EBV Strains Research Articles

Distribution of Epstein-Barr virus in the oral cavity of Thais with various oral mucosal conditions

ObjectivesWe aimed to examine the presence of EBV, EBV strains, and variants among 3 oral conditions including normal oral mucosa (NOM), oral potentially malignant disorders/oral cancer (OPMDs/OC) and non-OPMDs/OC in a group of Thais. Material and methodsOral exfoliated cells were obtained from 315 participants living in the northeastern and central regions of Thailand. The participants were divided into 3 groups encompassing the NOM, the OPMDs/OC and the non-OPMDs/OC groups. The presence of EBV was first determined by PCR using primers for LMP1 gene. Subsequently, EBV strains of EBNA3c and variants based on LMP1 sequences were determined by real-time PCR. ResultsThe prevalence of EBV in OPMDs/OC, non-OPMDs/OC and NOM were 72.0 %, 56.2 %, and 27.2 % respectively. EBV type A, B and AB were found in 52.1 %, 32.1 % and 15.8 % of all positive samples, respectively. The percentage of participants with EBV type A was more prominent in the NOM group (72.0 %) compared to the non-OPMDs/OC (54.8 %) and the OPMDs/OC group (41.8 %) whereas EBV type B was higher in the OPMDs/OC group (35.8 %) compared to the non-OPMDs/OC (31.5 %) and the NOM (24.0 %) groups. Regarding EBV variants, 30-bp deletion LMP1 variant (del-LMP1) which is more associated with malignant transformation was predominately found in the OPMDs/OC (32.8 %) and the non-OPMDs/OC (38.4 %) groups compared to the NOM group (20.0 %). ConclusionsHigh frequency of EBV was demonstrated in the OPMDs/OC group. EBV type A was more predominant in the NOM group whereas EBV type B was more prevalent in the OPMDs/OC group. The del-LMP1 variant was more common in the OPMDs/OC and the non-OPMDs/OC groups.

Effects of COVID-19 and vaccination on the human immune system: cases of lymphopenia and autoimmunity

Effects of COVID-19 and vaccination on the human immune system: cases of lymphopenia and autoimmunity

Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells.

Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.

Maternal HIV Infection as a Risk Factor for Primary Epstein-Barr Virus Infection in Kenyan Infants.

Human immunodeficiency virus (HIV) infection is known to be associated with EBV shedding in saliva suggesting an increased risk of EBV transmission to infants born to mothers with HIV at an earlier age. In this study we investigated (i) whether maternal HIV status was a risk factor for EBV in blood at delivery or for shedding in saliva and breast milk of 6- and 10-weeks post-partum mothers, (ii) if there was a difference in EBV strains shed between HIV+ and HIV- mothers, and (iii) if maternal HIV status was a determinant of EBV viral load in their infants. Samples were collected as part of a prospective cohort study that followed HIV-positive (HIV+) and HIV-negative (HIV-) pregnant women in Western Kenya through delivery and post-partum period. EBV viral load in blood was found to be significantly higher in mothers with HIV (p-value = 0.04). Additionally, a statistically significant difference was observed between EBV viral load in saliva samples and HIV status where HIV+ mothers had a higher EBV viral load in saliva at 6-weeks post-partum compared to HIV- mothers (p-value < 0.01). The difference in EBV shedding in breast milk was not found to be statistically significant. Furthermore, no difference in frequency of EBV strain was attributable to HIV- or HIV+ mothers. Interestingly, we found that infants born to HIV+ mothers had a higher EBV viral load at the time of their first EBV detection in blood than infants born to HIV- mothers and this was independent of age at detection. Overall, our study suggests that HIV infected mothers shed more virus in saliva than HIV-negative mothers and infants born to HIV+ mothers were at risk for loss of control of primary EBV infection as evidenced by higher EBV viral load following primary infection.

The genomic architecture of EBV and infected gastric tissue from precursor lesions to carcinoma

BackgroundEpstein-Barr virus (EBV)-associated gastric carcinomas (EBVaGCs) present unique molecular signatures, but the tumorigenesis of EBVaGCs and the role EBV plays during this process remain poorly understood.MethodsWe applied whole-exome sequencing, EBV genome sequencing, and whole-genome bisulfite sequencing to multiple samples (n = 123) derived from the same patients (n = 25), which covered saliva samples and different histological stages from morphologically normal epithelial tissues to dysplasia and EBVaGCs. We compared the genomic landscape between EBVaGCs and their precursor lesions and traced the clonal evolution for each patient. We also analyzed genome sequences of EBV from samples of different histological types. Finally, the key molecular events promoting the tumor evolution were demonstrated by MTT, IC50, and colony formation assay in vitro experiments and in vivo xenograft experiments.ResultsOur analysis revealed increasing mutational burden and EBV load from normal tissues and low-grade dysplasia (LD) to high-grade dysplasia (HD) and EBVaGCs, and oncogenic amplifications occurred late in EBVaGCs. Interestingly, within each patient, EBVaGCs and HDs were monoclonal and harbored single-strain-originated EBV, but saliva or normal tissues/LDs had different EBV strains from that in EBVaGCs. Compared with precursor lesions, tumor cells showed incremental methylation in promotor regions, whereas EBV presented consistent hypermethylation. Dominant alterations targeting the PI3K-Akt and Wnt pathways were found in EBV-infected cells. The combinational inhibition of these two pathways in EBV-positive tumor cells confirmed their synergistic function.ConclusionsWe portrayed the (epi) genomic evolution process of EBVaGCs, revealed the extensive genomic diversity of EBV between tumors and normal tissue sites, and demonstrated the synergistic activation of the PI3K and Wnt pathways in EBVaGCs, offering a new potential treatment strategy for this disease.

A comprehensive analysis of genetic diversity of EBV reveals potential high-risk subtypes associated with nasopharyngeal carcinoma in China.

Epstein–Barr virus (EBV), a widespread oncovirus, is associated with multiple cancers including nasopharyngeal carcinoma (NPC), gastric cancer and diverse lymphoid malignancies. Recent studies reveal that specific EBV strains or subtypes are associated with NPC development in endemic regions. However, these NPC specific subtypes were only identified in a portion of infected individuals due possibly to the limited samples size studied or the complicated population structures of the virus. To identify additional high-risk EBV subtypes, we conducted a comprehensive genetic analysis of 22 critical viral proteins by using the largest dataset of 628 EBV genomes and 792 sequences of single target genes/proteins from GenBank. The phylogenetic, principal component and genetic structure analyses of these viral proteins were performed through worldwide populations. In addition to the general Asia-Western/Africa geographic segregation, population structure analysis showed a ‘Chinese-unique’ cluster (96.57% isolates from China) was highly enriched in the NPC patients, compared to the healthy individuals (89.6% vs. 44.5%, P < 0.001). The newly identified EBV subtypes, which contains four Chinese-specific NPC-associated amino acid substitutions (BALF2 V317M, BNRF1 G696R, V1222I and RPMS1 D51E), showed a robust positive association with the risk of NPC in China (Odds Ratio = 4.80, 20.00, 18.24 and 32.00 for 1, 2, 3 and 4 substitutions, respectively, Ptrend <0.001). Interestingly, the coincidence of positively selected sites with NPC-associated substitutions suggests that adaptive nonsynonymous mutation on critical proteins, such as BNRF1, may interact with host immune system and contribute to the carcinogenesis of NPC. Our findings provide a comprehensive overview of EBV genetic structure for worldwide populations and offer novel clues to EBV carcinogenesis from the aspect of evolution.

The Role of Epstein-Barr Virus in Adults With Bronchiectasis: A Prospective Cohort Study.

BackgroundEpstein-Barr virus (EBV) is implicated in the progression of chronic obstructive pulmonary disease. We aimed to determine whether EBV correlates with bronchiectasis severity, exacerbations, and progression.MethodsWe collected induced sputum in healthy controls and spontaneous sputum at 3–6-month intervals and onset of exacerbations in bronchiectasis patients between March 2017 and October 2018. EBV DNA was detected with quantitative polymerase chain reaction.ResultsWe collected 442 sputum samples from 108 bronchiectasis patients and 50 induced sputum samples from 50 healthy controls. When stable, bronchiectasis patients yielded higher detection rates of EBV DNA (48.1% vs 20.0%; P = .001), but not viral loads (mean log10 load, 4.45 vs 4.76; P = .266), compared with controls; 64.9% of patients yielded consistent detection status between 2 consecutive stable visits. Neither detection rate (40.8% vs 48.1%; P = .393) nor load (mean log10 load, 4.34 vs 4.45; P = .580) differed between the onset of exacerbations and stable visits, nor between exacerbations and convalescence. Neither detection status nor viral loads correlated with bronchiectasis severity. EBV loads correlated negatively with sputum interleukin-1β (P = .002), CXC motif chemokine-8 (P = .008), and tumor necrosis factor–α levels (P = .005). Patients initially detected with, or repeatedly detected with, EBV DNA had significantly faster lung function decline and shorter time to next exacerbations (both P < .05) than those without. Detection of EBV DNA was unrelated to influenza virus and opportunistic bacteria (all P > .05). The EBV strains detected in bronchiectasis patients were phylogenetically homologous.ConclusionsPatients with detection of EBV DNA have a shorter time to bronchiectasis exacerbations. EBV may contribute to bronchiectasis progression.

Establishing a local bank and comparing efficacies of third party viral-specific cytotoxic T cells (VST) and cytokine induced killer cells (VSCIK)

Background & Aim Reactivation of dormant viruses is not uncommon after allogeneic haematopoietic stem cell transplants (allo-HSCT), which invariably carry risk of opportunistic infections. Although antiviral drugs are effective against a few viruses, drug therapy is limited by refractory strains and toxicities, while matched donor-derived VSTs have been successfully used to treat allo-HSCT patients with viral reactivation and there is promising data that minimally matched 3rd party banked VSTs are also effective and safe. Hence, we seek to establish a compact but effective bank of multi virus-specific cytotoxic cells for antiviral therapy not only for allo-HSCT patients but other immune compromised patients and patients with virus-associated malignancies more common in Asia. Methods, Results & Conclusion Previous studies have identified the common HLA types in Singaporeans: 96% of Singaporeans carry one or more of the common 6 HLA*A, 6 HLA*B and 5 HLA*C alleles. We are recruiting donors with these HLA types and screening their cells for reactivity against CMV, HHV6, AdV, EBV and BKV. Donor cells that express CD137 upon stimulation with commercially available virus-specific peptides will be further cultured with viral peptides, antibodies and cytokines to produce vsCIKs (with anti-CD137, IFNγ, OKT3 and IL2) or VSTs (with IL4 and IL7). Both cell types have, in clinical studies, been shown to be effective in modulating viral infections and elicit minimal or no graft-vs-host disease (GvHD). VsCIKs also have the advantage of retaining non-HLA restricted anti-tumour effects. As expected, both protocols produce a very small subset of naive T cells, which predicts low GvHD potential. Interestingly, vsCIKs consistently kill more target cells than VSTs, although a larger fraction of cells in VST cultures degranulate upon peptide stimulation. This incongruity warrants a more detailed study of the cytokines/enzymes released by the vsCIKs and VSTs, and the mechanisms involved. So far, no donor cells have shown reactivity against EBV or BKV peptides. This is despite a high percentage of EBV carriers among Singaporeans and the successful use of the same peptides in published reports. We are unsure if this is due to the unique repertoire of HLA types or the prevalent EBV strains in the local population. We have successfully generated CMV, HHV6 and AdV-specific vsCIKs and VSTs. However, further screening of viral peptides and mechanistic studies will be required to determine a suitable cell type for the proposed bank.

Abstract 3087: Identification of EBV infection and its life cycle in pan-cancer samples

Abstract Viral infection is associated with 10-15 percent of human cancers and EBV (human herpesvirus 4 (HHV-4)) contributes to cancer development in diverse cancer types. The life cycle of EBV is divided into latent and lytic cycles. Lytic replication cycle begins when early transcription factors are activated, and initiation complex is formed. Subsequently, lytic antigens are expressed once infected cells enter the lytic cycle. But the contribution of each cycle in cancer development is not well known yet. Hence we explored EBV integration and gene expression pattern by analyzing TCGA whole transcriptome sequencing (WTS) data.We investigated the integration of EBV by using FASTQ-formatted TCGA tumor WTS data. We designed a three-step EBV detection workflow. 1) Overall viral integration into human genome sequence was detected by VirusSeq. 2) “Spliced Transcripts Alignment to a Reference” algorithm was used for mapping against a hybrid reference genome of human and EBV. 3) To quantify the expression of EBV-related genes, we applied “RNA-Seq by Expectation Maximization” algorithm to obtain Transcripts Per Million values. Furthermore, we validated this detection workflow in EBV-positive cell lines from the public data repository and analyzed mutation profiles by using Mutation Annotation Format files.We investigated 851 WTS samples over 23 cancer types and scanned viral presentations by mapping against four types of EBV strains (Human HHV-4 complete wild-type, GD1, AG876, and artificial join). Consequently, we detected 88 samples mapped against the hybrid genome, and identified 46 of them were EBV-infected. By performing gene expression analysis, we recognized that 39/46 samples were expressing EBV lytic genes. Three types of cancers (Colorectal adenocarcinoma (COAD), Rectum adenocarcinoma (READ), and Stomach adenocarcinoma (STAD)) showed EBV lytic gene expression. Along with STAD, both BZLF1 and BALF2 were highly expressed in COAD; BZLF1 encodes an early transcription factor, and BALF2 is one of the viral genes constructs the initiation complex. Using our method, we also identified the expression of EBV lytic and latent genes in EBV-positive cell lines, hence validating our results. In addition, we found out an association between mutational events and EBV infection in COAD by profiling mutational signatures: increased C to T transitions and TP53 disruptions in EBV-infected samples. Overall, we could identify biologically relevant EBV-infected cancer samples considering EBV life cycle. We hereby drew two major conclusions based our findings. First, we established an analysis workflow to find out biologically meaningful EBV infection in cancer by quantifying gene expression of composing the EBV life cycle, beyond a simple viral integration. Second, we discovered EBV expression pattern in small number of colon cancer patients. Our integrative data will provide clues for comprehensively understanding of EBV-associated cancers. Citation Format: Hyojin Song, Hogune Im, Sung-Soo Yoon, Youngil Koh. Identification of EBV infection and its life cycle in pan-cancer samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3087.

Role of exosomes as a proinflammatory mediator in the development of EBV-associated lymphoma

AbstractEpstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkin's lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBV-encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-α, and arginase 1, suggesting the immune regulatory role of BART miRNAs. The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV+ B-cell lymphoma.

Early Epstein-Barr Virus Genomic Diversity and Convergence toward the B95.8 Genome in Primary Infection.

Over 90% of the world's population is persistently infected with Epstein-Barr virus. While EBV does not cause disease in most individuals, it is the common cause of acute infectious mononucleosis (AIM) and has been associated with several cancers and autoimmune diseases, highlighting a need for a preventive vaccine. At present, very few primary, circulating EBV genomes have been sequenced directly from infected individuals. While low levels of diversity and low viral evolution rates have been predicted for double-stranded DNA (dsDNA) viruses, recent studies have demonstrated appreciable diversity in common dsDNA pathogens (e.g., cytomegalovirus). Here, we report 40 full-length EBV genome sequences obtained from matched oral wash and B cell fractions from a cohort of 10 AIM patients. Both intra- and interpatient diversity were observed across the length of the entire viral genome. Diversity was most pronounced in viral genes required for establishing latent infection and persistence, with appreciable levels of diversity also detected in structural genes, including envelope glycoproteins. Interestingly, intrapatient diversity declined significantly over time (P < 0.01), and this was particularly evident on comparison of viral genomes sequenced from B cell fractions in early primary infection and convalescence (P < 0.001). B cell-associated viral genomes were observed to converge, becoming nearly identical to the B95.8 reference genome over time (Spearman rank-order correlation test; r = -0.5589, P = 0.0264). The reduction in diversity was most marked in the EBV latency genes. In summary, our data suggest independent convergence of diverse viral genome sequences toward a reference-like strain within a relatively short period following primary EBV infection.IMPORTANCE Identification of viral proteins with low variability and high immunogenicity is important for the development of a protective vaccine. Knowledge of genome diversity within circulating viral populations is a key step in this process, as is the expansion of intrahost genomic variation during infection. We report full-length EBV genomes sequenced from the blood and oral wash of 10 individuals early in primary infection and during convalescence. Our data demonstrate considerable diversity within the pool of circulating EBV strains, as well as within individual patients. Overall viral diversity decreased from early to persistent infection, particularly in latently infected B cells, which serve as the viral reservoir. Reduction in B cell-associated viral genome diversity coincided with a convergence toward a reference-like EBV genotype. Greater convergence positively correlated with time after infection, suggesting that the reference-like genome is the result of selection.

Inhibition of Ebola Virus by Anti-Ebola miRNAs in silico.

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate transcriptional and posttranscriptional gene regulation of the organisms. miRNA provides immune defense when the body is faced with challenges intracellular agents. miRNA molecules trigger gene silencing in eukaryotic cells. More than 3,000 different human miRNAs (hsa-miRs) have been identified thus far. During ontogenesis, viral or intracellular parasitic infections, miRNAs are differentially expressed to protect the host from intracellular invaders. In a viral infection context, miRNAs have been connected with the interplay between host and pathogen, and occupy a major role in pathogenesis. An in silico approach was used to analyze the four major Ebola Virus genome sequences including the recently characterized Ebola virus responsible for West African epidemic that has killed over 10,000 people. All totaled, 2,543 mature human miRNA sequences were retrieved through an miR-database, and the identification of mature miRNAs were aligned with full length sequences of the four major Ebola viruses via computational tools. We identified 32 miRNAs that exhibited significant inhibitory capacity to block more than one EBV strains. miR-607 showed capacity to quell all four major EBVs. Ten putative miRNAs were found to have near perfect identity at seed sequences with numerous targets of Ebola virus that may completely degrade the viral transcripts. We hypothesize that a miRNA-based vaccine can quell Ebola virus infection. Future approaches will focus on validation of these miRNAs in quelling the Ebola virus to further elucidate their biological functions in primate and other animal models.

The genetic diversity of Epstein-Barr virus in the setting of transplantation relative to non-transplant settings: A feasibility study.

This study examines EBV strains from transplant patients and patients with IM by sequencing major EBV genes. We also used NGS to detect EBV DNA within total genomic DNA, and to evaluate its genetic variation. Sanger sequencing of major EBV genes was used to compare SNVs from samples taken from transplant patients vs. patients with IM. We sequenced EBV DNA from a healthy EBV-seropositive individual on a HiSeq 2000 instrument. Data were mapped to the EBV reference genomes (AG876 and B95-8). The number of EBNA2 SNVs was higher than for EBNA1 and the other genes sequenced within comparable reference coordinates. For EBNA2, there was a median of 15 SNV among transplant samples compared with 10 among IM samples (p = 0.036). EBNA1 showed little variation between samples. For NGS, we identified 640 and 892 variants at an unadjusted p value of 5 × 10(-8) for AG876 and B95-8 genomes, respectively. We used complementary sequence strategies to examine EBV genetic diversity and its application to transplantation. The results provide the framework for further characterization of EBV strains and related outcomes after organ transplantation.

IS8-1 - Current Understanding of the Role of Epstein-Barr Virus in Lymphomagenesis

Ubiquitous EBV infects not only B cells but also T and NK cells, and is associated with various lymphoid malignancies. Since EBV was first isolated from Burkitt lymphoma in 1964, the spectrum of EBV-associated lymphoid malignancies is expanding from Burkitt lymphoma to EBV-positive T- and NK-cell lymphoproliferative disease/lymphoma. The extent of the association to EBV varies with each disease. Some are 100% associated, but others are associated only in 1/4 to 1/3 of patients. This fact suggests that EBV is not absolutely necessary for the establishment of a disease or for its maintenance. Although EBV can immortalize B cells in vitro, its in vivo role in lymphomagenesis has not been fully clarified. Also, its role in the lymphomagenesis of T or NK cells is unclear. Interestingly, a variety of populations are at high risk for EBV-associated malignancies. This suggests that genetic backgrounds are related with the pathogenesis of these diseases, although one cannot rule out the possibility that specific EBV strains are prevalent in certain areas. In this symposium, I summarize the lymphomagenesis and clinical features of EBV-associated lymphoid malignancies.

Abstract 3192A: Senescence may play an important role in explaining the mechanisms of increased radiosensitivity of HPV- and EBV-associated HNSCC

Abstract Background: Viral associated HPV and EBV squamous cell carcinomas are common in the oropharynx and nasopharynx respectively. In fact co-infection of these viruses is reported in 15-46% of nasopharyngeal carcinomas, 13% of oral cancers and 11% of other head and neck tumors. Viral associated HNSCC tumors are inherently radiosensitive when compared to tobacco associated malignancies. We had determined using stable clones generated in a HNSCC cell line that HPV, EBV and the combination was significantly more radiosensitive than the negative viral clone. However mechanisms of increased sensitivity had not been defined. The aim of the study is to elucidate the molecular mechanisms of increased radiosensitivity in viral associated HNSCC. Methods: To test our hypothesis four stable clones of FaDu cells (HPV-/EBV-, HPV+/EBV-, HPV-/EBV+, HPV+/EBV+) generated by transfection with HPV E6/7 ORF constructs or/and infection with recombinant EBV strain were irradiated with 4Gy. Cell cycle was analyzed using FACS and protein expression was evaluated by western blotting. Senescence associated beta-galactosidase (SABG) staining was used to determine the level of senescence. Results: Cell cycle analysis at various time points (0-72h) after 4Gy γ-radiation revealed the viral positive clones showed increased G1/S arrest compared to the HPV-/EBV- clone and resumed cycling earlier at 24h compared to recovery at 48h noted in the viral negative clone. Deregulated phosphorylation of DNA repair components such as ATM and ATR was observed in the viral clones while steady state phosphorylation was seen in the negative clone. Cell cycle and protein analysis did not show increased apoptosis in the viral clones. However p21 protein expression was significantly increased in the viral positive clones after radiation treatment (P&amp;lt;0.05) compared to negative clone. Given the increase in p21, a marker of cellular senescence, SABG staining was performed. There was an accumulation of senescent cells after radiation (at 72h: 80% increase for HPV-/EBV-; 217% increase for HPV+/EBV-; 215% increase for HPV-/EBV+ and 106% increase for HPV+/EBV+ over the respective baselines). There was a significant increase in the percentage of senescent cells in the irradiated HPV+/EBV- (P&amp;lt;0.01) and HPV-/EBV+ (P&amp;lt;0.05) clones compared to the irradiated HPV-/EBV- clone. Conclusion: We show that both HPV and EBV disrupt cell cycle control and DNA repair signaling. Radiation-induced upregulation of p21 expression in viral clones was associated with reduced clonogenicity, altered cell cycle progression and an increase in the percentage of cells with markers of premature cellular senescence. The faster recovery of cell cycle in the viral clones could result in incomplete DNA repair resulting in genomic instability and an increase in cellular senescence. Citation Format: Arunkumar Anandharaj, Oleksandr Ekshyyan, Xiaohua Rong, Dylan Hartel, Brandon Bauerle, Sean Nathan, Lynn Harrison, Rona Scott, Runhua Shi, Cherie-Ann O. Nathan. Senescence may play an important role in explaining the mechanisms of increased radiosensitivity of HPV- and EBV-associated HNSCC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3192A. doi:10.1158/1538-7445.AM2014-3192A

The MEC1 and MEC2 lines represent two CLL subclones in different stages of progression towards prolymphocytic leukemia.

The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cells of a chronic lymphocytic leukemia (CLL) patient at different stages of progression to prolymphocytoid transformation (PLL). This pair of lines is unique in several respects. Their common clonal origin was proven by the rearrangement of the immunoglobulin genes. The cells were driven to proliferation in vitro by the same indigenous EBV strain. They are phenotypically different and represent subsequent subclones emerging in the CLL population. Furthermore they reflect the clinical progression of the disease. We emphasize that the support for the expression of the EBV encoded growth program is an important differentiation marker of the CLL cells of origin that was shared by the two subclones. It can be surmised that proliferation of EBV carrying cells in vitro, but not in vivo, reflects the efficient surveillance that functions even in the severe leukemic condition. The MEC1 line arose before the aggressive clinical stage from an EBV carrying cell within the subclone that was in the early prolymphocytic transformation stage while the MEC2 line originated one year later, from the subsequent subclone with overt PLL characteristics. At this time the disease was disseminated and the blood lymphocyte count was considerably elevated. The EBV induced proliferation of the MEC cells belonging to the subclones with markers of PLL agrees with earlier reports in which cells of PLL disease were infected in vitro and immortalized to LCL. They prove also that the expression of EBV encoded set of proteins can be determined at the event of infection. This pair of lines is particularly important as they provide in vitro cells that represent the subclonal evolution of the CLL disease. Furthermore, the phenotype of the MEC1 cells shares several characteristics of ex vivo CLL cells.

Epstein–barr virus latent membrane protein 1 polymorphism in nasopharyngeal carcinoma and other oral cavity tumors in Russia

The genetic structure of EBV LMP1 alleles isolated from tumor, blood, and throat washing samples of 22 nasopharyngeal carcinoma patients, 17 patients with other non-EBV-related tumors of the oral cavity, and 19 blood donors have been studied in representatives of Central Russia and the Republics of Northern Caucasus, regions which are non-endemic for nasopharyngeal carcinoma. The analysis of the LMP1 alleles collected revealed that they practically matched previously described LMP1 variants; however, some characteristic features were also detected. In particular, the G212S substitution in LMP1 isolates investigated was not observed at all. Tumor samples obtained from nasopharyngeal carcinoma and other tumors of the oral cavity did not differ significantly either in the frequency of "high oncogenic" LMP1 alleles with 10 aa and/or 23 aa deletions (LMP1(China1) and/or LMP1(Med+)), nor in the number of 11 aa repeats and the frequency of 5 aa motif insertions. No differences in the frequency of amino acid substitutions between LMP1 alleles obtained from tumor and throat washing samples of both patient groups were also detected. The data obtained may indicate that in both nasopharyngeal carcinoma patients and patients with other tumors of the oral cavity, the EBV strains with similar LMP1 variants are found to persist. This observation allows us to suggest that in non-endemic areas, EBV strains with any LMP1 alleles can initiate the nasopharyngeal carcinoma development but only in those individuals who have a genetic predisposition to the disease and are subjected to specific environmental, and/or dietary factors present in certain geographic areas.

Genome-wide analyses of Epstein-Barr virus reveal conserved RNA structures and a novel stable intronic sequence RNA.

BackgroundEpstein-Barr virus (EBV) is a human herpesvirus implicated in cancer and autoimmune disorders. Little is known concerning the roles of RNA structure in this important human pathogen. This study provides the first comprehensive genome-wide survey of RNA and RNA structure in EBV.ResultsNovel EBV RNAs and RNA structures were identified by computational modeling and RNA-Seq analyses of EBV. Scans of the genomic sequences of four EBV strains (EBV-1, EBV-2, GD1, and GD2) and of the closely related Macacine herpesvirus 4 using the RNAz program discovered 265 regions with high probability of forming conserved RNA structures. Secondary structure models are proposed for these regions based on a combination of free energy minimization and comparative sequence analysis. The analysis of RNA-Seq data uncovered the first observation of a stable intronic sequence RNA (sisRNA) in EBV. The abundance of this sisRNA rivals that of the well-known and highly expressed EBV-encoded non-coding RNAs (EBERs).ConclusionThis work identifies regions of the EBV genome likely to generate functional RNAs and RNA structures, provides structural models for these regions, and discusses potential functions suggested by the modeled structures. Enhanced understanding of the EBV transcriptome will guide future experimental analyses of the discovered RNAs and RNA structures.

A monoclonal expansion of Epstein–Barr virus-infected natural killer cells after allogeneic peripheral blood stem cell transplantation

Here, we describe a Japanese woman showing a monoclonal expansion of EBV-infected natural killer (NK) cells after receiving allogeneic peripheral blood stem cell transplantation (PBSCT). The patient initially had T-cell-type chronic active EBV disease (CAEBV) and subsequently developed liver T-cell lymphoma. l-Asparaginase-containing chemotherapy led to a favorable lymphoma response. To eradicate CAEBV and the lymphoma, she further received allogeneic PBSCT from a human leukocyte antigen-matched sibling donor. After the PBSCT, the patient presented with transient lymphocytosis of NK cells, which were infected with a monoclonal EBV strain other than previously detected ones. These NK cells seemed to have been transmitted from the healthy donor to the recipient. The patient and donor remain well in spite of carrying these NK cells. This is the first report of an asymptomatic Japanese carrier harboring monoclonal EBV-infected NK cells.

Adoptive immunotherapy with unselected or EBV-specific T cells for biopsy-proven EBV+ lymphomas after allogeneic hematopoietic cell transplantation

AbstractWe evaluated HLA-compatible donor leukocyte infusions (DLIs) and HLA-compatible or HLA-disparate EBV-specific T cells (EBV-CTLs) in 49 hematopoietic cell transplantation recipients with biopsy-proven EBV-lymphoproliferative disease (EBV-LPD). DLIs and EBV-CTLs each induced durable complete or partial remissions in 73% and 68% of treated patients including 74% and 72% of patients surviving ≥ 8 days after infusion, respectively. Reversible acute GVHD occurred in recipients of DLIs (17%) but not EBV-CTLs. The probability of complete response was significantly lower among patients with multiorgan involvement. In responders, DLIs and EBV-CTLs regularly induced exponential increases in EBV-specific CTL precursor (EBV-CTLp) frequencies within 7-14 days, with subsequent clearance of EBV viremia and resolution of disease. In nonresponders, EBV-CTLps did not increase and EBV viremia persisted. Treatment failures were correlated with impaired T-cell recognition of tumor targets. Either donor-derived EBV-CTLs that had been sensitized with autologous BLCLs transformed by EBV strain B95.8 could not lyse spontaneous donor-derived EBV-transformed BLCLs expanded from the patient's blood or biopsied tumor or they failed to lyse their targets because they were selectively restricted by HLA alleles not shared by the EBV-LPD. Therefore, either unselected DLIs or EBV-specific CTLs can eradicate both untreated and Rituxan-resistant lymphomatous EBV-LPD, with failures ascribable to impaired T-cell recognition of tumor-associated viral antigens or their presenting HLA alleles.