12-O-Tetradecanoylphorbol-13-acetate Induces Epstein–Barr Virus Reactivation via NF-κB and AP-1 as Regulated by Protein Kinase C and Mitogen-Activated Protein Kinase

AbstractSignaling pathway components mediating Epstein–Barr virus (EBV) reactivation by 12-O-tetradecanoylphorbol-13-acetate (TPA) were characterized in terms of induction and modification of specific transacting factors. The consequences of protein kinase C (PKC) activation by TPA in inhibiting inducible nitric oxide synthase (iNOS) mRNA expression were analyzed in the EBV-infected gastric epithelial cell line GT38. Spontaneous expression of the EBV BZLF1 gene product ZEBRA became undetectable upon long-term culturing of GT38 cells, while iNOS mRNA expression increased. In such cells the PKC inhibitors 1-(5-isoquinolinesulphonyl)-2,5-dimethylpiperazine (H7) and staurosporine inhibited TPA-induced expression of BZLF1 and BRLF1 and reversed TPA-mediated inhibition of iNOS gene expression. The mitogen-activated protein kinase inhibitor PD98059 inhibited TPA-induced BZLF1 expression. Electrophoretic mobility shift assays demonstrated that transcription factors NF-κB and AP-1 were also activated by TPA in a time-dependent manner. The TPA-induced NF-κB activation was inhibited by prior treatment of the cells with the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). TPA-induced BZLF1 expression was also inhibited by the treatment with PDTC. Northern blot analyses characterized changes in levels of the c-jun and junB expressions of the AP-1 family. These results show that TPA induces EBV reactivation via NF-κB and AP-1 and that PKC is an important mediator in regulating gene expression leading to EBV reactivation after TPA treatment of GT38 cells

Increased Activity of 2',5'-Oligoadenylate Synthetase in Peripheral Blood Mononuclear Cells of Patients with Major Depressive Disorder

The aim of this study was to measure the activity of 2',5'-oligoadenylate synthetase (2',5'AS) which is mainly induced by interferon (IFN)-α and -β in patients with major depression and evaluate its relationship to the disease. 2',5'AS activity of 23 patients (male = 11, female = 12) with major depression and of 29 normal control subjects (male = 15, female = 14) was measured in peripheral blood mononuclear cells (PBMCs) by radioassay. The mean 2',5'AS activity in the PBMCs of the patients and that of the control subjects were 1.10 ± 0.69% and 0.72 ± 0.51%, respectively. The activity in the patients was statistically higher than that of the control subjects (P = 0.03). These results imply some abnormality in the IFN-2',5'AS system of patients with major depression

Rapid CRISPR/Cas9-Mediated Cloning of Full-Length Epstein-Barr Virus Genomes from Latently Infected Cells

Herpesviruses have relatively large DNA genomes of more than 150 kb that are difficult to clone and sequence. Bacterial artificial chromosome (BAC) cloning of herpesvirus genomes is a powerful technique that greatly facilitates whole viral genome sequencing as well as functional characterization of reconstituted viruses. We describe recently invented technologies for rapid BAC cloning of herpesvirus genomes using CRISPR/Cas9-mediated homology-directed repair. We focus on recent BAC cloning techniques of Epstein-Barr virus (EBV) genomes and discuss the possible advantages of a CRISPR/Cas9-mediated strategy comparatively with precedent EBV-BAC cloning strategies. We also describe the design decisions of this technology as well as possible pitfalls and points to be improved in the future. The obtained EBV-BAC clones are subjected to long-read sequencing analysis to determine complete EBV genome sequence including repetitive regions. Rapid cloning and sequence determination of various EBV strains will greatly contribute to the understanding of their global geographical distribution. This technology can also be used to clone disease-associated EBV strains and test the hypothesis that they have special features that distinguish them from strains that infect asymptomatically

A Replication-Defective Gammaherpesvirus Efficiently Establishes Long-Term Latency in Macrophages but Not in B Cells In Vivo ▿

Murine gammaherpesvirus 68 (γHV68 or MHV68) is genetically related to the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), providing a useful system for in vivo studies of the virus-host relationship. To begin to address fundamental questions about the mechanisms of the establishment of gammaherpesvirus latency, we previously generated a replication-defective γHV68 lacking the expression of the single-stranded DNA binding protein encoded by orf6. In work presented here, we demonstrate that this mutant virus established a long-term infection in vivo that was molecularly identical to wild-type virus latency. Thus, despite the absence of an acute phase of lytic replication, the mutant virus established a chronic infection in which the viral genome (i) was maintained as an episome and (ii) expressed latency-associated, but not lytic replication-associated, genes. Macrophages purified from mice infected with the replication-defective virus harbored viral genome at a frequency that was nearly identical to that of wild-type γHV68; however, the frequency of B cells harboring viral genome was greatly reduced in the absence of lytic replication. Thus, this replication-defective gammaherpesvirus efficiently established in vivo infection in macrophages that was molecularly indistinguishable from wild-type virus latency. These data point to a critical role for lytic replication or reactivation in the establishment or maintenance of latent infection in B cells

Identification of the Components in a <i>Vaccinium oldhamii</i> Extract Showing Inhibitory Activity against Influenza Virus Adsorption

We previously reported that extracts from plants of the Ericaceae genus Vaccinium, commonly known as the kind of blueberry, inhibited the early steps of influenza virus (IFV) infection to host cells, and that the activity was correlated with the total polyphenol content. Particularly potent inhibitory activity was observed for Vaccinium oldhamii. In this study, we identified the active components in Vaccinium oldhamii involved in the inhibition of IFV infection. We sequentially fractionated the Vaccinium oldhamii extract using a synthetic adsorbent resin column. High inhibitory activity was observed for the fractions eluted with 30%, 40%, and 50% ethanol, and three peaks (peak A, B, and C) considered to represent polyphenols were identified in the fractions by HPLC analysis. Among these peaks, high inhibitory activity was detected for peak A and B, but not for peak C. These peaks were analyzed by LC/MS, which revealed that peak A contained procyanidin B2 and ferulic acid derivatives, whereas peak B contained two ferulic acid O-hexosides, and peak C contained quercetin-3-O-rhamnoside and quercetin-O-pentoside-O-rhamnoside. It is already known that these polyphenols have anti-IFV activity, but we speculate that ferulic acid derivatives are the major contributors to the inhibition of the early steps of IFV replication, such as either adsorption or entry, observed for Vaccinium oldhamii

Congenital cytomegalovirus infection via a re-infected mother with original antigenic sin: A case report

A 27-year-old pregnant woman who was positive for anti-cytomegalovirus (CMV) antibodies gave birth to a congenitally CMV-infected neonate at 40 weeks of gestation. According to strain-specific serological analysis, which is able to determine the two types of CMV glycoprotein H (gH), the mother possessed anti-gH(To) antibodies only, but the neonate possessed anti-gH(AD) and anti-gH(To) antibodies at 4 weeks after birth. As the anti-gH(To) IgG was decreased in the neonate at 8 months post-delivery, these antibodies are thought to have been transferred from the mother as maternal antibodies. The anti-gH(AD) IgG level was maintained in the child even after 8 months post-delivery. Congenital infection with a CMV gH(AD) type strain was confirmed by strain-specific real-time PCR using a urine specimen from the child. On the other hand, anti-gH(AD) IgG was not detected even after 8 months post-delivery in a maternal specimen. The mother only produced antibodies against the CMV strain identified as the primary infection, which is characteristic of original antigenic sin. Keywords: Cytomegalovirus, Congenital infection, Glycoprotein H, Original antigenic si

Efficient Epstein-Barr Virus Progeny Production Mediated by Cancer-Derived LMP1 and Virally-Encoded microRNAs

Epstein-Barr virus (EBV) genomes, particularly their latent genes, are heterogeneous among strains. The heterogeneity of EBV-encoded latent membrane protein 1 (LMP1) raises the question of whether there are functional differences between LMP1 expressed by cancer-associated EBV and that by non-cancerous strains. Here, we used bacterial artificial chromosome (BAC)-cloned EBV genomes retaining all virally encoded microRNA (miRNA) genes to investigate the functions of cancer-derived LMP1 in the context of the EBV genome. HEK293 cells were stably transfected with EBV-BAC clone DNAs encoding either nasopharyngeal carcinoma (NPC)-derived CAO-LMP1 (LMP1CAO) or LMP1 from a prototype B95-8 strain of EBV (LMP1B95-8). When an EBV-BAC clone DNA encoding LMP1CAO was stably transfected into HEK293 cells, it generated many more stable transformants than the control clone encoding LMP1B95-8. Furthermore, stably transfected HEK293 cells exhibited highly efficient production of progeny virus. Importantly, deletion of the clustered viral miRNA genes compromised the ability to produce progeny viruses. These results indicate that cancer-derived LMP1 and viral miRNAs together are necessary for efficient production of progeny virus, and that the resulting increase in efficiency contributes to EBV-mediated epithelial carcinogenesis

Anti-influenza virus activity of two extracts of the blackcurrant (ribes nigrum L.) from new zealand and poland

We investigated the inhibitory effect of extracts of blackcurrant (Ribes nigrum L.) from New Zealand and Poland on 4 strains of influenza virus (IFV) by the inhibition of virus adsorption; pandemic flu from 2009-2010 (IFV-AH1pdm), Hong Kong flu (IFV-AH3), oseltamivir phosphate-resistant Russian flu (IFV-AH1tam(r)) and influenza virus type B (IFV-B). The inhibitory effect of the extracts of blackcurrant or blueberry on the infectivity of the virion were evaluated by the inhibition of virus adsorption on the cell surface (adsorption-inhibitory assay). Three percent solutions of the blackcurrant extracts from New Zealand and Poland were enough to disinfect more than half of IFV-AH1pdm and IFV-B, and 10% solutions from both regions disinfected all IFV strains completely. Our previous study showed that the antiviral effect of the blackcurrant differed according to viral species. Here we showed that although the antiviral effect of Blackcurrant was slightly different within viral strains from one species, the extract of Blackcurrant could disinfect all of 4 IFV strains we examined. The extracts of blackcurrant showed definite potential for use as a disinfectant and antiseptic agent to prevent IFV infection

Points of Recombination in Epstein-Barr Virus (EBV) Strain P3HR-1-Derived Heterogeneous DNA as Indexes to EBV DNA Recombinogenic Events In Vivo ▿

Deletions and rearrangements in the genome of Epstein-Barr virus (EBV) strain P3HR-1 generate subgenomic infectious particles that, unlike defective interfering particles in other viral systems, enhance rather than restrict EBV replication in vitro. Reports of comparable heterogeneous (het) DNA in EBV-linked human diseases, based on detection of an abnormal juxtaposition of EBV DNA fragments BamHI W and BamHI Z that disrupts viral latency, prompted us to determine at the nucleotide level all remaining recombination joints formed by the four constituent segments of P3HR-1-derived het DNA. Guided by endonuclease restriction maps, we chose PCR primer pairs that approximated and framed junctions creating the unique BamHI M/B1 and E/S fusion fragments. Sequencing of PCR products revealed points of recombination that lacked regions of extensive homology between constituent fragments. Identical recombination junctions were detected by PCR in EBV-positive salivary samples from human immunodeficiency virus-infected donors, although the W/Z rearrangement that induces EBV reactivation was frequently found in the absence of the other two. In vitro infection of lymphoid cells similarly indicated that not all three het DNA rearrangements need to reside on a composite molecule. These results connote a precision in the recombination process that dictates both composition and regulation of gene segments altered by genomic rearrangement. Moreover, the apparent frequency of het DNA at sites of EBV replication in vivo is consistent with a likely contribution to the pathogenesis of EBV reactivation

Protection from lethal herpes simplex virus type 1 infection by vaccination with a UL41-deficient recombinant strain

The UL41 gene of herpes simplex virus type 1 (HSV-1) encodes a virion host shut off protein which is involved in immune evasion. The growth and virulence of HSV-1 is markedly reduced by the deletion of UL41. In this report, the UL41-deleted recombinant HSV-1 strain VR∆41 was evaluated as a prophylactic live attenuated vaccine against lethal HSV-1 infection in a mouse model. Intraperitoneal (i.p.) inoculation with the VR∆41 strain clearly inhibited lethal wild-type HSV-1 (VR-3 strain) infection after both i.p. and intracerebral (i.c.) inoculations. Vaccination with the VR∆41 strain was safer than VR-3 vaccination and was able to protect against a wild-type challenge to the same degree as VR-3 vaccination. In contrast, i.p. inoculation with ultraviolet-irradiated VR-3 induced resistance against i.p. infection, but not against i.c. Although replication of the VR∆41 strain in mice was greatly reduced compared to that of the VR-3 strain, VR∆41 strain maintained the ability to spread to the central nervous system (CNS) from a peripheral inoculation site. These results indicated that the VR∆41 strain evoked a potent immune reaction through viral protein expression within CNS without the induction of lethal encephalitis. The entry of antigens into the CNS was essential for the establishment of protective immunity against the lethal HSV encephalitis. We concluded that only a live attenuated vaccine is able to afford a prophylactic effect against CNS infection with HSV. In order to fulfill this requirement, UL41-deleted viruses provide a strong candidate for use as a recombinant live vaccine