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。     

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     

In vitro infection of primary cultures of cryopreserved adult human hepatocytes with hepatitis B virus

Infection with HBeAg and HBV DNA positive serum in primary cultures of cryopreserved human hepatocytes in the presence of human whole blood in the medium was performed in 8 consecutive experiments. HBsAg and HBV DNA release into the medium was increased in the second week after infection. Via immunostaining, HBcAg was first observed in the nucleus of hepatocytes approximately 3 days after infection. A maximal percentage of HBcAg positive cells in 0.1% of cultured hepatocytes was detected on the 7th day. HBsAg was also first demonstrated on the 3rd day, and predominantly localized in the cytoplasm. About 5% of hepatocytes were HBsAg positive on the 12th day after infection. The percentage of positive cells did not appear to increase after this time. Using in situ cytohybridization and agarose gel electrophoresis and Southern blot analysis, HBV DNA was first detected on the 4th day. In addition, electron microscopic studies revealed the presence of 42 nm virus-like particles in the cytoplasm of infected cells in the second week after infection. This in vitro system provides a model for studying the mechanism of HBV infection, viral replication and maturation. However, further improvement of culture systems is needed, to increase the number of infected cells and for active HBV replication.     

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.     

肝硬变内HBV DNA及其五种抗原的表达及意义

取２２５例人肝硬变活检组织石蜡切片，检测了ＨＢＶＤＮＡ及其５种抗原。分别用免疫组化ＡＢＣ法检测ＨＢｘＡｇ、ｐｒｅ－Ｓ＿１和ｐｒｅ－Ｓ＿２抗原；用ＰＡＰ法检测ＨＢｓＡｇ和ＨＢｃＡｇ；用原位杂交方法检测ＨＢＶＤＮＡ；用免疫组化、原位杂交双标记方法检测ＨＢＶＤＮＡ和ＨＢｓＡｇ、ＨＢｘＡｇ或ＨＢｃＡｇ。结果显示，阳性检出率ＨＢｓＡｇ为７０．０％（１２８／１８３例），ｐｒｅ－Ｓ＿１抗原为６４．４％（８５／１３２例）、ｐｒｅ－Ｓ＿２抗原为６１．４％（８１／１３２例），ＨＢｘＡｇ为７５．３％（１１３／１５０例），ＨＢｃＡｇ为２２．４％（３９／１７４例），ＨＢＶＤＮＡ为６２．４％（５８／９３例）。双标阳性检出率ＨＢＶＤＮＡ和ＨＢｓＡｇ为３７．３％（１９／５１例），ＨＢＶＤＮＡ和ＨＢｘ－Ａｇ为８６．３％（４４／５１例），ＨＢＶＤＮＡ和ＨＢｃＡｇ为３９．２％（２０／５１例）。ＨＢＶＤＮＡ和ＨＢＶ５种抗原阳性病例中８０％以上均伴有肝细胞不典型增生。这一结果表明，在我国肝硬变的发生发展与ＨＢＶ慢性感染有密切的关系。     

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.     

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.     

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 DNA及HBV抗原在血清HBV标志物阴性肝炎肝组织中表达的研究

目的 研究HBV DNA及HBV抗原在血清HBV标志阴性的肝炎肝组织中的表达。方法 对45例HBV血清标志阴性阴性肝炎患者，进行肝组织HBV DNA的原位杂交及免疫组织化学染色检测。结果 原位杂交表明，HBV DNA阳性者7例，（阳性率15．56％），阳性信号主要存在于肝细胞的胞核中，少数位于胞浆内；免疫组化染色表明，HBsAg及HBcAg均呈阴性。结论 血清HBV标志阴性的肝炎肝组织中可检出HBV DNA，有利于提高对HBV感染的诊断。     

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.     

HBV viral load within subpopulations of peripheral blood mononuclear cells in HBV infection using limiting dilution PCR

Extrahepatic viral load in peripheral blood mononuclear cells (PBMCs) of patients with hepatitis B virus (HBV) is still under debate. In this study, HBV infection rates and viral titers were examined within all PBMC subpopulations using limiting dilution-PCR (LD-PCR). PBMCs of patients with acute or chronic hepatitis B were separated by magnetic beads in monocytes, B-cells, CD4+ T-cells, CD8+ T-cells, and NK cells. Using two-round nested PCR, HBV-DNA sequences were detected in all patients examined within each PBMC subpopulation. The frequencies of HBV-positive cells and viral loads were calculated by Poisson analysis of HBV PCR results from serial dilutions of cells and cell lysates. Highest infection rates were found in monocytes and B-cells followed by CD8 + T-cells, NK cells, and CD4+ T-cells. Concerning all subsets, frequencies of HBV-positive cells were 50- to 500-fold higher in chronic than in acute hepatitis B. Viral loads were mostly estimated at about one HBV genome per HBV-positive cell. Moreover, slightly elevated HBV titers were seen in B-cells, CD4+ T-cells, and NK cells in both acute and chronic hepatitis B. It was demonstrated that beside a generally more latent HBV infection in PBMCs, elevated HBV titers point to replication or selective viral uptake within particular PBMC subsets. Therefore, the data suggest that HBV-infected PBMCs may participate in persistence of HBV.     

肝脏内源性microRNA调控乙型肝炎病毒基因的表达与复制

目的探讨肝脏内源性microRNA对乙型肝炎病毒(HBV)复制与基因表达的影响。方法通过miRNA靶点分析软件寻找与HBV序列之间相关联的肝脏内源性microRNA,体外化学合成相应的microRNA分子,将合成寡核苷酸及对照与1.3倍HBV全基因组真核表达质粒pUC18-HBV1.3采用Lipofectamine2000共转染HepG2细胞,转染48h后收集细胞培养上清;通过ELISA检测HBsAg、HBeAg的表达水平;Western印迹检测HBcAg的表达水平;Trizol抽提转染细胞RNA,逆转录后用荧光定量PCR检测HBVmRNA的水平;提取细胞基因组DNA,Southern印迹检测HBV的复制中间体。经以上检测从HBV蛋白表达、转录和复制水平评价相应的microRNA作用效应。结果生物信息学方法提示miR-16和miR-122存在与HBV基因组作用的可能结合位点。经试验初步证实miR-16可下调HBV蛋白的表达及HBVDNA水平;miR-122可下调HBsAg、HBeAg的表达,上调HBVmRNA的水平。结论肝脏内源性microRNA可以调节HBV的复制与基因表达。     

Hepatitis B virus DNA in leukocytes of patients with hepatitis B virus-associated liver diseases

In order to determine the relationship between hepatitis B virus (HBV) infection of human white blood cells and different forms of HBV-associated liver diseases, we tested for HBV DNA in the sera and leukocytes of 11 healthy individuals without any serological markers of HBV infection and 91 patients with HBV infection and other gastrointestinal and urinary diseases by dot and Southern blot hybridization. HBV DNA was found in leukocytes of chronic HBV carriers, in acute and chronic hepatitis, and in patients with liver cirrhosis and hepatocellular carcinoma. Between 27 and 50% of individuals in different categories of patients examined were positive for leukocyte HBV DNA. HBV DNA was also detected in the sera of some of these patients but was absent in others. Serum HBV DNA-positive rates seemed to be highest in hepatitis B e antigen-positive asymptomatic carriers (8/10, 80%), and tended to drop to lower levels as the disease progressed to liver cirrhosis (0/8) while leukocyte HBV DNA-positive rates were highest in patients with cirrhosis (4/8, 50%). The results also show that in individuals who were serologically negative for hepatitis B surface antigen (HBsAg) and positive for antibodies to HBsAg and/or HBcAg, HBV DNA was absent in most of the sera (27/28, 96%) but it was present in leukocytes of some of these patients (7/28, 25%). In control experiments with 11 healthy individual, HBV DNA was not detected in either sera or leukocytes. In all the cases with leukocyte HBV DNA, the HBV DNA molecules were present in free forms with discrete sizes. The exceptions were a case of liver cirrhosis and a case of chronic hepatitis with possible HBV sequence integration into high molecular weight cellular DNA. Since HBV does infect human leukocytes, it may perhaps interfere with the immunological functions of the white blood cells, and thus play an important role in the pathogenesis of HBV-induced liver disease.     

Detection of hepatitis B virus DNA in hepatocellular carcinoma: methylation of integrated viral DNA

In order to determine whether integrated hepatitis B virus DNA sequences in primary liver tumours are methylated we have analysed tumour DNA digested with either MspI or HpaII restriction endonuclease by Southern hybridization. Our results demonstrate methylation in 11 of 17 tumour DNA samples. Where possible, we have examined the tumour tissues for expression of HBsAg and HBcAg using the indirect immunoperoxidase technique. One tumour was positive for both HBsAg and HBcAg and a second was positive for HBsAg alone. Both of these tumours were in the group in which methylation of integrated HBV DNA sequences could not be detected. We postulate that methylation of integrated HBV DNA sequences may influence HBV gene expression in hepatocellular carcinoma.     

Relationship between HBcAg in serum and liver and HBV replication in patients with HBsAg-positive chronic liver disease

The expression of hepatitis B core antigen (HBcAg) in serum and in hepatocytes was evaluated in relation to HBV replication. Fifty chronic HBsAg carriers with histological evidence of liver disease were studied, including 24 HBeAg-positive patients, 2 HBeAg/anti-HBe-negative patients, and 24 anti-HBe-positive cases, two of them with evidence of delta agent infection. Serum HBV-DNA was evaluated in all patients and related to HBcAg examined at the same time in frozen liver biopsies by immunofluorescence and to HBcAg detected in the corresponding serum by a recently developed radioimmunoassay. HBV-DNA was present in serum in 20 (83%) HBeAg-positive patients, all positive for serum HBeAg, whereas liver core antigen was detected in 14 (73%) of 19 cases. Among HBeAg-negative patients, 50% showed the presence of circulating DNA viral sequences, and HBcAg was identified in five of 26 (19%) cases in serum and in six of 24 (25%) in the liver respectively. In 15 patients, liver fragments permitted examination in parallel by immunofluorescence for HBcAg and molecular hybridization for viral DNA in liver cells. A DNA pattern characteristic of viral replication was found in cases with evidence of active virion production, independently from HBeAg and anti-HBe, and in these patients HBcAg was present both in serum and in hepatocytes. In two cases with free HBV-DNA, without evidence of replicative activity, core antigen was not detected in the liver, but in one patient HBcAg was found in the serum. A similar finding was also noted in another patient, in whom the hybridization pattern was consistent with integration of viral genome into high-molecular-weight cellular DNA. Whether serum HBcAg detected in these patients without HBV-DNA in serum reflects the presence of defective viral particles or of core antigen released as a viral protein remains to be determined.     

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。     

Frequent detection of hepatitis B virus DNA in hepatocellular carcinoma of patients with sustained virologic response for hepatitis C virus

Hepatocellular carcinoma (HCC) develops several years after the eradication of hepatitis C virus (HCV) by interferon therapy. Risk factors for the development of HCC are only partly understood. To elucidate the role of occult hepatitis B virus (HBV) infection in hepatocarcinogenesis in patients with sustained virologic response, the prevalences of HBV‐related makers were examined. Study group comprised 16 patients with sustained virologic response (group A) and 50 with HCV (group B). Anti‐HBc and anti‐HBs in serum were examined by enzyme‐linked immunoassay. HBV DNA in liver was examined by nested polymerase chain reaction, using primers specific for genes encoding for HBx, HBsAg, HBcAg, and HBV cccDNA. Sequence of the amplified HBV DNA for ‘a’ determinant of HBsAg was determined in HCC. Anti‐HBc was positive in 10 of 16 in group A and 25 of 50 in group B. HBV DNA in liver was detected in 12 of 16 in group A and 21 of 50 in group B (P = 0.044). In group A, HBV DNA in liver was detected frequently in patients without cirrhosis and in those with a longer period from the time of HCV eradication to the development of HCC. Mutation in ‘a’ determinant of HBsAg was found in three HCC of group A. Occult HBV infection may be one of the most important risk factors in hepatocarcinogenesis of Japanese patients with sustained virologic response. J. Med. Virol. 81:1009–1014, 2009. © 2009 Wiley‐Liss, Inc.     

Quantitative DNA analysis of low-level hepatitis B viremia in two patients with serologically negative chronic hepatitis B.

Low-level viremia due to hepatitis B virus (HBV) was demonstrated in the sera of two patients diagnosed previously as having non-B, non-C chronic hepatitis. Both patients had a "silent" HBV infection, because they were negative for both hepatitis B surface antigen (HBsAg) and anti-hepatitis B core antibody. The TaqMan chemistry polymerase chain reaction (PCR) amplified the HBV DNA, enabling quantitation of the virus in their sera. Their serum HBV DNA concentrations were low: the amount of each HBV S or X gene amplified showed there were approximately 10(3) copies/ml and HBV DNA was detected occasionally during clinical follow-up. Positive HBsAg staining in liver tissues was demonstrated by an immunoperoxidase technique. Vertical transmission of silent HBV from one patient to her daughter was confirmed. Direct nucleotide sequencing of the amplified HBV X region revealed several mutations, suggesting reduced viral replication. One patient had a T-to-C mutation at the extreme 5'-terminus of the direct repeat 2 region and the other exhibited a coexisting X region with a 155-nucleotide deletion. These findings suggest that HBV replication is suppressed considerably in patients with silent hepatitis B.     

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.