Detection of latent Epstein-Barr virus (EBV) DNA in paraffin sections of nasopharyngeal carcinomas expressing no EBV-encoded small RNAs using in situ PCR

Summary.  In situ hybridization (ISH) with EBER 1 (Epstein-Barrvirus (EBV)-encoded small RNA1) probes is widely used for in situ detection of EBV-infected cells. ISH with an EBER1 probe showed that 10 of 40 NPC cases were negative for EBER1 expression. For in situ detection of EBV DNA, we used in situ PCR method which can detect one copy of EBV DNA per cell. Of the 10 EBER1-negative cases, three cases including one each of well- and poorly differentiated carcinomas and undifferentiated carcinoma were EBV DNA-positive by in situ PCR. The remaining seven were truly negative for the presence of EBV DNA. All the EBV genome-negative NPC cases examined here were histologically classified as poorly differentiated or undifferentiated carcinomas which are known to be closely associated with EBV, indicating the existence of EBV DNA-negative NPC cases, regardless of histological type or differentiation. These results indicate that there are EBV genome-positive NPC cases expressing no EBER1 and that in situ PCR can be suitable for in situ detection of EBV-infected cells, especially those expressing no EBER1 in paraffin sections. Received April 14, 1997 Accepted June 5, 1997     

Epstein-Barr virus (EBV) infection and p53 protein expression in gastric carcinoma

In the presented studies p53 protein expression was evaluated in samples of gastric carcinoma originating from 32 selected adult patients (with documented diagnosis of adenocarcinoma of the stomach and without the presence of Helicobacter pylori infection). Among the patients 14 individuals carried EBV-positive gastric carcinoma (group 1) while the 18 remaining patients carried EBV-negative gastric carcinoma (group 2). EBV infection was detected testing the tissue material for the presence of EBER by RNA in situ hybridization (ISH) and testing sera of the patients for EBV-specific antibodies. Expression of p53 protein was analysed using immunohistochemistry. Presence of p53 protein was noted in 9 (64.3%) cases of EBV-positive gastric cancer (group 1) and in 10 (55.5%) cases of EBV-negative gastric cancer (group 2). No significant differences were detected in the frequencies of p53 protein expression in the two studied groups. The results permit to conclude that abnormalities in p53 in gastric cancer are independent of EBV infection, even if EBV may participate in development of the tumour.     

Epstein-Barr virus (EBV) and gastric carcinoma in Malaysian patients

Epstein-Barr virus (EBV) has consistently been detected in the tumour cells of nasopharyngeal carcinoma and lymphoepithelial-like carcinoma of the salivary glands, and have occasionally been found in similar tumours at other sites. Moreover, recent studies from various parts of the world including the Orient have shown about 10% of gastric carcinomas to be EBV-associated. We studied 50 gastric carcinomas from Malaysia to investigate its association with EBV in the Malaysian population. They comprised 37 intestinal and 13 diffuse type carcinomas from 32 male and 18 female patients, age range from 29 to 86 years with an ethnic distribution of Malay: Chinese: Indian with the ratio of 4: 27: 19. EBV gene and gene-expression were examined in sections of formalin-fixed, paraffin-embedded tissue using commercially available probes for detecting EBV encoded RNAs (EBERs) by in situ hybridization and monoclonal antibodies to EBV latent membrane protein-1 (LMP-1) by standard immunohistochemistry. Five of 50 gastric carcinomas showed EBER intranuclear positivity in all tumour cells but no cases expressed LMP-1. The EBV-associated cases were classified as intestinal type in 4 and diffuse type in one case and all were histologically unremarkable. EBV-positive tumours were found in 3 Chinese and 2 Indian patients with none in the small Malay group. Four EBV-positive tumours were in male patients, with age-range of 65 to 86 years. We conclude that our findings of about 10% of Malaysian gastric carcinomas being EBV-associated is in line with the results from other parts of the world and from other ethnic groups.     

Epstein-Barr virus (EBV)-associated undifferentiated carcinoma of the parotid gland

A case of undifferentiated carcinoma of the salivary gland occurring in the parotid gland of a southern Chinese was reported. Tumour cells showed immunofluorescence for Epstein-Barr virus (EBV)-associated nuclear antigen, and DNA hybridization demonstrated the presence of EBV-DNA in tumour tissue. The findings in this case, together with previous reports, suggest a causal relationship between EBV and salivary gland carcinoma. The relationships between EBV and undifferentiated epithelial tumours of the salivary glands, nasopharynx and thymus are also discussed.     

肝细胞肝癌组织中丙型肝炎病毒核心区基因及其表达 …

目的 采用多种方法检测丙型肝炎病毒（ＨＣＶ）核心区基因和表达产物在肝细胞肝癌（ＨＣＣ）组织中的分布及其在ＨＣＣ发生和发展中的作用。方法 对３９例ＨＣＣ患者进行免疫组化和原位杂匀的检测。ＩＳ－ＲＴ－ＰＣＲ法用于检测并定位ＨＣＶ－ＲＮＡ。微切组织的ＲＴ－ＰＣＲ法分别检测癌与癌旁组织中的ＨＣＶ－ＲＮＡ,以便能够控制扩增产物的来源。同时还进行 血清学ＥＬＩＳＡ和ＲＴ－ＰＣＲ的检测。结果 血清ＥＬＩＳＡ的阳     

Epstein-Barr virus (EBV) infection visualized by EGFP expression demonstrates dependence on known mediators of EBV entry

Summary.  Studies of Epstein-Barr virus (EBV) infection and pathogenesis are limited by the lack of recombinant EBV bearing a reporter gene. Currently such studies typically utilize cellular transformation and immortalization as indicators of virus infection which takes several weeks. To investigate the dependence on known cellular receptors for EBV infection, a novel enhanced green fluorescent protein (EGFP) expressing virus, designated EBfaV-GFP, was used to infect a variety of different cell types. EBfaV-GFP infects B lymphoma-derived cell lines, lymphoblastoid cell lines (LCLs) and primary B cells, as judged by expression of EGFP. Ability of clonal cells to be infected with EBfaV-GFP correlates with expression of the known EBV entry mediators CD21 and HLA-DR. EGFP-expressing LCLs were derived by infection of primary B cells with EBfaV-GFP. Cells of the Jurkat line, derived from T lymphocytes, could not be infected, and infected primary lymphocytes did not include appreciable numbers of CD2-positive cells, showing that EBV rarely infects T cells. Expression of EGFP by EBV provides the opportunity to rapidly visualize infection in living cells and better delineate populations of infected cells. Received October 22, 1998. Accepted January 25, 1999     

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 (EBV) in the oral mucosa of renal transplant patients

The purpose of this study was to determine the prevalence of Epstein-Barr virus (EBV)-DNA in the oral mucosa of renal transplant patients and observe the efficacy of mouth rinses with phosphate-buffered saline (PBS) to eliminate EBV present in the saliva. Lingual, gingival, and buccal cytobrushings were obtained from normal oral mucosa of 10 renal transplant patients and 10 normal subjects, and were examined through polymerase chain reaction (PCR), before and after rinses with PBS. EBV-DNA was detected in 86.6% of renal transplant recipients and in 46.6% of healthy subjects. No significant difference was observed between oral scrapes obtained before and after rinses with PBS with regard to detection of EBV-DNA. Our results suggest that the use of PCR to detect the presence of EBV-DNA in oral mucosa in the absence of specific lesions gives rise to the problem of identifying the viral replication sites. In addition, PBS was not effective at minimizing contamination by saliva.     

非霍奇金淋巴瘤EB病毒特异DNA序列的检测

目的　探讨EB病毒 (EBV)与中国南方地区非霍奇金淋巴瘤 (NHL)的相关性 ,以及EBV与不同类型NHL的关系。方法　采用PCR技术 ,检测 2 0 6例石蜡包埋的NHL组织及 2 3例反应性增生的淋巴组织中的EBV特异DNA序列。结果　(1 ) 2 0 6例NHL组织中 ,94例PCR扩增出EBV特异的DNA序列 ,阳性率 4 5 6 % ;对照组反应性增生的淋巴组织 2 3例中 ,5例阳性 ,阳性率 2 1 7% ;两者差异有显著性 (P <0 0 5 )。 (2 ) 2 0 6例NHL中B NHL 1 2 8例 ,EBV阳性者 4 8例 ,阳性率 37 5 % ;T NHL 78例 ,EBV阳性者 4 6例 ,阳性率 5 9 0 %。两者差异有显著性 (P <0 0 5 )。结论　EBV与中国南方地区NHL ,特别是T NHL有一定的相关性     

Epstein-Barr virus (EBV) infection in infancy.

BACKGROUND: Epstein-Barr virus (EBV) has been shown to be the cause of infectious mononucleosis (IM) and has more complicated associations with several malignant diseases. These EBV associated diseases provide a strong incentive for the development of an EBV vaccine. Most primary EBV infection during infancy and early childhood is mild or subclinical. Little is known about its infection in infancy. The pattern of EBV serological response during infancy may be important for vaccine management. OBJECTIVES: this study has served to clarify the epidemiology and serology of primary EBV infection during early infancy. STUDY DESIGN: longitudinal serum samples from 66 Hong Kong infants were tested for EBV antibodies by immunofluorescence. Cord blood and sequential serum samples from these infants were taken at birth and then at 4-month intervals up to 2 years of age. RESULTS: maternal antibodies were present at different levels in all cord blood specimens and in serum samples of 8 infants at 4-month of age. Evidenced by VCA-IgG seroconversion, 60.6% (40/66) infants were infected during the first 2 years of life. One episode occurred before 8 months of age but, thereafter and for the remaining 16 months of follow-up until the infants were 2 years of age, the infection occurred at essentially a constant rate affecting about 20% of the remaining seronegative infants every 4 months. CONCLUSIONS: the abrupt onset of the infection after a delay of 8 months is a remarkable feature of primary EBV infection during infancy, which implicates a protective role for maternal antibodies. Persisting maternal antibodies may additionally serve to contain the infection once it occurred. This may partly explain why, unlike during adolescence, primary EBV infection early in life is usually asymptomatic.     

Development of EBV-encoded small RNA targeted PCR to classify EBV positive diffuse large B-cell lymphoma (DLBCL) of the elderly

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) of the elderly has been included in the 2008 WHO classification of lymphoma as a new provisional entity. EBV-positive DLBCL of the elderly is newly classified due to the main occurrence usually in patients of older than 50-year-old. This study was performed in 91 DLBCL patients from January 2002 to December 2012 in Catholic university of St. Vincent Hospital. Age distribution of the patients was 14~87-year-old. Specimens were collected from lymph nodes (n = 45) and extra-lymph nodes (n = 46). EBV encoded small RNA1 in situ hybridization (EBER1-ISH) known as a standard method for the diagnosis of DLBCL. In this study, nested PCR of DNA polymerase gene and EBER PCR were conducted to detect EBV. Presence of EBV was indicated in 3 samples (3.30%) by EBER-ISH, 26 samples (28.57%) by nPCR, and 3 samples (3.30%) by EBER PCR. The concordant results were obtained from EBER1-ISH and EBER PCR. Two samples were classified as EBV-positive DLBCL of the elderly among 91 DLBCL patients. Previously, the incidence rate of DLBCL of the elderly in Asia has been reported as 5~11%, but the result in this study showed a slightly lower incidence rate. To our knowledge, this is the first report on EBV-positive DLBCL of the elderly in Suwon area, Korea. EBER1-ISH and EBER PCR developed in this study may be helpful in classification of EBV-positive DLBCL of the elderly in future.     

CD95 (Fas) ligand expression of Epstein-Barr virus (EBV)-infected lymphocytes: a possible mechanism of immune evasion in chronic active EBV infection

The Epstein-Barr virus (EBV) induces infectious mononucleosis (IM) and can be associated with chronic active EBV infection (CAEBV). Cytotoxic T lymphocytes (CTL) play an important role in excluding EBV-infected cells. Two cytotoxic mechanisms of CTL have been demonstrated: one perforin/granzyme-based and the other Fas (CD95)/Fas ligand (FasL)-based. To clarify these two pathways in CAEBV, we analyzed six patients with CAEBV and four patients with IM using immunohistochemical staining of the lymph nodes. In both CAEBV and IM, CD8⁺ T-cells increased in number, but CD56⁺ natural killer cells were rare. In four of six cases with CAEBV, approximately half the lymphocytes were positive for T cell-restricted intracellular antigens (TIA-1), which were recognized by the cytolytic granules of CTL. In IM, the number of TIA-1 positive cells was smaller than that in CAEBV. Fas-positive lymphocytes were frequently encountered in both CAEBV and IM. However, FasL-positive lymphocytes increased in three of six patients with CAEBV, but not in patients with IM. Except for one case with CAEBV, the number of perforin- and/or granzyme-positive cells was small in number in both CAEBV and IM cases. In double-staining FasL and EBV in situ hybridization, FasL-positive EBV-infected lymphocytes were detected in CAEBV but not in IM. In CAEBV, the Fas/FasL pathway and not perforin pathways appears to play an important role in the pathogenesis. The data suggest that EBV-infected lymphocytes may evade immune attack through the expression of FasL.     

Expression of the Epstein-Barr virus (EBV) receptor on the surface of cells infected with EBV derived from nasopharyngeal carcinoma

An Epstein-Barr virus (EBV) genome-positive epithelial hybrid cell line, NPC-KT, derived from the fusion of primary nasopharyngeal carcinoma cells with a human epithelial cell line of adenoid origin and a subline of EBV genome-positive Ramos cells, Ramos/NPC, converted after infection with NPC-KT EBV have been previously described (Takimoto et al., 1984; Takimoto et al., 1987). The NPC-KT cells produce virus (NPC virus) with both transforming and lytic properties. In this study, NPC-KT and Ramos/NPC cells were examined for the presence of the EBV receptor as measured by the capacity to absorb radio-labelled P3HR-1 and NPC viruses. It was determined that only P3HR-1 virus can attach to NPC-KT cells. Also, the relative concentration of NPC virus receptors on Ramos/NPC cells was found to be significantly reduced when compared to EBV genomenegative Ramos cells, whereas the relative concentration of receptors for P3HR-1 virus was similar to parental Ramos cells. The results suggest that there are differences at least in part of the receptors for P3HR-1 and NPC viruses.     

Comparative analysis of the expression of the Epstein-Barr virus (EBV) anti-apoptotic gene BHRF1 in nasopharyngeal carcinoma and EBV-related lymphoid diseases

Epstein-Barr virus (EBV) has been identified in a wide range of neoplastic and non-neoplastic disorders. The EBV open reading frame BHRF1 encodes a protein with partial sequence and functional homology to the anti-apoptotic onco-protein Bcl-2 and may therefore have a role in the proliferation of EBV positive cells. We have developed a rat monoclonal antibody against pBHRF1, which can detect BHRF1 in paraffin sections. While a number of mutant versions of BHRF1 were recognised, the monoclonal did not detect the BHRF1 homologue encoded by Herpesvirus papio or two mutants with deletions in the BH2 region. This novel rat monoclonal antibody (6A9) was used to examine tissue sections from 39 cases of non-keratinising undifferentiated nasopharyngeal carcinoma (NPC), 6 cases of metastatic NPC, 7 cases of EBV-positive NPC with squamous differentiation from Chinese patients, 15 cases of EBV-positive post-transplant lymphoproliferative disorder (PTLD), 6 EBV-containing lymphoblastoid cell lines, and 2 cases of oral hairy leukoplakia (OHL). In 11 cases of undifferentiated NPC, RT-PCR data were available for comparison with the immunohistochemistry. Both cases of OHL and two cases of LCL were positive for BHRF1 but none of the PTLD showed positive staining. All cases of undifferentiated NPC were positive for Bcl-2 but only one BHRF1 positive cell was identified in 1 of 39 cases of primary undifferentiated NPC. The 6A9 antibody produced less background staining and no nuclear positivity compared with the commercially available mouse monoclonal 5B11. It is concluded that BHRF1 can not be detected by immunohistochemistry in NPC and therefore it appears not to play a significant anti-apoptotic role in the progression of this EBV-associated tumour. The 6A9 monoclonal appears to be superior to 5B11 for the detection of pBHRF1 in tissue sections.     

Epstein-Barr virus (EBV) antibodies in children with non-Hodgkin's lymphomas

Antibody titres against Epstein-Barr virus (EBV) antigens in children suffering from non-Hodgkin's lymphoma (NHL) were determined. IgG antibody titres against the viral capsid antigen (VCA) and early antigen (EA) exceeded those found in healthy control subjects. On the other hand, antibody titres against EBV-determined nuclear antigen (EBNA complex) were generally lower than in the control group. The most striking phenomenon observed in the patient group was the frequent activation of latent virus infection as revealed by the periodical appearance of anti-EA and IgM class anti-VCA antibodies. Antibody titres against EBV antigens were generally lower among patients with progressing disease than in those with a more favourable course of the illness. The closest relation to EBV based on serological findings, was detected in lymphoblastic lymphomas of Burkitt-type histology, poorly differentiated lymphocytic lymphomas, and in lymphomas localized in the abdomen. The question whether EBV might be involved in a certain proportion of the cases examined is discussed and further approaches to elucidate this problem are suggested.