bluuebunny/crossref_metadata_2025_split huggingface dataset:
Performance of the 47-Kilodalton Membrane Protein versus DNA Polymerase I Genes for Detection of Treponema pallidum by PCR in Ulcers
Authors: Gayet-Ageron Angèle , Laurent Frédéric , Schrenzel Jacques , Charton Béatrice , Jimenez-Getaz Gisela , Tangomo Manuela , Ferry Tristan , Sednaoui Patrice , Lautenschlager Stephan , Toutous-Trellu Laurence , Martinez de Tejada Begoña , Cavassini Matthias , Emonet Stéphane , Perneger Thomas , Salord Hélène 
Published: December 2014

ABSTRACT

Treponema pallidum
PCR (
Tp
-PCR) is a direct diagnostic method for primary and secondary syphilis, but there is no recommendation regarding the best choice of target gene. In this study, we sequentially tested 272 specimens from patients with sexually transmitted ulcers using
Tp
-PCR targeting the
tpp47
and then
polA
genes. The two methods showed similar accuracies and an almost-perfect agreement.
Delayed Inflammatory and Cell Death Responses Are Associated with Reduced Pathogenicity in Lujo Virus-Infected Cynomolgus Macaques
Authors: Rasmussen Angela L. , Tchitchek Nicolas , Safronetz David , Carter Victoria S. , Williams Christopher M. , Haddock Elaine , Korth Marcus J. , Feldmann Heinz , Katze Michael G. 
Published: December 2014

ABSTRACTTo identify host factors associated with arenavirus virulence, we used a cynomolgus macaque model to evaluate the pathogenesis of Lujo virus (LUJV), a recently emerged arenavirus that caused an outbreak of severe viral hemorrhagic fever in southern Africa. In contrast to human cases, LUJV caused mild, nonlethal illness in macaques. We then compared this to contrasting clinical outcomes during arenavirus infection, specifically to samples obtained from macaques infected with three highly pathogenic lines of Lassa virus (LASV), the causative agent of Lassa fever (LF). We assessed gene expression in peripheral blood mononuclear cells (PBMC) and determined genes that significantly changed expression relative to that in uninfected animals over the course of infection. We detected a 72-h delay in the induction of host responses to infection during LUJV infection compared to that of the animals infected with LASV. This included genes associated with inflammatory and antiviral responses and was particularly apparent among groups of genes promoting cell death. We also observed early differential expression of a subset of genes specific to LUJV infection that accounts for the delayed inflammatory response. Cell type enrichment analysis suggested that host response induction delay and an LUJV-specific profile are due to a different proportion of natural killer cells responding in LUJV infection than that in the LASV-infected animals. Together, these data indicate that delayed proinflammatory and proapoptotic host responses to arenavirus infection could ameliorate disease severity. This conclusion provides insight into the cellular and molecular mechanisms of arenaviral hemorrhagic fever and suggests potential strategies for therapeutic development.IMPORTANCEOld World arenaviruses are significant human pathogens that often are associated with high mortality. However, mechanisms underlying disease severity and virulence in arenavirus hemorrhagic fever are largely unknown, particularly regarding host responses that contribute to pathogenicity. This study describes a comparison between Lujo and Lassa virus infection in cynomolgus macaques. Lujo virus-infected macaques developed only mild illness, while Lassa virus-infected macaques developed severe illness consistent with Lassa fever. We determined that mild disease is associated with a delay in host expression of genes linked to virulence, such as those causing inflammation and cell death, and with distinct cell types that may mediate this delay. This is the first study to associate the timing and directionality of gene expression with arenaviral pathogenicity and disease outcome and evokes new potential approaches for developing effective therapeutics for treating these deadly emerging pathogens.
Ataxia Telangiectasia Mutated Kinase Mediates NF-κB Serine 276 Phosphorylation and Interferon Expression via the IRF7-RIG-I Amplification Loop in Paramyxovirus Infection
Authors: Fang Ling , Choudhary Sanjeev , Tian Bing , Boldogh Istvan , Yang Chunying , Ivanciuc Teodora , Ma Yinghong , Garofalo Roberto P. , Brasier Allan R. 
Published: December 2014

ABSTRACT
Respiratory syncytial virus (RSV) is a primary etiological agent of childhood lower respiratory tract disease. Molecular patterns induced by active infection trigger a coordinated retinoic acid-inducible gene I (RIG-I)-Toll-like receptor (TLR) signaling response to induce inflammatory cytokines and antiviral mucosal interferons. Recently, we discovered a nuclear oxidative stress-sensitive pathway mediated by the DNA damage response protein, ataxia telangiectasia mutated (ATM), in cytokine-induced NF-κB/RelA Ser 276 phosphorylation. Here we observe that ATM silencing results in enhanced single-strand RNA (ssRNA) replication of RSVand Sendai virus, due to decreased expression and secretion of type I and III interferons (IFNs), despite maintenance of IFN regulatory factor 3 (IRF3)-dependent IFN-stimulated genes (ISGs). In addition to enhanced oxidative stress, RSV replication enhances foci of phosphorylated histone 2AX variant (γH2AX), Ser 1981 phosphorylation of ATM, and IKKγ/NEMO-dependent ATM nuclear export, indicating activation of the DNA damage response. ATM-deficient cells show defective RSV-induced mitogen and stress-activated kinase 1 (MSK-1) Ser 376 phosphorylation and reduced RelA Ser 276 phosphorylation, whose formation is required for IRF7 expression. We observe that RelA inducibly binds the native IFN regulatory factor 7 (IRF7) promoter in an ATM-dependent manner, and IRF7 inducibly binds to the endogenous retinoic acid-inducible gene I (RIG-I) promoter. Ectopic IRF7 expression restores RIG-I expression and type I/III IFN expression in ATM-silenced cells. We conclude that paramyxoviruses trigger the DNA damage response, a pathway required for MSK1 activation of phospho Ser 276 RelA formation to trigger the IRF7-RIG-I amplification loop necessary for mucosal IFN production. These data provide the molecular pathogenesis for defects in the cellular innate immunity of patients with homozygous ATM mutations.

IMPORTANCE
RNA virus infections trigger cellular response pathways to limit spread to adjacent tissues. This “innate immune response” is mediated by germ line-encoded pattern recognition receptors that trigger activation of two, largely independent, intracellular NF-κB and IRF3 transcription factors. Downstream, expression of protective antiviral interferons is amplified by positive-feedback loops mediated by inducible interferon regulatory factors (IRFs) and retinoic acid inducible gene (RIG-I). Our results indicate that a nuclear oxidative stress- and DNA damage-sensing factor, ATM, is required to mediate a cross talk pathway between NF-κB and IRF7 through mediating phosphorylation of NF-κB. Our studies provide further information about the defects in cellular and innate immunity in patients with inherited ATM mutations.
The Stress Granule Protein G3BP1 Recruits Protein Kinase R To Promote Multiple Innate Immune Antiviral Responses
Authors: Reineke Lucas C. , Lloyd Richard E. 
Published: December 2014

ABSTRACT
Stress granules (SGs) are cytoplasmic storage sites containing translationally silenced mRNPs that can be released to resume translation after stress subsides. We previously showed that poliovirus 3C proteinase cleaves the SG-nucleating protein G3BP1, blocking the ability of cells to form SGs late in infection. Many other viruses also target G3BP1 and inhibit SG formation, but the reasons why these functions evolved are unclear. Previously, we also showed a link between G3BP1-induced SGs and protein kinase R (PKR)-mediated translational control, but the mechanism of PKR interplay with SG and the antiviral consequences are unknown. Here, we show that G3BP1 exhibits antiviral activity against several enteroviruses, whereas truncated G3BP1 that cannot form SGs does not. G3BP1-induced SGs are linked to activation of innate immune transcriptional responses through NF-κB and JNK. The G3BP1-induced SGs also recruit PKR and other antiviral proteins. We show that the PXXP domain within G3BP1 is essential for the recruitment of PKR to SGs, for eIF2α phosphorylation driven by PKR, and for nucleating SGs of normal composition. We also show that deletion of the PXXP domain in G3BP1 compromises its antiviral activity. These findings tie PKR activation to its recruitment to SGs by G3BP1 and indicate that G3BP1 promotes innate immune responses at both the transcriptional and translational levels and integrates cellular stress responses and innate immunity.

IMPORTANCE
Stress granules appear during virus infection, and their importance is not well understood. Previously, it was assumed that they were nonfunctional artifacts associated with cellular stress. PKR is a well-known antiviral protein; however, its regulation in cells is not well understood. Our work links cellular stress granules with activation of PKR and other innate immune pathways through the activity of G3BP1, a critical stress granule component. The ability of stress granules and G3BP1 to activate PKR and other innate immune transcriptional responses indicates that G3BP1 is an antiviral protein. This work helps to refine a longstanding paradigm indicating stress granules are inert structures and explains why G3BP1 is subverted by many viruses to promote a productive infection.
Severe Acute Respiratory Syndrome-Associated Coronavirus Vaccines Formulated with Delta Inulin Adjuvants Provide Enhanced Protection while Ameliorating Lung Eosinophilic Immunopathology
Authors: Honda-Okubo Yoshikazu , Barnard Dale , Ong Chun Hao , Peng Bi-Hung , Tseng Chien-Te Kent , Petrovsky Nikolai 
Published: December 2014

ABSTRACT
Although the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) epidemic was controlled by nonvaccine measures, coronaviruses remain a major threat to human health. The design of optimal coronavirus vaccines therefore remains a priority. Such vaccines present major challenges: coronavirus immunity often wanes rapidly, individuals needing to be protected include the elderly, and vaccines may exacerbate rather than prevent coronavirus lung immunopathology. To address these issues, we compared in a murine model a range of recombinant spike protein or inactivated whole-virus vaccine candidates alone or adjuvanted with either alum, CpG, or Advax, a new delta inulin-based polysaccharide adjuvant. While all vaccines protected against lethal infection, addition of adjuvant significantly increased serum neutralizing-antibody titers and reduced lung virus titers on day 3 postchallenge. Whereas unadjuvanted or alum-formulated vaccines were associated with significantly increased lung eosinophilic immunopathology on day 6 postchallenge, this was not seen in mice immunized with vaccines formulated with delta inulin adjuvant. Protection against eosinophilic immunopathology by vaccines containing delta inulin adjuvants correlated better with enhanced T-cell gamma interferon (IFN-γ) recall responses rather than reduced interleukin-4 (IL-4) responses, suggesting that immunopathology predominantly reflects an inadequate vaccine-induced Th1 response. This study highlights the critical importance for development of effective and safe coronavirus vaccines of selection of adjuvants based on the ability to induce durable IFN-γ responses.

IMPORTANCE
Coronaviruses such as SARS-CoV and Middle East respiratory syndrome-associated coronavirus (MERS-CoV) cause high case fatality rates and remain major human public health threats, creating a need for effective vaccines. While coronavirus antigens that induce protective neutralizing antibodies have been identified, coronavirus vaccines present a unique problem in that immunized individuals when infected by virus can develop lung eosinophilic pathology, a problem that is further exacerbated by the formulation of SARS-CoV vaccines with alum adjuvants. This study shows that formulation of SARS-CoV spike protein or inactivated whole-virus vaccines with novel delta inulin-based polysaccharide adjuvants enhances neutralizing-antibody titers and protection against clinical disease but at the same time also protects against development of lung eosinophilic immunopathology. It also shows that immunity achieved with delta inulin adjuvants is long-lived, thereby overcoming the natural tendency for rapidly waning coronavirus immunity. Thus, delta inulin adjuvants may offer a unique ability to develop safer and more effective coronavirus vaccines.
COMMD1/Murr1 Reinforces HIV-1 Latent Infection through IκB-α Stabilization
Authors: Taura Manabu , Kudo Eriko , Kariya Ryusho , Goto Hiroki , Matsuda Kouki , Hattori Shinichiro , Vaeteewoottacharn Kulthida , McDonald Fiona , Suico Mary Ann , Shuto Tsuyoshi , Kai Hirofumi , Okada Seiji 
Published: December 2014

ABSTRACT
The transcription factor NF-κB is important for HIV-1 transcription initiation in primary HIV-1 infection and reactivation in latently HIV-1-infected cells. However, comparative analysis of the regulation and function of NF-κB in latently HIV-1-infected cells has not been done. Here we show that the expression of IκB-α, an endogenous inhibitor of NF-κB, is enhanced by latent HIV-1 infection via induction of the host-derived factor COMMD1/Murr1 in myeloid cells but not in lymphoid cells by using four sets of latently HIV-1-infected cells and the respective parental cells. IκB-α protein was stabilized by COMMD1, which attenuated NF-κB signaling during Toll-like receptor ligand and tumor necrosis factor alpha treatment and enhanced HIV-1 latency in latently HIV-1-infected cells. Activation of the phosphoinositol 3-kinase (PI3K)–JAK pathway is involved in COMMD1 induction in latently HIV-1-infected cells. Our findings indicate that COMMD1 induction is the NF-κB inhibition mechanism in latently HIV-1-infected cells that contributes to innate immune deficiency and reinforces HIV-1 latency. Thus, COMMD1 might be a double-edged sword that is beneficial in primary infection but not beneficial in latent infection when HIV-1 eradication is considered.

IMPORTANCE
HIV-1 latency is a major barrier to viral eradication in the era of combination antiretroviral therapy. In this study, we found that COMMD1/Murr1, previously identified as an HIV-1 restriction factor, inhibits the proteasomal degradation of IκB-α by increasing the interaction with IκB-α in latently HIV-1-infected myeloid cells. IκB-α protein was stabilized by COMMD1, which attenuated NF-κB signaling during the innate immune response and enhanced HIV-1 latency in latently HIV-1-infected cells. Activation of the PI3K-JAK pathway is involved in COMMD1 induction in latently HIV-1-infected cells. Thus, the host-derived factor COMMD1 is beneficial in suppressing primary infection but enhances latent infection, indicating that it may be a double-edged sword in HIV-1 eradication.
Nanobody Binding to a Conserved Epitope Promotes Norovirus Particle Disassembly
Authors: Koromyslova Anna D. , Hansman Grant S. 
Published: December 2014

ABSTRACT

Human noroviruses are icosahedral single-stranded RNA viruses. The capsid protein is divided into shell (S) and protruding (P) domains, which are connected by a flexible hinge region. There are numerous genetically and antigenically distinct noroviruses, and the dominant strains evolve every other year. Vaccine and antiviral development is hampered by the difficulties in growing human norovirus in cell culture and the continually evolving strains. Here, we show the X-ray crystal structures of human norovirus P domains in complex with two different nanobodies. One nanobody, Nano-85, was broadly reactive, while the other, Nano-25, was strain specific. We showed that both nanobodies bound to the lower region on the P domain and had nanomolar affinities. The Nano-85 binding site mainly comprised highly conserved amino acids among the genetically distinct genogroup II noroviruses. Several of the conserved residues also were recognized by a broadly reactive monoclonal antibody, which suggested this region contained a dominant epitope. Superposition of the P domain nanobody complex structures into a cryoelectron microscopy particle structure revealed that both nanobodies bound at occluded sites on the particles. The flexible hinge region, which contained ∼10 to 12 amino acids, likely permitted a certain degree of P domain movement on the particles in order to accommodate the nanobodies. Interestingly, the Nano-85 binding interaction with intact particles caused the particles to disassemble
in vitro
. Altogether, these results suggested that the highly conserved Nano-85 binding epitope contained a trigger mechanism for particle disassembly. Principally, this epitope represents a potential site of norovirus vulnerability.

IMPORTANCE
We characterized two different nanobodies (Nano-85 and Nano-25) that bind to human noroviruses. Both nanobodies bound with high affinities to the lower region of the P domain, which was occluded on intact particles. Nano-25 was specific for GII.10, whereas Nano-85 bound several different GII genotypes, including GII.4, GII.10, and GII.12. We showed that Nano-85 was able to detect norovirus virions in clinical stool specimens using a sandwich enzyme-linked immunosorbent assay. Importantly, we found that Nano-85 binding to intact particles caused the particles to disassemble. We believe that with further testing, Nano-85 not only will work as a diagnostic reagent in norovirus detection systems but also could function as a broadly reactive GII norovirus antiviral.
Acanthamoeba polyphaga Mimivirus Prevents Amoebal Encystment-Mediating Serine Proteinase Expression and Circumvents Cell Encystment
Authors: Boratto Paulo , Albarnaz Jonas Dutra , Almeida Gabriel Magno de Freitas , Botelho Lucas , Fontes Alide Caroline Lima , Costa Adriana Oliveira , Santos Daniel de Assis , Bonjardim Cláudio Antônio , La Scola Bernard , Kroon Erna Geessien , Abrahão Jônatas Santos 
Published: December 2014

ABSTRACT

Acanthamoeba
is a genus of free-living amoebas distributed worldwide. Few studies have explored the interactions between these protozoa and their infecting giant virus,
Acanthamoeba polyphaga
mimivirus (APMV). Here we show that, once the amoebal encystment is triggered, trophozoites become significantly resistant to APMV. Otherwise, upon infection, APMV is able to interfere with the expression of a serine proteinase related to amoebal encystment and the encystment can no longer be triggered.
Clustered MicroRNAs of the Epstein-Barr Virus Cooperatively Downregulate an Epithelial Cell-Specific Metastasis Suppressor
Authors: Kanda Teru , Miyata Mamiko , Kano Makoto , Kondo Satoru , Yoshizaki Tomokazu , Iizasa Hisashi 
Published: December 2014

ABSTRACT

The Epstein-Barr virus (EBV) encodes its own microRNAs (miRNAs); however, their biological roles remain elusive. The commonly used EBV B95-8 strain lacks a 12-kb genomic region, known as BamHI A rightward transcripts (BART) locus, where a number of BART miRNAs are encoded. Here, bacterial artificial chromosome (BAC) technology was used to generate an EBV B95-8 strain in which the 12-kb region was fully restored at its native locus [BART(+) virus]. Epithelial cells were stably infected with either the parental B95-8 virus or the BART(+) virus, and BART miRNA expression was successfully reconstituted in the BART(+) virus-infected cells. Microarray analyses of cellular gene expression identified N-myc downstream regulated gene 1 (
NDRG1
) as a putative target of BART miRNAs. The NDRG1 protein was barely expressed in B cells, highly expressed in epithelial cells, including primary epithelial cells, and strongly downregulated in the BART(+) virus-infected epithelial cells of various origins. Although
in vitro
reporter assays identified BART22 as being responsible for the NDRG1 downregulation, EBV genetic analyses revealed that BART22 was not solely responsible; rather, the entire BART miRNA cluster 2 was responsible for the downregulation. Immunohistochemical analyses revealed that the expression level of the NDRG1 protein was downregulated significantly in EBV-positive nasopharyngeal carcinoma specimens. Considering that
NDRG1
encodes an epithelial differentiation marker and a suppressor of metastasis, these data implicate a causative relationship between BART miRNA expression and epithelial carcinogenesis
in vivo
.

IMPORTANCE
EBV-related epithelial cancers, such as nasopharyngeal carcinomas and EBV-positive gastric cancers, encompass more than 80% of EBV-related malignancies. Although it is known that they express high levels of virally encoded BART miRNAs, how these miRNAs contribute to EBV-mediated epithelial carcinogenesis remains unknown. Although a number of screenings have been performed to identify targets of viral miRNAs, many targets likely have not been identified, especially in case of epithelial cell infection. This is the first study to use EBV genetics to perform unbiased screens of cellular genes that are differentially expressed in viral miRNA-positive and -negative epithelial cells. The result indicates that multiple EBV-encoded miRNAs cooperatively downregulate NDRG1, an epithelial differentiation marker and suppressor of metastasis. The experimental system described in this study should be useful for further clarifying the mechanism of EBV-mediated epithelial carcinogenesis.
Modified Vaccinia Virus Ankara-Infected Dendritic Cells Present CD4
+
T-Cell Epitopes by Endogenous Major Histocompatibility Complex Class II Presentation Pathways
Authors: Thiele Frank , Tao Sha , Zhang Yi , Muschaweckh Andreas , Zollmann Tina , Protzer Ulrike , Abele Rubert , Drexler Ingo 
Published: December 2014

ABSTRACT

CD4
+
T lymphocytes play a central role in the immune system and mediate their function after recognition of their respective antigens presented on major histocompatibility complex II (MHCII) molecules on antigen-presenting cells (APCs). Conventionally, phagocytosed antigens are loaded on MHCII for stimulation of CD4
+
T cells. Certain epitopes, however, can be processed directly from intracellular antigens and are presented on MHCII (endogenous MHCII presentation). Here we characterized the MHCII antigen presentation pathways that are possibly involved in the immune response upon vaccination with modified vaccinia virus Ankara (MVA), a promising live viral vaccine vector. We established CD4
+
T-cell lines specific for MVA-derived epitopes as tools for
in vitro
analysis of MHCII antigen processing and presentation in MVA-infected APCs. We provide evidence that infected APCs are able to directly transfer endogenous viral proteins into the MHCII pathway to efficiently activate CD4
+
T cells. By using knockout mice and chemical inhibitory compounds, we further elucidated the molecular basis, showing that among the various subcellular pathways investigated, proteasomes and autophagy are key players in the endogenous MHCII presentation during MVA infection. Interestingly, although proteasomal processing plays an important role, neither TAP nor LAMP-2 was found to be involved in the peptide transport. Defining the molecular mechanism of MHCII presentation during MVA infection provides a basis for improving MVA-based vaccination strategies by aiming for enhanced CD4
+
T-cell activation by directing antigens into the responsible pathways.

IMPORTANCE
This work contributes significantly to our understanding of the immunogenic properties of pathogens by deciphering antigen processing pathways contributing to efficient activation of antigen-specific CD4
+
T cells. We identified autophagosome formation, proteasomal activity, and lysosomal integrity as being crucial for endogenous CD4
+
T-cell activation. Since poxvirus vectors such as MVA are already used in clinical trials as recombinant vaccines, the data provide important information for the future design of optimized poxviral vaccines for the study of advanced immunotherapy options.