Identification Of Epstein-Barr Virus Research Articles

Identification of Epstein-Barr virus after topical treatment for oral hairy leukoplakia: A preliminary study.

This study evaluated the presence of Epstein-Barr virus type 1 (EBV-1) DNA in patients living with HIV, before and after three different topical therapy protocols for oral hairy leukoplakia (OHL). The sample consisted of five patients treated with topical solution of 25% podophyllin resin; six with 25% podophyllin resin plus 5% acyclovir cream; and four with 25% podophyllin resin plus 1% penciclovir cream. DNA was extracted from OHL scrapings and amplified by the PCR using specific primers for EBV-1 (EBNA-1). Clinical healing of OHL lesions was observed across all treatment groups over time. At baseline, EBNA-1 was detected in all OHL lesions. After treatment, OHL samples from three patients treated with 25% podophyllin resin plus 5% acyclovir cream and from one patient treated with 25% podophyllin resin plus 1% penciclovir cream exhibited negative EBNA-1 viral gene encoding. Despite the clinical resolution of OHL, 11 patients (73.3%) showed EBNA-1 positivity immediately after the lesion disappeared. Three patients (20%) treated with podophyllin resin displayed both EBNA-1 positivity and a recurrence of OHL, in contrast to no recurrence in the other two groups. These findings suggest potential associations between treatment formulations, EBNA-1 persistence, and the recurrence of OHL lesions.

Association of Epstein–Barr Virus and Cytomegalovirus Infections with Esophageal Carcinoma

Background: Given the fact that viral infections play an important role, either directly or indirectly, in around 20 percent of human cancers, this study aimed at investigating the potential association of Epstein–Barr virus (EBV) and cytomegalovirus (CMV) infections in esophageal cancer that is the sixth most common cause of cancer-related deaths. Methods: In this case-control study, a total of 200 paraffin-embedded biopsies of cancerous and benign esophageal tissues were gathered from the biopsy bank of Imam Reza Hospital, Tabriz, Iran in 2017. All samples were first deparaffinized, and then subjected to commercial DNA extraction. The quality of extracted DNA was evaluated by amplification of the beta globulin gene. Identification of EBV and CMV DNA was performed using primers designed for the EBER region of EBV and the immediate early (IE) region of the CMV genome, respectively. Results: The mean age of the subjects in the test and control groups was 52.2 (17.1) and 59.9 (18.9), respectively. The distribution of gender (male/female) in patient and control groups was 54/46 and 53/47, respectively. Our results showed that the frequency of EBV (P < 0.001) and CMV (P < 0.001) in cancerous samples was statistically higher than control group. Moreover, in the cancerous group the rate of EBV was significantly higher in the esophageal adenocarcinomas (EAC) sample (12 out of 70) than esophageal squamous cell carcinomas (ESCC) (0 out of 30) (P = 0.016) but, in the ESCC group, 17 out of 30 subjects were positive for CMV which was significantly higher in comparison with EAC patients (1 out of 70) (P < 0.001). Conclusions: Findings indicated that EBV and CMV might be contributed to the pathogenesis of EAC and ESCC types of esophageal carcinoma, respectively, although further studies are warranted.

Detection of Epstein-Barr virus encoded RNA in fixed cells and tissues using CRISPR/Cas-mediated RCasFISH

Identification of Epstein-Barr virus (EBV)-infected cells is critical for the diagnosis and clinical management of EBV-associated diseases. EBV-encoded RNA (EBER) located in the nucleus is a reliable marker due to its high levels of expression and inherent stability in tissue specimens. EBER in situ hybridization has long been the gold standard for detecting tumor-associated latent EBV infection and is valuable in determining the primary site and radiation fields of EBV-related malignancies. However, reliable detection is somewhat restricted by diffused signal and time-consuming procedure of this method, especially when proteins and RNA needed to be labeled simultaneously. Here, we optimized and validated our CRISPR-dCas9 mediated in situ RNA imaging tool—RCasFISH that enabled us to detect EBER rapidly and was compatible with IHC methods in fixed cells and tissue sections. Our approach could provide an attractive alternative for the molecular diagnosis of latent EBV infection.

Identification of Epstein-Barr Virus in the Human Placenta and Its Pathologic Characteristics.

Epstein-Barr virus (EBV), a common pathogen in humans, is suspected as the cause of multiple pregnancy-related pathologies including depression, preeclampsia, and stillbirth. Moreover, transmission of EBV through the placenta has been reported. However, the focus of EBV infection within the placenta has remained unknown to date. In this study, we proved the expression of latent EBV genes in the endometrial glandular epithelial cells of the placenta and investigated the cytological characteristics of these cells. Sixty-eight placentas were obtained from pregnant women. Tissue microarray was constructed. EBV latent genes including EBV-encoding RNA-1 (EBER1), Epstein-Barr virus nuclear antigen 1 (EBNA1), late membrane antigen (LMP1), and RPMS1 were detected with silver in situ hybridization and/or mRNA in situ hybridization. Nuclear features of EBV-positive cells in EBV-infected placenta were compared with those of EBV-negative cells via image analysis. Sixteen placentas (23.5%) showed positive expression of all 4 EBV latent genes; only the glandular epithelial cells of the decidua showed EBV gene expression. EBV infection status was not significantly correlated with maternal, fetal, or placental factors. The nuclei of EBV-positive cells were significantly larger, longer, and round-shaped than those of EBV-negative cells regardless of EBV-infection status of the placenta. For the first time, evidence of EBV gene expression has been shown in placental tissues. Furthermore, we have characterized its cytological features, allowing screening of EBV infection through microscopic examination.

High Throughput Sequencing-Based Pathogen Discovery In Multiple Myeloma

BackgroundMultiple myeloma is a malignancy of antibody-secreting, terminally differentiated B cells with a predilection for the bone marrow. Considering that microbial infections are common triggers for antibody production, we hypothesize that known and/or previously undiscovered viruses may be present in myeloma tissues and may influence myeloma prognosis, even if the viruses themselves do not cause myeloma.Virus-harboring tissues should presumably contain both human and pathogenic DNA/RNA, but viruses may not be detected if they are present at frequencies that are too low for detection by standard assays. Therefore, high-throughput sequencing, followed by computational subtraction of human sequence reads should result in the identification of non-human (and candidate pathogenic) sequences. The Multiple Myeloma Research Consortium has generated such a dataset, thus providing a unique opportunity to address this question. MethodsWe have developed a computational subtraction pipeline, PathSeq, for pathogen discovery. It consists of 3 main modules. Quality filtering is carried out in the “pre-subtraction module”. In the “subtraction module”, the quality filtered reads are analyzed by alignment to a series of human genome and transcriptomes databases using the BWA aligner and BLAST. In the “post-subtraction module”, residual nucleotide and conceptually translated sequences are aligned to microbial databases. We also generate large contigs composed of unmapped reads that do not possess significant alignment similarity to any sequence in the reference databases. Such contigs may be suggestive of novel microorganisms.Whole genome sequencing data obtained from an initial set of 26 tumor-normal sample pairs (reported in Chapman et al, Nature 2010) were analyzed by PathSeq. In addition, analysis of mRNA sequencing data from 84 tumors is ongoing. Comparative marker selection analysis was also performed on array-based gene expression profiling data from 20 of the initial 26 tumors. ResultsPathSeq analyses revealed the presence of Epstein-Barr virus (EBV) sequence reads in 7 cases and human herpesvirus 6B (HHV-6B) reads in 2 cases. In most instances, the viral sequences were found in both tumor and normal samples from the same patient. The lowest viral-human read ratio was 1 in 1 billion, demonstrating the sensitivity of the PathSeq algorithm for pathogen identification.Comparative marker selection analysis comparing gene expression profiles between the 7 tumors with EBV reads with 13 tumors without viral sequences revealed a tentative virus associated signature, with SHISA2 (which encodes a protein that modulates FGF and Wnt signaling) as the gene whose expression is most significantly associated with virus positivity. ConclusionsWe have described the identification of EBV and HHV6B sequences in DNA samples obtained from myeloma tissues. Our data are preliminary and it is possible that the viral sequences are simply vestiges of non-active herpesviral infection present in the tumor microenvironment. Therefore, these findings need to be confirmed by our ongoing PathSeq analysis of RNA sequencing data. In addition, efforts are ongoing to identify novel pathogens, as described above.The translational utility of knowledge about the influence of host-viral relationships on the prognosis of myeloma, if they exist, transcends the need to prove causation. Therefore if viral sequences are found in RNA sequencing data, future plans will include comparison of our data to published prognosis-related gene expression signatures in myeloma. Disclosures:No relevant conflicts of interest to declare.

Su1863 Genome-Wide Identification of Epstein-Barr Virus (EBV) Driven Gastric Cancer-Related DNA Methylation by Medip-Chip Profiling of EBV Infected AGS Cells

Su1863 Genome-Wide Identification of Epstein-Barr Virus (EBV) Driven Gastric Cancer-Related DNA Methylation by Medip-Chip Profiling of EBV Infected AGS Cells

Identification of Epstein-Barr Virus RK-BARF0-Interacting Proteins and Characterization of Expression Pattern

The Epstein-Barr virus (EBV) BamHI A transcripts are a family of transcripts that are differentially spliced and can be detected in multiple EBV-associated malignancies. Several of the transcripts may encode proteins. One transcript of interest, RK-BARF0, is proposed to encode a 279-amino-acid protein with a possible endoplasmic reticulum-targeting sequence. In this study, the properties of RK-BARF0 were examined through identification of cellular-interacting proteins through yeast two-hybrid analysis and characterization of its expression in EBV-infected cells and tumors. In addition to the interaction previously identified with cellular Notch, it was determined that RK-BARF0 also bound cellular human I-mfa domain-containing protein (HIC), epithelin, and scramblase. An interaction between RK-BARF0 and Notch or epithelin induced proteasome-dependent degradation of Notch and epithelin but not of HIC or scramblase. Low levels of endogenous Notch expression in EBV-positive cell lines may correlate with RK-BARF0 expression. However, a screen of EBV-positive cell lines and tumors with an affinity-purified alpha-RK-BARF0 antiserum did not consistently detect RK-BARF0. These data suggest that while RK-BARF0 may have important cellular functions during EBV infection, and while the phenotype of EBV-positive cells suggest its expression, RK-BARF0 levels may be too low to detect.

Identification of Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) target proteins by proteome analysis: activation of EBNA2 in conditionally immortalized B cells reflects early events after infection of primary B cells by EBV.

The Epstein-Barr virus (EBV) is a ubiquitous B-lymphotropic herpesvirus associated with several malignant tumors, e.g., Burkitt's lymphoma and Hodgkin's disease, and is able to efficiently immortalize primary B lymphocytes in vitro. The growth program of infected B cells is initiated and maintained by the viral transcription factor EBV nuclear antigen 2 (EBNA2), which regulates viral and cellular genes, including the proto-oncogene c-myc. In our study, patterns of protein expression in B cells with and without EBNA2 were analyzed by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. For this purpose, we used a conditional immortalization system for EBV, a B cell line (EREB2-5) that expresses an estrogen receptor-EBNA2 fusion protein. In order to discriminate downstream targets of c-Myc from c-Myc-independent EBNA2 targets, we used an EREB2-5-derived cell line, P493-6, in which c-Myc is expressed under the control of a tetracycline-regulated promoter. Of 20 identified EBNA2 target proteins, 11 were c-Myc dependent and therefore most probably associated with proliferation, and one of these proteins was a posttranslationally modified protein, i.e., hypusinylated eIF5a. Finally, to estimate the relevance of EBNA2 targets during early EBV infection, we analyzed the proteomes of primary B cells before and after infection with EBV. The protein expression pattern induced upon EBV infection was similar to that following EBNA2 activation. These findings underscore the value of EREB2-5 cells as an appropriate model system for the analysis of early events in the process of EBV-mediated B-cell immortalization.

Identification of Epstein-Barr Virus RK-BARF0-Interacting Proteins and Characterization of Expression Pattern

Identification of Epstein-Barr Virus RK-BARF0-Interacting Proteins and Characterization of Expression Pattern

Microdissection combined with the polymerase chain reaction to identify potentiating viral co-infection in patients with HIV/AIDS with ocular infection

Background: In the presence of several coexisting infections, superimposed tissue necrosis or tissue metaplasia, it may be difficult to recognize standard histologic morphology on hematoxylin-eosin slides. Tissue microdissection combined with the polymerase chain reaction (PCR-MD) offers the advantages of high specificity and relative speed. The objective of this study was to describe the use of PCR-MD in identifying potentiating viral co-infection in patients with HIV/AIDS with retinitis and choroiditis.Methods: Eyes from two patients with HIV/AIDS with several ocular infections were studied by a variety of techniques, including standard histologic examination, immunochemistry, electron microscopy and in situ hybridization. PCR-MD was used to identify coexisting viral infections.Results: Histologic examination showed cytomegalovirus retinitis in both cases. Use of PCR-MD allowed the identification of Epstein-Barr virus within a zone of fulminant varicella-zoster virus retinitis in one patient. PCR-MD confirmed the presence of human herpesvirus 8 in the second patient, who had ocular lymphoma.Interpretation: PCR-MD can be used to demonstrate coexisting viral infection in ocular specimens from patients with unusually fulminant courses. Co-infections may contribute to the observed clinical course and should be considered in patients with rapid progression or unusual presentation.

No Significant Association of Epstein-Barr Virus Infection with Invasive Breast Carcinoma

We studied 48 cases of invasive breast carcinoma for evidence of Epstein-Barr virus (EBV), which is associated with many human malignancies. In situ hybridization studies to detect the presence of EBV-encoded small nonpolyadenylated RNA (EBER)-1 were performed in paraffin sections. Immunohistochemical studies to detect EBV nuclear antigen (EBNA)-1, latent membrane protein (LMP)-1, and the transactivating immediate-early BZLF1 (ZEBRA) protein were also performed in paraffin sections. The presence of EBV genomic DNA was studied by polymerase chain reaction (PCR) amplification using sets of primers flanking the EBNA-4 and the EBV-LMP-1 genes in frozen tissues. Southern blot analysis using a probe flanking the EBV terminal repeat region was then attempted in cases that were PCR-positive. Five of 48 cases (10%) of breast carcinoma showed focal EBER-positive tumor cells. Twelve cases (25%) were positive for EBNA-1 by immunohistochemistry, all but one different from the EBER-positive cases. None of the cases were positive for LMP-1 or ZEBRA protein by immunohistochemistry. PCR studies for EBNA-4 and LMP-1 were each positive in five cases (including three cases in common). However, Southern blot studies successfully performed in all but one of the PCR-positive cases were completely negative. The identification of EBV by any methodology was not correlated with tumor size, grade, or lymph node status. This study demonstrated evidence of EBV infection in tissues involved by invasive breast carcinomas in a significant subset of cases. However, the lack of localization of EBV infection to a significant population of the tumor cells in any case, the negativity by Southern blot hybridization, and the lack of expression of multiple antigens in any case strongly argue against a significant role for EBV in the pathogenesis of breast carcinoma.

Evidence for a complement-mediated inhibition and an antibody-dependent cellular cytotoxicity of dermal fibroblasts in alopecia areata.

It has been suggested that prolonged antigenic stimulation contributes to the development of epidermotropic cutaneous T cell lymphoma (CTCL), mycosis fungoides and Sézary syndrome, characterized by a cutaneous infiltration of proliferating helper T cells. Since Epstein-Barr virus (EBV) antibodies were increased in CTCL sera, we investigated a possible etiologic role for EBV in epidermotropic CTCL by looking for the EBV genome in 25 cutaneous biopsies of mycosis fungoides or Sézary syndrome and 12 reactional inflammatory skin lesions. The use of a non-isotopic in situ hybridization procedure based on the detection of Epstein-Barr encoded RNAs with biotinylated oligonucleotide probes (EBER) revealed 32% of the lesions with CTCL to be positive for EBV (3 in dermis, 3 in epidermis, 2 both in dermis and epidermis), as compared to no detection of the EBV genome in the reactional inflammatory skin lesions. Moreover, a combined hybridization (EBER probe) and immunochemistry technique (anti-CD3 or anti-Kil monoclonal antibody) permitted the identification of EBV in T cells of dermis and in keratinocytes, respectively. The identification of EBV in epidermotropic CTCL suggests that this virus could play a role in the development of these CTCLs, either as an etiological agent or more probably as a chronic activating agent. Indeed, the infection of keratinocytes by EBV could activate them and so induce the production of in situ cytokines (IL1a, IL6, TNFa) playing a role in the development of tumoral infiltrate.