Epstein-Barr Virus Strains Research Articles

Distribution, persistence and interchange of Epstein-Barr virus strains among PBMC, plasma and saliva of primary infection subjects.

Our study aimed at investigating the distribution, persistence and interchange of viral strains among peripheral blood mononuclear cells (PBMC), plasma and saliva of primary Epstein-Barr virus (EBV) infection subjects. Twelve infectious mononucleosis (IM) patients and eight asymptomatic individuals (AS) with primary EBV infection were followed longitudinally at several time points for one year from the time of diagnosis, when blood and saliva samples were collected and separated into PBMC, plasma and saliva, representing circulating B cell, plasma and epithelial cell compartments, respectively. To survey the viral strains, genotyping assays for the natural polymorphisms in two latent EBV genes, EBNA2 and LMP1, were performed and consisted of real-time PCR on EBNA2 to distinguish type 1 and 2 viruses, fluorescent-based 30-bp typing assay on LMP1 to distinguish deletion and wild type LMP1, and fluorescent-based heteroduplex tracking assays on both EBNA2 and LMP1 to distinguish defined polymorphic variants. No discernible differences were observed between IM patients and AS. Multiple viral strains were acquired early at the start of infection. Stable persistence of dominant EBV strains in the same tissue compartment was observed throughout the longitudinal samples. LMP1-defined strains, China 1, China 2 and Mediterranean+, were the most common strains observed. EBNA2-defined groups 1 and 3e predominated the PBMC and saliva compartments. Concordance of EBNA2 and LMP1 strains between PBMC and saliva suggested ready interchange of viruses between circulating B cell and epithelial cell pools, whilst discordance of viral strains observed between plasma and PBMC/saliva indicated presence of viral pools in other undetermined tissue compartments. Taken together, the results indicated that the distribution, persistence and interchange of viral strains among the tissue compartments are more complex than those proposed by the current model of EBV life cycle.

Genome diversity of Epstein-Barr virus from multiple tumor types and normal infection.

ABSTRACTEpstein-Barr virus (EBV) infects most of the world's population and is causally associated with several human cancers, but little is known about how EBV genetic variation might influence infection or EBV-associated disease. There are currently no published wild-type EBV genome sequences from a healthy individual and very few genomes from EBV-associated diseases. We have sequenced 71 geographically distinct EBV strains from cell lines, multiple types of primary tumor, and blood samples and the first EBV genome from the saliva of a healthy carrier. We show that the established genome map of EBV accurately represents all strains sequenced, but novel deletions are present in a few isolates. We have increased the number of type 2 EBV genomes sequenced from one to 12 and establish that the type 1/type 2 classification is a major feature of EBV genome variation, defined almost exclusively by variation of EBNA2 and EBNA3 genes, but geographic variation is also present. Single nucleotide polymorphism (SNP) density varies substantially across all known open reading frames and is highest in latency-associated genes. Some T-cell epitope sequences in EBNA3 genes show extensive variation across strains, and we identify codons under positive selection, both important considerations for the development of vaccines and T-cell therapy. We also provide new evidence for recombination between strains, which provides a further mechanism for the generation of diversity. Our results provide the first global view of EBV sequence variation and demonstrate an effective method for sequencing large numbers of genomes to further understand the genetics of EBV infection. IMPORTANCE Most people in the world are infected by Epstein-Barr virus (EBV), and it causes several human diseases, which occur at very different rates in different parts of the world and are linked to host immune system variation. Natural variation in EBV DNA sequence may be important for normal infection and for causing disease. Here we used rapid, cost-effective sequencing to determine 71 new EBV sequences from different sample types and locations worldwide. We showed geographic variation in EBV genomes and identified the most variable parts of the genome. We identified protein sequences that seem to have been selected by the host immune system and detected variability in known immune epitopes. This gives the first overview of EBV genome variation, important for designing vaccines and immune therapy for EBV, and provides techniques to investigate relationships between viral sequence variation and EBV-associated diseases.

Варианты онкогенаLMP1вируса Эпштейна–Барр в клеточных линиях различного происхождения

It is well known that the Epstein-Barr virus (EBV) is a widespread infection in the human population. Typically, infection occurs in early childhood without serious consequences for infected people. At the same time, a secondary infection with an additional EBV strain occurs quite often. During the in vitro cultivation of peripheral blood lymphocytes from persons infected with multiple strains of the virus, only one of these strains with higher transforming potential becomes dominant, while the others are eliminated. Under certain conditions, such a highly transforming EBV strain apparently is able to be the etiologic agent of EBVassociated diseases. To find out the range of highly transforming EBV strains prevalent among Russians, cell lines from patients with EBV-associated and non-associated tumors, as well as healthy individuals, were established. The structural analysis of the latent membrane protein 1 gene (LMP1), a key oncogene of the virus, isolated from established cell lines and peripheral blood lymphocytes of blood donors was carried out, and data obtained were compared with the respective data for LMP1 isolates, amplified from cell lines established from African and Japanese patients with Burkitt's lymphoma. The data obtained show a genetic relationship between Russian LMP1 isolates regardless the fact whether they come from patients with tumors or healthy individuals and differ significantly from LMP1 isolates from Burkitt's lymphoma patients. Thus, the results of the study suggest that in nonendemic region for EBV-associated pathology, Russia, any strain of EBV with any structure of LMP1 with concomitant effect of additional factors may become an etiologic agent for EBV-associated neoplasia.

Epstein-Barr Virus Strain Variation.

What is wild-type Epstein-Barr virus and are there genetic differences in EBV strains that contribute to some of the EBV-associated diseases? Recent progress in DNA sequencing has resulted in many new Epstein-Barr virus (EBV) genome sequences becoming available. EBV isolates worldwide can be grouped into type 1 and type 2, a classification based on the EBNA2 gene sequence. Type 1 transforms human B cells into lymphoblastoid cell lines much more efficiently than type 2 EBV and molecular mechanisms that may account for this difference in cell transformation are now becoming understood. Study of geographic variation of EBV strains independent of the type 1/type 2 classification and systematic investigation of the relationship between viral strains, infection and disease are now becoming possible. So we should consider more directly whether viral sequence variation might play a role in the incidence of some EBV-associated diseases.

New Insight into Epstein-Barr Virus-Associated Biological Behavior of Aggressive Natural Killer Cell Leukemia

Aggressive natural killer cell leukemia (ANKL) is a rare and highly aggressive subtype of mature NK-cell neoplasms. Similar with extranodal NK/T-cell lymphoma, nasal type (ENKL), another subtype of NK-cell neoplasm, ANKL is also an Asian-prevalent and Epstein-Barr virus (EBV)-related neoplasm. In contrast, our knowledge of ANKL, especially about EBV biological behavior in this rare leukemia, lags far behind that of ENKL and other EBV-related hematopoietic malignancies, such as Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and post-transplant lymphoproliferative disorder (PTLD). Dissection of the virus-host crosstalk in ANKL could contribute to better understanding the mechanism and finding out effective therapy for this neoplasm.In the present study, we investigated EBV-associated biological behavior in serial ANKL patients, including the clinical presentation, EBV genomic DNA, EBV antigens expression, cytogenetic-molecular aberrations, and leukemia-associated microenvironment. A total of 28 ANKL patients were collected upon review of the clinical database in Nanfang hospital. Different items of EBV infection evidence consisted of EBV viremia (n=9), EBV genomic DNA (n=20), and EBER/EBNA/LMP1/LMP2A expression (n=23). EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) was the predominant clinical feature. Bone marrow smear was infiltrated with large granular lymphocyte (LGL) with LMP1/LMP2a-positive bulb, indicating the presence of EBV viral inclusions bodies. Positron emission tomographic (PET)-computed tomographic (CT) scan revealed bone marrow, liver and spleen as the most frequently involved organs, compared with nose and nasopharynx in ENKL. Cytogenetic analysis demonstrated 7q10-32 (n=4) was the “hotspot” of chromosome aberrations in ANKL. PCR analysis with EBNA-2/LMP1 specific primers on reserved DNA samples (n=20) revealed ANKL cells were infected with type-1 EBV strain with wide-type LMP1 (n=20), compared with 30bp-deleted LMP1 gene in ENKL. Integrated mutation analysis (n=20) identified recurrent mutations in Src homology 2 (SH2) domains of STAT5a (n=7) and p16inka (exon 3/4, n=20), but no mutation in SH2 domains of ID2, STAT1, and STAT3. Immunochemical (IHC) analysis on formalin-fixed paraffin-embedded tissues (n=23) revealed latency type-3 EBV expression in ANKL cells, with latency antigens of EBER, EBNA, LMP-1, and LMP-2. Furthermore, LMP-1/LMP-2-positive leukemia/lymphoma-associated macrophages (LAMs, n=23) were enriched in ANKL microenvironment. Notably, EBV-positive LAMs were significantly associated with poor prognosis and disease progression. Univariate analysis revealed significant difference (p<0.05) in overall survival (OS) between High-Ratio of CD68+LAMs/CD56+ANKLs (HMA, n=13) and Low-Ratio LAM/ANKLs cohorts (LMA, n=10). Furthermore, IHC analysis on paired presentation and progression samples (n=3) showed that EBV-positive LAMs increased in pace with disease progression.Conclusions: Our data demonstrate that type-1 EBV strain as being latency type-3 expression infected both leukemia cells and microenvironment and thus linked the pathogen-microenvironment-host crosstalk in ANKL. EBV-infected leukemia-associated microenvironment, particularly LAMs, might not only play a critical role in prognosis classification but also contribute to the leukemogenesis of NK transformation in ANKL, which it’s still poorly understood and deserves more research efforts. DisclosuresZhou:Guangzhou Pearl River of Science and Technology New Star Project: Research Funding.

The V-val subtype Epstein-Barr virus nuclear antigen 1 promotes cell survival after serum withdrawal.

Epstein-Barr virus (EBV) can establish latent infection and has been associated with various human cancers. Epstein-Barr nuclear antigen 1 (EBNA1) is the only viral protein that is expressed in all EBV-associated malignant tissues. The N- and C-terminal domains of EBNA1, which are connected by internal glycine/alanine-rich short repeat sequences of various sizes, show sequence divergence across EBV strains isolated from around the world. At least five subtypes have been described, according to the amino acid at residue 487: P-ala, P-thr, V-val, V-pro, and V-leu. Whether the variations of EBNA-1 contribute to the pathogenesis of EBV or simply reflect the geographical distribution of EBV remain to be investigated. Furthermore, the cell effects conferred by EBNA1 subtypes that differ from that of the B95.8 prototype, which belongs to the P-ala subtype, remain to be elucidated. In this study, PCR was amplified with the full-length V-val EBNA1 gene from the CG3 cell line, an EBV-carrying lymphoblastoid cell line derived from a Taiwanese chronic myeloid leukemia patient. Plasmids expressing His-tagged EBNA1 fusion proteins in E. coli were constructed and used to raise antibodies in rabbit. The V-val EBNA1 gene was then cloned into a eukaryotic expression vector and successfully expressed in the transfected cultured cells. Expression of V-val EBNA1 rendered 293 cells able to undergo serum‑independent cell proliferation, providing them with anti-apoptotic abilities, which are two characteristics of cancer cells. These data suggested that use of EBNA1 originally derived from tumor cells, rather than the more commonly utilized prototype, when investigating the potential role of EBNA1 in the oncogenesis of EBV-associated malignancies, is crucial.

Functional analysis and molecular characterization of spontaneously outgrown human lymphoblastoid cell lines.

In vitro, the infection of human B-cells with the lymphotropic gammaherpesvirus Epstein-Barr virus (EBV) induces formation of permanently growing lymphoblastoid cell lines (LCL). In a spontaneously outgrown LCL (cell line CSIII), we detected nucleotide sequence variations of the EBV nuclear antigen 1 (EBNA1) RNA that was different from the reference sequence of EBNA1 in the prototypic EBV strain B95-8. In the present study, we molecularly and functionally characterized this virus isolate in comparison to LCL with the prototypic nucleotide sequence. Although we detected high functional similarity between CSIII and the other LCL, our data suggest that the lytic cycle might be ineffective in the CSIII LCL. DNA microarray analysis indicated that RNA binding motif, single stranded interacting protein 1 (RBMS1), which is typically expressed in latency III of EBV to prevent the lytic cycle, was the most overexpressed gene in CSIII LCL.

Genomic diversity of Epstein-Barr virus genomes isolated from primary nasopharyngeal carcinoma biopsy samples.

Undifferentiated nasopharyngeal carcinoma (NPC) has a 100% association with Epstein-Barr virus (EBV). However, only three EBV genomes isolated from NPC patients have been sequenced to date, and the role of EBV genomic variations in the pathogenesis of NPC is unclear. We sought to obtain the sequences of EBV genomes in multiple NPC biopsy specimens in the same geographic location in order to reveal their sequence diversity. Three published EBV (B95-8, C666-1, and HKNPC1) genomes were first resequenced using the sequencing workflow of target enrichment of EBV DNA by hybridization, followed by next-generation sequencing, de novo assembly, and joining of contigs by Sanger sequencing. The sequences of eight NPC biopsy specimen-derived EBV (NPC-EBV) genomes, designated HKNPC2 to HKNPC9, were then determined. They harbored 1,736 variations in total, including 1,601 substitutions, 64 insertions, and 71 deletions, compared to the reference EBV. Furthermore, genes encoding latent, early lytic, and tegument proteins and glycoproteins were found to contain nonsynonymous mutations of potential biological significance. Phylogenetic analysis showed that the HKNPC6 and -7 genomes, which were isolated from tumor biopsy specimens of advanced metastatic NPC cases, were distinct from the other six NPC-EBV genomes, suggesting the presence of at least two parental lineages of EBV among the NPC-EBV genomes. In conclusion, much greater sequence diversity among EBV isolates derived from NPC biopsy specimens is demonstrated on a whole-genome level through a complete sequencing workflow. Large-scale sequencing and comparison of EBV genomes isolated from NPC and normal subjects should be performed to assess whether EBV genomic variations contribute to NPC pathogenesis. This study established a sequencing workflow from EBV DNA capture and sequencing to de novo assembly and contig joining. We reported eight newly sequenced EBV genomes isolated from primary NPC biopsy specimens and revealed the sequence diversity on a whole-genome level among these EBV isolates. At least two lineages of EBV strains are observed, and recombination among these lineages is inferred. Our study has demonstrated the value of, and provided a platform for, genome sequencing of EBV.

A Molecular Basis for the Interplay between T Cells, Viral Mutants, and Human Leukocyte Antigen Micropolymorphism

Mutations within T cell epitopes represent a common mechanism of viral escape from the host protective immune response. The diverse T cell repertoire and the extensive human leukocyte antigen (HLA) polymorphism across populations is the evolutionary response to viral mutation. However, the molecular basis underpinning the interplay between HLA polymorphism, the T cell repertoire, and viral escape is unclear. Here we investigate the T cell response to a HLA-B*35:01- and HLA-B*35:08-restricted (407)HPVGEADYFEY(417) epitope from Epstein-Barr virus and naturally occurring variants at positions 4 and 5 thereof. Each viral variant differently impacted on the epitope's flexibility and conformation when bound to HLA-B*35:08 or HLA-B*35:01. We provide a molecular basis for understanding how the single residue polymorphism that discriminates between HLA-B*35:01/08 profoundly impacts on T cell receptor recognition. Surprisingly, one viral variant (P5-Glu to P5-Asp) effectively changed restriction preference from HLA-B*35:01 to HLA-B*35:08. Collectively, our study portrays the interplay between the T cell response, viral escape, and HLA polymorphism, whereby HLA polymorphism enables altered presentation of epitopes from different strains of Epstein-Barr virus.

Detection of the Predominant Strain of Epstein-Barr Virus in Systemic Autoimmune and Thalassemia Patients

EBV, is a member of the herpesvirus family and one of the most common human viruses, Epidemiological data suggest that EBV is associated with polytransfused blood βthalassemia and several autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis. We examined the presence of IgM antibodies against EBV in serum of 35 Thalassemic patients, 75 autoimmune patients among as 35 rheumatoid arthritis patients, 20 Systemic lupus erythrematosus and 20 autoimmune hypothyroid diseases, and 20 healthy controls by ELISA assay then detected the predominant strain in positive samples. The results show that the highest EBV infection percent was in SLE 15% whilst the lowest infection percent was in Thalassemia 5.7%., and according to gender, the results showed that the highest infection percentage recorded in females with rheumatoid arthritis 30 %, whilst the infection does not appear in males with rheumatoid arthritis and autoimmune thyroid disease and females of thalassemia patients. On the other hand, this study reveals that EBV-1 is the predominant strain in autoimmune diseases and thalassemia in Iraq.

Genome-Wide Analysis of Wild-Type Epstein–Barr Virus Genomes Derived from Healthy Individuals of the 1000 Genomes Project

Most people in the world (∼90%) are infected by the Epstein–Barr virus (EBV), which establishes itself permanently in B cells. Infection by EBV is related to a number of diseases including infectious mononucleosis, multiple sclerosis, and different types of cancer. So far, only seven complete EBV strains have been described, all of them coming from donors presenting EBV-related diseases. To perform a detailed comparative genomic analysis of EBV including, for the first time, EBV strains derived from healthy individuals, we reconstructed EBV sequences infecting lymphoblastoid cell lines (LCLs) from the 1000 Genomes Project. As strain B95-8 was used to transform B cells to obtain LCLs, it is always present, but a specific deletion in its genome sets it apart from natural EBV strains. After studying hundreds of individuals, we determined the presence of natural EBV in at least 10 of them and obtained a set of variants specific to wild-type EBV. By mapping the natural EBV reads into the EBV reference genome (NC007605), we constructed nearly complete wild-type viral genomes from three individuals. Adding them to the five disease-derived EBV genomic sequences available in the literature, we performed an in-depth comparative genomic analysis. We found that latency genes harbor more nucleotide diversity than lytic genes and that six out of nine latency-related genes, as well as other genes involved in viral attachment and entry into host cells, packaging, and the capsid, present the molecular signature of accelerated protein evolution rates, suggesting rapid host–parasite coevolution.

Study of Epstein-Barr virus expression in Burkitt's lymphoma by polymerase chain reaction and in situ hybridization: A study in Iran.

Background:The association of Epstein-Barr virus (EBV) with Burkitt's lymphoma (BL) is variable in different geographic regions. In developing countries, the association of EBV with BL is regarded to be of an endemic-type in equatorial Africa (> 95%) and sporadic-type in the developed countries (15-30%). The purpose of this study is to assess the frequency of EBV infection in BL, in Iran. The study also aims to compare Ribonucleic acid (RNA) in situ hybridization (RISH), the standard diagnostic method, with the polymerase chain reaction (PCR)-based method for diagnosing BL.Materials and Methods:In this epidemiological study, the paraffinized specimens of 18 cases of BL were selected. Next, the ISH of EBV-encoded RNA (EBER-RISH) and PCR assays that were based on Epstein Barr Nuclear Antigen 2 (EBNA2) amplification were used. The EBV strain was determined by PCR. The data were analyzed using the SPSS10 software and by performing Pearson correlation coefficient formula at a significant level of 0.05.Results:EBV RNA was detected in 50% of the BL specimens. Type 1 and 2 accounted for 70 and 30% of the cases, respectively. Regarding RISH as the standard method for EBV diagnosis, the PCR assays showed a sensitivity and specificity of 100 and 88.9%, respectively.Conclusion:According to the obtained findings, the frequency of EBV in BL was 50% and PCR and RISH showed high concordance and sensitivity in EBV detection. Therefore, PCR can be used as a faster method for EBV detection in high-risk geographical regions.

Epstein-Barr virus IL-10 gene expression by a recombinant murine gammaherpesvirus in vivo enhances acute pathogenicity but does not affect latency or reactivation.

BackgroundMany viral genes affect cytokine function within infected hosts, with interleukin 10 (IL-10) as a commonly targeted mediator. Epstein-Barr virus (EBV) encodes an IL-10 homologue (vIL-10) expressed during productive (lytic) infection and induces expression of cellular IL-10 (cIL-10) during latency. This study explored the role of vIL-10 in a murine gammaherpesvirus (MHV) model of viral infection.MethodsThe EBV vIL-10 gene was inserted into MHV-76, a strain which lacks the ability to induce cIL-10, by recombination in transfected mouse cells. Mice were infected intranasally with the recombinant, vIL-10-containing MHV-76 or control virus strains and assayed at various days post infection for lung virus titer, spleen cell number, percentage of latently infected spleen cells and ability to reactivate virus from spleen cells.ResultsRecombinant murine gammaherpesvirus expressing EBV vIL-10 rose to significantly higher titers in lungs and promoted an increase in spleen cell number in infected mice in comparison to MHV strains lacking the vIL-10 gene. However, vIL-10 expression did not alter the quantity of latent virus in the spleen or its ability to reactivate.ConclusionsIn this mouse model of gammaherpesvirus infection, EBV vIL-10 appears to influence acute-phase pathogenicity. Given that EBV and MHV wild-type strains contain other genes that induce cIL-10 expression in latency (e.g. LMP-1 and M2, respectively), vIL-10 may have evolved to serve the specific role in acute infection of enlarging the permissive host cell population, perhaps to facilitate initial survival and dissemination of viral-infected cells.

Sequence variants of LMP1 oncogene in patients with oral cavity tumors associated and not associated with Epstein-Barr virus

The role of Epstein-Barr virus (EBV), ubiquitous lymphotropic human herpesvirus 4, in etiology of nasopharyngeal carcinoma (NPC) has not been completely clarified. The mechanism of carcinogenesis in this disease (closely associated with EBV) is also unclear. The aim of the present study was to compare the structure of the LMP1 oncogene of EBV in isolates of the virus obtained from patients with two types of oral cavity tumors, including (a) associated (NPC) and (b) not associated (other tumors of the same anatomical region, OTOC) with EBV. A comparative analysis of the deductive C-terminal amino acid sequences of the LMP1 variants was carried out based on the LMP1 sequence data from samples of the tumor, blood, and oropharynx lavages from patients with NPC and OTOC. It was demonstrated that, in the compared groups of patients, all structural characteristics of LMP1 were close, and existing differences between the compared parameters were statistically insignificant. Thus, it was demonstrated for the first time that genetically related EBV strains with structurally similar LMP1 variants persist in patients with NPC and OTOC in Russia, which most likely reflects the polymorphism of EBV strains that circulate in the population. Based on the data obtained, it is possible to assume that the risk of the occurrence of NPC in NPC non-endemic world regions (including Russia) depends not so much on the EBV strain (and on the variant of the LMP1 that it contains) as on the genetic predisposition to the disease of individuals infected by this virus and the effect of other (still unknown) agents.

Spontaneous Lytic Replication and Epitheliotropism Define an Epstein-Barr Virus Strain Found in Carcinomas

The Epstein-Barr virus (EBV) is found in a variety of tumors whose incidence greatly varies around the world. A poorly explored hypothesis is that particular EBV strains account for this phenomenon. We report that M81, a virus isolated from a Chinese patient with nasopharyngeal carcinoma (NPC), shows remarkable similarity to other NPC viruses but is divergent from all other known strains. M81 exhibited a reversed tropism relative to common strains with a reduced ability to infect B cells and a high propensity to infect epithelial cells, which is in agreement with its isolation from carcinomas. M81 spontaneously replicated in B cells invitro and invivo at unusually high levels, in line with the enhanced viral replication observed in NPC patients. Spontaneous replication and epitheliotropism could be partly ascribed to polymorphisms within viral proteins. We suggest considering M81 and its closely related isolates as an EBV subtype with enhanced pathogenic potential.

Epstein-barr virus diversity in immunocompetent healthy persons: Reassessment of the distribution of genetic variants

Epstein-Barr virus (EBV) has many strains; however, it remains unclear whether a causal relationship exists between different regions and viral genetic variants in healthy persons. This study was designed to examine the relationship between EBV strains in tonsils and adenoids and peripheral blood lymphocytes of the same individuals using different measurements of EBV strain polymorphism. This study examined whether EBV contains two or three copies of a tandem repeat sequence in the first intron of the BZLF-1 gene. The genotype of the virus from P3HR-1, designated Z*, yielded a 415-bp product, and this was distinguished from the smaller, 386-bp product obtained with the B95-8 virus, designated the Z genotype. Simultaneous sequence infections with Z and Z* genotypes were also detected in one of the tonsils examined, suggesting that more than one strain or variant of EBV genotype may be present in a specimen from the same subject. Co-infection with Z and Z* was recognized in two subjects, so variation of the EBV gene may be seen in at least two different strains of EBV. It was seen that Z and Z* strain-infected cells are constantly in flux through lymph nodes and/or the blood stream in healthy persons; therefore, these results indicated that EBV genome variants probably show no specific tissue distribution.

Evaluation of Novel Acyclic Nucleoside Phosphonates against Human and Animal Gammaherpesviruses Revealed an Altered Metabolism of Cyclic Prodrugs upon Epstein-Barr Virus Reactivation in P3HR-1 Cells

Acyclic nucleoside phosphonates (ANPs), such as (S)-1-[(3-hydroxy-2-phosphonomethoxy)propyl)]cytosine (HPMPC), are an important group of broad-spectrum antiviral agents with activity against DNA viruses. In this report, we present the in vitro potencies of novel ANPs against gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus (EBV), and three animal gammaherpesviruses. 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine (HPMP-5-azaC), (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-3-deazaadenine (3-deaza-HPMPA), and their cyclic derivatives have emerged as highly potent antigammaherpesvirus agents. Interestingly, cyclic prodrugs of ANPs exhibited reduced activities against EBV strain P3HR-1, but not against EBV strain Akata. Cell culture metabolism studies with HPMPC and cyclic HPMPC revealed that these differences were attributable to an altered drug metabolism in P3HR-1 cells after EBV reactivation and, more specifically, to a reduced hydrolysis of cyclic HPMPC by cyclic CMP phosphodiesterase. We did not correlate this effect with phosphodiesterase downregulation, or to functional mutations. Instead, altered cyclic AMP levels in P3HR-1 cells indicated a competitive inhibition of the phosphodiesterase by this cyclic nucleotide. Finally, both HPMPC and HPMP-5-azaC emerged as highly effective inhibitors in vivo through significant inhibition of murine gammaherpesvirus replication and dissemination. With the current need for potent antigammaherpesvirus agents, our findings underline the requirement of appropriate surrogate viruses for antiviral susceptibility testing and highlight HPMP-5-azaC as a promising compound for future clinical development.

Complete genomic sequence of Epstein-Barr virus in nasopharyngeal carcinoma cell line C666-1

BackgroundNasopharyngeal carcinoma is a distinct type of head and neck cancer which is consistently associated with Epstein-Barr virus (EBV). The C666-1 cell line is the only in vitro native EBV-infected NPC cell model commonly used for study of the viral-host interaction. Nevertheless, the complete EBV genome sequence in this in vitro EBV-infected NPC model has not been characterized.ObjectiveTo determine the complete EBV genome sequence in C666-1 cells.MethodsThe C666-1 genome was sequenced by 100-bases pair-end massive parallel sequencing. Bioinformatics analysis was performed to extract the EBV sequences and construct an EBV consensus sequence map. PCR amplification and Sanger DNA sequencing were used for sequence validation and gap filling. A phylogenetic analysis of EBV strain in C666-1 cells and other reported EBV strains was performed.ResultsA 171,317 bp complete EBV genome of C666-1 was successfully constructed (GenBank accession number: KC617875). Phylogenetic analysis of EBV genome in C666-1 revealed that the C666-1 EBV strain is closely related to the reported strains in NPC primary tumors.ConclusionC666-1 contains a representative NPC-associated EBV genome and might serve as an important model for studying the roles or function of viral proteins in NPC tumorigenesis.

Epstein-Barr virus gene polymorphism in different parts of the same nasopharyngeal carcinoma patient

In most previous studies, comparisons of Epstein-Barr virus (EBV) gene polymorphisms and genotypes were made between strains from tumors and normal throat washings (TWs) from different individuals. However, it remains controversial whether different EBV subtypes are present in different parts of the same NPC patient. In order to address this question, in this study, we compared the genotypes in sets of paired throat washings (TW) and paraffin-embedded tissues of 20 patients with nasopharyngeal carcinoma (NPC), and we found the same genotype in throat washings (TWs) and tumor cells from the same individual in most cases. The subtypes of EBER and EBNA1 genes were furthermore sequenced, and identical EBV strains were identified in tumor tissues and TWs. In conclusion, different sites of the same individual are infected by the same EBV strains, except for a few differences in occasional cases, suggesting that the EBV subtype detected in throat washings is a reasonable guide to the subtype present in the carcinoma tissue.

Variations of Epstein-Barr virus nuclear antigen 1 in Epstein-Barr virus-associated gastric carcinomas from Guangzhou, southern China.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein consistently expressed in all EBV-associated malignancies, and play a critical role in the onset, progression, and/or maintenance of these tumors. Based on the signature changes at amino acid residue 487, EBNA1 is classified into five distinct subtypes: P-ala, P-thr, V-leu, V-val and V-pro. In the present study, the sequence variations of EBNA1 in EBV-associated gastric carcinoma (EBVaGC) and throat washing (TW) samples of healthy EBV carriers in Guangzhou, southern China, where nasopharyngeal carcinoma (NPC) is endemic, were analyzed by PCR and DNA sequencing. V-val subtype was the most predominant (53.6%, 15/28) in EBVaGC, followed by P-ala (42.9%, 12/28) and V-leu (32.1%, 9/28) subtypes. In TWs of healthy EBV carriers, V-val subtype was also predominant (85.7%, 18/21). The sequence variations of EBNA1 in EBVaGC were similar to those in TW of healthy EBV carriers (p>0.05), suggesting that the EBV strains in EBVaGC might originate from the viral strains prevalent within the background population. The predominance of V-val subtype in EBVaGC in Guangzhou was similar to that in EBVaGC in northern China and Japan, but was different from that in EBVaGC in America, suggesting that the variations of EBNA1 in EBVaGC represent geographic-associated polymorphisms rather than tumor-specific mutations. In addition, the EBNA1 variations in EBVaGC in gastric remnant carcinoma were also determined. V-leu subtype was detected in all 4 (100%) cases, although 2 cases occurred as mixed infection with P-ala subtype. This is different from the predominant V-val subtype in EBVaGC in conventional gastric carcinoma, suggesting that V-leu might be a subtype that adapts particularly well to the microenvironment within the gastric stump and enters the remnant gastric mucosa epithelia easily. This, to our best knowledge, is the first investigation of EBNA1 polymorphisms in EBVaGC from endemic area of NPC.