A global phylogenetic analysis of Japanese tonsil-derived Epstein-Barr virus strains using viral whole-genome cloning and long-read sequencing.

Epstein-Barr virus (EBV) establishes lifelong latent infection in the majority of healthy individuals, while it is a causative agent for various diseases, including some malignancies. Recent high-throughput sequencing results indicate that there are substantial levels of viral genome heterogeneity among different EBV strains. However, the extent of EBV strain variation among asymptomatically infected individuals remains elusive. Here, we present a streamlined experimental strategy to clone and sequence EBV genomes derived from human tonsillar tissues, which are the reservoirs of asymptomatic EBV infection. Complete EBV genome sequences, including those of repetitive regions, were determined for seven tonsil-derived EBV strains. Phylogenetic analyses based on the whole viral genome sequences of worldwide non-tumour-derived EBV strains revealed that Asian EBV strains could be divided into several distinct subgroups. EBV strains derived from nasopharyngeal carcinoma-endemic areas constitute different subgroups from a subgroup of EBV strains from non-endemic areas, including Japan. The results could be consistent with biased regional distribution of EBV-associated diseases depending on the different EBV strains colonizing different regions in Asian countries.

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.

Epstein-Barr virus strain variation and cancer.

Epstein-Barr virus (EBV) is a human tumor virus and is etiologically linked to various malignancies. Certain EBV-associated diseases, such as Burkitt lymphomas and nasopharyngeal carcinomas, are endemic and exhibit biased geographic distribution worldwide. Recent advances in deep sequencing technology enabled high-throughput sequencing of the EBV genome from clinical samples. Rapid cloning and sequencing of cancer-derived EBV genomes, followed by reconstitution of infectious virus, have also become possible. These developments have revealed that various EBV strains are differentially distributed throughout the world, and that the behavior of cancer-derived EBV strains is different from that of the prototype EBV strain of non-cancerous origin. In this review, we summarize recent progress and future perspectives regarding the association between EBV strain variation and cancer.

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.

Prevalence and viral load of oncogenic human papillomavirus (HPV) in pterygia in multi-ethnic patients in the Malay Peninsula.

PURPOSE: The aim of the study was to determine the prevalence of human papillomavirus (HPV) in primary and recurrent pterygia samples collected from different ethnic groups in the equatorial Malay Peninsula. METHODS: DNA was extracted from 45 specimens of freshly obtained primary and recurrent pterygia from patients and from 11 normal conjunctival swabs from volunteers with no ocular surface lesion as control. The presence of HPV DNA was detected by nested PCR. PCR-positive samples were subjected to DNA sequencing to determine the HPV genotypes. Real-time PCR with HPV16 and HPV18 type-specific TaqMan probes was employed to determine the viral DNA copy number. RESULTS: Of 45 pterygia samples with acceptable DNA quality, 29 (64.4%) were positive for HPV DNA, whereas all the normal conjunctiva swabs were HPV negative. Type 18 was the most prevalent (41.4% of positive samples) genotype followed by type 16 (27.6%). There was one case each of the less common HPV58 and HPV59. Seven of the samples harboured mixed infections of both HPV16 and HPV18. All the four known recurrent pterygia samples were HPV-positive, whereas the sole early-stage pterygium sample in the study was HPV-negative. There was no significant association between HPV-positive status with gender or age. A high proportion of patients from the Indian ethnic group (five of six) were HPV-positive, whereas the Malay patients were found to have higher HPV positivity than the Chinese. The viral load of HPV18 samples ranged between 2 × 10(2) and 3 × 10(4) copies per µg, whereas the viral load of HPV16 specimen was 4 × 10(1) to 10(2) copies per µg. CONCLUSION: This report describes for the first time the quantitative measurement of HPV viral DNA for pterygium samples. The high prevalence of oncogenic HPVs in our samples suggests a possible role for HPV in the pathogenesis of pterygia. Moreover, the relatively low HPV viral load is concordant with the premalignant nature of this ocular condition.

In vivo and in vitro studies suggest a possible involvement of HPV infection in the early stage of breast carcinogenesis via APOBEC3B induction.

High prevalence of infection with high-risk human papilloma virus (HPV) ranging from 25 to 100% (average 31%) was observed in breast cancer (BC) patients in Singapore using novel DNA chip technology. Early stage of BC demonstrated higher HPV positivity, and BC positive for estrogen receptor (ER) showed significantly higher HPV infection rate. This unique association of HPV with BC in vivo prompted us to investigate a possible involvement of HPV in early stages of breast carcinogenesis. Using normal breast epithelial cells stably transfected with HPV-18, we showed apparent upregulation of mRNA for the cytidine deaminase, APOBEC3B (A3B) which is reported to be a source of mutations in BC. HPV-induced A3B overexpression caused significant γH2AX focus formation, and DNA breaks which were cancelled by shRNA to HPV18 E6, E7 and A3B. These results strongly suggest an active involvement of HPV in the early stage of BC carcinogenesis via A3B induction.

Epstein-Barr virus induces erosive arthritis in humanized mice.

Epstein-Barr virus (EBV) has been implicated in the pathogenesis of rheumatoid arthritis (RA) on the basis of indirect evidence, such as its presence in affected joint tissues, antigenic cross reactions between EBV and human proteins, and elevated humoral and cellular anti-EBV immune responses in patients. Here we report development of erosive arthritis closely resembling RA in humanized mice inoculated with EBV. Human immune system components were reconstituted in mice of the NOD/Shi-scid/IL-2Rγ(null) (NOG) strain by transplantation with CD34(+) hematopoietic stem cells isolated from cord blood. These humanized mice were then inoculated with EBV and examined pathologically for the signs of arthritis. Erosive arthritis accompanied by synovial membrane proliferation, pannus formation, and bone marrow edema developed in fifteen of twenty-three NOG mice transplanted with human HSC and inoculated with EBV, but not in the nine NOG mice that were transplanted with HSC but not inoculated with EBV. This is the first report of an animal model of EBV-induced arthritis and strongly suggest a causative role of the virus in RA.

Novel mouse xenograft models reveal a critical role of CD4+ T cells in the proliferation of EBV-infected T and NK cells.

Epstein-Barr virus (EBV), a ubiquitous B-lymphotropic herpesvirus, ectopically infects T or NK cells to cause severe diseases of unknown pathogenesis, including chronic active EBV infection (CAEBV) and EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). We developed xenograft models of CAEBV and EBV-HLH by transplanting patients' PBMC to immunodeficient mice of the NOD/Shi-scid/IL-2Rγ(null) strain. In these models, EBV-infected T, NK, or B cells proliferated systemically and reproduced histological characteristics of the two diseases. Analysis of the TCR repertoire expression revealed that identical predominant EBV-infected T-cell clones proliferated in patients and corresponding mice transplanted with their PBMC. Expression of the EBV nuclear antigen 1 (EBNA1), the latent membrane protein 1 (LMP1), and LMP2, but not EBNA2, in the engrafted cells is consistent with the latency II program of EBV gene expression known in CAEBV. High levels of human cytokines, including IL-8, IFN-γ, and RANTES, were detected in the peripheral blood of the model mice, mirroring hypercytokinemia characteristic to both CAEBV and EBV-HLH. Transplantation of individual immunophenotypic subsets isolated from patients' PBMC as well as that of various combinations of these subsets revealed a critical role of CD4+ T cells in the engraftment of EBV-infected T and NK cells. In accordance with this finding, in vivo depletion of CD4+ T cells by the administration of the OKT4 antibody following transplantation of PBMC prevented the engraftment of EBV-infected T and NK cells. This is the first report of animal models of CAEBV and EBV-HLH that are expected to be useful tools in the development of novel therapeutic strategies for the treatment of the diseases.

CD40 signaling activated by Epstein-Barr virus promotes cell survival and proliferation in gastric carcinoma-derived human epithelial cells.

CD40 signaling plays a critical role in the survival and proliferation of EBV-infected lymphocytes. Here we show that CD40 is constitutively expressed in the human gastric carcinoma-derived cell lines AGS, MKN28, and MKN74, and expression of CD40L is induced by in vitro infection with EBV. Blocking the interaction between CD40 and CD40L with CD40Ig, a fusion protein of CD40 and IgG, impaired proliferation of EBV-infected AGS cells and enhanced their calcium ionophore-induced apoptosis. These results suggest that CD40 signaling plays a critical role in the survival and proliferation of EBV-infected epithelial cells, as well as in the virus-infected lymphocytes.

A new humanized mouse model of Epstein-Barr virus infection that reproduces persistent infection, lymphoproliferative disorder, and cell-mediated and humoral immune responses.

The functional human immune system, including T, B, and natural killer lymphocytes, is reconstituted in NOD/Shi-scid/IL-2Rgamma(null) (NOG) mice that receive hematopoietic stem cell transplants. Here, we show that these humanized mice can recapitulate key aspects of Epstein-Barr virus (EBV) infection in humans. Inoculation with approximately 1 x 10(3) TD(50) (50% transforming dose) of EBV caused B cell lymphoproliferative disorder, with histopathological findings and latent EBV gene expression remarkably similar to that in immunocompromised patients. Inoculation with a low dose of virus (

The latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus induces expression of the putative oncogene Bcl-3 through activation of the nuclear factor-kappaB.

The Epstein-Barr virus (EBV)-encoded oncoprotein latent membrane protein 1 (LMP1) has an essential role in B-lymphocyte transformation by the virus and is expressed in certain EBV-associated tumors and lymphoproliferative disorders. By using the Flp-In/TREx-inducible expression system, we introduced LMP1 into two human cell lines, Jurkat and HEK-293, and found that in both of them the putative cellular oncogene Bcl-3 is rapidly induced following the expression of LMP1. Bcl-3 was also induced in Ramos cells after in vitro EBV infection and after transfection with an LMP1 expression vector. This LMP1-induced Bcl-3 expression is considered to be mediated by the transcription factor NF-kappaB, because (1) deletion of a critical NF-kappaB-binding site in the Bcl-3 promoter abolished its responsiveness to LMP1, (2) an IkappaB mutant that specifically inhibits NF-kappaB activity suppressed the LMP1-induced activation of the Bcl-3 promoter, and (3) an LMP1 mutant lacking its effector domain CTAR2, required for the activation of NF-kappaB, is severely impaired in its ability to induce Bcl-3. Western blot analyses showed that all EBV-infected and LMP1-expressing lymphoid cell lines express Bcl-3. These results suggest the possibility that Bcl-3 is involved in the pathogenesis of certain EBV-associated malignancies and lymphoproliferative disorders.

Humanized NOD/SCID/IL2Rgamma(null) mice transplanted with hematopoietic stem cells under nonmyeloablative conditions show prolonged life spans and allow detailed analysis of human immunodeficiency virus type 1 pathogenesis.

In a previous study, we demonstrated that humanized NOD/SCID/IL2Rgamma(null) (hNOG) mice constructed with human hematopoietic stem cells (HSCs) allow efficient human immunodeficiency virus type 1 (HIV-1) infection. However, HIV-1 infection could be monitored for only 43 days in the animals due to their short life spans. By transplanting HSCs without any myeloablation methods, the mice successfully survived longer than 300 days with stable engraftment of human cells. The mice showed high viremia state for more than the 3 months examined, with systemic HIV-1 infection and gradual decrease of CD4+ T cells analogous to that in humans. These capacities of the hNOG mice are very attractive for modeling mechanisms of AIDS progression and therapeutic strategy.

Epstein-Barr virus (EBV)-encoded RNA 2 (EBER2) but not EBER1 plays a critical role in EBV-induced B-cell growth transformation.

Epstein-Barr virus (EBV)-encoded RNA 1 (EBER1) and EBER2 are untranslated RNAs and the most abundant viral transcripts in latently EBV-infected cells. We previously reported that EBERs play a critical role in efficient EBV-induced growth transformation of primary B cells. To investigate whether EBER1 and EBER2 have distinct roles in B-cell growth transformation, recombinant EBVs carrying either EBER1 or EBER2 were generated. The transforming ability of recombinant EBVs expressing EBER2 was as high as that of EBVs expressing both EBER1 and EBER2. In contrast, the transforming ability of recombinant EBVs carrying EBER1 was impaired and was similar to that of EBV lacking both EBER1 and EBER2. Lymphoblastoid cell lines (LCLs) established with EBVs carrying EBER2 proliferated at low cell densities, while LCLs established with EBVs carrying EBER1 did not. Interleukin 6 (IL-6) production in LCLs expressing EBER2 was more abundant than in those lacking EBER2. The growth of LCLs lacking EBER2 was enhanced by the addition of recombinant IL-6 to the cell culture, while the growth of EBER2-expressing LCLs was inhibited by a neutralizing anti-IL-6 antibody. These results demonstrate that EBER2, but not EBER1, contributes to efficient B-cell growth transformation. We conclude that EBER1 and EBER2, despite their structural similarity, have different functions in latently infected lymphoblastoid cells.

Hematopoietic stem cell-engrafted NOD/SCID/IL2Rgamma null mice develop human lymphoid systems and induce long-lasting HIV-1 infection with specific humoral immune responses.

Critical to the development of an effective HIV/AIDS model is the production of an animal model that reproduces long-lasting active replication of HIV-1 followed by elicitation of virus-specific immune responses. In this study, we constructed humanized nonobese diabetic/severe combined immunodeficiency (NOD/SCID)/interleukin-2 receptor gamma-chain knockout (IL2Rgamma(null)) (hNOG) mice by transplanting human cord blood-derived hematopoietic stem cells that eventually developed into human B cells, T cells, and other monocytes/macrophages and 4 dendritic cells associated with the generation of lymphoid follicle-like structures in lymphoid tissues. Expressions of CXCR4 and CCR5 antigens were recognized on CD4+ cells in peripheral blood, the spleen, and bone marrow, while CCR5 was not detected on thymic CD4+ T cells. The hNOG mice showed marked, long-lasting viremia after infection with both CCR5- and CXCR4-tropic HIV-1 isolates for more than the 40 days examined, with R5 virus-infected animals showing high levels of HIV-DNA copies in the spleen and bone marrow, and X4 virus-infected animals showing high levels of HIV-DNA copies in the thymus and spleen. Furthermore, we detected both anti-HIV-1 Env gp120- and Gag p24-specific antibodies in animals showing a high rate of viral infection. Thus, the hNOG mice mirror human systemic HIV infection by developing specific antibodies, suggesting that they may have potential as an HIV/AIDS animal model for the study of HIV pathogenesis and immune responses.

Critical role of Epstein-Barr Virus (EBV)-encoded RNA in efficient EBV-induced B-lymphocyte growth transformation.

It was demonstrated that Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) were nonessential for B-lymphocyte growth transformation. We revisited this issue by producing a large quantity of EBER-deleted EBV by using an Akata cell system. Although the EBER-deleted virus efficiently infected B lymphocytes, its 50% transforming dose was approximately 100-fold less than that of the EBER-positive EBV. We then engineered the genome of EBER-deleted virus and generated a recombinant virus with the EBER genes reconstituted at their native locus. The resultant EBER-reconstituted EBV exhibited restored transforming ability. In addition, lymphoblastoid cell lines established with the EBER-deleted EBV grew significantly more slowly than those established with wild-type or EBER-reconstituted EBV, and the difference between the growth rates was especially highlighted when the cells were plated at low cell densities. These results clearly demonstrate that EBERs significantly contribute to the efficient growth transformation of B lymphocytes by enhancing the growth potential of transformed lymphocytes.

Production of high-titer Epstein-Barr virus recombinants derived from Akata cells by using a bacterial artificial chromosome system.

An Epstein-Barr virus (EBV) genome in Burkitt's lymphoma-derived cell line Akata was cloned into a bacterial artificial chromosome (BAC) vector. The BAC clone, designated AK-BAC, was rapidly and precisely modified by means of efficient homologous recombination in Escherichia coli. This system was used to produce recombinant EBVs with transgenes. An expression cassette of green fluorescent protein (GFP) was inserted into AK-BAC, and the resultant BAC clone, AK-BAC-GFP, was transfected into Akata cells. We found that transfected BAC plasmids efficiently formed episomes in EBV-positive Akata cells. Mixtures of wild-type and AK-BAC-GFP viruses were then produced and used to infect EBV-negative Akata cells. We obtained cell clones that harbored only AK-BAC-GFP but no wild-type episome. These cell clones produced infectious viruses after stimulating virus production, and the recombinant viruses of AK-BAC-GFP efficiently immortalized primary B lymphocytes. We further revised the method so that any kind of cDNA could be rapidly inserted into the unique I-PpoI site that had been artificially introduced into AK-BAC. The AK-BAC system will have a broad range of applications, such as genetic analyses of various viral gene products and development of viral vectors for human gene therapy.

Genes from Pseudomonas sp. strain BS involved in the conversion of L-2-amino-Delta(2)-thiazolin-4-carbonic acid to L-cysteine.

DL-2-amino-Delta(2)-thiazolin-4-carbonic acid (DL-ATC) is a substrate for cysteine synthesis in some bacteria, and this bioconversion has been utilized for cysteine production in industry. We cloned a DNA fragment containing the genes involved in the conversion of L-ATC to L-cysteine from Pseudomonas sp. strain BS. The introduction of this DNA fragment into Escherichia coli cells enabled them to convert L-ATC to cysteine via N-carbamyl-L-cysteine (L-NCC) as an intermediate. The smallest recombinant plasmid, designated pTK10, contained a 2.6-kb insert DNA fragment that has L-cysteine synthetic activity. The nucleotide sequence of the insert DNA revealed that two open reading frames (ORFs) encoding proteins with molecular masses of 19.5 and 44.7 kDa were involved in the L-cysteine synthesis from DL-ATC. These ORFs were designated atcB and atcC, respectively, and their gene products were identified by overproduction of proteins encoded in each ORF and by the maxicell method. The functions of these gene products were examined using extracts of E. coli cells carrying deletion derivatives of pTK10. The results indicate that atcB and atcC are involved in the conversion of L-ATC to L-NCC and the conversion of L-NCC to cysteine, respectively. atcB was first identified as a gene encoding an enzyme that catalyzes thiazolin ring opening. AtcC is highly homologous with L-N-carbamoylases. Since both enzymes can only catalyze the L-specific conversion from L-ATC to L-NCC or L-NCC to L-cysteine, it is thought that atcB and atcC encode L-ATC hydrolase and N-carbamyl-L-cysteine amidohydrolase, respectively.