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 in vitro and in vivo 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.

ESBL-producing E. coli gradually became coresistant to other broad-spectrum antibiotics, notably ciprofloxacin. UTIs caused by such resistant organisms led to a longer hospital stay and more antibiotic use. Reinforcement of infection control measures, especially hand washing in childcare settings and antibiotic stewardship, is critical to reduce the spread of ESBL-producing E. coli.

Reverse genetics is a powerful tool to study single-stranded RNA viruses. Despite tremendous efforts having been made to improve the methodology for constructing flavivirus cDNAs, the cause of toxicity of flavivirus cDNAs in bacteria remains unknown. Here we performed mutational analysis studies to identify Escherichia coli promoter (ECP) sequences within nucleotides (nt) 1 to 3000 of the dengue virus type 2 (DENV2) and Japanese encephalitis virus (JEV) genomes. Eight and four active ECPs were demonstrated within nt 1 to 3000 of the DENV2 and JEV genomes, respectively, using fusion constructs containing DENV2 or JEV segments and empty vector reporter gene Renilla luciferase. The Flavivirus genus consists of more than 70 members that are categorized into several antigenic groups (46). Most flaviviruses are transmitted by mosquito or tick vectors and cause serious human and animal diseases (46). They include dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV). DENV and JEV cause some of the most serious arthropod-borne viral illnesses. There are four different serotypes of dengue virus, DENV1, DENV2, DENV3, and DENV4. Dengue cases have been reported in over 100 countries, and an estimated 2.5 billion people live in areas in which dengue is epidemic (26, 27, 49). DENV infection often leads to dengue fever, dengue hemorrhagic fever, and dengue shock syndrome (24,28,48). JEV transmission has been observed in the Southern Hemisphere and has the potential to become a worldwide public health threat. JEV can cause permanent neuropsychiatric sequelae and is sometimes fatal in children (56,60,61).Flaviviruses are enveloped RNA viruses that consist of single-stranded, positive-sense, 10.5-to 11-kb genomic RNA. The genome is associated with multiple copies of capsid proteins that are translated as a single polyprotein. After entering a host cell, the translated polyprotein is then cleaved into three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by host proteases and a single virus-encoded protease to initiate viral replication (12,18). Introduction of flavivirus genomic RNA into susceptible cell lines can result in the production of infectious virus particles (1). This phenomenon has prompted the study of flavivirus virology via introduction of flavivirus genomic RNA that has been transcribed in vitro from fulllength flavivirus infectious cDNA.Reverse genetics is a powerful method for studying the viral replication of positive-strand RNA viruses (8). Unfortunately, the instability of full-length flavivirus cDNA in Escherichia coli has been a major hurdle in a attempt to construct flavivirus cDNAs (reviewed in references 4, 54, and 63). Several strategies have been developed to avoid or overcome the instability of infectious flavivirus cDNA. The instability of plasmids containing full-length YFV was avoided using an in vitro ligation approach involving two plasmids (5...

Epstein Barr virus (EBV) infection expands CD8+ T cells specific for lytic antigens to high frequencies during symptomatic primary infection, and maintains these at significant numbers during persistence. Despite this, the protective function of these lytic EBV antigen-specific cytotoxic CD8+ T cells remains unclear. Here we demonstrate that lytic EBV replication does not significantly contribute to virus-induced B cell proliferation in vitro and in vivo in a mouse model with reconstituted human immune system components (huNSG mice). However, we report a trend to reduction of EBV-induced lymphoproliferation outside of lymphoid organs upon diminished lytic replication. Moreover, we could demonstrate that CD8+ T cells against the lytic EBV antigen BMLF1 can eliminate lytically replicating EBV-transformed B cells from lymphoblastoid cell lines (LCLs) and in vivo, thereby transiently controlling high viremia after adoptive transfer into EBV infected huNSG mice. These findings suggest a protective function for lytic EBV antigen-specific CD8+ T cells against EBV infection and against virus-associated tumors in extra-lymphoid organs. These specificities should be explored for EBV-specific vaccine development.

The Epstein-Barr virus (EBV) is etiologically associated with the development of multiple types of tumors, but it is unclear whether this diversity is due to infection with different EBV strains. We report a comparative characterization of SNU719, GP202, and YCCEL1, three EBV strains that were isolated from gastric carcinomas, M81, a virus isolated in a nasopharyngeal carcinoma and several well-characterized laboratory type A strains. We found that B95-8, Akata and GP202 induced cell growth more efficiently than YCCEL1, SNU719 and M81 and this correlated positively with the expression levels of the viral BHRF1 miRNAs. In infected B cells, all strains except Akata and B95-8 induced lytic replication, a risk factor for carcinoma development, although less efficiently than M81. The panel of viruses induced tumors in immunocompromised mice with variable speed and efficacy that did not strictly mirror their in vitro characteristics, suggesting that additional parameters play an important role. We found that YCCEL1 and M81 infected primary epithelial cells, gastric carcinoma cells and gastric spheroids more efficiently than Akata or B95-8. Reciprocally, Akata and B95-8 had a stronger tropism for B cells than YCCEL1 or M81. These data suggest that different EBV strains will induce the development of lymphoid tumors with variable efficacy in immunocompromised patients and that there is a parallel between the cell tropism of the viral strains and the lineage of the tumors they induce. Thus, EBV strains can be endowed with properties that will influence their transforming abilities and the type of tumor they induce.

The N-terminal domains of the herpesvirus large tegument proteins encode a conserved cysteine protease with ubiquitin- and NEDD8-specific deconjugase activity. The proteins are expressed during the productive virus cycle and are incorporated into infectious virus particles, being delivered to the target cells upon primary infection. Members of this viral enzyme family were shown to regulate different aspects of the virus life cycle and the innate anti-viral response. However, only few substrates have been identified and the mechanisms of these effects remain largely unknown. In order to gain insights on the substrates and signaling pathways targeted by the viral enzymes, we have used co-immunoprecipitation and mass spectrometry to identify cellular proteins that interact with the Epstein-Barr virus encoded homologue BPLF1. Several members of the 14-3-3-family of scaffold proteins were found amongst the top hits of the BPLF1 interactome, suggesting that, through this interaction, BPLF1 may regulate a variety of cellular signaling pathways. Analysis of the shared protein-interaction network revealed that BPLF1 promotes the assembly of a tri-molecular complex including, in addition to 14-3-3, the ubiquitin ligase TRIM25 that participates in the innate immune response via ubiquitination of cytosolic pattern recognition receptor, RIG-I. The involvement of BPLF1 in the regulation of this signaling pathway was confirmed by inhibition of the type-I IFN responses in cells transfected with a catalytically active BPLF1 N-terminal domain or expressing the endogenous protein upon reactivation of the productive virus cycle. We found that the active viral enzyme promotes the dimerization and autoubiquitination of TRIM25. Upon triggering of the IFN response, RIG-I is recruited to the complex but ubiquitination is severely impaired, which functionally inactivates the RIG-I signalosome. The capacity to bind to and functionally inactivate the RIG-I signalosome is shared by the homologues encoded by other human herpesviruses.

Infections with Epstein–Barr virus (EBV) are associated with cancer development, and EBV lytic replication (the process that generates virus progeny) is a strong risk factor for some cancer types. Here we report that EBV infection of B-lymphocytes (in vitro and in a mouse model) leads to an increased rate of centrosome amplification, associated with chromosomal instability. This effect can be reproduced with virus-like particles devoid of EBV DNA, but not with defective virus-like particles that cannot infect host cells. Viral protein BNRF1 induces centrosome amplification, and BNRF1-deficient viruses largely lose this property. These findings identify a new mechanism by which EBV particles can induce chromosomal instability without establishing a chronic infection, thereby conferring a risk for development of tumours that do not necessarily carry the viral genome.

Mice devoid of the IL-15 system lose over 90% of CD8αα+ TCRαβ and TCRγδ intestinal intraepithelial lymphocytes (iIELs). Previous work revealed that IL-15Rα and IL-15 expressed by parenchymal cells, but not by bone marrow-derived cells, are required for normal CD8αα+ iIEL homeostasis. However, it remains unclear when and how the IL-15 system affects CD8αα+ iIELs through their development. This study found that IL-15Rα is dispensable for the thymic stage of CD8αα+ TCRαβ and TCRγδ iIEL development but is required for the maintenance and/or differentiation of the putative lineage marker negative precursors in the intestinal epithelium, especially for the most mature CD8 single positive subset. Moreover, the IL-15 system directly supports the survival of mature CD8αα+ iIEL in vivo. Taken together, this study suggests that regulation of CD8αα+ iIEL homeostasis by the IL-15 system does not occur in the thymus but involves mature cells and putative precursors in the intestine.