Epstein–Barr virus in Reed–Sternberg G-like cells in non Hodgkin's lymphomas

In the course of our study on Hodgkin's disease (HD), ten cases of non-Hodgkin's lymphomas (NHL) containing Hodgkin and Reed-Sternberg-like (MRS) cells were encountered. Many of these cases had initially been diagnosed as HD, but on careful review of the histology, with the aid of immunophenotyping studies, they were reclassified as NHL. The presence of Epstein–Barr virus (EBV) in these HRS-like cells was investigated using a combination of EBER in situ hybridization (ISH) and immunostaining for the detection of EBV-encoded latent membrane protein (LMP). HRS-like cells in four cases (two lymphoplasmacytoid lymphomas, one Richter's transformation of lymphoplasmacytoid lymphoma, and one immunoblastic lymphoma of T-cell type) were found to be EBV-positive. In two of these cases, a second biopsy taken up to 10 years later also contained EBV in the HRS-like cells. In three of the four cases, HRS-like cells expressed the activation antigen CD30, but the expression of B- or T-cell antigens was variable. All cases of T-cell-rich B-cell lymphomas were negative for EBV. In conclusion, EBV may play a role in the development of HRS-like cells i some cases of NHL. The relationship of HRS-like cells to HRS cells of HD is discussed.     

Prevalence of Epstein–Barr virus in oral squamous cell carcinoma

Forty-six samples of oral squamous cell carcinoma (OSCC) were evaluated for the prevalence of Epstein–Barr virus (EBV) infection by the polymerase chain reaction (PCR), Southern blot hybridization, and in situ hybridization (ISH). EBV DNA was detected in 7 (15·2 per cent) out of 46 samples by a combination of PCR and Southern blot hybridization methods. All seven positive samples showed well-differentiated carcinoma, thus suggesting a possible relationship between EBV infection and the degree of differentiation of carcinoma tissue. Latent infection membrane protein 1 (LMP1) was detected immunohistochemically in six of the EBV-positive OSCCs. However, no signal of the EBV-encoded small RNA (EBER)-1 was demonstrated by the ISH method. No significant relationship was observed between EBV infection and lymph node metastasis. A follow-up study (range from 4·4 to 79 months; mean 34·9 months) showed no recurrence or death to occur in the EBV-positive patients, which thus suggested a good prognosis for EBV-positive OSCC patients. Copyright © 1999 John Wiley & Sons, Ltd.     

Epstein–Barr virus infection in non-carcinomatous gastric epithelium

Gastric tissue specimens from 20 patients with chronic atrophic gastritis, one of whom also had an early gastric carcinoma, were studied for evidence of Epstein–Barr virus (EBV) infection by Southern blot analysis, DNA and RNA in situ hybridization, and immunohistochemistry for the presence of the EBV-determined nuclear antigen 1 (EBNA-1) and the latent membrane protein 1 (LMP-1). EBV DNA was detected in two cases with chronic atrophic gastritis and in the case with early gastric carcinoma by Southern blot hybridization. DNA in situ hybridization showed EBV genomes in the epithelial cells of two other cases with chronic atrophic gastritis and in non-carcinomatous and carcinomatous epithelial cells of the early gastric carcinoma case. EBNA-1 was detected in all cases. LMP-1 was detected in areas of intestinal metaplasia in eight patients with chronic atrophic gastritis. EBV-encoded small RNA 1 (EBER-1) expression was limited to carcinoma cells. These results show that gastric epithelium is frequently infected with EBV and suggest that prolonged EBV persistence may contribute to the development of gastric carcinoma. © 1997 John Wiley & Sons, Ltd.     

Cytomegalovirus and Epstein–Barr virus coinfection in three toddlers with prolonged illnesses

Cytomegalovirus (CMV) and Epstein–Barr virus (EBV) usually cause primary and latent infections during childhood; thus, coinfection with these viruses occurs occasionally in children. However, its clinical impact has not been established, and may be underestimated. Three cases of coinfection involving these two viruses in toddlers are described: a 14‐month‐old male with infectious mononucleosis, an 18‐month‐old female with hemophagocytic lymphohistiocytosis, and a 13‐month‐old female with acute hepatitis. All three patients had prolonged illnesses. Serial serological testing and quantitation of viral DNA for CMV and EBV using peripheral blood from the patients suggested primary infections with both viruses. In all three cases, the viral load of EBV and CMV in the early stage of disease exceeded 6.4 × 10³ and 8.8 × 10² copies/ml of whole blood, respectively, suggesting that the viruses were associated with the clinical condition. Recognizing that coinfection with these viruses may modulate the clinical course of disease is important. J. Med. Virol. 81:1399–1402, 2009. © 2009 Wiley‐Liss, Inc.     

ABSENCE OF EPSTEIN–BARR VIRAL ENCODED RNA (EBER) IN PRIMARY CUTANEOUS T-CELL LYMPHOMA

Epstein-Barr virus (EBV) has been associated with various extracutaneous lymphoproliferative diseases and it has been suggested that EBV may have a similar aetiological role in cutaneous T-cell lymphoma. In this study, in situ hybridization was used to investigate the presence of EBV encoded RNAs (EBER-1 and EBER-2) in 37 biopsies from 28 cases of primary cutaneous T-cell lymphoma originating from the U.K. The results showed that EBV had no demonstrable pathogenic role in the lymphomas studied, as EBER was not detected in any case.     

Disappearance of the Epstein–Barr virus in a relapse of Hodgkin's disease

Hodgkin's disease (HD) is associated with the Epstein–Barr virus (EBV) in approximately half of cases. This is a report of a case of nodular sclerosing HD of the B-cell type that was associated with EBV in the initial manifestation, but was found to be EBV-negative in the relapse of the tumour. Both tumours displayed similar clinical, pathological, and immunohistochemical features. This finding implies that in a given individual EBV can be lost from malignant tumours and therefore shows that the EBV infection is not required to maintain neoplastic growth of HD tumour cells. © 1997 John Wiley & Sons, Ltd.     

Modulation of interleukin-6 expression in Hodgkin and Reed–Sternberg cells by Epstein–Barr virus

Variable proportions of Hodgkin's disease (HD) cases are associated with the Epstein–Barr virus (EBV), but the role of EBV in HD is not entirely clear. Hodgkin and Reed–Sternberg (HRS) cells of EBV-associated HD are characterized by expression of the EBV gene product LMP1. In other cellular environments, LMP1 has been shown to induce interleukin (IL)-6. In this study, 105 HD cases were tested for differences in IL-6 expression among LMP1-positive and -negative cases. Isotopic in situ hybridization and correlation with the presence of EBV gene products revealed significantly higher proportions of cases with IL-6-expressing tumour cells in LMP1-positive (31 of 37, 84 per cent) as compared with LMP1-negative HD cases (35 of 68, 51 per cent). Thus, although not exclusive to EBV-positive HRS cells, IL-6 expression appears to be upregulated in EBV-associated HD. IL-6 receptor (CD126) expression was tested by in situ hybridization and found in a broad spectrum of cell types, regularly including HRS cells. Superinduction of IL-6 expression may be among the mechanisms by which EBV confers a growth advantage on virus-infected HRS cells and by which the virus may contribute to the morphological and clinical peculiarities of HD. © 1997 John Wiley & Sons, Ltd.     

The dominance of China 1 in the spectrum of Epstein–Barr virus strains from Cantonese patients with nasopharyngeal carcinoma

Nasopharyngeal carcinoma is a disease with a remarkable geographic and ethnic distribution, and has a high incidence in southern China. Infection with Epstein–Barr virus (EBV) is an important contributing factor. The profile of EBV strains in Cantonese patients from Guangdong, the nasopharyngeal carcinoma endemic region in southern China, is described on the sequence variations in latent membrane protein 1 carboxyl‐terminus. The results show that China 1 was the dominant EBV strain detected in both the tumor biopsies and samples of throat washings, whereas multiple strains, including China 1, China 2, B95‐8, and Med, were detected in blood samples. In addition, a new strain named China 4 was found in blood samples. These findings suggest that the host population is susceptible to the predominant China 1 strain in the nasopharyngeal carcinoma endemic region of China, but its relationship with the host remains to be characterized further. J. Med. Virol. 81:1253–1260, 2009. © 2009 Wiley‐Liss, Inc.     

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。     

Frequent expansion of Epstein–Barr virus (EBV) infected cells in germinal centres of tonsils from an area with a high incidence of EBV-associated lymphoma

Burkitt's lymphoma (BL) and Hodgkin's disease (HD) occurring in developing regions are frequently associated with Epstein–Barr virus (EBV) infection and have a high incidence in childhood. Recent genotyping studies indicate that the tumour cells of both neoplasms represent B cells that contain somatically mutated immunoglobulin heavy chain genes. This implies that the precursors of these neoplasms have participated in the germinal centre (GC) reaction. We therefore presumed that normal lymphoid tissues from children living in developing regions would harbour an increased number of EBV-infected cells within the GC, when compared with children living in industrialized nations. To test this hypothesis, hyperplastic tonsils from 40 children living in Bahia (Brazil) and 40 from German children were analysed for the presence of EBV-encoded small nuclear RNA (EBER) and EBV-encoded proteins by in situ hybridization and immunohistology, respectively. Although the overall EBV infection rate was similar in both groups (50 per cent of Bahian vs. 45 per cent of German cases), a significantly higher number of EBER-positive lymphoid cells were found in the GCs of 8/20 EBV-positive tonsils from Brazil (9–89 cells/GC; mean: 14·5 cells/GC per case), while only 3/18 tonsils from Germany displayed a few EBER positive cells (1–9 cells/GC; mean: 0·5 cell/GC per case) in this compartment (p < 0·007). In addition, the EBV-infected GC cells in Bahian samples resembled centroblasts, exhibited mitotic activity, and in two cases showed expression of EBV-encoded latent membrane protein (LMP)-1, findings not present in German samples. These data show that latently EBV-infected cells participate more frequently in GC reactions in developing regions than in industrialized countries and may abnormally express the oncogenic protein LMP-1. This could in part explain the higher incidence in this region of EBV association with lymphomas related to GC cells or their progeny, such as BL and HD. Copyright © 1999 John Wiley & Sons, Ltd.     

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.     

New variations of Epstein–Barr virus‐encoded small RNA genes in nasopharyngeal carcinomas,gastric carcinomas,and healthy donors in northern China

It has been generally believed that the Epstein–Barr virus (EBV)‐encoded small RNA 1 and 2 (EBER1 and EBER2) genes are conserved as two families that correlated with type 1 (B95‐8) and type 2 (AG876 or P3HR‐1) EBV strains. EBER polymorphism and its association with EBV‐associated disease have not received much attention. To explore the variations of EBER genes in different malignancies and healthy donors, the sequences of EBER genes were analyzed in 154 EBV‐positive samples, including 47 nasopharyngeal carcinoma (NPC), 50 EBV‐associated gastric carcinoma (EBVaGC) biopsies and 57 throat washing (TW) samples from healthy donors in northern China, where NPC is non‐endemic. Three main distinct variants of EBER genes, designated as EB‐6m, EB‐8m, and EB‐10m, were identified. EB‐6m had a previously identified EBER sequence identical to P3HR‐1 and was found in 33/47 (70.2%) NPC, 48/50 (96.0%) EBVaGC, and 54/57 (94.7%) TW isolates. EB‐8m and EB‐10m were new EBER variants with more mutations in EBER2 genes. They were found in 13/47 (27.7%) NPC cases, whereas in only 1/50 (2.0%) EBVaGC cases and not found in TW cases. The distributions were significantly different (P < 0.05). Other five isolates (one NPC, one EBVaGC and three TW cases) showed different sequences and could not be assigned to any of the three groups. Type 1 EBV strains showed heterogeneous in terms of EBER variants. These results suggest that EBER locus can be useful to identify different EBV isolates, and it would be interesting to evaluate the association of EBER polymorphisms with EBV‐associated tumors. J. Med. Virol. 82: 829–836, 2010. © 2010 Wiley‐Liss, Inc.     

Epstein–Barr virus infection induces genome‐wide de novo DNA methylation in non‐neoplastic gastric epithelial cells

Epstein–Barr virus (EBV)‐positive gastric cancer (GC) shows a higher DNA methylation epigenotype. EBV infection can causally induce genome‐wide aberrant DNA methylation, as previously demonstrated by in vitro infection experiments in the low‐methylation GC cell line MKN7. However, whether EBV exerts DNA methylation remodelling properties in non‐neoplastic epithelial cells remains unclear. Here we performed post‐infection time‐series DNA methylation analyses using the immortalized normal gastric epithelial cell line GES1. Genome‐wide analysis using Illumina's Infinium 450 k BeadArray demonstrated global de novo DNA methylation from post‐infection day 17, which was completed by 28 days in a manner similar to that observed in MKN7 cells. De novo methylation of all types of GC‐specific methylation marker genes was observed, indicating that EBV infection is sufficient for gastric epithelial cells to acquire an EBV‐positive GC epigenotype. Pyrosequencing demonstrated that methylation of the viral genome preceded that of the host cellular genome, suggesting the existence of well‐ordered mechanisms that induce methylation. Spatiotemporal representation with differential models revealed dynamic alterations of DNA methylation in promoter regions, occurring from lower‐CpG peripheral regions and extending to higher‐CpG core regions. In summary, EBV infection exerted powerful pressure to induce global de novo DNA methylation in non‐neoplastic cells within a month in a spatiotemporally well‐ordered manner. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Epstein–Barr virus can infect rabbits by the intranasal or peroral route: An animal model for natural primary EBV infection in humans

Epstein–Barr virus (EBV) is spread universally in humans, and it causes infectious mononucleosis and sometimes induces serious EBV‐associated disease. The detailed mechanism of primary infection in humans has remained unclear, because it is difficult to examine the dynamics of EBV in vivo. In this study, a natural EBV‐infection rabbit model by intranasal or peroral inoculation is described. Ten male rabbits were examined for EBV‐DNA or mRNA expression and anti‐EBV antibodies in blood. Four of 10 rabbits showed the evidence of EBV infection; detection of EBV‐DNA or EBV‐related genes mRNA in peripheral blood mononuclear cells, increased EBV antibodies in the plasma, and the presence of lymphocytes expressing EBER1 and EBV‐related gene proteins in the lymphoid tissues of a rabbit. Three of four infected rabbits were detected transiently EBV‐DNA and/or mRNA of EBV‐related genes such as EBNA1, EBNA2, BZLF1, and EA in blood, while in one of four, EBV‐DNA and/or mRNA were detected for more than 200 days after viral inoculation. The level of EA‐IgG increased and its level was maintained in all infected rabbits, whereas those of VCA‐IgM and VCA‐IgG increased transiently, and EBNA‐IgG was not elevated. Pathological examination of a rabbit infected transiently revealed some scattered lymphocytes expressing EBER1, LMP1, and EBNA2 in the spleen and lymph nodes. EA expression was also observed in the spleen. These findings suggest that EBV can infect the rabbit by the intranasal or peroral route, and that this rabbit model is useful for examining the pathophysiology of natural primary EBV infection in humans. J. Med. Virol. 82:977–986, 2010. © 2010 Wiley‐Liss, Inc.     

Epstein–Barr virus BZLF1 gene promoter variants in pediatric patients with acute infectious mononucleosis: Its comparison with pediatric lymphomas

Epstein–Barr virus genotypes can be distinguished by polymorphic variations in the genes encoding EBNA2, 3A, 3B, and 3C. The immediate early gene BZLF1 plays a key role in modulating the switch from latency to lytic replication and therefore enabling viral propagation. The aim of this study was to investigate and compare BZLF1 promoter sequence (Zp) variation in pediatric infectious mononucleosis (IM) and in pediatric EBV positive lymphoma biopsies. Zp was sequenced from peripheral blood mononuclear cells (PBMC) and throat swabs from 10 patients with IM at the time of diagnosis (D0) and during convalescence; and from 13 lymphoma biopsies. Zp ? P and Zp ? V3 variants were found in eight and one IM patients, as well as in five and six tumor biopsies, respectively. A correlation between viral genotype and Zp variant was found significant for Zp ? V3 and EBV2 (P = 0.0002). One IM patient harbored two concomitant Zp variants. Regardless of anatomical compartment or stage of disease all IM patients displayed the same Zp variant along the course of the study. No new infections or adaptative selection of different variants was evidenced. A new Zp variant (Zp ? V3 + 49) was described in two Hodgkin lymphomas, but not in IM. This is the first study to describe Zp variants compartmentalization in children with acute EBV infection and convalescence in a developing country; and comparing them with Zp variants in pediatric lymphomas from the same geographic area. J. Med. Virol. 81:1912–1917, 2009. © 2009 Wiley‐Liss, Inc.     

Association of distinctive Epstein–Barr virus variants with gastric carcinoma in Guangzhou,southern China

To investigate the clinicopathologic features, Epstein–Barr virus (EBV) latency pattern and genome polymorphism of EBV‐associated gastric carcinoma (EBVaGC) in Guangzhou, an endemic area of nasopharyngeal carcinoma (NPC), an in situ hybridization assay of EBV‐encoded small RNA‐1 (EBER‐1) was used to identify the presence of EBV in 676 consecutive gastric carcinoma cases. EBV‐encoded proteins EBNA1, EBNA2, LMP1, and ZEBRA were detected by immunohistochemistry. EBV genome polymorphism was also analyzed by PCR and DNA sequencing. Of the 676 cases, 45 EBV‐positive cases (6.7%) were identified, including 37 (8.5%) male and 8 (3.3%) female cases. EBNA1 was detected in 42 cases (93.3%), while EBNA2, LMP1, and ZEBRA were all negative. In the EBV genome polymorphism analysis, type A strain, prototype F, type I, XhoI?, and del‐LMP1 variants were predominant among EBVaGC patients, accounting for 44 (97.8%), 37 (82.2%), 45 (100%), 34 (75.6%), and 42 (93.3%) cases, respectively. Moreover, a new hotspot mutation in the BamHI‐W1/I1 boundary region (148,972 T → C) was found in 39 (86.7%) of the 45 cases. The predominant EBV variants in EBVaGC in Guangzhou are prototype F, type I, and XhoI?, which are different from those in NPC in this area (predominant variant‐type “f”) and in EBVaGC in Latin American countries (predominant type “i” and XhoI+), suggesting that the EBV variants are not only geographically distributed but also disease restricted, and the pathogenic role of EBV in different EBV associated epithelial malignancies in different areas may be distinct. J. Med. Virol. 82:658–667, 2010. © 2010 Wiley‐Liss, Inc.     

Inhibition of the LKB1–AMPK pathway by the Epstein–Barr virus‐encoded LMP1 promotes proliferation and transformation of human nasopharyngeal epithelial cells

The association of Epstein–Barr virus (EBV) infection with the development of nasopharyngeal carcinoma (NPC) is well established. Latent membrane protein 1 (LMP1), the major oncogene encoded by EBV, is believed to play a crucial role in NPC pathogenesis by virtue of its ability to constitutively activate multiple cell signalling pathways. The LKB1–AMPK pathway is a master regulator of cellular metabolism that, via modulation of energy metabolism, has tumour suppressor activity. In this study we identify a novel ability of LMP1 to inhibit the LKB1–AMPK pathway through phosphorylation of LKB1 at serine 428 with subsequent suppression of the phosphorylation of AMPK and its substrates, ACC and Raptor. We show that MEK/ERK–MAPK signalling, activated by the CTAR1 domain of LMP1, is responsible for LKB1–AMPK inactivation. In addition, reactivation of AMPK signalling by AMPK activator, AICAR, abolished LMP1‐induced cellular transformation (proliferation and anchorage‐independent growth) in nasopharyngeal epithelial cells. Immunohistochemical staining revealed that a low level of phosphorylated AMPK is common in primary NPC specimens, and that this correlated significantly with the expression of LMP1. AICAR treatment inhibited the proliferation and anchorage‐independent growth of NPC cells as well as potentiating the cytotoxic effect of the chemotherapeutic drug 5‐fluorouracil. The current findings demonstrate that LMP1‐mediated AMPK inactivation contributes to the proliferation and transformation of epithelial cells, thereby implicating the LKB1–AMPK pathway in the EBV‐driven pathogenesis of NPC. Our findings also suggest that AMPK activators could be used to enhance the efficacy of conventional chemotherapeutic agents in the treatment of local and metastatic NPC. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.