Integrative profiling of Epstein-Barr virus transcriptome using a multiplatform approach.

BACKGROUND: Epstein-Barr virus (EBV) is an important human pathogenic gammaherpesvirus with carcinogenic potential. The EBV transcriptome has previously been analyzed using both Illumina-based short read-sequencing and Pacific Biosciences RS II-based long-read sequencing technologies. Since the various sequencing methods have distinct strengths and limitations, the use of multiplatform approaches have proven to be valuable. The aim of this study is to provide a more complete picture on the transcriptomic architecture of EBV. METHODS: In this work, we apply the Oxford Nanopore Technologies MinION (long-read sequencing) platform for the generation of novel transcriptomic data, and integrate these with other's data generated by another LRS approach, Pacific BioSciences RSII sequencing and Illumina CAGE-Seq and Poly(A)-Seq approaches. Both amplified and non-amplified cDNA sequencings were applied for the generation of sequencing reads, including both oligo-d(T) and random oligonucleotide-primed reverse transcription. EBV transcripts are identified and annotated using the LoRTIA software suite developed in our laboratory. RESULTS: This study detected novel genes embedded into longer host genes containing 5'-truncated in-frame open reading frames, which potentially encode N-terminally truncated proteins. We also detected a number of novel non-coding RNAs and transcript length isoforms encoded by the same genes but differing in their start and/or end sites. This study also reports the discovery of novel splice isoforms, many of which may represent altered coding potential, and of novel replication-origin-associated transcripts. Additionally, novel mono- and multigenic transcripts were identified. An intricate meshwork of transcriptional overlaps was revealed. CONCLUSIONS: An integrative approach applying multi-technique sequencing technologies is suitable for reliable identification of complex transcriptomes because each techniques has different advantages and limitations, and the they can be used for the validation of the results obtained by a particular approach.

Astaxanthin Exerts Anabolic Effects via Pleiotropic Modulation of the Excitable Tissue.

Astaxanthin is a lipid-soluble carotenoid influencing lipid metabolism, body weight, and insulin sensitivity. We provide a systematic analysis of acute and chronic effects of astaxanthin on different organs. Changes by chronic astaxanthin feeding were analyzed on general metabolism, expression of regulatory proteins in the skeletal muscle, as well as changes of excitation and synaptic activity in the hypothalamic arcuate nucleus of mice. Acute responses were also tested on canine cardiac muscle and different neuronal populations of the hypothalamic arcuate nucleus in mice. Dietary astaxanthin significantly increased food intake. It also increased protein levels affecting glucose metabolism and fatty acid biosynthesis in skeletal muscle. Inhibitory inputs innervating neurons of the arcuate nucleus regulating metabolism and food intake were strengthened by both acute and chronic astaxanthin treatment. Astaxanthin moderately shortened cardiac action potentials, depressed their plateau potential, and reduced the maximal rate of depolarization. Based on its complex actions on metabolism and food intake, our data support the previous findings that astaxanthin is suitable for supplementing the diet of patients with disturbances in energy homeostasis.

Supraoptimal Iron Nutrition of Brassica napus Plants Suppresses the Iron Uptake of Chloroplasts by Down-Regulating Chloroplast Ferric Chelate Reductase.

Iron (Fe) is an essential micronutrient for plants. Due to the requirement for Fe of the photosynthetic apparatus, the majority of shoot Fe content is localised in the chloroplasts of mesophyll cells. The reduction-based mechanism has prime importance in the Fe uptake of chloroplasts operated by Ferric Reductase Oxidase 7 (FRO7) in the inner chloroplast envelope membrane. Orthologue of Arabidopsis thaliana FRO7 was identified in the Brassica napus genome. GFP-tagged construct of BnFRO7 showed integration to the chloroplast. The time-scale expression pattern of BnFRO7 was studied under three different conditions: deficient, optimal, and supraoptimal Fe nutrition in both leaves developed before and during the treatments. Although Fe deficiency has not increased BnFRO7 expression, the slight overload in the Fe nutrition of the plants induced significant alterations in both the pattern and extent of its expression leading to the transcript level suppression. The Fe uptake of isolated chloroplasts decreased under both Fe deficiency and supraoptimal Fe nutrition. Since the enzymatic characteristics of the ferric chelate reductase (FCR) activity of purified chloroplast inner envelope membranes showed a significant loss for the substrate affinity with an unchanged saturation rate, protein level regulation mechanisms are suggested to be also involved in the suppression of the reduction-based Fe uptake of chloroplasts together with the saturation of the requirement for Fe.

The developmental and iron nutritional pattern of PIC1 and NiCo does not support their interdependent and exclusive collaboration in chloroplast iron transport in Brassica napus.

MAIN CONCLUSION: The accumulation of NiCo following the termination of the accumulation of iron in chloroplast suggests that NiCo is not solely involved in iron uptake processes of chloroplasts. Chloroplast iron (Fe) uptake is thought to be operated by a complex containing permease in chloroplast 1 (PIC1) and nickel-cobalt transporter (NiCo) proteins, whereas the role of other Fe homeostasis-related transporters such as multiple antibiotic resistance protein 1 (MAR1) is less characterized. Although pieces of information exist on the regulation of chloroplast Fe uptake, including the effect of plant Fe homeostasis, the whole system has not been revealed in detail yet. Thus, we aimed to follow leaf development-scale changes in the chloroplast Fe uptake components PIC1, NiCo and MAR1 under deficient, optimal and supraoptimal Fe nutrition using Brassica napus as model. Fe deficiency decreased both the photosynthetic activity and the Fe content of plastids. Supraoptimal Fe nutrition caused neither Fe accumulation in chloroplasts nor any toxic effects, thus only fully saturated the need for Fe in the leaves. In parallel with the increasing Fe supply of plants and ageing of the leaves, the expression of BnPIC1 was tendentiously repressed. Though transcript and protein amount of BnNiCo tendentiously increased during leaf development, it was even markedly upregulated in ageing leaves. The relative transcript amount of BnMAR1 increased mainly in ageing leaves facing Fe deficiency. Taken together chloroplast physiology, Fe content and transcript amount data, the exclusive participation of NiCo in the chloroplast Fe uptake is not supported. Saturation of the Fe requirement of chloroplasts seems to be linked to the delay of decomposing the photosynthetic apparatus and keeping chloroplast Fe homeostasis in a rather constant status together with a supressed Fe uptake machinery.

Hemokinin-1 Gene Expression Is Upregulated in Trigeminal Ganglia in an Inflammatory Orofacial Pain Model: Potential Role in Peripheral Sensitization.

A large percentage of primary sensory neurons in the trigeminal ganglia (TG) contain neuropeptides such as tachykinins or calcitonin gene-related peptide. Neuropeptides released from the central terminals of primary afferents sensitize the secondary nociceptive neurons in the trigeminal nucleus caudalis (TNC), but also activate glial cells contributing to neuroinflammation and consequent sensitization in chronic orofacial pain and migraine. In the present study, we investigated the newest member of the tachykinin family, hemokinin-1 (HK-1) encoded by the Tac4 gene in the trigeminal system. HK-1 had been shown to participate in inflammation and hyperalgesia in various models, but its role has not been investigated in orofacial pain or headache. In the complete Freund's adjuvant (CFA)-induced inflammatory orofacial pain model, we showed that Tac4 expression increased in the TG in response to inflammation. Duration-dependent Tac4 upregulation was associated with the extent of the facial allodynia. Tac4 was detected in both TG neurons and satellite glial cells (SGC) by the ultrasensitive RNAscope in situ hybridization. We also compared gene expression changes of selected neuronal and glial sensitization and neuroinflammation markers between wild-type and Tac4-deficient (Tac4-/-) mice. Expression of the SGC/astrocyte marker in the TG and TNC was significantly lower in intact and saline/CFA-treated Tac4-/- mice. The procedural stress-related increase of the SGC/astrocyte marker was also strongly attenuated in Tac4-/- mice. Analysis of TG samples with a mouse neuroinflammation panel of 770 genes revealed that regulation of microglia and cytotoxic cell-related genes were significantly different in saline-treated Tac4-/- mice compared to their wild-types. It is concluded that HK-1 may participate in neuron-glia interactions both under physiological and inflammatory conditions and mediate pain in the trigeminal system.

Investigation of Cytokine Changes in Osteoarthritic Knee Joint Tissues in Response to Hyperacute Serum Treatment.

One option to fight joint degradation and inflammation in osteoarthritis is the injection of activated blood products into the synovial space. It has been demonstrated that hyperacute serum is the most proliferative among plasma products, so we investigated how the cytokine milieu of osteoarthritic knee joint reacts to hyperacute serum treatment in vitro. Cartilage, subchondral bone, and synovial membrane explanted from osteoarthritic knees were stimulated by interleukin-1 beta (IL-1ß) and the concentration of 39 biomarkers was measured in the co-culture supernatant after hyperacute serum treatment. The IL-1ß stimulation triggered a strong inflammatory response and enhanced the concentrations of matrix metalloproteinase 3 and 13 (MMP-3 and MMP-13), while hyperacute serum treatment reduced inflammation by decreasing the concentrations of IL-1ß, tumor necrosis factor alpha (TNF-α), interleukin-6 receptor alpha (IL-6Rα), and by increasing the level of interleukin-1 antagonist (IL-1RA) Cell viability increased by day 5 in the presence of hyperacute serum. The level of MMPs-1, 2, and 9 were higher on day 3, but did not increase further until day 5. The concentrations of collagen 1 alpha 1 (COL1A1) and osteonectin were increased and receptor activator of nuclear factor kappa-B ligand (RANKL) was reduced in response to hyperacute serum. We concluded that hyperacute serum treatment induces cell proliferation of osteoarthritic joint tissues and affects the cytokine milieu towards a less inflamed state.

The Composition of Hyperacute Serum and Platelet-Rich Plasma Is Markedly Different despite the Similar Production Method.

Autologous blood derived products, such as platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) are widely applied in regenerative therapies, in contrast to the drawbacks in their application, mainly deriving from the preparation methods used. Eliminating the disadvantages of both PRP and PRF, hyperacute serum (HAS) opens a new path in autologous serum therapy showing similar or even improved regenerative potential at the same time. Despite the frequent experimental and clinical use of PRP and HAS, their protein composition has not been examined thoroughly yet. Thus, we investigated and compared the composition of HAS, serum, PRP and plasma products using citrate and EDTA by simple laboratory tests, and we compared the composition of HAS, serum, EDTA PRP and plasma by Proteome Profiler and ELISA assays. According to our results the natural ionic balance was upset in both EDTA and citrate PRP as well as in plasma. EDTA PRP contained significantly higher level of growth factors and cytokines, especially platelet derived angiogenic and inflammatory proteins, that can be explained by the significantly higher number of platelets in EDTA PRP. The composition analysis of blood derivatives revealed that although the preparation method of PRP and HAS were similar, the ionic and protein composition of HAS could be advantageous for cell function.

Chloroplasts preferentially take up ferric-citrate over iron-nicotianamine complexes in Brassica napus.

MAIN CONCLUSION: Fe uptake machinery of chloroplasts prefers to utilise Fe(III)-citrate over Fe-nicotianamine complexes. Iron uptake in chloroplasts is a process of prime importance. Although a few members of their iron transport machinery were identified, the substrate preference of the system is still unknown. Intact chloroplasts of oilseed rape (Brassica napus) were purified and subjected to iron uptake studies using natural and artificial iron complexes. Fe-nicotianamine (NA) complexes were characterised by 5 K, 5 T Mössbauer spectrometry. Expression of components of the chloroplast Fe uptake machinery was also studied. Fe(III)-NA contained a minor paramagnetic Fe(II) component (ca. 9%), a paramagnetic Fe(III) component exhibiting dimeric or oligomeric structure (ca. 20%), and a Fe(III) complex, likely being a monomeric structure, which undergoes slow electronic relaxation at 5 K (ca. 61%). Fe(II)-NA contained more than one similar chemical Fe(II) environment with no sign of Fe(III) components. Chloroplasts preferred Fe(III)-citrate compared to Fe(III)-NA and Fe(II)-NA, but also to Fe(III)-EDTA and Fe(III)-o,o'EDDHA, and the Km value was lower for Fe(III)-citrate than for the Fe-NA complexes. Only the uptake of Fe(III)-citrate was light-dependent. Regarding the components of the chloroplast Fe uptake system, only genes of the reduction-based Fe uptake system showed high expression. Chloroplasts more effectively utilize Fe(III)-citrate, but hardly Fe-NA complexes in Fe uptake.

The potential of currently unavailable herpes virus vaccines.

INTRODUCTION: Despite overwhelming experimental work, there are no licensed vaccines against the most frequent Alphaherpesviruses, namely herpes simplex virus 1 and 2 (HSV1 and 2) nor against the Epstein-Barr virus (EBV), a member of the subfamily Gammaherpesvirus. AREAS COVERED: Since the DNAs of both HSVs reside in the regional sensory ganglia in a latent state (i.e. as circularized episomal molecules), a corresponding vaccine might be useful for immunotherapy rather than for prevention of primary infection. Here we describe the design of a purified subunit vaccine as well as the preparation and efficacy of a recombinant fusion protein consisting of the gD ectodomain from our domestic attenuated HSV1 strain HSZP. The EBV vaccines considered so far, were destined for prevention of infectious mononucleosis (IM) or to prevent formation of EBV related tumors. To design the EBV peptide vaccine, at least 15 carefully selected immunogenic epitopes coming from 12 virus coded proteins were bound to synthetic micro-particle carriers along with a non-specific pathogen recognizing receptor (PRR) stimulating both the T as well as B lymphocytes. EXPERT COMMENTARY: The efficacy of a novel EBV peptide in the rabbit model was based on criteria such as antibody formation (EA-D detected by ELISA, early and capsid proteins tested by immunoblot), presence of LMP1 antigen and of viral DNA in peripheral white blood cells. Out of 19 peptide combinations used for vaccination, at least 6 showed a satisfactory protective effect.

Low CD23 expression correlates with high CD38 expression and the presence of trisomy 12 in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by a neoplastic B-cell population coexpressing CD5 and CD23; however, the expression of CD23 is variable. In human, two isotypes of CD23 have been identified and related to different functions. The aim of our study was to investigate the relative expression of the two CD23 isotypes in CLL and find possible correlation with other prognostic factors. The expression of CD23 isotypes was analyzed in 54 cases of CLL by polymerase chain reaction (PCR) and quantitative real-time PCR. The immunophenotype of CLL cells was characterized by flow cytometry. We demonstrated a higher CD23a than CD23b expression of CLL cells. Our results also revealed two subsets of CLL cases with a distinct CD23 isotype expression pattern. Thirty-two percent of the cases (group CLL1) showed both low mRNA level of CD23 isotypes and high protein levels of CD20 and CD38 in contrast to group CLL2 with high CD23 mRNA levels. By correlating these results to the presence of prognostic factors determined by fluorescence in situ hybridization, we found that the majority of the cases of group CLL1 (14/17) carried trisomy 12. In summary, our results confirm a high CD23a/CD23b ratio of the CLL cells and demonstrate that in a subset of CLL cases, low CD23 expression together with high CD20 and CD38 expressions may serve as a surrogate for trisomy 12. Copyright © 2015 John Wiley & Sons, Ltd.

Analysis of Viral Promoter Usage in EBV-Infected Cell Lines: A Comparison of qPCR Following Conventional RNA Isolation and Nuclear Run-On Assay.

To interpret the results of an epigenetic analysis in gene expression studies, it is essential to characterize the activity of the relevant promoters. According to the literature, real-time PCR assay is the most widely used method for the determination of latent EBV promoter usage. Here we describe two alternative approaches to measure the activity of viral promoters in cell lines carrying latent EBV episomes. The widespread typical approach relies on total cellular RNA isolation, whereas the nuclear run-on assay described here is based on the initial isolation of nuclei, followed by in vitro transcription in the presence of biotinylated-UTP, and purification of RNA transcripts using avidin-coated magnetic beads. Finally, both methods apply reverse transcription-based real-time PCR (i.e., quantitative polymerase chain reaction, qPCR) to quantitatively measure the amount of specific transcripts. We shall describe these methods step by step and demonstrate their use for the determination of EBER1 promoter activity in EBV-positive cell lines.

Terminal Repeat Analysis of EBV Genomes.

Epstein-Barr virus (EBV) was the first human virus associated directly with human malignancies. During EBV infection of various host cells the double-stranded linear EBV DNA carried by the virions undergoes circularization. Since there are variable numbers of terminal repetitions (TRs) at the ends of the linear EBV genome, the resulting circular episomes enclose a variable number of TRs. Thus, in cells carrying viral episomes, the sizes of the terminal restriction enzyme fragments of EBV is affected by the number of TRs (Raab-Traub and Flynn Cell 47:883-889, 1986). Southern blot analysis revealed that in monoclonal proliferations, arising from a single cell, there was only a single band representing the joined EBV termini, whereas multiple terminal restriction enzyme fragments that differ in size were characteristic for oligoclonal or polyclonal proliferations. Using suitable probes, one can distinguish the episomal form from the linear EBV genomes that are formed during lytic EBV replication or during integration into the host genome. TR analysis is a useful tool for the determination of EBV clonality in different clinical samples and in cell lines carrying EBV genomes. A single terminal restriction enzyme fragment may indicate EBV infection at an early phase of clonal cell proliferation, whereas polyclonal EBV genomes may derive from multiple infections of proliferating cells.

Characterization of EBV Promoters and Coding Regions by Sequencing PCR-Amplified DNA Fragments.

DNA sequencing approaches originally developed in two directions, the chemical degradation method and the chain-termination method. The latter one became more widespread and a huge amount of sequencing data including whole genome sequences accumulated, based on the use of capillary sequencer systems and the application of a modified chain-termination method which proved to be relatively easy, fast, and reliable. In addition, relatively long, up to 1000 bp sequences could be obtained with a single read with high per-base accuracy. Although the recent appearance of next-generation DNA sequencing (NGS) technologies enabled high-throughput and low cost analysis of DNA, the modified chain-terminating methods are often applied in research until now. In the following, we shall present the application of capillary sequencing for the sequence characterization of viral genomes in case of partial and whole genome sequencing, and demonstrate it on the BARF1 promoter of Epstein Barr virus (EBV).

Analysis of Viral Epigenotypes Using Chromatin Immunoprecipitation.

Chromatin Immunoprecipitation (ChIP) is a method used to detect DNA-protein interactions in vivo. ChIP has been widely applied to assess the abundance of various epigenetic regulators, including modified histones, in various regions of cellular and viral chromatin. During the procedure, DNA binding proteins are covalently cross-linked to DNA, and the isolated chromatin is broken into pieces of 300-500 bps in length on average. Thereafter, using specific antibody directed against the protein of interest the covalently cross-linked DNA is pulled down together with Protein A or G carrying beads that bind the Fc fragment of the antibody. After the reversal of crosslinks and DNA isolation, one may analyze the precipitated DNA fragments by quantitative and qualitative methods to assess the relative abundance of the examined protein in a region or within the genome studied in vivo. In addition to the analysis of transcription factor binding, ChIP has proved to be a reliable method to map histone modifications across cellular and viral epigenomes.

Epigenetic Regulation.

Some of the key epigenetic regulatory mechanisms appeared early during evolution, and the acquisition of novel epigenetic regulators apparently facilitated certain evolutionary transitions. In this short review we focus mainly on the major epigenetic mechanisms that control chromatin structure and accessibility in mammalian cells. The enzymes methylating CpG dinucleotides and those involved in the active demethylation of 5-metylcytosine (5mC) are outlined together with the members of the methyl binding protein (MBP) family that bind to and "interpret" the 5mC mark. The enzymes involved in reversible, covalent modifications of core histone proteins that affect chromatin structure are also described briefly. Proteins that build up Polycomb group (PcG) and Trithorax group (TrxG) protein complexes may also modify histones. By establishing heritable chromatin states, PcG and TrxG complexes contribute - similarly to cytosine methylation - to the transmission of cell type-specific gene expression patterns from cell generation to cell generation. Novel players involved in epigenetic regulation, including variant histones, pioneer transcription factors, long noncoding RNA molecules and the regulators of long-distance chromatin interactions are introduced as well, followed by the characterization of various chromatin types.

Epstein-Barr virus-host cell interactions: an epigenetic dialog?

Here, we wish to highlight the genetic exchange and epigenetic interactions between Epstein-Barr virus (EBV) and its host. EBV is associated with diverse lymphoid and epithelial malignancies. Their molecular pathogenesis is accompanied by epigenetic alterations which are distinct for each of them. While lymphoblastoid cell lines derived from B cells transformed by EBV in vitro are characterized by a massive demethylation and euchromatinization of the viral and cellular genomes, the primarily malignant lymphoid tumor Burkitt's lymphoma and the epithelial tumors nasopharyngeal carcinoma and EBV-associated gastric carcinoma are characterized by hypermethylation of a multitude of cellular tumor suppressor gene loci and of the viral genomes. In some cases, the viral latency and oncoproteins including the latent membrane proteins LMP1 and LMP2A and several nuclear antigens affect the level of cellular DNA methyltransferases or interact with the histone modifying machinery. Specific molecular mechanisms of the epigenetic dialog between virus and host cell remain to be elucidated.

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.

Survey of Epstein Barr virus (EBV) immunogenic proteins and their epitopes: implications for vaccine preparation.

The Epstein-Barr virus (Human herpesvirus 4) encodes approximately 80 proteins, from which 15 possess at least 90 antigenic epitopes. Many of them stimulate the T cell receptors (TCR), but a few interact with the B cell receptors (BCR). Activation of B-cells and subsequent antibody production has not only been related to at least 3 envelope glycoproteins (mostly gp350) but also to latency associated membrane proteins (LMPs). The majority of EBV epitopes (over 80) inducing either cytotoxic and/or helper T lymphocytes were located on non-structural and/or latency associated polypeptides. The former interaction mediated by CD8plus/T cells is restricted by the HLA I molecules, predominantly of HLA-A subclass. In acute infectious mononucleosis (IM) patients (about 40 %) a considerable proportion of HLA B8 restricted CTL reactivity is directed against a single peptide (RAKFKQLL) of transactivator protein BZLF1/Zta. The EBV vaccines designed so far fall into two categories: those preventing any kind of infection (including prophylaxis of EBVassociated malignancies) and those designed for therapeutic purposes (to be used in subjects already infected). Preventive vaccines protecting against acute disease (such as IM) contain, as a rule, the gp350 polypeptide(s) encoded by the BLLF1 gene. Vaccines destined for tumor prevention rather consist of peptides derived from latency associated nuclear proteins (EBNA 2, 3 and 6) and/or from oncogenic latent membrane proteins (LMP1/LMP2a). Whereas the former generates antibodies preventing virus entry, the latter would potentiate the cell mediated response. In addition to recently described and purified individual recombinant immunogenic EBV polypeptides and/or their mixes, new perspectives were opened by construction of random overlapping strongly immunogenic scrambled polypeptide(s). Further novel approaches are based on carefully selected antigenic peptides (oligopeptides) coming from both, structural as well as non-structural or latencyassociated proteins bound to suitable carriers. Any constructs based on latency-associated proteins might be useful either for immunoprophylactic therapy following bone marrow and/or heart transplantations or for the prevention of EBVrelated tumors such as lymphomas and nasopharyngeal carcinoma. Due to the growing importance of the selected immunogenic epitopes as future vaccine components, at least the half of them has been patented not only as the natural amino acid sequence but also in different variations.

Epstein-Barr virus (HHV-4) inoculation to rabbits by intranasal and oral routes results in subacute and/or persistent infection dissimilar to human disease.

OBJECTIVE: We report the infection of New Zealand white rabbits with Epstein-Barr virus (EBV). METHODS: EBV prepared in B95-8 (producer) cells was inoculated to rabbits by combined intranasal and oral routes. Blood and white blood cell (WBC) samples were taken before infection, then on days 8, 28 and 98 post-infection (p.i.). RESULTS: Administration of either 3 × 10(8) (group A, 11 rabbits) or 1 × 10(9) (group B, 10 rabbits) EBV DNA copies per animal induced subacute and/or persistent infection. The IgG antibodies in plasma were detected by ELISA as well as by immunoblot (IB). The IB bands showed mainly antibodies to the BZRF1/Zta transactivation polypeptide (69.2%), the p54 early protein (53.4%) and to the p23 capsid protein (35.8%). No anti-EBNA1 antibody was detected throughout. Viral DNA could be detected by PCR in WBCs and/or spleen of 7 out of 21 infected rabbits (30%), while 60-80% of them showed serologic response. The transiently present EBV DNA was accompanied by LMP1 antigen. CONCLUSIONS: Rabbits developed persistent EBV infection in the absence of EBNA1 antibodies and by the lack of typical infectious mononucleosis-like syndrome. The absence of EBNA1 antibody may reflect the lack of EBNA1 in B cells of EBV-inoculated rabbits.

The role of DNA hypomethylation, histone acetylation and in vivo protein-DNA binding in Epstein-Barr virus-induced CD23 upregulation.

We analyzed epigenetic marks at the CD23 regulatory regions in well characterized Epstein-Barr virus (EBV)-carrying cell lines covering the major latency types. Bisulfite sequencing showed that DNA methylation is not a major regulator of EBV-induced CD23 transcription, although a wide hypomethylated DNA sequence in the regulatory regions is always present in the cell lines with high CD23 expression. Acetylated histone H3 levels at the CD23b promoter showed strong correlation with CD23b expression, while a weaker correlation could be observed at the CD23a core promoter. DMS in vivo footprinting at the intronic EBV-responsive enhancer and the intermediate-affinity CBF1 site at the CD23a core promoter did not reveal any significant sign of in vivo protein-DNA interactions, despite the presence of strong, characteristic footprints in the same DMS-treated DNA samples at the two CBF1 sites of the LMP2A-promoter. Our in vivo results suggest a minor role for DNA methylation, while a more important role for histone acetylation in the regulation of EBV-induced CD23 expression. Furthermore, our in vivo footprinting results support the complex model of CD23 induction by EBV, rather than a simple model with direct transactivation of CD23 by EBNA-2.