EBV Genome Research Articles

EBV + B cell-derived exosomes promote EBV-associated T/NK-cell lymphoproliferative disease immune evasion by STAT3/IL-10/PD-L1 pathway.

EBV-associated T/NK-cell lymphoproliferative diseases (EBV-T/NK-LPDs) are characterized by the clonal proliferation of EBV-positive ( +) T/NK cells. EBV is typically latent in B cells and the mechanism by which the EBV genome invades T/NK cells remains unknown. Recent studies have demonstrated that exosomes derived from EBV + B cells play a pivotal role in immunosuppressive microenvironment remodeling. Moreover, the existence of an immunosuppressive microenvironment is known to be critical in the development of EBV-T/NK-LPDs. Hence, we hypothesized that exosomes derived from EBV + B cells might promote the development of EBV-T/NK-LPDs by stimulating immune evasion. In this study, we utilized paraffin sections to clarify the STAT3/IL-10/PD-L1-associated immunosuppressive microenvironment in EBV-T/NK-LPDs. Further, we extracted exosomes from BL2009 (EBV + B cell lymphoma) and CA46 (EBV- B cell lymphoma) cell lines to co-culture with cutaneous T-cell lymphoma (CTCL) cell lines, to verify the changes in the above immune evasion pathway. The paraffin sections of EBV-T/NK-LPDs showed high-expression levels of IL-10/PD-L1, which might be related to the phosphorylation of STAT3. Exosomes derived from EBV + B cells could significantly activate the STAT3/IL-10/PD-L1 pathway. After being treated with C188-9, EBV + B cell-derived exosomes were no longer able to stimulate the expression of IL-10/PD-L1 in CTCL cells. EBV-T/NK-LPDs have a STAT3/IL-10/PD-L1 overactivation-associated immunosuppressive microenvironment. Our study elucidated part of this mechanism. Exosomes derived from EBV + B could induce phosphorylation of STAT3 in CTCL cells, leading to the overexpression of IL-10/PD-L1. Our findings might shed light on new directions for understanding immune evasion in EBV-T/NK-LPDs.

Geographic EBV variants confound disease-specific variant interpretation and predict variable immune therapy responses

AbstractEpstein-Barr virus (EBV) is a potent carcinogen linked to hematologic and solid malignancies and causes significant global morbidity and mortality. Therapy using allogeneic EBV-specific lymphocytes shows promise in certain populations, but the impact of EBV genome variation on these strategies remains unexplored. To address this, we sequenced 217 EBV genomes, including hematologic malignancies from Guatemala, Peru, Malawi, and Taiwan, and analyzed them alongside 1307 publicly available EBV genomes from cancer, nonmalignant diseases, and healthy individuals across Africa, Asia, Europe, North America, and South America. These included, to our knowledge, the first natural killer (NK)/T-cell lymphoma (NKTCL) EBV genomes reported outside of East Asia. Our findings indicate that previously proposed EBV genome variants specific to certain cancer types are more closely tied to geographic origin than to cancer histology. This included variants previously reported to be specific to NKTCL but were prevalent in EBV genomes from other cancer types and healthy individuals in East Asia. After controlling for geographic region, we did identify multiple NKTCL-specific variants associated with a 7.8-fold to 21.9-fold increased risk. We also observed frequent variations in EBV genomes that affected peptide sequences previously reported to bind common major histocompatibility complex alleles. Finally, we found several nonsynonymous variants spanning the coding sequences of current vaccine targets BALF4, BKRF2, BLLF1, BXLF2, BZLF1, and BZLF2. These results highlight the need to consider geographic variation in EBV genomes when devising strategies for exploiting adaptive immune responses against EBV-related cancers, ensuring greater global effectiveness and equity in prevention and treatment.

Regulation mechanism of EBV-encoded EBER1 and LMP2A on YAP1 and the impact of YAP1 on the EBV infection status in EBV-associated gastric carcinoma

This study aims to explore the role and regulatory mechanism of Yes-associated protein 1 (YAP1) in the development of Epstein-Barr virus-associated gastric cancer (EBVaGC). Here we showed that EBV can upregulate the expression and activity of YAP1 protein through its encoded latent products EBV-encoded small RNA 1 (EBER1) and latent membrane protein 2A (LMP2A), enhancing the malignant characteristics of EBVaGC cells. In addition, we also showed that overexpression of YAP1 induced the expression of EBV encoding latent and lytic phase genes and proteins in the epithelial cell line AGS-EBV infected with EBV, and increased the copy number of the EBV genome, while loss of YAP1 expression reduced the aforementioned indicators. Moreover, we found that YAP1 enhanced EBV lytic reactivation induced by two known activators, 12-O-tetradecanoylhorbol-13-acetate (TPA) and sodium butyrate (NaB). These results indicated a bidirectional regulatory mechanism between EBV and YAP1 proteins, providing new experimental evidence for further understanding the regulation of EBV infection patterns and carcinogenic mechanisms in gastric cancer.

Antibody Profiling of Pan-Cancer Viral Proteome Reveals Biomarkers for Nasopharyngeal Carcinoma Diagnosis and Prognosis

Diagnosing, predicting disease outcome, and identifying effective treatment targets for virus-related cancers are lacking. Protein biomarkers have the potential to bridge the gap between prevention and treatment for these types of cancers. While it has been shown that certain antibodies against EBV proteins could be used to detect nasopharyngeal carcinoma (NPC), antibodies targeting are solely a tiny part of the about 80 proteins expressed by the EBV genome. Furthermore, it remains unclear what role other viruses play in NPC since many diseases are the result of multiple viral infections. For the first time, this study measured both IgA and IgG antibody responses against 646 viral proteins from 23 viruses in patients with NPC and control subjects using nucleic acid programmable protein arrays. Candidate seromarkers were then validated by ELISA using 1665 serum samples from three clinical cohorts. We demonstrated that the levels of five candidate seromarkers (EBV-BLLF3-IgA, EBV-BLRF2-IgA, EBV-BLRF2-IgG, EBV-BDLF1-IgA, EBV-BDLF1-IgG) in NPC patients were significantly elevated than controls. Additional examination revealed that NPC could be successfully diagnosed by combining the clinical biomarker EBNA1-IgA with the five anti-EBV antibodies. The sensitivity of the six-antibody signature at 95% specificity to diagnose NPC was comparable to the current clinically-approved biomarker combination, VCA-IgA, and EBNA1-IgA. However, the recombinant antigens of the five antibodies are easier to produce and standardize compared to the native viral VCA proteins. This suggests the potential replacement of the traditional VCA-IgA assay with the 5-antibodies combination to screen and diagnose NPC. Additionally, we investigated the prognostic significance of these seromarkers titers in NPC. We showed that NPC patients with elevated BLLF3-IgA and BDLF1-IgA titers in their serum exhibited significantly poorer disease-free survival, suggesting the potential of these two seromarkers as prognostic indicators of NPC. These findings will help develop serological tests to detect and treat NPC in the future.

EBV DNA methylation profiles and its application in distinguishing nasopharyngeal carcinoma and nasal NK/T-cell lymphoma

BackgroundAs an oncovirus, EBV is associated with multiple cancers, including solid tumors and hematological malignancies. EBV methylation plays an important role in regulating tumor occurrence. However, the EBV methylation profiles in EBV-associated tumor tissues are poorly understood.ResultsIn this study, EBV methylation capture sequencing was conducted in several different tumor tissue samples, including NPC, EBVaGC, lung LELC and parotid LELC. Besides, EBV capture sequencing and following qMSP were performed on nasopharyngeal brushing samples from NPC and nasal NKTCL patients. Our results showed that the EBV genome among different types of tumors displayed specific methylation patterns. Among the four types of tumors from epithelial origin (NPC, EBVaGC, lung LELC and parotid LELC), the most significant differences were found between EBVaGC and the others. For example, in EBVaGC, all CpG sites within 1,44,189–1,45,136 bp of the EBV genome sequence on gene RPMS1 were hyper-methylated compared to the others. Differently, significant differences of EBV CpG sites, particularly those located on gene BILF2, were observed between NPC and nasal NKTCL patients in nasopharyngeal brushing samples. Further, the methylated level of BILF2 was further detected using qMSP, and a diagnostic model distinguishing NPC and nasal NKTCL was established. The AUC of the model was 0.9801 (95% CI 0.9524–1.0000), with the sensitivity and specificity of 98.81% (95% CI 93.63–99.94%) and 76.92% (95% CI 49.74–91.82%), respectively.ConclusionsOur study reveals more clues for further understanding the pathogenesis of EBV, and provides a possibility for distinguishing EBV-related tumor by detecting specific EBV CpG sites.

Epstein‐Barr virus in tonsillar tissue of Iranian children with tonsillar hypertrophy: Quantitative measurement by real‐time PCR

AbstractBackground and ObjectivesEpstein‐Barr virus (EBV) infection is ubiquitous all around the world. Tonsils seem to be candidate replication sites for EBV, and these tissues can be infected acutely or chronically. Some studies reported an association between EBV infection and tonsillar hypertrophy. In this study, we aimed to evaluate the presence and copy number of the EBV genome in tonsil tissue specimens of patients with tonsillar hypertrophy.MethodsA cross‐sectional study was performed on 50 fresh tonsil tissue samples from children, who underwent tonsillectomy because of tonsillar hypertrophy. Patients' tonsil tissues were evaluated using real‐time polymerase chain reaction for EBV genome and viral load. Finally, the results were analyzed using SPSS software.ResultsEBV genome was detected in 58% (29/50) of tonsillar tissues. The relationship between EBV genome detection rate and age groups was in the statistical significance range (P = 0.051). Among 29 positive cases, the average EBV viral load was (3.1 × 105) copy/g ± (0.5 × 105) copy/g. No significant difference was observed among different sex and age groups for EBV viral load.ConclusionHerein, EBV genome detection could support the colonization of EBV in the tonsils, which may have a direct or indirect association with the pathogenesis of tonsillar hypertrophy.

A distinct isoform of lymphoid enhancer binding factor 1 (LEF1) epigenetically restricts EBV reactivation to maintain viral latency.

As a human tumor virus, EBV is present as a latent infection in its associated malignancies where genetic and epigenetic changes have been shown to impede cellular differentiation and viral reactivation. We reported previously that levels of the Wnt signaling effector, lymphoid enhancer binding factor 1 (LEF1) increased following EBV epithelial infection and an epigenetic reprogramming event was maintained even after loss of the viral genome. Elevated LEF1 levels are also observed in nasopharyngeal carcinoma and Burkitt lymphoma. To determine the role played by LEF1 in the EBV life cycle, we used in silico analysis of EBV type 1 and 2 genomes to identify over 20 Wnt-response elements, which suggests that LEF1 may bind directly to the EBV genome and regulate the viral life cycle. Using CUT&RUN-seq, LEF1 was shown to bind the latent EBV genome at various sites encoding viral lytic products that included the immediate early transactivator BZLF1 and viral primase BSLF1 genes. The LEF1 gene encodes various long and short protein isoforms. siRNA depletion of specific LEF1 isoforms revealed that the alternative-promoter derived isoform with an N-terminal truncation (ΔN LEF1) transcriptionally repressed lytic genes associated with LEF1 binding. In addition, forced expression of the ΔN LEF1 isoform antagonized EBV reactivation. As LEF1 repression requires histone deacetylase activity through either recruitment of or direct intrinsic histone deacetylase activity, siRNA depletion of LEF1 resulted in increased histone 3 lysine 9 and lysine 27 acetylation at LEF1 binding sites and across the EBV genome. Taken together, these results indicate a novel role for LEF1 in maintaining EBV latency and restriction viral reactivation via repressive chromatin remodeling of critical lytic cycle factors.

Sequence variation of the Epstein-Barr virus nuclear antigen 1 (EBNA1) gene in chronic lymphocytic leukemia and healthy volunteer subjects.

Epstein-Barr virus-related malignancies have been linked to variations in the sequences of EBV genes, notably EBNA1. Therefore, the purpose of this study was to examine the DBD/DD domain and USP7 binding domain sequences at the C-terminus of the EBNA1 gene in patients with chronic lymphocytic leukemia (CLL). This study included 40 CLL patients and 21 healthy volunteers. Using commercial kits, total DNA was extracted from buffy coat samples, and each sample was tested for the presence of the EBV genome. The C-terminus of EBNA1 was then amplified from positive samples, using nested PCR. Sanger sequencing was used to identify mutations in the PCR products, and the results were analyzed using MEGA11 software. The mean ages of CLL patients and healthy individuals were 61.07 ± 10.2 and 59.08 ± 10.3, respectively. In the EBNA-1 amplicons from CLL patients and healthy individuals, 38.5% and 16.7%, respectively, harbored mutations in the DBD/DD domain of the C-terminal region of the EBNA1 gene (P = 0.378). The mutation frequency at locus 97,320 was significantly higher in CLL patients than in healthy individuals (P = 0.039). Three EBV subtypes based on residue 487 were detected. The frequency of alanine, threonine, and valine in both groups was 88, 8, and 4 percent, respectively (P = 0.207). Moreover, all of the isolates from healthy donors had alanine at this position. The findings indicated that the presence of threonine or valine at residue 487 as well as a synonymous substitution at residue 553 in the C-terminal region of EBNA1 might be involved in the pathogenesis of EBV in CLL patients.

FISH for EBV genome in a patient with diffuse large B-cell lymphoma harboring t(14;18)(q32;q21)

FISH for EBV genome in a patient with diffuse large B-cell lymphoma harboring t(14;18)(q32;q21)

Detection of Epstein–Barr virus infection in thymic epithelial tumors by nested PCR and Epstein–Barr-encoded RNA ISH

BackgroundEpstein–Barr virus (EBV) is well known to be associated with a lot of tumors, including lymphoma, nasopharyngeal carcinoma, EBV-associated gastric carcinoma, and some other carcinomas with similar lymphoepithelioma-like features. However, the association between EBV and thymic epithelial tumors (TETs) is inconclusive as reports in this regard are not entirely consistent and the methods employed are of different sensitivity and specificity. The geographical difference of the patients is also one of the reasons for the different points of view.MethodsIn our study, we examined 72 thymomas, including 3 cases of type A thymomas, 27 cases of type AB, 6 cases of type B1, 26 cases of type B2 and 10 cases of type B3 thymomas, and 15 thymic carcinomas to detect the viral genome at both DNA and RNA levels. The genome DNA of fresh tissues was first screened by nested polymerase chain reaction (PCR), which could be regarded as the most sensitive method to detect small amounts of DNA. Then all the tissue blocks were further submitted for viral localization by Epstein–Barr-encoded RNA (EBER) ISH. Group parameters were assessed using the chi-square test at a significance level of p < 0.05.ResultsNested PCR results showed that none of type A, eight (29.6%) type AB, one (16.7%) type B1, fifteen (57.7%) type B2, and four (40.0%) type B3 were positive for EBV genome. However, none of them detected EBER expression except for one case of type B2 thymoma. Fourteen (93.3%) thymic carcinomas were positive for EBV by nested PCR, of which three displayed weak nuclear signals within the tumor cells by EBER ISH.ConclusionsThese results showed that nested PCR was a sensitive method for screening the EBV genome in thymic epithelial tumors. As the malignancy of thymoma increases, the rate of EBV infection became higher. Thymic carcinomas were well associated with the Epstein–Barr virus.There was significant association between the EBV infection rate and thymoma type (p < 0.05). We further analyzed the association between EBV infection and myasthenia gravis. However, it showed no significant difference(p = 0.2754), although the EBV infection rate was higher in the thymomas with myasthenia gravis.

Saliva biopsy: Detecting the difference of EBV DNA methylation in the diagnosis of nasopharyngeal carcinoma.

Saliva sampling is a non-invasive method, and could be performed by donors themselves. However, there are few studies reporting biomarkers in saliva in the diagnosis of NPC. A total of 987 salivary samples were used in this study. First, EBV DNA methylation was profiled by capture sequencing in the discovery cohort (n = 36). Second, a q-PCR based method was developed and five representative EBV DNA CpG sites (11 029 bp, 45 849 bp, 57 945 bp, 66 226 bp and 128 102 bp) were selected and quantified to obtain the methylated density in the validation cohort1 (n = 801). Third, a validation cohort2 (n = 108) was used to further verify the differences of EBV methylation in saliva. A significant increase of EBV methylation was found in NPC patients compared with controls. The methylated score of EBV genome obtained by capture sequencing could distinguish NPC from controls (sensitivity 90%, specificity 100%). Further, the methylated density of EBV DNA CpG sites revealed by q-PCR showed a good diagnostic performance. The sensitivity and specificity of detecting a single CpG site (11 029 bp) could reach 75.4% and 99.7% in the validation cohort1, and 78.2% and 100% in the validation cohort2. Besides, the methylated density of the CpG site was found to decrease below the COV in NPC patients after therapy, and increase above the COV after recurrence. Our study provides an appealing alternative for the non-invasive detection of NPC without clinical setting. It paves the way for conducting a home-based large-scale screening in the future.

Epstein-Barr virus in breast carcinoma and in triple negative cases impact on clinical outcomes.

The contribution of viral infection in tumors pathogenesis has currently attracted attention. Epstein-Barr virus is an infectious agent involved in numerous human malignancies, including breast cancer. Although, their prognostic impact in breast tumor is rarely investigated. Therefore, we sought in our study to evaluate the prevalence of EBV in Tunisian breast carcinoma and to examine their potential association with clinicopathological features and overall survival. Our retrospective study included 100 formalin fixed paraffin embedded samples from Tunisian breast carcinoma. EBV infection was evaluated by immunohistochemical analysis, using monoclonal antibody against latent membrane protein 1 (LMP-1) and polymerase chain reaction. A subset of PCR positive specimens was subjected to in situ hybridization for the detection of EBER expression. Biomarker's expression was evaluated by immunohistochemistry method. Statistical analysis was also explored. The expression status of ER, PR and HER2 was 81%, 71.4% and 33.7% respectively. The triple negative profile was present in 10.84% of cases. LMP-1 expression was negative in all breast cancer specimens. PCR assay showed that 44% of patients were positive for EBV genome. None of the 15 PCR positive cases showed positive results for EBV by ISH. According to the molecular phenotype, there was a statistically significant difference in EBV DNA prevalence between breast cancer subgroups including TN (67%), Lum B (64%), HER2+(50%) and Lum A (30%). Bivariate analysis showed that EBV DNA was significantly associated with HER2+(p=0.035), tumor size (p=0.018) and high SBR grade (p=0.009). Multiple logistic regression analysis confirms the positive correlation of EBV with tumor size (p=0.048) and SBR grade (p=0.042). Kaplan-Meier analysis showed that patients with EBV+had significantly shorter overall survival than those with EBV- (p=0.032). Our study demonstrated the presence of EBV DNA in Tunisian breast carcinoma. EBV DNA was associated with aggressive features and poor overall survival. Further investigations will be required in large samples size to clarify the potential role of EBV in breast tumor progression.

Epstein-Barr virus-associated lymphoproliferative disorders after BNT162b2 mRNA COVID-19 vaccination

Epstein-Barr virus-associated lymphoproliferative disorders (EBV-LPD) is a rare disease characterized by persistent or recurrent inflammation accompanied by EBV infection of T or NK cells that is not self-limiting, and it is fatal, if untreated. After receiving the first dose of the BNT162b2 mRNA COVID-19 vaccine, a 79-year-old male presented to the hospital with a 2-week history of fever. Laboratory results indicated pancytopenia, elevated liver transaminase levels, hyperferritinemia, and hypofibrinogenemia. Computed tomography revealed hepatosplenomegaly, but lymphadenopathy was not observed. A bone marrow biopsy, a random skin biopsy, and a liver biopsy revealed no malignancy, but an infectious evaluation revealed EBV viremia (5.19 Log IU/ml). Flow cytometry and RT-PCR revealed that the EBV genome was localized in NK cells, suggesting the diagnosis of EBV-NK-LPD. We administered prednisolone, intravenous immunoglobulin, and etoposide, but the EBV-DNA load failed to decrease, and he died 2 months later. Recently, case reports of COVID-19 vaccination-related hemophagocytic lymphohistiocytosis have been published. Although the mechanisms and risk factors for EBV-LPD after BNT162b2 mRNA COVID-19 vaccination remain unknown, it is important to note the possibility of reactivation of EBV after COVID-19 vaccination to initiate early and targeted therapy.

Epstein-barr virus/Helicobacter pylori coinfection and gastric cancer: the possible role of viral gene expression and shp1 methylation.

Among the various factors involved in the development of gastric cancer (GC), infectious agents are one of the most important causative inducers. This study aimed to investigate the possible role of EBV gene expression on SHP1 methylation in co-infection with Helicobacter pylori in patients with GC. Formalin-fixed paraffin-embedded samples were obtained from 150 patients with gastrointestinal disorders. The presence of the H. pylori and EBV genome were examined by PCR. The expression level of viral gene transcripts and methylation status of the SHP1 cellular gene was assessed by quantitative real-time PCR and methyl-specific PCR. EBV and H. pylori coinfection were reported in 5.6% of patients. The mean DNA viral load was significant in patients coinfected with cagA-positive H. pylori (P= 0.02). The expression of BZLF1 and EBER was associated with GC. Also, the expression level of BZLF1in GC tissues was significantly higher in coinfection (P = 0.01). SHP1 methylation frequency was higher in the GC group than in the control group (P = 0.04). The correlation between the methylation rate and the H. pylori infection was highly significant (P<0.0001). The strongest positive correlation was observed in GC specimens between SHP1 methylation and H. pylori cagA-positive strains (p= 0.003). Our results suggested that cagA might involve in the elevation of EBV lytic gene expression and SHP1 methylation, and the development of gastric cancer. Understanding the mechanism of EBV H. pylori - cagA + coinfection, as well as host epigenetic changes, can play an important role in diagnosing and preventing gastric cancer.

Epstein-Barr Virus Detection in the Central Nervous System of HIV-Infected Patients.

Simply detecting Epstein–Barr virus deoxyribonucleic acid (EBV-DNA) is insufficient to diagnose EBV-associated diseases. The current literature around EBV-DNA detection from cerebrospinal fluid (CSF) in human immunodeficiency virus (HIV)-positive non-lymphoma patients was systematically reviewed and a meta-analysis reporting the estimated pooled prevalence in this population when PCR methods are employed, targeting different sequence segments within the EBV genome, was conducted. Using a combination of three key concepts—Epstein–Barr virus detection, central nervous system disease, and human cerebrospinal fluid—and their MeSH terms, the PubMed database was searched. A total of 273 papers reporting the detection of EBV in CNS were screened, of which 13 met the inclusion criteria. The meta-analysis revealed a pooled prevalence of EBV-DNA in CSF of 20% (CI: 12–31%). The highest pooled prevalence was from studies conducted on the African population at 39% (CI: 27–51%). The investigation of the presence of EBV-DNA in the CSF was also very varied, with several gene targets used. While most patients from the articles included in this review and meta-analysis were symptomatic of CNS disorders, the pathogenicity of EBV in non-lymphoma HIV patients when detected in CSF has still not been determined. The presence of EBV-DNA in the CNS remains a concern, and further research is warranted to understand its significance in causing CNS disorders.

Abstract 774: Microdissected gene expression profiling of recurrent nasopharyngeal carcinoma

Abstract Nasopharyngeal cancer (NPC) is an EBV-driven epithelial carcinoma with a recurrence rate of 30%. Biological pathways and predictive markers for recurrence remain understudied. Further, NPC tumors are associated with an intense inflammatory infiltrate, masking gene expression signals from the tumor epithelial compartment. We perform laser-capture microdissection on a preliminary cohort of 12 tumors, including primary tumors from recurrent and non-recurrent cases matched for stage of disease. Smart-3SEQ, a novel 3’ end RNA-Seq technique which allows for the accurate quantification of transcript abundance in FFPE samples, was performed to obtain gene expression libraries. Tumor epithelial and microenvironment regions from were separately microdissected, giving a total of 42 histologically-pure gene expression libraries. Raw reads were mapped to the human hg19 and EBV genomes and gene expression counts were obtained with Subread. Differential gene expression and gene set enrichment was performed in R with DESeq2 and fgsea respectively. We observed that primary NPC tumors that did not recur were significantly enriched for immune processes, including leukocyte mediated immunity, innate immune response and lymphocyte activation (padj &amp;lt; 1E-9 for all). The top differentially expressed genes in this group included mediators of inflammation such as STAT1 (padj = 0.0204). Critically, these gene expression changes were observed within the tumor epithelial compartment. In contrast, primary NPC tumors that eventually recurred were enriched for known oncogenes including BCL2 (padj = 0.000420). These findings suggest that heterogeneity in gene expression between NPC tumors play an important role in treatment response. Further work to define gene expression subtypes of NPC tumors will be helpful for patient stratification and personalized care. Citation Format: Joshua Tay, Wei Keat Teo, Chuan Keng Goh, Bing Cheng Wu, Kwok Seng Loh. Microdissected gene expression profiling of recurrent nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 774.

Facts and Hopes in the Relationship of EBV with Cancer Immunity and Immunotherapy.

Epstein-Barr virus (EBV), the first identified human tumor virus, infects and takes up residency in almost every human. However, EBV genome-positive tumors arise in only a tiny minority of infected people, presumably when the virus-carrying tumor cells are able to evade immune surveillance. Traditional views regard viral antigens as the principal targets of host immune surveillance against virus-infected cells. However, recent findings indicate that EBV-infected/-transformed B cells elicit both cytotoxic CD8+ and CD4+ T-cell responses against a wide range of overexpressed cellular antigens known to function as tumor-associated antigens (TAA), in addition to various EBV-encoded antigens. This not only broadens the ways by which the immune system controls EBV infection and prevents it from causing cancers, but also potentially extends immune protection toward EBV-unrelated cancers by targeting shared TAAs. The goal of this review is to incorporate these new findings with literature data and discuss future directions for improved understanding of EBV-induced antitumor immunity, as well as the hopes for rational immune strategies for cancer prevention and therapy.

Mono a Mano: ZBP1's Love-Hate Relationship with the Kissing Virus.

Z-DNA binding protein (ZBP1) very much represents the nuclear option. By initiating inflammatory cell death (ICD), ZBP1 activates host defenses to destroy infectious threats. ZBP1 is also able to induce noninflammatory regulated cell death via apoptosis (RCD). ZBP1 senses the presence of left-handed Z-DNA and Z-RNA (ZNA), including that formed by expression of endogenous retroelements. Viruses such as the Epstein–Barr “kissing virus” inhibit ICD, RCD and other cell death signaling pathways to produce persistent infection. EBV undergoes lytic replication in plasma cells, which maintain detectable levels of basal ZBP1 expression, leading us to suggest a new role for ZBP1 in maintaining EBV latency, one of benefit for both host and virus. We provide an overview of the pathways that are involved in establishing latent infection, including those regulated by MYC and NF-κB. We describe and provide a synthesis of the evidence supporting a role for ZNA in these pathways, highlighting the positive and negative selection of ZNA forming sequences in the EBV genome that underscores the coadaptation of host and virus. Instead of a fight to the death, a state of détente now exists where persistent infection by the virus is tolerated by the host, while disease outcomes such as death, autoimmunity and cancer are minimized. Based on these new insights, we propose actionable therapeutic approaches to unhost EBV.

Methyl-qPCR: a new method to investigate Epstein\u2013Barr virus infection in post-transplant lymphoproliferative diseases

Epstein–Barr virus DNA viral load is used as a surrogate marker to start Rituximab in transplant recipients at risk of developing PTLD. However, an elevated EBV DNAemia does not discriminate lymphoproliferation and replication. We designed a new molecular assay (methyl-qPCR) to distinguish methylated versus unmethylated viral genomes. In blood, viral genomes were highly methylated in EBV primary infections, PTLD and 4/5 transplant recipients with high viral load. The only patient with under-methylated EBV genomes did not respond to rituximab. Methyl-qPCR is a convenient method to discriminate between latent and lytic EBV genomes and could be useful in treatment decisions.

The presence of cardiotropic viral genomes is not increased in atrial tissue of atrial fibrillation patients.

BackgroundInfections with potentially cardiotropic viruses are associated with the development of atrial fibrillation (AF). However, whether direct viral infection of the atria is involved in the pathogenesis of AF is unclear. We have therefore analysed the presence of cardiotropic viral genomes in AF patients.MethodsSamples of left atrial tissue were obtained from 50 AF patients (paroxysmal, n = 20; long-standing persistent/permanent, n = 30) during cardiac surgery and from autopsied control patients (n = 14). Herein, the presence of PVB19, EBV, CMV, HHV‑6, adenovirus and enterovirus genomes was determined by polymerase chain reaction. The densities of CD45+ and CD3+ cells and fibrosis in the atria were quantified by (immuno)histochemistry.ResultsOf the tested viruses only the PVB19 genome was detected in the atria of 10% of patients, paroxysmal AF (2 of 20) and long-standing persistent/permanent AF (3 of 30). Conversely, in 50% of controls (7 of 14) PVB19 genome was found. No significant association was found between PVB19 and CD45+ and CD3+ cells, or between the presence of PVB19 and fibrosis, in either control or AF patients.ConclusionThe presence of viral genomes is not increased in the atria of AF patients. These results do not support an important role for viral infection of the atria in the pathogenesis of AF.Supplementary InformationThe online version of this article (10.1007/s12471-022-01660-4) contains supplementary material, which is available to authorized users.