Morphologic, immunohistochemical, and ultrastructural studies of the production of hepatitis B virus in vitro

Well differentiated human hepatoblastoma Hep G2 cells after transfection with cloned hepatitis B virus (HBV) genomes produce replicative HBV DNA intermediates, high levels of HBsAg, HBeAg and HBcAg as well as mature Dane particles. To analyze the replication cycle of HBV, we studied the expression of HBV antigens with monoclonal antibodies by immunomorphologic methods in the transfected cells at various time intervals after plating. HBcAg and HBeAg were detected in the cytoplasm and less frequently in the nuclei of transfected cells. The percentage of positive cells increased with time after plating and reached a plateau of about 50% positive cells at 10 days. HBsAg and the large and middle HBsAg polypeptides were observed in the cytoplasm of transfected cells and a maximum of 20 to 30% positive cells was reached during the 3rd week after plating. Examination of viable cells in suspension revealed HBcAg/HBeAg and HBsAg expression on the cell surface. Electron microscopy demonstrated characteristic core particles in the nuclei and cytoplasm and Dane particles in cytoplasmic vesicles and culture media of transfected cells. The HBV producing cells did not show any evidence of a cytopathic effect. These observations demonstrate significant similarities between the HBV DNA transfected cells and infected human hepatocytes which support active HBV replication in vivo. Taken together, the results suggest that the cultured cells may serve as a model to elucidate a number of unsolved problems of the molecular and cellular pathobiology of hepatitis B.     

In situ hybridization study of HBV DNA in chronic active hepatitis

Liver biopsies from 50 patients with chronic active hepatitis B were analysed immunohistochemically for HBcAg and HBsAg, and with in situ hybridization for HBV DNA. Double labelling technique for detecting HBV DNA and viral antigens simultaneously was also performed in some of these cases. The results showed that localization of HBV DNA in hepatocytes could be classified into whole cytoplasmic, submembranous, nucleic and intermembranous types. The last type suggests that HBV DNA might be transmitted directly to the adjacent hepatocytes through the cell membrane. By double labelling technique, it was disclosed that most hepatocytes with high level of HBV replication did not contain HBcAg or HBsAg. Conversely and most liver cells strongly positive for HBAg have low or negligible level of viral replication. Additionally, in a few cases, HBV DNA was found in the cytoplasm of bile ductule epithelia and sinusoidal endothelia.     

原位杂交法检测肝组织中丁型和乙型肝炎病毒核酸

利用国外引进的重组质粒获得纯化基因片段，分别以随机引物法和ＰＣＲ法制备地高辛素标记的ＨＢＶＤＮＡ探针和ＨＤＶｃＤＮＡ探针。用原位杂交法检测了石蜡包埋的肝组织切片ＢＶＤＮＡ和ＨＤＶＲＮＡ。４９例感染肝组织分为两组：丁肝组２３例；单纯乙肝组２６例，ＨＢＶＤＮＡ的检出率丁肝组（７８．２６％）与乙肝组（７６．９２％）无统计学差异；而ＨＤＶＮＡ的检出率丁肝组（６０．８７％）明显高于乙肝组（１５．３８％）。ＨＢＶＤＮＡ可见于受染肝细胞的胞核或胞浆内，而ＨＤＶＲＮＡ绝大部分见于肝细胞胞核。两种病毒核酸阳性细胞在肝组织中的分布特点大致相同：弥漫或散在地分布于肝小叶或假小叶内，或局灶性分布于小叶周边。ＨＤＶＲＮＡ阳性的肝组织都或多或少地同时存在ＨＢＶＤＮＡ。同一例肝组织中，ＨＢＶＤＮＡ阳性细胞从数量和颗粒密度上似略高于ＨＤＶＲＮＡ。将乙肝组和丁肝组两组病人肝内ＨＢ－ｓＡｇ、ＨＢｃＡｇ和ＨＢＶＤＮＡ及血清ＨＢｅＡｇ作了比较，各指标阳性率虽有差异，但均无统计学意义。因此，未发现ＨＤＶ感染对ＨＢＶ的复制有明显抑制作用。此结果对以往用血清学或免疫组化方法对ＨＤＶ的研究有所补充和深入，亦可为研究其它类型病毒性肝炎之间的重叠感染所借鉴。     

HBV体外感染卵巢颗粒细胞

目的建立HBV体外感染颗粒细胞模型,研究HBV在颗粒细胞中的复制情况,为深入研究HBV经卵细胞母婴垂直传播提供研究平台。方法原代颗粒细胞体外培养后用HBV阳性血清感染。收集培养上清,在不同时点检测HBsAg、HBeAg定量,实时定量PCR检测HBVDNA。免疫组化检测培养细胞中的HBsAg和HBcAg。巢式PCR检测细胞中的HBVDNA及HBV-mRNA。原位杂交检测细胞内的HBVDNA。结果成功建立了HBV体外感染颗粒细胞模型,在培养上清中可以持续96h检测到HBsAg和HBV DNA,在细胞内检测到HBsAg和HBcAg的阳性信号,PCR扩增显示细胞内有HBVD-NA及HBV-mRNA的存在,原位杂交证实细胞内HBVDNA阳性。结论 HBV能够在体外感染颗粒细胞,并在其内复制,该结果为深入研究HBV经卵细胞传播机制提供了很好的研究平台。     

Hepatitis B virus infection and replication in primary cultured human granulosa cells

To investigate the infection and replication of hepatitis B virus (HBV) in primary cultured human granulosa cells. Human granulosa cells were cultured with HBV-positive serum. Media were collected and assayed for HBsAg and HBeAg by ELISA, and HBV DNA by quantitative PCR. HBsAg and HBcAg were detected by immunocytochemistry in cultured cells. HBV DNA and RNA were extracted and amplified by nested PCR. Intracellular HBV DNA was localized by in situ hybridization. By co-cultivation of human GCs with HBV-positive serum, a system was established to study HBV infection and replication in GCs. HBsAg in medium could be detected from 4 to 96 h, and HBV DNA could be detected from 12 to 96 h after exposure. HBsAg and HBcAg showed positive signals by immunocytochemistry. A 206-bp fragment was amplified by nested PCR to detect HBV DNA and RNA in granulosa cells. HBV DNA was detected in GC nuclei by in situ hybridization. HBV can infect and replicate in human primary granulosa cells. This culture system could enable us to study infection of ova by HBV.     

Detection of HBV DNA in hepatocellular carcinoma by in situ hybridization using a biotin-labelled probe

Eight cases of hepatocellular carcinoma were hybridized in situ with a biotin-labelled HBV DNA probe on formalin fixed paraffin embedded sections. HBV DNA was detected in 218 cases both in cancer and pericancerous tissue of the liver, and both carcinomas were well differentiated. In three cases, HBV DNA was only present in pericancerous tissue and no HBV DNA could be identified in the remaining three cases. The positive rate of HBV DNA was 25% in tumor and 62.5% in the pericancerous area of liver. HBcAg was negative in all the eight tumors, nevertheless, HBsAg was present in one case. Both HBsAg and HBcAg were positive in the peri-cancerous tissue of liver in 6 out of 8 cases. In the remaining two, one was only HBsAg positive while the other was HBcAg positive. HBV DNA was identified mainly in the cytoplasm of tumor cells. In certain cells it was seen in the perinuclear cytoplasm or beneath the nuclear envelope. Only in a few cells, HBV DNA was distributed in the nuclei diffusely. Since HBV DNA was present both in the cytoplasm and nuclei, suggesting that HBV DNA was present in either integrated or free forms.     

Co-expression of markers for hepatitis delta and hepatitis B viruses in human liver

Delta hepatitis (HDV) infection can only occur in the presence of hepatitis B (HBV) infection, as HDV requires a coat of HBV surface antigen (HBsAg) for assembly of complete virus. A number of studies have examined the variation of HBV markers in serum and liver during establishment of HDV infection, but none has systematically examined the relationship between the two viruses in individual hepatocytes. Liver biopsies from five patients with HDV/HBV infection were stained for HBsAg, HBV core antigen (HBcAg) and hepatitis D (delta) antigen (HDAg). Double immunostaining was performed with a combination of indirect immunoperoxidase and alkaline phosphatase/antialkaline phosphatase techniques. HDV and HBV antigens were expressed in all five liver biopsies. Co-localization of HBsAg was seen in up to 39% of HDAg positive cells, and HBcAg in up to 8% of HDAg positive cells. HBcAg was detectable in approximately 9% of HBsAg positive cells, and HBsAg in approximately 12% of HBcAg positive cells. HDV can replicate without HBV but ultimately requires HBV to produce complete virus and subsequently infect other cells. In this study the majority of HDV positive cells did not appear to contain HBV markers. This might suggest delta virus replication without assembly, or possibly sequential production/assembly of the virus.     

Flowcytometric quantitation of hepatitis B viral antigens in hepatocytes from regular and fine-needle biopsies

The aim of the study was to investigate the use of flow cytometry, as an alternative for immunohistochemistry, for the detection of viral antigens in the liver of patients with chronic hepatitis B virus (HBV) infection. Hepatocytes were obtained from regular- and fine-needle biopsy from HBV positive (n=17) and negative (n=7) patients and quantified by flow cytometry for intracellular hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg). Number of HBsAg positive hepatocytes ranged from 0 to 83%. A significant correlation was found between the percentage of infected hepatocytes and the intracellular expression level of HBsAg (R=0.841, p<0.001). The specificity and sensitivity of flow cytometry was similar to immunohistochemistry. Of the patients on anti-viral treatment with undetectable serum HBV DNA (<400 copies/ml), two had high HBsAg expression in the liver. HBcAg staining was found in 3 out of 15 patients, with 2-3% positive hepatocytes. The results obtained with fine-needle aspiration biopsy (n=12) were comparable to regular biopsy. In conclusion, flowcytometric quantitation of HBV antigens is sensitive and provides relevant information on the course of infection. The minimally invasive fine-needle biopsy provides a useful alternative for regular-needle biopsy for monitoring intrahepatic antiviral responses during therapy.     

Hepatitis B virus (HBV) infections in turtles

Thirty turtles (15 Clemys mutica and 15 Geoclemys reevesii) which were inoculated with human sera those were positive for hepatitis B surface antigen (HBsAg) and hepatitis B "e" antigen (HBeAg) were found to be infected with hepatitis B virus (HBV). The levels of HBV infection markers, such as HBsAg and antibody to HBsAg (anti-HBsAg), were retinely monitored in the turtles' serum for 46 weeks. Within two weeks of the inoculation, 42% of the turtles tested were positive for HBsAg, and their reciprocal titers as measured by reverse passive hemagglutination (RPHA) and enzyme linked immunoabsorbance assay (ELISA) ranged from 16 to 96. Within 20 weeks, the remaining turtles tested HBsAg positive, as confirmed by ELISA. At 20 weeks, all but one of the turtles exhibited changes in HBV blood marker from HBsAg to anti-HBs; the one exception was positive for both HBsAg and anti-HBs. At the 47th week, 7 animals were killed and their organs were examined for HBV infected cells utilizing an immunofluorescent technique. Numerous fluorescent cells which reacted with human anti-HBs nad anti-HBc were observed in the following organs: pancreas, liver, kidney, and brain. Histopathologically, edematous changes in hepatocytes and minor cellular infiltration attributed to an inflammatory response were noted. Liver and kidney cells from the infected animals were cultured, and HBV antigen positive cells for HBsAg and HBcAg were detected in the cultures. Throughout the experiment, HBsAg was detected in the supernatant by ELISA. Virus particles which were indistinguishable from Dane particles were seen in the cytoplasmic vacuoles of the cultured cells by electron microscopy. Finally, the presence of HBV DNA was established by molecular hybridization techniques in the culture supernatants of kidney cells from the infected turtles.     

Replication of hepatitis B virus in culture systems with adult human hepatocytes

We developed a technique for isolation and primary culture of adult human hepatocytes from surgical liver biopsy specimens by in situ perfusion and a shaking method. Cultured hepatocytes were maintained in monolayers for more than three weeks and showed morphological and functional characteristics in vivo. The cultured human hepatocytes were inoculated with hepatitis B virus (HBV). Hepatitis B surface antigen (HBsAg) in the medium was detected for about three weeks after inoculation, which was longer than that reported in previous studies. In one case of high attachment efficiency, hepatitis B e antigen (HBeAg) was detected in the medium five to eight days after inoculation. HBsAg and HBeAg were also detected in the extracts of inoculated human hepatocytes. Immunofluorescence study revealed HBsAg in 20-30% of hepatocytes and hepatitis B core antigen (HBcAg) in 2-3% of the cultured human hepatocytes four days after inoculation. Free HBV DNA was identified in the human hepatocytes for at least two weeks after inoculation, although single-stranded HBV DNA was not detected. These studies suggest that HBsAg was actively produced and that HBV replicated in a small number of inoculated adult human hepatocytes in primary culture. However, further improvement of culture systems is needed for active replication of HBV in vitro.     

HBV感染与IgA肾病肾小管-间质病变的关系

目的 探讨ＩｇＡ肾病ＨＢＶ感染与肾小管间质病变的关系。方法 利用原位分子杂交（ＧＨＢＶ ＤＮＡ）、免疫组化（ＨＢＡｇ、ＣＤ３、ＣＤ８）以及ＨＢＶ ＤＮＡ－ＨＢＡｇ和ＨＢＡｇ－ＣＤ４３双标记技术,对９１例ＩｇＡ肾病肾穿刺标本进行研究。结果 肾组织内ＨＢＡｇ阳性率为６９．２％。ＨＢＶ ＤＮＡ原位杂交阳性率为４２．９％。ＨＢＶ ＤＮＡ阳性的病例,双重标记染色发现ＨＢＶ ＤＮＡ阳性肾小管上皮细胞可表达ＨＢ     

HBV感染人胎盘滋养层细胞机制的初步探讨

目的 采用透射电镜观察HBV体外感染人胎盘滋养层细胞的超微结构变化.方法 HBV体外感染人胎盘滋养层细胞.ELISA检测培养上清中HBsAg,PCR检测细胞培养上清和滋养层细胞中的HBV DNA.HBV荧光定量PCR检测细胞培养上清中HBV DNA量(拷贝/ml).透射电镜观察滋养层细胞的超微结构.结果 感染组滋养层细胞培养上清中HBsAg在PBS清洗后12 h时A值为0.942,96 h时上升为1.264.PCR检测感染组细胞培养上清和感染组细胞HBV DNA均为阳性.PBS彻底清洗后0、12、36、60、84 h感染组细胞培养上清的HBV DNA分别为:＜103,3×104,6×105,5×105,3×105拷贝/ml.透射电镜观察到感染组滋养层细胞膜附近存在包涵素(Clathrin)形式的内吞小体形成,并且发现内吞小体内存在病毒颗粒样结构.在感染组细胞粗面内质网内发现HBsAg特异性的纤维丝状结构.结论 HBV可能经包涵素依赖的细胞内吞形式进入滋养层细胞,进而实现感染细胞或通过胞释作用将病毒排至细胞的对侧而实现穿越滋养层细胞屏障.     

HBV抗原在乙型肝炎肝硬化患者脑组织中的表达及意义

目的 探讨惭型肝炎肝硬化患者脑组织中乙型肝炎病毒（HBV）感染的状况及意义。方法 应用免疫组织化学链霉菌亲生物素蛋白-过氧化物酶连接法（S-P法）对70例乙型肝炎肝硬化死亡患者脑组织进行了HBsAg、HBcAg检测，分析HBV抗原的表达与临床、病理的关系。结果 HBV抗原患者30例（42.89%)，其中HBsAg阳性24例（34.29%)，HBcAg阳性18例（25.71%)。抗原主要定位于细胞质，分布于神经元、神经胶质细胞及血管内皮细胞内，阳性细胞呈单个霰 在或灶性、小片状分布。HBV抗原的表达与血清HBV水平无关，而与肝性脑病（HE）的有无及HE患者脑组织病理损害的程度相关。结论 乙型肝炎肝硬化患者脑组织中存在HBV感染。并可能存在复制，脑组织中HBV感染，可能对乙型肝炎肝硬化患者HE的发生发展具有重要作用。     

HBV感染者血清HBcAg SP RIA测定的临床分析

目的:研究HBcAg在HBV感染者中的阳性检出率、分布规律及与其他乙肝标志物的关系,探讨其在反映HBV复制及传染性和观察疗效方面的临床价值.方法:采用SP RIA对461例HBV感染者进行血清乙肝六项指标测定,按其不同阳性结果分9种模式对比分析.结果:HBV感染者中HBcAg阳性总检出率达50.75%,而未感染者及69例抗-HBs单项阳性者中无阳性检出.结论:SP RIA测定血清HBcAg在HBV感染者中的阳性检出具有特异性,并可作为疑有抗-HBe阳性"逆转"为HBeAg阳性者的筛选检测,对判断HBV复制程度、病程、疗效及预后评估均有临床价值.     

Successful establishment and evaluation of a new animal model for studying the hepatitis B virus YVDD mutant

The treatment of infection with lamivudine-resistant mutants of hepatitis B virus (HBV) with mutations in the YMDD motif has become a crucial issue in the clinic. In this work, the plasmids pcDNA3.1 (+)-HBV/C-YVDD and pcDNA3.1 (+)-HBV/C-YMDD were constructed and injected into BALB/c mice using a hydrodynamics-based procedure to investigate viral replication and expression of HBV lamivudine-resistant YVDD mutants in vivo. Compared with the YMDD group, HBsAg levels were higher in sera of mice in the YVDD group, but HBeAg levels were lower on day 1 after injection. Levels of HBcAg in hepatocytes were higher in the YVDD group on day 1, whereas the HBsAg levels were lower. The levels of HBV mRNA in the liver were higher in mice in the YVDD group on day 1 after injection. The results showed that injection with these plasmids resulted in efficient initiation of replication of HBV in mice and also suggested that the combined mutations in YVDD mutants could affect the replication process.     

The degrees of hepatocyte nuclear but not cytoplasmic expression of hepatitis B core antigen reflect the level of viral replication in chronic hepatitis B virus infection.

Although immunodetection of hepatitis B core antigen (HBcAg) in the liver has long been recognized as a marker of active hepatitis B virus (HBV) replication, the correlation between the level of viral replication and the degrees of expression of HBcAg in hepatocytes remains to be established. In this study, we examined the semiquantitative relationship between the level of HBV DNA in serum and the degree of expression of HBcAg in the hepatocyte nucleus or cytoplasm in 80 adults with chronic hepatitis B. Expression of HBcAg in hepatocytes was studied by the avidin-biotin immunoperoxidase method, and the results were scored on a scale of 0 to 4, values corresponding to the positivity of 0, 1 to 10, 11 to 25, 26 to 50, and > 50%, respectively, of hepatocytes examined. Serum HBV DNA was tested by a liquid hybridization assay, and the results were scored on a scale of 0 to 5, values corresponding to undetectable levels and levels of < or = 50, 51 to 100, 101 to 150, 151 to 200, and > 200 pg/ml, respectively. The results revealed a highly significant, positive correlation between the level of HBV DNA in serum and the degree of expression of HBcAg in nuclei (Spearman rank correlation coefficient [rs] = 0.653, P < 0.001). The mean scores (95% confidence intervals) of levels of HBV DNA in sera of patients whose levels of expression of HBcAg in nuclei received a score of 0 (n = 33), 1 or 2 (n = 35), and 3 or 4 (n = 12) were 1.3 (1.1 to 1.5), 2.5 (2.1 to 2.9), and 3.9 (3.1 to 4.7), respectively. However, no correlation between the level of HBV DNA in serum and the degree of expression of HBcAg in the cytoplasm was noted (rs = 0.026, P > 0.8). In conclusion, the degree of expression of HBcAg in the hepatocyte nucleus but not the cytoplasm can accurately reflect the level of viral replication in patients with chronic hepatitis B.     

IgA肾病肾组织内乙型肝炎病毒感染的发病机制研究

目的：探讨乙型肝炎病毒感染致IgA肾病肾损伤的发病机制。方法： 随机选取48例IgA肾病肾穿刺组织，参照Meadow病变分级标准分为Ⅰ-Ⅴ级5个实验组，应用Envision免疫组织化学方法检测各级肾组织内HBsAg和HBcAg；同时用直接IS-PCR技术检测其中18例IgA肾病肾组织内HBV DNA。结果： 48例IgA肾病肾组织内HBcAg和HBsAg总的阳性检出率分别为75.00%(36/48)和43.75%(21/48)；18例IgA肾病肾组织内HBV DNA阳性检出率为61.11%(11/18)；3者均表现为肾小管阳性检出率高于肾小球(P＜0.05)，但各级之间，HBcAg、HBsAg和HBV DNA检出率均无显著差异(P＞0.05)。结论： HBV参与了IgA肾病的发生，其导致肾组织损伤的机制可能主要是由细胞免疫或一系列细胞因子介导，并非病毒直接所致；肾小管上皮细胞可能是HBV感染的靶对象。     

Intrahepatic expression of hepatitis B virus antigens: effect of hepatitis C virus infection.

Coinfection with hepatitis B and C viruses (HBV, HCV) is not uncommon, but the expression of HBV antigens in the liver of patients with concomitant HCV infection has not been investigated. This study aimed to evaluate the effects of concomitant HCV infection on the intrahepatic expression of HBV antigens in chronic hepatitis. HBV surface and core antigens (HBsAg, HBcAg) were immunohistochemically evaluated and semiquantitatively scored in liver biopsy specimens from patients with chronic hepatitis, comprising 17 cases with dual HBV/HCV infection and 25 with HBV infection alone. The prevalence of HBV Ag expression proved significantly lower in the group with dual infection. In the presence of active HBV replication (HBV DNA-positive serum) the prevalence of HBsAg and HBcAg immunoreaction was similar in the two groups, though a significantly lower percentage of cells expressed HBcAg in the group of coinfected patients. HBV Ag was not detected at all among HBV DNA-negative/HCV RNA-positive cases. In conclusion, these observations suggest that HCV might influence HBV antigen expression in the liver and that either partial or complete suppression might occur.     

EsiRNAs inhibit Hepatitis B virus replication in mice model more efficiently than synthesized siRNAs

RNA interference (RNAi) has been proved to be a promising strategy to combat Hepatitis B virus (HBV) infection by way of cell culture and animal model studies. In this work, esiRNAs (endoribonuclease-prepared siRNAs) targeting all of the four open reading frames (ORFs) of HBV genome were prepared. In vitro experiment showed that esiHBVP suppressed HBsAg expression most effectively. Its capacity to suppress HBV replication in vivo was then tested. A single dose of 1 microg esiHBVP was able to reduce HBsAg and HBeAg level in the mouse serum by 90 and 89% one day after injection, while the same amount of chemically synthesized siRNA only reduced that by 33 and 45%. Immunostaining of HBcAg showed that esiHBVP inhibited HBcAg expression more potently than chemically synthesized siRNA. Quantification of HBV DNA in the mouse serum showed 1 microg eiHBVP treatment reduced serum HBV DNA copy number to 18% that of the untreated control, while 1 microg siRNA treatment only reduced that to 63%. In conclusion, the data presented here proved that esiRNA is much more efficient in suppressing HBV replication than chemically synthesized siRNA, and it might be a better therapeutic agent to fight against HBV infection.