Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis B virus

Studies of hepatitis B virus (HBV) mutants have been hampered by the lack of a small animal model with long-term infection of cloned HBV. Using a mouse model in which liver cells were highly replaced with human hepatocytes that survived over a long time with mature human hepatocyte function, we performed transmission experiments of HBV. Human serum containing HBV and the virus produced in HepG2 cell lines that transiently or stably transfected with 1.4 genome length HBV DNA were inoculated. Genetically modified e-antigen-negative mutant strain also was produced and inoculated into the mouse model. A high-level (approximately 10(10) copies/mL) viremia was observed in mice inoculated with HBV-positive human serum samples. The level of viremia tended to be high in mice with a continuously high human hepatocyte replacement index. High levels and long-lasting viremia also were observed in mice injected with the in vitro generated HBV. The viremia continued up to 22 weeks until death or killing. Passage experiments showed that the serum of these mice contained infectious HBV. Genetically engineered hepatitis B e antigen-negative mutant clone also was shown to be infectious. Lamivudine effectively reduced the level of viremia in these infected mice. In conclusion, this mouse model of HBV infection is a useful tool for the study of HBV virology and evaluation of anti-HBV drugs. Our results indicate that HBeAg is dispensable for active viral production and transmission.     

HLA class I antigens on the hepatocyte membrane during recovery from acute hepatitis B virus infection and during interferon therapy in chronic hepatitis B virus infection

In a chimpanzee model of acute type B hepatitis, at the time of onset of hepatitis B virus replication and before the development of immunity to hepatitis B virus, interferon is present in the plasma. This is followed by an increase in the display of HLA class I, but not class II proteins, on the hepatocyte membrane. In chronic hepatitis B virus infection, there is a low density of HLA class I protein display on the infected hepatocyte. Administration of alpha-interferon enhances HLA display and in many cases is followed by a transaminase elevation, seroconversion of HBe antigen to antibody and disappearance of hepatitis B virus DNA from serum, changes implying clearance of infected hepatocytes. Successful response to interferon therapy may be predicted by a rapidly rising serum beta 2-microglobulin, a component of the HLA class I molecule, during the first 2 weeks of therapy, before the rise in transaminases.     

小鼠对乙肝病毒小基因嵌合DNA疫苗免疫应答

目的:探讨含CD80的乙肝病毒小基因嵌合DNA疫苗的免疫应答.方法:以乙肝病毒小基因嵌合DNA疫苗pcHBV-CD80(含CMV启动子、HBV-preS2片段、HBV-C片段和CD80)、pcHBV(含CMV启动子、HBV-preS2片段、HBV-C片段),以及质粒pcDNA-CD80、pcDNA3.1和生理盐水,im免疫小鼠2次(间隔2 wk),末次接种后2 wk时检测特异性抗体、γ-IFN水平和淋巴细胞转化率.结果:质粒pcHBV-CD80和pcHBV免疫后均可检测到特异性的抗体,抗-preS2在pcHBV-CD80组和DcHBV组分别为1 5125.52 nkat/L和14463.56 nkat/L;抗-HB c在pcHBV-CD80组和DcHBV组分别为7607.35 nkat/L和7711.21 nkat/L,两组间无明显差异;但p cHBV-CD80更能增加γ-IFN的产生(1266.7 ng/L vs 986.3 ng/L,P＜0.01),并增强免疫小鼠的激淋巴细胞转化.结论:含CD80的乙肝病毒小基因嵌合DNA疫苗可诱导特异性的体液免疫和细胞免疫应答.     

Effects of hepatitis B virus infection on human sperm chromosomes

AIM: To evaluate the level of sperm chromosome aberrations in male patients with hepatitis B, and to directly detect whether there are HBV DNA integrations in sperm chromosomes of hepatitis B patients. METHODS: Sperm chromosomes of 14 tested subjects (5 healthy controls, 9 patients with HBV infection, including 1 with acute hepatitis B, 2 with chronic active hepatitis B, 4 with chronic persistent hepatitis B, 2 chronic HBsAg carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free golden hamster ova and human spermatozoa, and the frequencies of aberration spermatozoa were compared between subjects of HBV infection and controls. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes. RESULTS: The total frequency of sperm chromosome aberrations in HBV infection group (14.8 %, 33/223) was significantly higher than that in the control group (4.3 %, 5/116). Moreover, the sperm chromosomes in HBV infection patients commonly presented stickiness, clumping, failure to staining, etc, which would affect the analysis of sperm chromosomes. Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis. In 9 (9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots, others presented 2 to 4 signals. There was significant difference of fluorescence intensity among the signal spots. The distribution of signal sites among chromosomes was random. CONCLUSION: HBV infection can bring about mutagenic effects on sperm chromosomes. Integrations of viral DNA into sperm chromosomes which are multisites and nonspecific, can further increase the instability of sperm chromosomes. This study suggested that HBV infection can create extensively hereditary effects by alteration genetic constituent and/or induction chromosome aberrations, as well as the possibility of vertical transmission of HBV via the germ line to the next generation.     

Adaptive response in hepatitis B virus infection

Hepatitis B virus (HBV) is a major cause of acute and chronic liver inflammation worldwide. The immune response against the virus represents a key factor in determining infection outcome, in terms of both viral clearance and the perpetuation of liver damage. Significant advances have recently been achieved regarding the functions of antiviral CD8+ T cells, leading to a better understanding of their abnormalities during chronic infection as well as the pathways to be manipulated to reverse the immune impairment of chronic infection. In this review, we aimed to analyse the patterns of adaptive immunity that develop during acute infection and the profiles in chronic infection. In addition to CD8+ T cells, which are the best‐described subset to date, we reviewed and commented on the direct and indirect roles of CD4+ T cells and B cells.     

The impact of prior hepatitis B virus infection on liver histology and the response to interferon therapy in chronic hepatitis C

summary.   Patients with chronic hepatitis C frequently have antibodies to the hepatitis B core antigen (anti-HBc), indicative of prior hepatitis B virus (HBV) infection. In these patients, persistence of HBV may exacerbate liver injury and diminish the response to treatment. The aim of this study was to evaluate the relationship between previous HBV infection and liver histology and the sustained virologic response (SVR) to interferon (IFN)-based therapy in patients with chronic hepatitis C. A total of 132 HBsAg-negative, treatment-naive patients were evaluated. Using multiple logistic regression analysis, the impact of anti-HBc-positivity on the rate of SVR was determined. Progression to bridging fibrosis or cirrhosis was assessed using Cox proportional hazards regression and Kaplan–Meier survival analysis. The median age of the patients was 47 years (IQR, 37–60), 57% were male, and 73% had genotypes 1, 4, 5, or 6. Fifty-one patients (39%) were anti-HBc-positive. The prevalence of moderate to severe necroinflammatory activity ( P  = 0.36) and progression to bridging fibrosis or cirrhosis (log-rank P  = 0.83) was similar between anti-HBc-positive and -negative patients. After a median of 48 weeks (IQR, 26–52) of therapy (IFN, n  = 116; IFN and ribavirin, n  = 16), 23 patients (17%) achieved a SVR; the rate of response was similar in anti-HBc-positive and -negative patients (18% vs 17%, P  = 1.00). After controlling for age, gender, genotype, fibrosis, and treatment regimen, anti-HBc status did not independently affect the rate of SVR (anti-HBc-positive vs negative: odds ratio, 1.36; 95% confidence interval, 0.45 to 4.06; P  = 0.58). In conclusion, previous HBV infection does not affect liver histology or the response to IFN-based therapy in patients with chronic hepatitis C.     

Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures

Hepatitis C virus (HCV) remains a major public health problem, affecting approximately 130 million people worldwide. HCV infection can lead to cirrhosis, hepatocellular carcinoma, and end-stage liver disease, as well as extrahepatic complications such as cryoglobulinemia and lymphoma. Preventative and therapeutic options are severely limited; there is no HCV vaccine available, and nonspecific, IFN-based treatments are frequently ineffective. Development of targeted antivirals has been hampered by the lack of robust HCV cell culture systems that reliably predict human responses. Here, we show the entire HCV life cycle recapitulated in micropatterned cocultures (MPCCs) of primary human hepatocytes and supportive stroma in a multiwell format. MPCCs form polarized cell layers expressing all known HCV entry factors and sustain viral replication for several weeks. When coupled with highly sensitive fluorescence- and luminescence-based reporter systems, MPCCs have potential as a high-throughput platform for simultaneous assessment of in vitro efficacy and toxicity profiles of anti-HCV therapeutics.     

乙型肝炎病毒基因组变异对干扰素治疗的影响

目的 研究乙型肝炎病毒基因组Ｘ区和前Ｃ／Ｃ区多部位变异对干扰素（ＩＦＮ）治疗的影响。方法 利用套式聚合酶链反应产物直接测序技术,测定１７例慢性乙型肝炎患者ＩＦＮ治疗前病毒核苷酸序列。结果 ５例Ｔ＾１７６２－Ａ＾１７６４变异株感染者均对ＩＦＮ治疗有应答,而在１２例无Ｔ＾１７６２－Ａ＾１７６４突变患者中仅３例有应答。在８例Ａ＾１８９６变异株感染者中,５例同时伴Ｃ区Ｂ细胞表位ａａ１０７￣１１８突变,其中     

Susceptibility of chimeric mice with livers repopulated by serially subcultured human hepatocytes to hepatitis B virus

We previously identified a small population of replicative hepatocytes in long-term cultures of human adult parenchymal hepatocytes (PHs) at a frequency of 0.01%-0.09%. These hepatocytes were able to grow continuously through serial subcultures as colony-forming parenchymal hepatocytes (CFPHs). In the present study, we generated gene expression profiles for cultured CFPHs and found that they expressed cytokeratin 19, CD90 (Thy-1), and CD44, but not mature hepatocyte markers such as tryptophan-2,3-dioxygenase (TO) and glucose-6-phosphatase (G6P), confirming that these cells are hepatic progenitor-like cells. The cultured CFPHs were resistant to infection with human hepatitis B virus (HBV). To examine the growth and differentiation capacity of the cells in vivo, serially subcultured CFPHs were transplanted into the progeny of a cross between albumin promoter/enhancer-driven urokinase plasminogen activator-transgenic mice and severe combined immunodeficient (SCID) mice. The cells were engrafted into the liver and were able to grow for at least 10 weeks, ultimately reaching a maximum occupancy rate of 27%. The CFPHs in the host liver expressed differentiation markers such as TO, G6P, and cytochrome P450 subtypes and could be infected with HBV. CFPH-chimeric mice with a relatively high replacement rate exhibited viremia and had high serum levels of hepatitis B surface antigen. CONCLUSION: Serially subcultured human hepatic progenitor-like cells from postnatal livers successfully repopulated injured livers and exhibited several phenotypes of mature hepatocytes, including susceptibility to HBV. In vitro-expanded CFPHs can be used to characterize the differentiation state of human hepatic progenitor-like cells.     

Immunological response in chronic hepatitis C virus infection during interferon alpha therapy

BACKGROUND/AIMS: Chronic hepatitis C infection is a serious therapeutic problem. Interferon therapy is one of the possible methods leading to HCV infection elimination in some patients. The aim of the study was the estimation of administration of interferon alpha 2a and its effect on the lymphocyte subpopulations in peripheral blood in patients with chronic hepatitis C. METHODOLOGY: A cytometric analysis was completed concerning the level of CD19+, CD3+, CD4+, CD8+, CD4+ + HLA DR+, CD16+ + 56+ in 21 patients in the 0, 4, 24, 48 weeks of interferon alpha 2a treatment, with the dose of 18 MU/week/48 weeks. RESULTS: Virus elimination was obtained in 26% of patients (responders, R) and a higher CD8+ level and a decrease in CD4+ was observed during interferon administration in those patients. A slight increased NK cell level was noticed mainly in patients who did not eliminate HCV (non-responders, NR). In R patients, lower lymphocyte values of CD19+, NK, CD3+ + HLA DR+, and CD4+ were observed in comparison to NR, which could suggest that they play a role in the process of HCV elimination. Significant immunological changes in peripheral blood were observed mainly in the first 4 weeks of interferon alpha therapy. CONCLUSIONS: Our studies did not reveal whether any of examined lymphocyte subpopulations in peripheral blood could be considered as a predictive factor of a positive reaction to interferon alpha treatment. However, there are significant differences in the levels of lymphocytes in R and NR.     

Effects of hepatitis E virus infection on interferon production via ISG15

AIM To assess the effects of hepatitis E virus(HEV) on the production of type Ⅰ interferons(IFNs) and determine the underlying mechanisms.METHODS We measured the production of interferon(IFN)-alpha and-beta(-α/β) in genotype 3 HEV-infected C3 A cells at different time points(0, 8, 12, 24, 48, 72 and 120 h) by enzyme-linked immunosorbent assay(ELISA). The expression levels of IFN-stimulated gene(ISG)15 in HEVinfected C3A cells at different time points were tested by western blotting. The plasmid-expressing open reading frame 3(ORF3) or control plasmids(green fluorescent protein-expressing) were transfected into C3A cells, and the levels of IFN-α/β and ISG15 were evaluated, respectively. Furthermore, the plasmid-expressing ISG15 or small interfering RNA-inhibiting ISG15 was transfected into infected C3A cells. Then, the production of IFN-α/β was also measured by ELISA.RESULTS We showed that genotype 3 HEV could enhance the production of IFN-α/β and induce elevation of ISG15 in C3A cells. HEV ORF3 protein could enhance the production of IFN-α/β and the expression of ISG15. Additionally, ISG15 silencing enhanced the production of IFN-α/β. Overexpression of ISG15 resulted in the reduction of IFN-α/β.CONCLUSION HEV may promote production of IFN-α/β and expression of ISG15 via ORF3 in the early stages, and increased ISG15 subsequently inhibited the production of IFN-α/β.     

Early dynamics of hepatitis B virus in chimeric mice carrying human hepatocytes monoinfected or coinfected with genotype G

Of the 8 genotypes of HBV (genotypes A-H), genotype G is unique in that it has an insertion in the core gene and two stop codons in the precore region preventing the synthesis of hepatitis B e antigen. Most individuals with genotype G are coinfected with other genotypes, typically genotype A. Mice with severe combined immunodeficiency disease carrying human hepatocytes were infected with HBV particles propagated in Huh7 cells in culture. Mice monoinfected with genotype G did not raise detectable HBV DNA in serum, although products of the core gene emerged 4 to 8 weeks after inoculation. When they were superinfected with genotype A at week 10, however, HBV DNA of genotype A developed, which was replaced almost completely by that of genotype G within 10 weeks. Such a rapid takeover was also observed in mice initially infected with genotype A or C and superinfected with genotype G. Similar viral dynamics occurred in mice simultaneously coinfected with genotypes G and A. Takeover was markedly enhanced in mice inoculated with a serum passage containing genotype G with a trace of genotype A. Coinfection of mice with genotypes G and A induced abundant cellular steatosis along with increased fibrosis in the liver, which was not detected in mice monoinfected with genotype A or G. CONCLUSION: Genotype G can monoinfect chimeric mice at very low levels, and its replication increases maredly when coinfected with other genotypes. Coinfection with genotype G could enhance fibrosis under immunocompromised states.     

商陆抗病毒蛋白在急性人乙型肝炎病毒感染小鼠体内的抑制作用

目的 探讨商陆抗病毒蛋白(PAP)在急性HBV感染小鼠模型体内抗HBV的效果.方法 通过尾静脉注射具有复制能力的HBV真核表达质粒来建立小鼠急性HBV感染模型.根据注射后第1天小鼠血清HBsAg和HBeAg水平,从35只小鼠中选出24只进行配对,分为PAP治疗组(腹腔注射0.25 mg/kg PAP)和对照组.于PAP注射前、注射后第1、3、5、7天,时间分辨免疫荧光分析法检测小鼠血清HBsAg、HBeAg和抗-HBc水平,荧光定量PCR检测小鼠血清HBV DNA水平.注射后第7天HE染色检测肝组织病理变化,免疫组织化学检测肝组织HBsAg、HBeAg表达.采用配对t检验进行统计学分析.结果 与对照组相比,在PAP腹腔注射后第1、3、5天对HBsAg的抑制率分别为23%(t=116.3,P<0.05)、47%(t=38.2,P<0.05)、68%(t=23.7,P<0.05),对HBeAg的抑制率分别为36%(t=34.2,P<0.05)、55%(t=61.6,P<0.05)、83%(t=98.8,P<0.05),对HBV DNA的抑制率分别为70.7%(t=6.6,P<0.05)、86.9%(t=5.9,P<0.05)、95.2%(t=36.6,P<0.05)、95.3%(t=19.7,P<0.05).结论 0.25 mg/kg剂量的PAP在急性HBV感染小鼠模型体内对血清和肝组织的HBsAg、HBeAg的表达及HBV DNA的复制均有明显的抑制效果.