Detection of Epstein-Barr virus in lymphoid tissues of patients with infectious mononucleosis by in situ hybridization.

Although the immunological response during infectious mononucleosis (IMN) has been studied in detail, little is known about the spread of Epstein-Barr virus (EBV) in lymphoid organs or the topographical distribution of the infected cells. In this study, EBV was detected in 11 lymph nodes, 4 tonsils, and 1 spleen of 16 patients with IMN. The predominant cell type positive for the EBV genome was identified as small lymphocytes localized chiefly within typical T areas, preferentially in perifollicular and interfollicular regions of the lymph node. A few endothelia of epithelioid venules were also found to be positive. Furthermore, a small number of sinus lining cells of lymph nodes exhibited labelling. Altogether, only a small number of cells, not exceeding 1 per cent of all cells, were infected with EBV. Our results show that only a small number of lymphocytes carry the EBV and that besides B lymphocytes, other cell constituents of lymphatic tissues are infected by EBV during IMN.     

Identification of Epstein-Barr virus-infected cells in tonsils of acute infectious mononucleosis by in situ hybridization

In situ hybridization with 35S-labeled Epstein-Barr virus (EBV) probes was applied to paraffin sections of tonsils from seven patients with clinical, serologic, and morphologic evidence of acute infectious mononucleosis. EBV genomes were demonstrated in activated lymphoid B blasts in the interfollicular and perifollicular zones in all these cases. However, in no case could EBV be identified in epithelial cells. These results are at variance with the current concept which attributes a central role to the tonsillar epithelium in primary EBV infection.     

Epidemiological analysis of infectious mononucleosis as primary Epstein-Barr virus infection]

We report the characteristics of 45 Epstein-Barr virus (EBV)-infected patients visiting our hospital from 1989 to 1991 in comparison with 102 patients seen from 1981 to 1988. Cases of infectious mononucleosis (IM) increased especially in patients over 30 years old. They tended to have nonspecific symptoms and signs for IM such as general fatigue. Immunologically showed twice higher positivity for anti-EBV VCA-IgM antibody in the recent 3 years when compared to the previous 8 years. The patients with anti-EBNA antibody decreased in the recent 3 years. This may be due to early diagnosis as IM. EBV also causes atypical diseases. non-IM in this report, including hematological or neurological disorders. We indicated here the increased number and advanced age of patients with IM and early diagnosis as IM in the recent 3 years compared to the previous 8 years.     

EB病毒和巨细胞病毒双重感染的传染性单核细胞增多症的临床特征

目的 探讨EB病毒（EBV）和巨细胞病毒（CMV）双重感染的传染性单核细胞增多症（IM）的临床特征.方法 回顾性总结21例由EBV和CMV双重感染导致IM的临床特点,并与单独EBV感染导致的IM病例进行比较.结果 ①两组间临床特点比较：双重感染组的发热持续时间显著长于EBV感染组（P＜0.01）,双重感染组的肝肿大发生率和脾肿大发生率均显著高于EBV感染组（分别P＜0.05,P＜0.01）.IM并发症,如血小板减少症、肺炎和贫血的发生率在双重感染组均显著高于EBV感染组（P＜0.01）.②各组间实验室指标比较：异型淋巴细胞百分比和肝功能异常的发生率在双重感染组均显著高于EBV感染组（P＜0.01）.结论 EBV和CMV双重感染IM常临床症状较重,发热持续时间较长,并发症如血小板减少症、肺炎和贫血的发生率较高.     

Clonal Epstein-Barr virus in lymphoepithelioma-like carcinoma of the stomach: demonstration of viral genome by in situ hybridization and Southern blot analysis.

Lymphoepithelioma (LE), originally described in the nasopharynx, is an undifferentiated carcinoma with heavy lymphocytic infiltrate. The tumor is common in Southeast Asia, particularly in southern China, where the Epstein-Barr virus (EBV) association has been documented more consistently than in Western countries. Tumors histologically similar to LE have been described also in other anatomical sites, mostly of fore-gut origin, such as salivary gland, tonsil, lung, thymus, and more recently stomach. We are reporting a case of poorly differentiated gastric adenocarcinoma with marked lymphocytic infiltrate resembling LE (LE-like carcinoma) in a Chinese without evidence of nasopharyngeal carcinoma. In situ hybridization for EBV revealed that the tumor cells but not the lymphoid cells harbored the virus. Tumor cells both in syncytial and glandular areas were positive for EBV. By Southern blot analysis EBV was demonstrated in the DNA extracted from the tumor, while the adjacent normal gastric tissue was negative. Moreover, analysis of the EBV termini revealed a clonal episomal form of the virus. Our case further supports the hypothesis that EBV is associated with LE-like gastric carcinoma. It also strongly suggests that EBV infection has preceded, and thus most likely contributed to, the clonal expansion in this tumor.     

Duck hepatitis B virus DNA in liver, spleen, and pancreas: analysis by in situ and Southern blot hybridization

Tissues from a 10-week-old Pekin duck, experimentally infected at 1 day of age with duck hepatitis B virus (DHBV), were examined for the presence of replicative levels of DHBV DNA by in situ and Southern blot hybridization. Hepatocytes, pancreatic lymphoid follicle, exocrine, and endocrine cells, and splenic mononuclear cells all contained DHBV DNA localized predominantly to the cytoplasm of infected cells. Duck hepatitis B surface antigen distribution in the same tissues correlated well with the presence of DHBV DNA in many of these cells. In hepatocytes and pancreatic islet cells, 60% of DHBV DNA was present as single-stranded DNA, indicating the likelihood of ongoing virus replication in these cell types and providing further evidence that hepadnavirus DNA replication occurs largely within the cell cytoplasm. In contrast, DHBV in mononuclear cells within splenic germinal centers was wholly double-stranded, suggesting that limited, if any, DHBV DNA replication was occurring in this cell type. These data provide further information about the pathogenesis and cell-specific sites of DHBV infection.     

传染性单核细胞增多症的临床病理及EB病毒定位性研究

目的 研究传染性单核细胞增多症(IM)的临床特征、病理特点、免疫表型和EB病毒原位感染特征,以提高对IM的认识和诊断水平.方法 采用HE染色以及免疫组织化学、原位杂交技术,结合临床资料分析,对15例IM进行了临床病理、免疫表型和EB病毒感染的研究.结果 (1)IM多见于儿童和青年人(中位年龄18岁),起病急,常有发热(12例),伴浅表淋巴结肿大,多数在短期内痊愈.(2)病变以T区增生为主,斑驳状改变常见,细胞混杂,种类多样,可见B细胞分化谱(活化淋巴样母细胞、免疫母细胞、浆样细胞、浆细胞),包膜不厚,间质不多.(3)病变中以CD3阳性的小T淋巴细胞为主,部分活化的淋巴样母细胞和免疫母细胞表达CD20和CD30,信号强弱不等,散在分布.(4)所有病例都有EB病毒编码的小RNA(EBER)阳性细胞,数量多少不一(10～100个/HPF),大中小淋巴细胞均可阳性,主要分布在T区,也见于套区、初级滤泡和生发中心内.结论 进一步确认了IM是EB病毒引起的一种急性自限性淋巴组织增生性疾病.IM在临床、病理、免疫表型和EB病毒感染方面都具有特点,只有综合考虑这4方面的信息才能减少错误,做出更准确的诊断.     

Acute cerebellar syndrome in infectious mononucleosis: documentation of two cases with Epstein-Barr virus infection

Acute cerebellar ataxia has been described occasionally with infectious mononucleosis. Two additional cases are reported with serologic identification of Epstein-Barr virus (EBV) infection in blood and cerebrospinal fluid. As with previously described cases, the outcome was benign, and examination and laboratory studies did not indicate diffuse neurologic involvement. Visual and brainstem auditory-evoked responses were normal. Electroencephalograms (EEG) demonstrated 14 and 6 per second positive spikes in both patients. This pattern is considered a normal variant and has been recorded from depth electrodes and reported with deep midline lesions. These cases support the prognosis of benign cerebellar involvement in infectious mononucleosis and suggest that evidence of EBV infection be sought in patients with acute ataxia. The significance of 14/sec and 6/sec positive EEG spikes is uncertain.     

Absence of latent Epstein-Barr virus in thymic epithelial tumors as demonstrated by Epstein-Barr-encoded RNA(EBER) in situ hybridization

BACKGROUND: Several studies have established that Epstein-Barr virus (EBV) is associated with lympho-proliferative disorders such as Burkitt's lymphoma and Hodgkin's disease. EBV is also present in undifferentiated nasopharyngeal carcinomas and in tumors of similar morphology (lymphoepithelioma-like carcinomas) arising in a variety of organs, predominantly in stomach, salivary gland and thymus. As reports of EBV-positive thymic epithelial tumors (TET) have been divergent and as different methods have been used to detect EBV, the aim of this study was to investigate the possible role of EBV in TET of Danish patients. MATERIAL AND METHODS: Archival material of 157 cases of TET (105 thymomas and 52 thymic carcinomas, including 4 lymphoepithelioma-like thymic carcinomas (LELTC)) was analyzed for EBV by applying a sensitive and specific method for detecting latently EBV-infected cells (in situ hybridization for EBV-encoded RNA (EBER)). RESULTS: All investigated cases were EBER negative. CONCLUSIONS: EBV does not seem to be implicated in the pathogenesis of TET. However, a review of the literature showed that 28% of LELTC were EBER ISH positive. As they occurred in young people (mean 18 years), at an age when the patients were susceptible to infection by EBV, it is suggested that EBV merely acts as an innocent bystander.     

Variable expression of Epstein-Barr virus genome as demonstrated by in situ hybridization in central nervous system lymphomas in immunocompromised patients

In situ hybridization, using a biotinylated sequence from the internal repeat (IR1) region of Epstein-Barr virus (EBV), was performed on two well characterized EBV-infected cell lines, B95-8 (productively infected) and Namalwa (latently infected), and on eighteen formalin-fixed paraffin-embedded primary central nervous system (CNS) lymphomas. Ten of the lymphomas were from immunocompetent patients, and eight were from immunocompromised patients (five had acquired immunodeficiency syndrome (AIDS), two had renal allografts, and one had X-linked immunoproliferative (XLP) disease). Both fresh and paraffin-embedded B95-8 cells showed detectable hybridization signal in 5 to 10% of cells, with other cells showing lower signal. Fresh Namalwa cells showed signal in every cell and in 40% of paraffin-embedded cells. Evidence of EBV genome was seen in seven of eight lymphomas from immunocompromised patients and in none of the lymphomas from immunocompetent ones. In the EBV-positive lymphomas, three patterns of hybridization were recognized: +3, more than 60% of tumor cells positive, +2, 20 to 60% of tumor cells positive, and +1, less than 20% of tumor cells positive. There was no definite relationship between survival after diagnosis and hybridization pattern type. While the signal in Namalwa cells was uniform, a wide variation in the degree and intensity of signal was noted among the seven positive tumors and even in different areas of the same tumor. This heterogeneity raises the possibility of lytic or secondary infection in a small number of the latently infected tumor cells.     

肺癌组织中EB病毒感染的检测

目的探讨原发性肺癌中EB病毒（Epstein—Barr virus，EBV）的存在情况及EBV与原发性肺癌的关系。方法唐山市人民医院和开滦医院病理科储存的2001--2006年肺癌手术切除石蜡包埋肺癌组织108份，癌旁组织22份，以EBV阳性鼻咽癌组织为阳性对照，用原位杂交法（ISH）检测肺癌患者石蜡包埋组织标本中EBV编码的小RNA（EBERl），并采用图像分析法进行形态学定量。结果癌组织及癌旁组织EBERl的阳性率分别为33．3％（36／108）和4．5％（1／22），二者间差异有统计学意义（P〈0．01）。鳞癌、腺癌、小细胞癌及大细胞癌中EBV感染率分别是35．9％（14／39）、31．6％（12／38）、31．0％（9／29）和1／2。EBV感染与患者年龄、性别和组织学类型无关，但与肺癌的部位、癌组织分化程度有关，右肺明显高于左肺，中低分化癌明显高于高中分化癌。结论唐山地区原发性肺癌组织中EBV感染率为33．3％，EBV感染可能是肺癌的潜在病因之一，在癌组织分化的不同阶段有不同的作用。     

Detection of Epstein-Barr virus infection in the epithelial cells and lymphocytes of non-neoplastic tonsils by in situ hybridization and in situ PCR

Summary.  Non-neoplastic tonsils were analyzed for detection of Epstein-Barr virus (EBV)-positive cells by in situ hybridization and in situ PCR. EBV-encoded small nuclear RNA 1(EBER1)-positive cells were found in 28.2% of the tonsils and were evenly localized in the extrafollicular area and within germinal centers. Latent membrane protein 1 (LMP1)-positive cells were also dispersed in the extrafollicular and germinal center. Using in situ DNA-DNA hybridization, the EBV-positive signals were observed in the upper epithelial cell layers of the tonsils. In addition, in situ PCR detected EBV DNA-positive cells in the lower epithelial cell layers and lymphoid cells. Accepted December 1, 1997 Received September 16, 1997     

Lymphoepithelioma-like carcinoma of the uterine cervix: a case report studied by in situ hybridization and polymerase chain reaction for Epstein-Barr virus

Lymphoepithelioma-like carcinomas have been reported outside the nasopharynx in many sites, including the uterine cervix. The association with the Epstein-Barr virus in the latter site is still controversial. To date, Epstein-Barr virus genome has only been demonstrated in Asian patients. We report a case of lymphoepithelioma-like carcinoma of the uterine cervix in a white woman in whom the Epstein-Barr virus infection was tested for by in situ hybridization and polymerase chain reaction. The results of both techniques were negative. Our case and a review of the literature support the contention that cervical lymphoepithelioma-like carcinoma is not associated with Epstein-Barr virus infection in non-Asian patients.     

Neonatal adenovirus infection: A case report with in situ hybridization confirmation of ascending intrauterine infection

Adenovirus infection is a rare, but serious infection, during the neonatal period. the actual mode of infection at birth is currently unknown, however several mechanisms have been proposed. We describe a case of fatal neonatal adenovirus pneumonia in a 25-wk gestational age infant. Adenovirus was confirmed by electron microscopy and by in situ hybridization. the maternal cervical/endocervical smear taken prior to the delivery contained epithelial cells with changes suggestive of adenovirus, which was confirmed by in situ hybridization on the smear. These findings suggest that ascending viral infection is a factor in the pathogenesis of neonatal adenovirus infection. the identification and reporting of adenovirus may be important during pregnancy in order to avoid delay in delivery of the fetus once membranes have ruptured. © 1995 Willey- Liss, Inc.     

Immunohistological studies of lymphoproliferative lesions in a fatal case of Epstein-Barr virus infection.

A fatal case of infectious mononucleosis occurred in a young adult. Abnormal serological features were noted in his mother, although there was no other family history suggesting an inherited defect of immune response to Epstein-Barr virus (EBV). The cellular infiltrate observed in tissues obtained at necropsy was analysed with a range of specific monoclonal and polyclonal antibodies. Polyclonal plasmacytoid B cell proliferation had occurred in many tissues. These cells were positive for EBV nuclear antigen, but viral particles were not seen on ultrastructural examination, and the virus was not isolated, suggesting a non-permissive infection.     

Detection of the Epstein-Barr virus in primary gastric lymphoma by in situ hybridization

The Epstein-Barr virus (EBV) has been shown to be associated with numerous human malignancies including Burktt's lymphoma and nasopharyngeal lymphoepithelioma. In addition, some typical gastric adenocarcinomas were also recently reported to demonstrate EBV relevance. The present study was designed to detect EBV in primary gastric lymphoma, using the in situ hybridization (ISH) method, in which oligo-nucleotide probes for the EBERl RNA and the EBV DNA W region have been used. Of the 49 cases of primary gastric lymphoma studied, which all showed B cell immunopheno-type, EBER1 sequences could only be found in four cases, including two low-grade cases and two high-grade cases of histological subtypes while the number of positive cells was less than 50% of the tumor cells. In one case of low-grade mucosa associated lymphoid tissue (MALT) lymphoma, the EBER1 -positive neoplastic cells were found in the regional lymph node, but the primary site of the stomach showed no positive signals. The EBV presence was further confirmed by the EBV DNA ISH. Using the ISH method, rare or occasional positive lymphoid cells (probably non-tumorous bystander cells) could be detected in 10 other cases including all histological subtypes. The present study shows that only a small proportion of primary gastric lymphoma is associated with EBV, and such positive cases could be found in both high- and low-grade histological subtypes. It is also suggested that the EBV presence in the neoplastic cells of some cases of primary gastric lymphoma is most likely a secondary phenomenon.     

The systemic distribution of Epstein-Barr virus genomes in fatal post-transplantation lymphoproliferative disorders. An in situ hybridization study.

The systemic distribution of Epstein-Barr virus (EBV) genomes was studied in paraffin-embedded tissues from 12 fatal cases of Post-transplantation lymphoproliferative disease (PTLD), using an in situ hybridization technique employing an alpha-35S-dCTP-radiolabeled BamHI-W fragment of EBV DNA. The presence of EBV was documented in various PTLD-involved organs. The hybridization signal for the virus localized predominantly in the abnormal lymphoid cells, but signals also were detected in hepatocytes and/or adrenal cortical cells in five cases. The distribution of autoradiographic label within the lymphoid cells was focal and its intensity varied from field to field suggesting a nonuniformity of the viral genomic load in the infected tissues. Recruitment of EBV genome-bearing cells was not observed into inflammatory mononuclear infiltrates found in organs without histopathologic evidence of PTLD.     

Presence of infective Epstein-Barr virus in the urine of patients with infectious mononucleosis

The presence of Epstein-Barr virus (EBV) in the blood and urine of 20 patients with infectious mononucleosis (IM) was investigated together with the clinical course of the disease, and in 9 patients up to 2–7 months after recovery. EBV DNA, analyzed by the polymerase chain reaction (PCR), was detected in the blood of all 20 patients from the first sample obtained and detected between 3 to 42 days from the beginning of symptoms and up to 2–3 months after recovery. In the urine, EBV DNA was detected in 15 out of 16 (93%) patients in the first sample obtained and detected between 3 to 50 days during the clinical course of the disease. In four patients EBV DNA was detected in the urine up to 3 months after full recovery. Seventeen out of 26 (65%) urine samples including 3 which were obtained 2–7 months after recovery infected B cells as assessed by PCR. Nine out of 12 (75%) urine samples tested induced Epstein-Barr nuclear antigen (EBNA) in the infected B-cell line. In addition to the persistence of EBV in the blood of IM patients, these studies show for the first time the presence of infective EBV in the urine during the clinical course of the disease and up to 7 months after full clinical recovery. © 1994 Wiley-Liss, Inc.