Anti-Epstein-Barr Viral Agents from the Medicinal Herb-Derived Fungus Alternaria alstroemeriae Km2286.

Chromatographic separation on the liquid-state fermented products produced by the fungal strain Alternaria alstroemeriae Km2286 isolated from the littoral medicinal herb Atriplex maximowicziana Makino resulted in the isolation of compounds 1-9. Structures were determined by spectroscopic analysis as four undescribed perylenequinones, altertromins A-D (1-4), along with altertoxin IV (5), altertoxin VIII (6), stemphyperylenol (7), tenuazonic acid (8), and allo-tenuazonic acid (9). Compounds 1-6 exhibited antiviral activities against Epstein-Barr virus (EBV) with EC50 values ranging from 0.17 ± 0.07 to 3.13 ± 0.31 µM and selectivity indices higher than 10. In an anti-neuroinflammatory assay, compounds 1-4, 6, and 7 showed inhibitory activity of nitric oxide production in lipopolysaccharide-induced microglial BV-2 cells, with IC50 values ranging from 0.33 ± 0.04 to 4.08 ± 0.53 µM without significant cytotoxicity. This is the first report to describe perylenequinone-type compounds with potent anti-EBV and anti-neuroinflammatory activities.

The Ethyl Acetate Subfraction of Polygonum cuspidatum Root Containing Emodin Affect EBV Gene Expression and Induce EBV-Positive Cells Apoptosis.

Epstein-Barr virus (EBV), a human herpesvirus, is several human lymphoid malignancies-associated. Our earlier study found the effect of Polygonum cuspidatum root on promoting EBV-positive apoptosis. Therefore, this study investigated the effects of the Polygonum cuspidatum ethyl acetate subfraction containing emodin on EBV gene expression and anti-EBV tumor cells. Resultantly, the the Polygonum cuspidatum ethyl acetate subfraction containing emodin (F3a) promoted Raji cell death (50% cytotoxic concentration, CC50: 12.08 µg/mL); the 12.5 µg/mL F3a effect transcribed BRLF1 and BNLF1 and increased latent membrane protein 1 (LMP1), which may reduce the intracellular phospho-extracellular signal-regulated kinase (ERK) and phospho-inhibitor of Nuclear factor kappa B α (IκBα). Meanwhile, the Raji cells increased the intracellular reactive-oxygen species (ROS), activated the apoptosis-related proteins, cleaved caspase 3 and poly(ADP-ribose)polymerase (PARP), and increased the apoptosis percentage. Therefore, the Polygonum cuspidatum ethyl acetate subfraction containing emodin could be a therapeutic drug for EBV-related tumors.

Natural killer cells play an important role in virus infection control: Antiviral mechanism, subset expansion and clinical application.

With the global spread of coronavirus disease 2019 (COVID-19), the important role of natural killer (NK) cells in the control of various viral infections attracted more interest, via non-specific activation, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and activating receptors, as well as specific activation, such as memory-like NK generation. In response to different viral infections, NK cells fight viruses in different ways, and different NK subsets proliferate. For instance, cytomegalovirus (CMV) induces NKG2C + CD57 + KIR+ NK cells to expand 3-6 months after hematopoietic stem cell transplantation (HSCT), but human immunodeficiency virus (HIV) induces KIR3DS1+/KIR3DL1 NK cells to expand in the acute phase of infection. However, the similarities and differences among these processes and their molecular mechanisms have not been fully discussed. In this article, we provide a summary and comparison of antiviral mechanisms, unique subset expansion and time periods in peripheral blood and tissues under different conditions of CMV, HIV, Epstein-Barr virus (EBV), COVID-19 and hepatitis B virus (HBV) infections. Accordingly, we also discuss current clinical NK-associated antiviral applications, including cell therapy and NK-related biological agents, and we state the progress and future prospects of NK cell antiviral treatment.

Magnesium Restores Activity to Peripheral Blood Cells in a Patient With Functionally Impaired Interleukin-2-Inducible T Cell Kinase.

Interleukin-2-inducible T cell kinase (ITK) is critical for T cell signaling and cytotoxicity, and control of Epstein-Barr virus (EBV). We identified a patient with a novel homozygous missense mutation (D540N) in a highly conserved residue in the kinase domain of ITK who presented with EBV-positive lymphomatoid granulomatosis. She was treated with interferon and chemotherapy and her disease went into remission; however, she has persistent elevation of EBV DNA in the blood, low CD4 T cells, low NK cells, and nearly absent iNKT cells. Molecular modeling predicts that the mutation increases the flexibility of the ITK kinase domain impairing phosphorylation of the protein. Stimulation of her T cells resulted in reduced phosphorylation of ITK, PLCγ, and PKC. The CD8 T cells were moderately impaired for cytotoxicity and degranulation. Importantly, addition of magnesium to her CD8 T cells in vitro restored cytotoxicity and degranulation to levels similar to controls. Supplemental magnesium in patients with mutations in another protein important for T cell signaling, MAGT1, was reported to restore EBV-specific cytotoxicity. Our findings highlight the critical role of ITK for T cell activation and suggest the potential for supplemental magnesium to treat patients with ITK deficiency.

Epstein-Barr virus (EBV) reactivation and therapeutic inhibitors.

Epstein-Barr virus (EBV) is a ubiquitous human virus which infects almost all humans during their lifetime and following the acute phase, persists for the remainder of the life of the individual. EBV infects B lymphocytes leading to their immortalisation, with persistence of the EBV genome as an episome. In the latent phase, EBV is prevented from reactivating through efficient cytotoxic cellular immunity. EBV reactivates (lytic phase) under conditions of psychological stress with consequent weakening of cellular immunity, and EBV reactivation has been shown to occur in a subset of individuals with each of a variety of cancers, autoimmune diseases, the autoimmune-like disease, chronic fatigue syndrome/myalgic encephalitis and under other circumstances such as being an inpatient in an intensive care unit. Chronic EBV reactivation is an important mechanism in the pathogenesis of many such diseases, yet is rarely tested for in immunocompetent individuals. This review summarises the pathogenesis of EBV infection, EBV reactivation and its role in disease, and methods which may be used to detect it. Known inhibitors of EBV reactivation and replication are discussed, including drugs licensed for treatment of other herpesviruses, licensed or experimental drugs for various other indications, compounds at an early stage of drug development and nutritional constituents such as vitamins and dietary supplements.

Plasma magnesium is inversely associated with Epstein-Barr virus load in peripheral blood and Burkitt lymphoma in Uganda.

BACKGROUND: Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma (eBL). EBV control was improved by magnesium (Mg2+) supplementation in XMEN, an X-linked genetic disease associated with Mg2+ deficiency, high circulating EBV levels (viral loads), and EBV-related lymphomas. We, therefore, investigated the relationship between Mg2+ levels and EBV levels and eBL in Uganda. METHODS: Plasma Mg2+ was measured in 45 women with low or high circulating EBV levels, 40 pediatric eBL cases, and 79 healthy children. Mg2+ uptake by T-lymphocytes was evaluated in samples from healthy donors. RESULTS: Plasma Mg2+ deficiency (plasma level <1.8 mg/dl) was more likely in women with high- vs. low-EBV levels (76.0% vs. 35%; odds ratio [OR] 11.3, 95% CI 2.14-60.2), controlling for age, and in eBL cases than controls (42.0% vs. 13.9%; OR 3.61, 95% CI 1.32-9.88), controlling for sex, age group, and malaria status. Mg2+ uptake by T-lymphocytes was related to extracellular Mg2+ concentration. INTERPRETATION: Plasma Mg2+ deficiency is associated with high EBV levels and eBL.

Effect of high dose vitamin C on Epstein-Barr viral infection.

Background Many natural compounds were tested for the ability to suppress viral replication. The present manuscript details an analysis of high dose vitamin C therapy on patients with EBV infection. Material and Methods The data were obtained from the patient history database at the Riordan Clinic. Among people in our database who were treated with intravenous vitamin C (7.5 g to 50 g infusions) between 1997 and 2006, 178 patients showed elevated levels of EBV EA IgG (range 25 to 211 AU) and 40 showed elevated levels of EBV VCA IgM (range 25 to 140 AU). Most of these patients had a diagnosis of chronic fatigue syndrome, with the rest being diagnosed as having mononucleosis, fatigue, or EBV infection. Results Our data provide evidence that high dose intravenous vitamin C therapy has a positive effect on disease duration and reduction of viral antibody levels. Plasma levels of ascorbic acid and vitamin D were correlated with levels of antibodies to EBV. We found an inverse correlation between EBV VCA IgM and vitamin C in plasma in patients with mononucleosis and CFS meaning that patients with high levels of vitamin C tended to have lower levels of antigens in the acute state of disease. In addition, a relation was found between vitamin D levels and EBV EA IgG with lower levels of EBV early antigen IgG for higher levels of vitamin D. Conclusions The clinical study of ascorbic acid and EBV infection showed the reduction in EBV EA IgG and EBV VCA IgM antibody levels over time during IVC therapy that is consistent with observations from the literature that millimolar levels of ascorbate hinder viral infection and replication in vitro.

Herpesvirus infections and allergic sensitization in children of families with anthroposophic and non-anthroposophic lifestyle - the ALADDIN birth cohort.

BACKGROUND: Growing up in families with an anthroposophic lifestyle has been associated with reduced risk of allergic disease in children. The aim of this report was to assess whether children with this lifestyle are infected earlier with Epstein-Barr virus (EBV), which has been associated with reduced risk of allergic disease, and three other herpesviruses potentially involved in allergy development, namely Human herpesvirus 6 (HHV6), Human herpesvirus 7 (HHV7) and cytomegalovirus (CMV). METHODS: Within the ALADDIN (Assessment of Lifestyle and Allergic Disease During Infancy), birth cohort study 157 children were categorized according to lifestyle into anthroposophic and non-anthroposophic. IgG-levels for EBV, HHV6, HHV7 and CMV were determined in plasma samples collected at ages 12 and 24 months and from parents. IgE levels against seven common allergens were analyzed at 24 months. RESULTS: No significant differences in seroprevalence of EBV, HHV7 or CMV were detected at any age between the two lifestyle groups. The seroprevalence of HHV6 was significantly lower in the anthroposophic group at 24 months of age (74.6% vs. 87.5%, p-value 0.048). Further, no significant associations between allergic sensitization and seropositivity to any of the viruses were detected; however, an interaction effect of lifestyle could not be ruled out. CONCLUSIONS: Our results indicate that there is no strong influence of exposure to the anthroposophic lifestyle on the time for infection with EBV, HHV6, HHV7 or CMV. These infections can therefore not be assumed to be important factors in the allergy-protective effect of this lifestyle.

Inhibition of Epstein-Barr virus reactivation in nasopharyngeal carcinoma cells by dietary sulforaphane.

Epstein-Barr virus (EBV) has been associated with several human malignancies including nasopharyngeal carcinoma (NPC). Reactivation of latent EBV has been considered to contribute to the carcinogenesis of NPC. Blocking the EBV lytic cycle has been shown effective in the treatment of EBV-associated diseases. We have searched for natural dietary compounds inhibiting EBV reactivation in NPC cells. Among them, sulforaphane (SFN) was found to be effective in the inhibition of EBV reactivation in latent EBV-positive NPC cells, NA and HA. SFN is a histone deacetylase (HDAC) inhibitor and has been recognized as an antioxidant and antitumor compound for chemoprevention. However, its antiviral effect is less well elucidated. In this study, after determination of the cytotoxicity of SFN on various epithelial cells, we showed that SFN treatment inhibits EBV reactivation, rather than induction, by detection of EBV lytic gene expression in EBV-positive NPC cells. We also determined that the number of cells supporting the EBV lytic cycle is decreased using immunofluorescence and flow cytometric analysis. Moreover, we have found that this inhibitory effect decreases virus production. To elucidate the inhibitory mechanism of SFN on the EBV lytic cycle, luciferase reporter assays were carried out on the Zta and Rta promoters. The results show that SFN inhibits transactivation activity of the EBV immediate-early gene Rta but not Zta. Together, our results suggest that SFN has the capability to inhibit EBV lytic cycle and the potential to be taken as a dietary compound for prevention of EBV reactivation.

Thymoquinone efficiently inhibits the survival of EBV-infected B cells and alters EBV gene expression.

Epstein--Barr virus (EBV) is a human virus with oncogenic potentials that is implicated in various human diseases and malignancies. In this study, the modulator activity of the potent herbal extract drug thymoquinone on EBV was assessed in vitro. Thymoquinone was tested for cytotoxicity on human cells of lymphoblastoid cells, Raji Burkitt's lymphoma, DG-75 Burkitt's lymphoma, peripheral blood mononuclear cells, and periodontal ligament fibroblast. Apoptosis induction was analyzed via TUNEL assay and activity studies of caspase-3. The effect of thymoquinone on EBV gene expression was determined using real-time polymerase chain reaction. We report here, for the first time, a promising selective inhibitory affect of thymoquinone on EBV-infected B cell lines in vitro, compared with lower activity on EBV negative B cell line and very low toxicity on human peripheral blood mononuclear cells and periodontal ligament fibroblasts. Moreover, the drug was found to efficiently suppress the RNA expression of EBNA2, LMP1, and EBNA1 genes. Specifically, EBNA2 expression levels were the most affected indicating that this gene might have a major contribution to thymoquinone potency against EBV infected cells. Overall, our results suggest that thymoquinone has the potential to suppress the growth of EBV-infected B cells efficiently.

Effect of Pinus massoniana Lamb. bark extract on lytic cycle of Epstein-Barr virus.

Pinus massoniana bark extract (PMBE) at a concentration of 60 microg/mL or more inhibits the expression of Epstein-Barr virus (EBV) lytic proteins, such as Rta, Zta, and EA-D. EBV lytic cycle was blocked by inhibiting the transcription of immediate-early genes. The results suggest that the PMBE has anti-EBV activity. Thus, the extract is potentially useful in preventing the lytic development of EBV in vitro.

Parthenolide induces apoptosis and lytic cytotoxicity in Epstein-Barr virus-positive Burkitt lymphoma.

Burkitt lymphoma (BL) has been reported to be strongly associated with Epstein-Barr virus (EBV) infection. The fact that EBV is generally present in cancer cells but rarely found in healthy cells represents an opportunity for targeted cancer therapy. One approach is to activate the lytic replication cycle of the latent EBV. Nuclear factor (NF)-κB is thought to play an essential role in EBV lytic infection. Elevated NF-κB levels inhibit EBV lytic replication. Parthenolide (PN) is a sesquiterpene lactone found in medicinal plants, particularly in feverfew (Tanacetum parthenium). The aim of the present study was to analyze the effect of PN on the survival of Raji EBV-positive lymphoma cells. Raji cells were treated with 0, 4 or 6 µmol/l PN for 48 h. MTT assay and western blot analysis were performed to evaluate the findings. Results showd that PN suppressed the growth of the EBV-positive BL cell line, Raji, and activated the transcription of BZLF1 and BRLF1 by inhibiting NF-κB activity. Most notably, when PN was used in combination with ganciclovir (GCV), the cytotoxic effect of PN was amplified. These data suggest that the induction of lytic EBV infection with PN in combination with GCV may be a viral­targeted therapy for EBV-associated BL.

[Subacute inflammatory demyelinating polyradiculopathy associated with Epstein-Barr viral infection: a clinical case].

The clinical case of treatment a patient for subacute inflammatory demyelinating polyradiculopathy associated with Epstein-Barr viral infection is presented. It is showed that the period of rehabilitation after the indicated disease lasts long enough. It requires the differentiated approach in acute and recovery treatment period with using of different rehabilitation measures.

Inhibition of the Epstein-Barr virus lytic cycle by protoapigenone.

Epstein-Barr virus (EBV) expresses two transcription factors, Rta and Zta, during the immediate-early stage of the lytic cycle to activate the transcription of early and late genes. This study finds that 0.31 mM protoapigenone from Thelypteris torresiana (Gaud.) inhibits the expression of EBV lytic proteins, including Rta, Zta, EA-D and VCA, in P3HR1 cells after lytic induction with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate. The lack of expression of EBV lytic proteins after protoapigenone treatment is attributed to the inhibition of the transactivation function of Zta because protoapigenone reduces the transactivation activity of Zta and Gal4-Zta, which contains the transactivation domain of Zta fused with Gal4. In contrast, protoapigenone does not affect the ability of Rta to activate a promoter that contains an Rta-response element, showing that the inhibition is unrelated to Rta. Furthermore, in a lactate dehydrogenase assay, protoapigenone is not toxic to P3HR1 cells at the concentrations that inhibit the function of Zta, showing that protoapigenone is valuable for studying the function of Zta and preventing EBV lytic proliferation.

Characteristics of Epstein-Barr virus-associated gastric cancer: a study of 235 cases at a comprehensive cancer center in U.S.A.

BACKGROUND: Epstein-Barr virus (EBV) has been shown to be associated with gastric cancer. However, inconsistent findings have been reported regarding the distribution of EBV infected cells (in normal gastric epithelium vs. intestinal metaplastic cells vs. in neoplastic cells) and the characteristics of EBV-associated gastric cancer. Lymph node positive EBV-associated gastric cancer has not been systematically studied. The aims of this study were to evaluate EBV-associated gastric cancer, to assess the distribution of EBV infected cells including all positive lymph nodes, and to define the characteristics of EBV-associated gastric cancer. DESIGN: The study included primary gastric cancer patients who underwent surgical resection with no preoperative treatment at M.D. Anderson Cancer Center between 1987 and 2006. Formalin-fixed paraffin-embedded tissue from these resection specimens were assessed for EBV by in situ hybridization, the gold standard for EBV detection in tissue. EBV status was analyzed along with clinicopathologic parameters including age, gender, tumor type, lymph node status, and pathologic stage of the tumor. RESULTS: Among 235 patients, 12 had intranuclear expression of EBV. EBV staining was seen only in tumor cells and no detectable EBV was observed in normal gastric mucosa, intestinal metaplasia or stromal cells. Eight of 12 patients with EBV-associated gastric cancer had regional lymph node metastasis. Of note, metastatic tumor cells in all of the involved lymph nodes of these 8 cases contained EBV. The epidemiologic data showed 11 of the 12 patients with EBV-associated gastric cancer were men, ranging in age from 54 to 78 years (mean age, 60 years; median age, 62.1 years). The age distribution for non-EBV associated gastric cancer patients ranged from 21 to 93 years (mean age, 67 years; median age, 66.4 years). CONCLUSION: Our study demonstrated that EBV is present exclusively in gastric cancer cells. The detection of EBV in tumor cells in all of the lymph nodes involved with metastatic gastric carcinoma suggests simultaneous replication of EBV and tumor cells. The predominantly male gender and relatively younger age observed for the EBV-infected gastric cancer cases suggest an association between this disease and other factors, such as life style.

Epstein-Barr virus (EBV) reactivation in allogeneic stem-cell transplantation: relationship between viral load, EBV-specific T-cell reconstitution and rituximab therapy.

BACKGROUND: Monitoring of Epstein-Barr virus (EBV) reactivation after allogeneic hematopoietic stem-cell transplantation markedly improved with quantitative real-time polymerase chain reaction amplification of EBV DNA and visualization of EBV-specific CD8+ T cells with peptide-human leukocyte antigen (HLA) class I tetramers. We decided to combine these methods to evaluate posttransplant EBV reactivation and rituximab therapy. METHODS: We followed 56 patients treated with an HLA-genoidentical sibling (n=32), an HLA-matched unrelated donor (MUD, n=19), or an unrelated cord-blood transplant (n=5). EBV DNA was quantified in plasma and in peripheral blood mononuclear cells (PBMC). Patient CD8+ T cells were stained with a panel of eight tetramers. RESULTS: EBV DNA was detected in half of the patients, mainly in the MUD group (17/19). In 19 patients, viral DNA was detected only in the cellular compartment. All patients who controlled reactivation without rituximab and despite a viral load of greater than 500 genome equivalents (gEq)/150,000 PBMC mounted an EBV-specific CD8+ T-cell response in greater than 1.4% of CD3+CD8+ T cells. Plasmatic EBV genome was found in nine patients preceded by a high cellular viral load. Three of these patients controlled the reactivation before or without the introduction of rituximab, and they all developed a significant and increasing EBV-specific T-cell response. Patients with EBV-specific T cells at the onset of reactivation controlled viral reactivation without rituximab. CONCLUSION: This study emphasizes the benefit of an early and close monitoring of EBV reactivation and CD8+-specific immune responses to initiate rituximab only when necessary and before the immune response becomes overwhelmed by the viral burden.

Comparison of gene expression profiles of lymphoma cell lines from transformed follicular lymphoma, Burkitt's lymphoma and de novo diffuse large B-cell lymphoma.

To determine the specific gene expression in B-cell lymphoma subtypes, we compared expression profiles of cell lines from transformed follicular lymphoma (tFL), Epstein-Barr virus-negative (EBV(-)) Burkitt's lymphoma (BL) and EBV(+)BL. Complementary DNAs were synthesized from these cell lines and hybridized with the Atlas Human 1.2 Array membrane. Hierarchical clustering analysis based upon the levels of 43 genes highlighted characteristic expression patterns of the 3 lymphoma subtypes. Genes expressed at higher levels in tFL than EBV(-)BL and EBV(+)BL included calcium/calmodulin-dependent protein kinase I (CAMK1) and mitogen-activated protein kinase 10 (MAPK10). EBV(-)BL was characterized by high-level expression of amyloid beta precursor protein (APP), heat shock 27 kD protein 1 (HSPB1) and mothers against decapentaplegic homolog 1 (MADH1). Gardner-Rasheed feline sarcoma viral oncogene homolog (FGR) was the most significant gene to delineate EBV(+)BL. A subtype prediction algorithm using 34 genes correctly classified 22 (92%) of 24 lymphomas into FL/tFL, EBV(-)BL or EBV(+)BL. By comparison with normal reference B-cell materials, the expression patterns of the selected genes were characteristic of lymphomas. We extended the clustering analysis to cell lines from de novo diffuse large B-cell lymphoma (DLBCL). The DLBCL cell lines were either separated from the former 3 lymphoma subtypes or segregated with EBV(+)BL, possibly reflecting variable genetic abnormalities. The associations of CAMK1 with tFL, APP and MADH1 with EBV(-)BL, FGR with EBV(+)BL, and BCL2 with tFL and DLBCL were confirmed by real-time quantitative reverse transcriptase-mediated polymerase chain reaction assays. This study has provided new molecular markers, expressions of which are closely associated with B-cell lymphoma subtypes.