimpanzees is strictly constrained because of ethical reasons. Many methods were used to establish small-animal models of HBV infection. Tupaia is the only nonprimate animalthat can be infected by HBV. Use of HBV-related duck hepatitis virus and marmot hepatitis virus infection model contributed to evaluation of mechanism of HBV replication and HBV treatment methods. In recent years, development of human–mouse chimeric model provided possibility of using common experimental animals to carry out HBV research.These models feature their own advantages and disadvantages and can be complementary in some ways. This

Establishment and primary application of a mouse model with hepatitis B virus replication

AIM： To establish a rapid and convenient animal model with hepatitis B virus （HBV） replication.METHODS： A naked DNA solution of HBV-replicationcompetent plasmid was transferred to BALB/C mice via the tail vein, using a hydrodynamic in vivo transfection procedure. After injection, these mice were sacrificed on d 1, 3, 4, 5, 7 and 10. HBV DNA replication intermediates in the liver were analyzed by Southern blot hybridization. The expression of hepatitis B core antigen （HBcAg） and hepatitis B surface antigen （HBsAg） in the liver was checked by immunohistochemistry. Serum HBsAg and hepatitis B e antigen （HBeAg） was detected by enzyme- linked immunosorbent assay （ELISA）. Inhibition of HBV replication was compared in HBV replication model mice treated intraperitoneally with polyinosinic-polytidylin acid （polyIC） or phosphate-buffered saline （PBS）.RESULTS： After hydrodynamic in vivo transfection, HBV DNA replication intermediates in the mouse liver were detectable on d 1 and abundant on d 3 and 4, the levels were slightly decreased and remained relatively stable between d 5 and 7, and were almost undetectable on d 10. The expression patterns of HBcAg and HBsAg were similar to that of HBV replication intermediate DNA, except that they reached a peak on d 1 after injection. No obvious differences in HBV DNA replication intermediates were observed in the left, right and middle lobes of the liver. After treatment with polyIC, the level of HBV intermediate DNA in the liver was lower than that in the control mice injected with PBS.CONCLUSION： A rapid and convenient mouse model with a high level of HBV replication was developed and used to investigate the inhibitory effect of polyIC on HBV replication, which provides a useful tool for future functional studies of the HBV genome.

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.

Preliminary study on the production of transgenic mice harboring hepatitis B virus X gene

AIM To establish transgenic mice lineage xharboring hepatitis B virus X gene and to provide an efficient animal model for studying the exact role of the HBx gene in the process of hepatocarcinogenesis. METHODS The HBx transgenic mice were produced by microinjecting the construct with X gene of HBV (subtype adr) DNA fragment into fertilized eggs derived from inbred C57 BL/6 strain; transgenic mice were identified by using Nested PCR; expression and phenotype of HBx gene were analyzed in liver from transgenic mice at the age of 8 weeks by RT PCR, pathologic examination and periodic acid schiff staining (PAS), respectively. RESULTS Five hundred and fourteen fertilized eggs of C57 BL/6 mice were microinjected with recombinant retroviral DNA fragment, and 368 survival eggs injected were transferred to the oviducts of 18 pseudopregnant recipient mice, 8 of them became pregnant and gave birth to 20 F1 offspring. Of 20 offsprings, four males and two females carried the hybrid gene (HBx gene). Four male mice were determined as founder, named X1, X5, X9 and X15. These founders were back crossed to set up F1 generations with other inbred C57BL/6 mice or transgenic littermates, respectively. Transmission of HBx gene in F1 offspring of X1, X5 and X9 except in X15 followed Mendelian rules. The expression of HBx mRNA was detected in liver of F1 offspring from the founder mice (X1 and X9), which showed vacuolation lesion and glycogen positive foci. CONCLUSION Transgenic mice harboring HBx gene were preliminarily established.

Establishment of transgenic mouse harboring hepatitis B virus (adr subtype) genomes

INTRODUCTIONHepatitis B virus (HBV) belongs to the group ofhepatovirus, a major pathogen of human acute andchronic hepatitis B[1 4], which has a very closeassociation with human hepatocellular carcinoma(HCC)[5-8], For example, a statistical data from ahospital in Shanghai showed that 80% of HCCpatients were positive for HBsAg ( personalcommunication).

Follow up of infection of chacma baboons with inoculum containing a and non-a genotypes of hepatitis B virus

AIM: To determine whether one genotype (A or non-A genotypes of HBV) predominated over the other during the course of HBV infection.METHODS: Four baboons were inoculated with HBV. DNA was extracted from serum obtained at monthly intervals postinoculation for 52 weeks and HBV DNA was amplified using primers specific for the core region containing an insert characteristic of genotype A (nt 2 354-2 359, numbering from the EcoRI site). The amplicons were cloned into PCRScriptTM and a minimum of 15 clones per time point were sequenced in both directions.RESULTS: Both genotypes persisted for the entire followup period of 52 weeks. Genotype non-A predominated in two baboons and genotype A in one baboon. Neither genotype predominated in the fourth baboon, as shown at a 5 % level of testing.CONCLUSION: No conclusions concerning the dominance of either genotype or the natural progression or replication rates of HBV could be drawn because the pattern of the genotypes found may have been caused by sampling fluctuations at the time of DNA extraction and cloning as a result of the very low viral loads in the baboon sera.

Research Progress of Model Establishment in Treating Hepatitis B

Hepatitis B virus is a major liver disease caused by virus infection. Viral hepatitis is popular in China,mainly caused by hepatitis B. Experimental animal model is a necessary platform for the research on mechanism of viral infection and pathogenicity,for treatment and vaccine development. Up to date,a great progress in the development of viral hepatitis animal models has been achieved in spite of the most of findings are limited to hepatitis B. Here,we summarized the recent findings of viral hepatitis animal models,focusing on the tree shrew animal model.

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.

DUCK HEPATITIS B VIRUS MODEL FOR SCREENING OF ANTIVIRAL AGENTS FROM MEDICINAL HERBS

The effects of the extracts of 20 Chinese medicinal herbs and an antiviral drug foscarnet on duck hepatitis B virus (DHBV) endogenous DNA polymerase (DNAp) activity were compared. The extracts of P. urinaria showed a dose-dependent inhibition on DHBV DNAp. And those of other herbs showed little inhibition effect. Primary duck hepatocyte (PDH) cultures were used for evaluating effects of the extract of P. urinaria, foscarnet and acyclovir (ACV) on DHBV, and all the drugs or the extracts showed inhibition of DHBV DNA replication. Furthermore, in vivo trials were carried out. Peking ducks infected with LJ-76 strain of DHBV were treated with the extract of P. urinaria or ACV and compared with placebo treated control ducks. The treatment results in the loss or reduction of circulating viral DHBV DNA and DHBsAg.

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.

食蟹猴实验感染HBV的血清学反应

目的：研究食蟹猴感染乙型肝炎病毒（ HBV）后的血清学反应。方法：实验室内人工繁育的1～3日龄或成年食蟹猴观察1个月经病毒学筛选后证实为健康动物后各随机分为对照组、感染组，感染组接种HBV携带者血清0．5 ml（ HBV-DNA≥108拷贝）单只笼养，各组从接种后1～12周每日观察行为变化，每1周取血样检测HBV-M、HBV-DNA、肝功能及对于HBsAg阳性食蟹猴在B超引导下取肝组织常规HE染色检测肝组织炎症程度。结果：成年猴接种后未引发HBsAg阳性反应，有3只幼年猴出现HBsAg、HBcAb及2只出现HBV-DNA反应，ALT在攻毒出现HBsAg阳性后1周开始升高，1个月后达到高峰，其值为180 U/L，以后渐降，持续1个月后接近正常。 AST一周后高于正常参考值呈低平曲线，高峰较ALT迟后1个月， HBsAg阳性食蟹猴HE染色可见部分肝组织呈轻微肝炎病变。结论：攻毒后HBV-M、HBV-DNA、ALT、AST及肝组织病理改变提示HBV感染后能产生应答及肝细胞炎症反应。

HBV感染小鼠模型的研究进展

HBV由于其较强的种属特异性,目前尚缺乏理想的实验动物模型来阐明其具体的发病机制。目前有关HBV感染模型的研究大多集中在小鼠方面,并已取得了很大进展。综述了HBV转基因小鼠、HBV转染小鼠和人鼠嵌合肝脏HBV小鼠等模型的优缺点,提出合理使用这些模型有助于更好地阐明HBV的致病机制。

中国流行株C基因型HBV稳定复制表达小鼠模型的建立

目的构建稳定复制中国流行株C基因型HBV的小鼠模型。方法将携带1.3倍C基因型HBV基因组（adr血清型）的重组腺相关病毒r AAV8-1.3HBV-C转导人肝癌细胞Hu H7,采用ELISA法评估HBV抗原（HBs Ag、HBe Ag）在肝癌细胞中的表达。筛选高表达的重组病毒,经尾静脉注射入6~8周龄C57BL/6小鼠体内,建立HBV-C复制小鼠模型（实验组,n=8）;同时建立文献已报道HBV-D复制小鼠模型（对照组,n=7,r AAV8-1.3HBV-D,ayw血清型）。动态监测两组小鼠血清（眼底静脉丛采血）HBV DNA载量和HBs Ag、HBe Ag的抗原表达量;于第9周处死小鼠,HE染色观察肝组织病理改变,免疫组化染色分析HBs Ag和HBc Ag的表达。结果重组病毒r AAV8-1.3HBV-C体外转导人肝癌细胞Hu H7,72h后细胞上清中可检测到HBs Ag和HBe Ag的表达。小鼠注射重组病毒后第2、3、5、7、9周,血清HBV DNA存在稳定复制,血清HBe Ag表达水平较稳定,但血清HBs Ag表达存在波动。两组小鼠肝组织未见明显的炎性细胞浸润及组织结构异常,但可检测到HBs Ag和HBc Ag蛋白。结论利用高嗜肝性重组8型腺相关病毒载体携带1.3倍C基因型HBV基因组体内转导C57BL/6小鼠,成功地建立了稳定复制并持续表达C基因型HBV的小鼠模型。

HBV的小鼠模型研究进展

乙型肝炎病毒感染是全球范围内影响人类健康的重要问题,目前人们对HBV及其所致疾病有了相当深入的认识.但由于缺乏合适的动物模型,乙型肝炎病毒的生物学研究和治疗进展缓慢.小鼠作为一种实验室常用的动物,遗传免疫背景清楚明确,已经成为人们研究乙肝的重要工具.本文简要综述了小鼠模型在乙型肝炎研究中的进展.

HBV动物模型研究进展

HBV感染动物模型是研究HBV致病机制、筛选新型有效抗HBV药物和治疗方法的重要工具,然而HBV感染具有高度组织特异性以及种属特异性,这给HBV感染动物模型的建立带来了困难。近年来,随着分子生物学、实验动物学、病毒学及免疫学等相关学科技术的进步,HBV感染或复制动物模型取得了明显进展。目前应用于HBV(包括与HBV具有相似特性的动物肝炎病毒)研究的动物模型主要包括黑猩猩、树鼩、土拨鼠及鸭HBV感染模型,HBV转基因小鼠、高压水动力注射介导的小鼠HBV复制模型和重组腺相关病毒载体介导的小鼠HBV复制模型,以及人源化人-鼠嵌合肝脏HBV感染模型,此外,钠离子-牛磺胆酸共转运蛋白转基因小鼠感染模型是近年的研究热点。本文就上述HBV动物模型的研究进展进行综述。

HBV感染与复制模型的建立及应用

乙型肝炎是危害人类健康的重要传染病,目前的抗病毒治疗,如干扰素、核苷和核苷酸类药物仍无法治愈慢性乙型肝炎。因此,亟待阐明HBV复制和致病机制,探索新的治疗靶点,进而研发新的治疗药物或方案。合适的HBV感染与复制模型是上述研究的基础。由于HBV具有严格的种属限制性及组织亲嗜性,使得HBV感染与复制模型的研发受到一定限制。在国家传染病科技重大专项资助下,国内研究者建立了一系列的细胞和动物模型,就此并结合近年来国内外研究进展进行简要综述。

利用RNA干扰技术干扰HBeAg表达的研究

构建针对乙肝病毒HBeAg前c/c区(HBV prec/c)的短发夹环RNA(shRNA)质粒(psiHBV),观察其在体内外对HBV复制的抑制作用。构建RNA干扰真核表达载体psiHBV,与1.5倍HBV真核表达质粒pHBV1.5共转染HeLa细胞,用微粒子化学发光分析仪(MEIA)分别检测细胞上清和细胞裂解液中HBeAg表达水平;用半定量PCR检测prec/cmRNA的转录情况。随后用水动力学方法,向小鼠尾静脉注射pHBV1.5建立小鼠急性乙型肝炎病毒感染模型。采用此感染模型,将pHBV1.5与在体外实验筛选到有明显抑制作用的shRNA表达载体(psiHBV4)共注射,注射后第6天用同样方法检测其干扰效果。结果显示,成功构建了针对HBV前c/c区的shRNA表达载体psiHBV4、psiHBV5、psiHBV6及无关shRNA表达载体psiC,筛选到在体外对HBeAg有明显的抑制作用的psiHBV4载体;注射pHBV1.5的动物血清高表达HBsAg和HBeAg。而共注射干扰性psiHBV4明显抑制了HBeAg的表达,与单纯感染组相比有显著差异,RT-PCR显示肝内HBV C mRNA水平亦明显降低。上述结果表明,siRNA能特异抑制HBV的复制和表达,对乙型肝炎的治疗有潜在应用前景。

HBV感染性克隆长度对其复制能力的影响

目的比较不同长度HBV感染性克隆复制能力的差异,为HBV复制相关研究提供依据。方法采用分子克隆的方法体外分别扩增HBV基因组的相应片段,并进行连接,分别构建含1.1、1.2、1.3倍HBV基因组的可复制性克隆。体外转染Huh7细胞系评价抗原分泌和病毒复制情况,使用高压水注射建立急性感染小鼠模型评价病毒分泌情况以及抗原在小鼠肝脏内的表达情况。结果所构建的克隆体外转染Huh-7细胞后均可分泌高浓度的HBsAg,而1.3倍HBV基因组的可复制性克隆HBeAg的分泌能力明显强于其他克隆,转录水平也最高。高压尾静脉注射小鼠后肝组织内均可检测到HBcAg的分布,血清中可以检测到HBV DNA并随时间发生动态变化。其蛋白表达水平和病毒分泌能力均以1.3倍HBV基因组的可复制性克隆较高。结论所构建的克隆在体外及体内均可进行复制,其中含HBV全基因组1.3倍体的可复制性克隆的复制能力最强,可以较好反映来源病毒株的天然复制能力。