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

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 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.

Differentially expressed genes in hepatocellular carcinoma induced by woodchuck hepatitis B virus in mice

INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-5[1-5].Previously,a variety of studies have described the differences in gene expression which distinguished tumor from nontumor[6-11].Cloning of the genes,especially the genes associated with HBV and HCV,is still very important to account for the development of liver cancer.

Inhibition of hepatitis B virus by oxymatrine in vivo

AIM To investigate the anti-HBV effect ofoxymatrine (oxy) in vivo.METHODS HBV transgenic mice were producedby micro-injection of a 4.2kb fragmentcontaining the complete HBV genomes.Expression level of HBsAg and HBcAg in thetransgenic mice liver was determined byimmunohistochemical assay.RESULTS Four groups (6 mice in each group)were injected intraperitoneally with oxy at thedosage of 100,200, and 300 mg/kg or with salineonce a day for 30 days. Both HBsAg and HBcAgwere positive in livers of all the six mice in thecontrol group (injected with saline), and werepositive in livers of two mice in 100 mg/kg groupand 300mg/kg group. In 200mg/kg group,HBsAg and HBcAg were negative in livers of allthe six mice. Based on the results, 200 mg/kg isthe ideal dosage to explore the effect of oxy atdifferent time points. According to the oxytreatment time, mice were divided into fourgroups: 10 d, 20 d, 30 d and 60 d (4 mice in eachgroup). Each mouse underwent liver biopsy twoweeks before the treatment of oxy. Down-regulation of HBsAg and HBcAg appeared aftertreatment of oxymatrine for 10 d and 20 d, Dane-like particles disappeared after the treatment ofoxy for 20d under electron microscopy,however, the expression level of HBsAg andHBcAg returned to normal 60 d later after oxytreatment.CONCLUSION oxymatrine can reduce thecontents of HBsAg and HBcAg in transgenic miceliver, longer treatment time and larger dosagedo not yield better effects.

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.

Tumor radioimmunoimaging of chimeric antibody in nude mice with hepatoma xenograft

IM To study the radioimmunoimaging (RAII) using the human/mouse chimeric Ab to evaluate its targeting activity in animal models.METHODS To chimeric Ab was labeled with 131I. RAII was performed at different intervals after injection of radiolabeled Abs in nude mice with human hepatoma xenograft, and tissue distribution of radioactivity was measured. Comparison was made in the chimeric Ab between the single segment Ab and previous murine mAb against HBxAg.RESULTS The experimental objects developed tumorpositive image after 2 days of radiolabeled Abs injection, and the peak accumulation of radioactivity fell on the 7th day. The tumor/liver ratioactivity of the chimeric Ab, single segment Ab, antiHBx mAb, and the control group was 281±021, 244±016, 460±019, and 096±014, respectively.CONCLUSION The genetic engineering Abs have a considerable targeting activity which can be used as a novel humanized vector in the targeting treatment of liver cancer..

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.

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.

Liver chimeric mice with tupaia hepatocyte transplantation as an animal model for hepatitis B virus infection and antiviral therapy

The human liver chimeric mouse is a milestone animal model for hepatitis B virus(HBV)infection.Such mice with primary human hepatocyte(PHH)transplantation are adequate to support chronic HBV infection for several weeks and to evaluate antiviral drugs.However,the drawbacks of PHHs include lack of available donors,poor expansion in vitro and ethical issues that limit the application of human liver chimeric mice,necessitating the search for alternatives.Here,we transplanted primary tupaia hepatocytes(PTHs)into the livers of immunodeficient mice and achieved high liver chimerism within six weeks.These tupaia liver chimeric mice are adequate to support chronic infection of the four common HBV genotypes A,B,C and D for 36 weeks,as well as evaluate of antiviral drugs,including hepatitis B immune globulin(HBIG),monoclonal antibody and nucleoside analogues(NAs),for preventative therapy and treatment post infection.In conclusion,the tupaia liver chimeric mouse model provides a convenient,efficient and stable animal model for chronic HBV infection and long-term drug evaluation.

针对HBV X基因区三个关键位点的反义寡核苷酸体内抑瘤研究

目的 :研究HBVX基因区关键区段的反义寡核苷酸 (asON)对人肝癌裸小鼠模型的抑瘤生长及体内抗HBV作用。方法 :PCR法分别合成了互补于HBVX基因的翻译起始区Xp、DR2、ENⅡ区的反义寡核苷酸及无关对照序列 ,进行硫代化修饰 ,以HBVDNA转染的HepG2 .2 .15细胞接种裸小鼠 ,2 4h内同部位一次性用药 10 0 μg ,观察并比较 3种反义硫代寡核苷酸的体内抑瘤作用。ELLSA法检测反义核酸作用裸小鼠血清中HBsAg和HBeAg含量的变化。结果 :3种药物作用裸小鼠组均表现为出瘤潜伏期延长 ,出瘤率明显低于未用药瘤细胞对照组 (P <0 .0 5 ) ,且瘤体生长缓慢。互补于Xp、DR2、ENⅡ区的反义寡核苷酸一次性给药方式对荷瘤鼠HBsAg和HBeAg的表达无明显抑制作用 ;无关序列不影响荷瘤鼠瘤体生长及HBV抗原表达。结论 :针对HBVX基因区关键部位的反义寡核苷酸可抑制荷瘤裸小鼠人肝癌的生长 。

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。

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。

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。

参仙乙肝灵联合HBsAg基因修饰的树突状细胞对HBV转基因小鼠免疫应答及肝细胞损伤的影响

【目的】观察补肾解毒中药参仙乙肝灵联合乙肝表面抗原(HBs Ag)基因修饰的树突状细胞(DC/HBs Ag)诱导乙肝病毒(HBV)转基因小鼠的免疫应答及其对肝细胞损伤的影响。【方法】HBV转基因(Tg)小鼠尾静脉注射DC/HBs Ag免疫,使用参仙乙肝灵灌胃给药,共4周。采用酶联免疫吸附(ELISA)法检测HBV Tg小鼠脾脏T细胞内细胞因子白细胞介素-2(IL-2)、干扰素-γ(IFN-γ)分泌水平及谷丙转氨酶(ALT)、谷草转氨酶(AST)值;采用乳酸脱氢酶释放法检测HBV Tg小鼠脾脏HBs Ag特异性T淋巴细胞体外杀伤活性;采用ELISA检测HBV Tg小鼠血清HBs Ag表达情况。【结果】联合治疗能显著增加小鼠脾脏T细胞内细胞因子IL-2、IFN-γ水平(P<0.05或P<0.01),增加HBV Tg小鼠脾脏HBs Ag特异性T淋巴细胞杀伤活性(P<0.05或P<0.01),增加HBs Ag表达抑制率(P<0.05或P<0.01),作用均优于DC/HBs Ag免疫给药,并能明显减少肝细胞损伤。【结论】参仙乙肝灵对DC/HBs Ag诱导HBV Tg小鼠免疫应答具有一定的提升作用;同时能够减轻肝脏损害,在IFN-γ的抗HBV活性不受影响情况下,使HBV清除过程独立于肝脏损伤过程。