bluuebunny/crossref_metadata_2025_split huggingface dataset:
Francisella tularensisGenes Required for Inhibition of the Neutrophil Respiratory Burst and Intramacrophage Growth Identified by Random Transposon Mutagenesis of Strain LVS
Authors: Schulert Grant S. , McCaffrey Ramona L. , Buchan Blake W. , Lindemann Stephen R. , Hollenback Clayton , Jones Bradley D. , Allen Lee-Ann H. 
Published: February 2009

ABSTRACTFrancisella tularensisis a facultative intracellular pathogen and the causative agent of tularemia. We have shown thatF. tularensissubspeciesholarcticastrain LVS prevents NADPH oxidase assembly and activation in human neutrophils, but how this is achieved is unclear. Herein, we used random transposon mutagenesis to identify LVS genes that affect neutrophil activation. Our initial screen identifiedcarA, carB, andpyrB, which encode the small and large subunits of carbamoylphosphate synthase and aspartate carbamoyl transferase, respectively. These strains are uracil auxotrophs, and their growth was attenuated on cysteine heart agar augmented with sheep blood (CHAB) or in modified Mueller-Hinton broth. Phagocytosis of the uracil auxotrophic mutants triggered a respiratory burst in neutrophils, and ingested bacteria were killed and fragmented in phagosomes that contained superoxide. Conversely, phagocytosis did not trigger a respiratory burst in blood monocytes or monocyte-derived macrophages (MDM), and phagosomes containing wild-type or mutant bacteria lacked NADPH oxidase subunits. Nevertheless, the viability of mutant bacteria declined in MDM, and ultrastructural analysis revealed that phagosome egress was significantly inhibited despite synthesis of the virulence factor IglC. Other aspects of infection, such as interleukin-1β (IL-1β) and IL-8 secretion, were unaffected. The cultivation ofcarA, carB, orpyrBon uracil-supplemented CHAB was sufficient to prevent neutrophil activation and intramacrophage killing and supported escape from MDM phagosomes, but intracellular growth was not restored unless uracil was added to the tissue culture medium. Finally, all mutants tested grew normally in both HepG2 and J774A.1 cells. Collectively, our data demonstrate that uracil auxotrophy has cell type-specific effects on the fate ofFrancisellabacteria.
Modulation of Toll-Like Receptor 2 (TLR2) and TLR4 Responses byAspergillus fumigatus
Authors: Chai Louis Y. A. , Kullberg Bart Jan , Vonk Alieke G. , Warris Adilia , Cambi Alessandra , Latgé Jean-Paul , Joosten Leo A. B. , van der Meer Jos W. M. , Netea Mihai G. 
Published: February 2009

ABSTRACTToll-like receptor (TLR)-based signaling pathways in the host may be modulated by pathogens during the course of infection. We describe a novel immunomodulatory mechanism in whichAspergillus fumigatusconidia induce attenuation of TLR2- and TLR4-mediated interleukin (IL)-6 and IL-1β proinflammatory responses in human mononuclear cells with suppression of IL-1β mRNA transcription. Background TLR2 and TLR4 mRNA transcription was not influenced.A. fumigatusconidia induced TLR2 internalization and uptake into the phagosome with a resultant decrease in surface receptor expression.A. fumigatushyphae, on the other hand, selectively downregulated the TLR4-mediated response. These novel immunosuppressive effects may facilitate the invasiveness ofA. fumigatus.
Molecular Analyses of
Vibrio cholerae
O1 Clinical Strains, Including New Nontoxigenic Variants Isolated in Mexico during the Cholera Epidemic Years between 1991 and 2000
Authors: Lizárraga-Partida Marcial Leonardo , Quilici Marie-Laure 
Published: February 2009

ABSTRACT

We studied the evolution of
Vibrio cholerae
O1 during the 1991 to 2000 cholera epidemic in Mexico by biochemical, serological, and molecular characterization of strains collected during this period. Strains were divided into toxigenic and nontoxigenic groups according to the presence or absence of genes encoding cholera toxin. As previously reported, we characterized two populations among toxigenic strains, which were present from the first year of the epidemic. BglI rRNA analysis revealed that these strains had ribotype profiles, denoted M5 and M6 in our study, that were identical to those previously designated Koblavi B5 or Popovic 5 and Popovic 6a or Tamayo B21a, respectively. Ribotype M5 was isolated between 1991 and 1993. This ribotype had a low level of genetic variation as detected by pulsed-field gel electrophoresis (PFGE). Ribotype M6 persisted from 1991 to 2000. However, PFGE profiles suggested that two epidemiologically unrelated strains coexisted within this single ribotype from 1995 until the end of the epidemic. We identified three new BglI ribotypes, Mx1, Mx2, and Mx3, from nontoxigenic
V. cholerae
O1 strains isolated between 1998 and 2000; one of them grouped strains positive for the toxin-coregulated pilus island. They differed from nontoxigenic clones isolated in Latin America and on the U.S. Gulf Coast and are probably autochthonous Mexican
V. cholerae
O1 variants. Most of these new variants were isolated from states surrounding the Gulf of Mexico, where the highest incidence of cholera in the country was recorded. Thus, the Mexican Gulf Coast, like the U.S. Gulf Coast, may act as an environmental reservoir of
V. cholerae
O1.
Sensitivity of PCR Targeting
Mycobacterium ulcerans
by Use of Fine-Needle Aspirates for Diagnosis of Buruli Ulcer
Authors: Phillips R. O. , Sarfo F. S. , Osei-Sarpong F. , Boateng A. , Tetteh I. , Lartey A. , Adentwe E. , Opare W. , Asiedu K. B. , Wansbrough-Jones M. 
Published: February 2009

ABSTRACT

In a previous study, we reported that the sensitivity of PCR targeting the IS
2404
insertion sequence of
Mycobacterium ulcerans
was 98% when it was applied to 4-mm punch biopsy samples of Buruli lesions. Fine-needle aspiration (FNA) is a less traumatic sampling technique for nonulcerated lesions, and we have studied the sensitivity of PCR using FNA samples. Fine-needle aspirates were taken with a 21-gauge needle from 43 patients diagnosed clinically with
M. ulcerans
disease. Four-millimeter punch biopsies were obtained for microscopy, culture, and PCR targeting the IS
2404
insertion sequence. The sensitivity of PCR using samples obtained by FNA was 86% (95% confidence interval [95% CI], 72 to 94%) compared with that for PCR using punch biopsy samples. In this study, the sensitivities of culture and microscopy for punch biopsy samples were 44% (95% CI, 29 to 60%) and 26% (95% CI, 14 to 41%), respectively. This demonstrates that PCR on an FNA sample is a viable minimally invasive technique to diagnose
M. ulcerans
lesions.
One-Step, Multiplex, Real-Time PCR Assay with Molecular Beacon Probes for Simultaneous Detection, Differentiation, and Quantification of Human T-Cell Leukemia Virus Types 1, 2, and 3
Authors: Besson Guillaume , Kazanji Mirdad 
Published: February 2009

ABSTRACT

A single-tube, multiplex, real-time PCR assay with molecular beacons was established in which various probes were used for the simultaneous detection, differentiation, and quantification of human T-cell leukemia virus types 1, 2, and 3 (HTLV-1, HTLV-2, and HTLV-3, respectively) and of simian T-cell leukemia virus types 1 and 3 (STLV-1 and STLV-3, respectively). The quantitative amplification of the standards with MT4 (HTLV-1) and C19 (HTLV-2) cell lines and a molecular clone of HTLV-3 was linear, with the simplex and multiplex methods having similar efficiencies. A maximum difference of 0.9 (mean, 0.4; range, 0.0 to 0.9) was found between threshold cycle values in single and multiplex reactions. The efficiency with each probe in the multiplex reaction was close to 100%, indicating strong linear amplification. The albumin gene was used to standardize the copy number. Comparable results for the detection and quantification of HTLV-1 were obtained with our new methods and with other real-time PCR methods described previously. With our new multiplex assay, however, we were able to detect and quantify HTLV-2 and -3 and STLV-1 and -3 in clinical specimens, with an excellent dynamic range of 10
6
to 10
0
copies per assay, which the other assays could not do. Thus, it will be possible to determine a wide range of HTLV types in both standard and clinical samples, with a detection of 1 to 10 HTLV copies in samples containing at least 100 cells. Furthermore, our system can provide evidence for multiple infections with the three HTLV types, with separate proviral load results. Our new method also could be used for epidemiological studies in Africa and in countries where HTLVs and STLVs are endemic.
Major
Mycobacterium tuberculosis
Lineages Associate with Patient Country of Origin
Authors: Reed Michael B. , Pichler Victoria K. , McIntosh Fiona , Mattia Alicia , Fallow Ashley , Masala Speranza , Domenech Pilar , Zwerling Alice , Thibert Louise , Menzies Dick , Schwartzman Kevin , Behr Marcel A. 
Published: February 2009

ABSTRACT

Over recent years, there has been an increasing acknowledgment of the diversity that exists among
Mycobacterium tuberculosis
clinical isolates. To facilitate comparative studies aimed at deciphering the relevance of this diversity to human disease, an unambiguous and easily interpretable method of strain classification is required. Presently, the most effective means of assigning isolates into a series of unambiguous lineages is the method of Gagneux et al. (S. Gagneux et al., Proc. Natl. Acad. Sci. USA 103:2869-2873, 2006) that involves the PCR-based detection of large sequence polymorphisms (LSPs). In this manner, isolates are classified into six major lineages, the majority of which display a high degree of geographic restriction. Here we describe an independent replicate of the Gagneux study carried out on 798 isolates collected over a 6-year period from mostly foreign-born patients resident on the island of Montreal, Canada. The original trends in terms of bacterial genotype and patient ethnicity are remarkably conserved within this Montreal cohort, even though the patient distributions between the two populations are quite distinct. In parallel with the LSP analysis, we also demonstrate that “clustered” tuberculosis (TB) cases defined through restriction fragment length polymorphism (RFLP) analysis (for isolates with ≥6 IS
6110
copies) or RFLP in combination with spoligotyping (for isolates with <6 IS
6110
copies) do not stray across the LSP-defined lineage boundaries. However, our data also demonstrate the poor discriminatory power of either RFLP or spoligotyping alone for these low-IS
6110
-copy-number isolates. We believe that this independent validation of the LSP method should encourage researchers to adopt this system in investigations aimed at elucidating the role of strain variation in TB.
Apoptosis in Murine Norovirus-Infected RAW264.7 Cells Is Associated with Downregulation of Survivin
Authors: Bok Karin , Prikhodko Victor G. , Green Kim Y. , Sosnovtsev Stanislav V. 
Published: February 2009

ABSTRACTNoroviruses (NVs) are recognized as a major cause of nonbacterial gastroenteritis in humans. Studies of the human NVs continue to be hampered by the inability to propagate them in any cell culture system. Until recently, most data concerning NV replication were derived from studies of feline calicivirus and rabbit hemorrhagic disease virus, which are cultivable members of the familyCaliciviridae. From such studies, it was proposed that caliciviruses induce apoptosis to facilitate the dissemination of viral progeny in the host. The discovery that MNV type 1 (MNV-1) grows in RAW264.7 cells provided the first cell culture system for use in studying the role of apoptosis in NV infection. We first showed that MNV-1 replication triggered apoptosis in infected RAW264.7 cells and then demonstrated that cell death was associated with activation of caspase-9 and caspase-3 through the mitochondrial pathway. This process was dependent on virus replication, since inactivated virus failed to induce signs of apoptosis. In order to better understand the apoptotic process induced by MNV-1 infection of RAW264.7 cells, we investigated the expression profiles of MNV-1-infected versus mock-infected cells. Survivin, a member of the inhibitor of apoptosis protein family, was found to be significantly downregulated in an inverse relationship with the virus genome replication. This study showed that, unlike other viruses that upregulate survivin, MNV-1 is the first virus found to downregulate the levels of survivin. We observed that MNV-1 replication in RAW264.7 cells activated caspases, resulting in apoptosis through the mitochondrial pathway, possibly as a result of downregulation of survivin.
Phosphorylation of the Human Papillomavirus Type 16 E1^E4 Protein at T57 by ERK Triggers a Structural Change That Enhances Keratin Binding and Protein Stability
Authors: Wang Qian , Kennedy Alan , Das Papia , McIntosh Pauline B. , Howell Steven A. , Isaacson Erin R. , Hinz Steven A. , Davy Clare , Doorbar John 
Published: February 2009

ABSTRACT
The E1^E4 protein of human papillomavirus type 16 (HPV16) causes cytokeratin reorganization in the middle and upper epithelial layers and is thought to contribute to multiple facets of the virus life cycle. Although little is known as to how HPV16 E1^E4 (16E1^E4) functions are controlled following the first expression of this protein, the finding that low-risk E1^E4 proteins can be phosphorylated in vivo suggests an important role for kinases. Here, we show that 16E1^E4 is phosphorylated by cyclin-dependent kinase 1 (CDK1) and CDK2, extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and PKC α, with CDK1/2 serine 32 and ERK threonine 57 phosphorylations representing the two primary events seen in cells in cycle. Interestingly, T57 phosphorylation was found to trigger a structural change in the 16E1^E4 protein that compacts the central fold region, leading to an increase in 16E1^E4 stability and overall abundance in the cell. When compared to wild-type 16E1^E4, a T57D phosphomimic was found to have greatly enhanced keratin-binding ability and an ability to modulate the binding of the unphosphorylated form, with keratin binding protecting the T57-phosphorylated form of 16E1^E4 from proteasomal degradation. In HPV16 genome-containing organotypic rafts, the T57-phosphorylated form was specifically detected in the intermediate cell layers, where productive infection occurs, suggesting that T57 phosphorylation may have a functional role at this stage of the viral life cycle. Interestingly, coexpression with 16E5 and ERK activation enhanced T57 phosphorylation, suggesting that E1^E4 and E5 may work together in vivo. Our data suggest a model in which the expression of 16E5 from the major E1^E4-E5 mRNA promotes T57 phosphorylation of E1^E4 and keratin binding, with dephosphorylation occurring following the switch to late poly(A) usage. Other forms of E1^E4, with alternative functional roles, may then increase in prevalence in the upper layers of the epithelium.
Proteinase 2AproIs Essential for Enterovirus Replication in Type I Interferon-Treated Cells
Authors: Morrison Juliet M. , Racaniello Vincent R. 
Published: February 2009

ABSTRACTThePicornaviridaefamily comprises a diverse group of small RNA viruses that cause a variety of human and animal diseases. Some of these viruses are known to induce cleavage of components of the innate immune system and to inhibit steps in the interferon pathway that lead to the production of type I interferon. There has been no study of the effect of picornaviral infection on the events that occur after interferons have been produced. To determine whether members of theEnterovirusgenus can antagonize the antiviral activity of interferon-stimulated genes (ISGs), we pretreated cells with alpha interferon (IFN-α) and then infected the cells with poliovirus type 1, 2, or 3; enterovirus type 70; or human rhinovirus type 16. We found that these viruses were able to replicate in IFN-α-pretreated cells but that replication of vesicular stomatitis virus, aRhabdovirus, and encephalomyocarditis virus (EMCV), a picornavirus of theCardiovirusgenus, was completely inhibited. Although EMCV is sensitive to IFN-α, coinfection of cells with poliovirus and EMCV leads to EMCV replication in IFN-α-pretreated cells. The enteroviral 2A proteinase (2Apro) is essential for replication in cells pretreated with interferon, because amino acid changes in this protein render poliovirus sensitive to IFN-α. The addition of the poliovirus 2Aprogene to the EMCV genome allowed EMCV to replicate in IFN-α-pretreated cells. These results support an inhibitory role for 2Aproin the most downstream event in interferon signaling, the antiviral activities of ISGs.
The Human Cytomegalovirus UL112-113 Locus Can Activate the Full Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication Cycle
Authors: Wells Richard , Stensland Laurence , Vieira Jeffrey 
Published: February 2009

ABSTRACT
Human cytomegalovirus (HCMV) infection of a cell containing latent Kaposi's sarcoma-associated herpesvirus (KSHV) results in the activation of KSHV lytic replication and the production of infectious virus. In this study, we examined the HCMV genes identified as having a role in the activation of HCMV early genes for their ability to activate KSHV lytic replication. It was found that the UL112-113 locus was able to activate the complete KSHV lytic cycle, while the UL122-123 locus, encoding the IE1 and IE2 proteins, known to be strong transactivators, did not.
The Mouse Cytomegalovirus Immediate-Early 1 Gene Is Not Required for Establishment of Latency or for Reactivation in the Lungs
Authors: Busche Andreas , Marquardt Anja , Bleich Andre , Ghazal Peter , Angulo Ana , Messerle Martin 
Published: February 2009

ABSTRACT
The immediate-early protein IE1 of human and mouse cytomegalovirus (MCMV) is one of the first proteins expressed during the productive infection cycle and upon reactivation from latency. The CMV IE1 proteins have been found to inhibit histone deacetylases, suggesting a role in the epigenetic regulation of viral gene expression. Consequently, the IE1 protein is considered to have a profound effect on reactivation, because small amounts of IE1 may be decisive for the switch to lytic replication. Here we asked if an MCMV Δie1 mutant is able both to establish latency and to reactivate from the lungs of latently infected mice. Since the Δie1 mutant was known to be attenuated during acute infection, we first defined conditions that led to comparable levels of viral genomes during latent infection with mutant and wild-type (wt) MCMV. Viral genome copy numbers dropped considerably at the onset of the latent infection but then remained steady for both viruses even after several months. Reactivation of the Δie1 mutant and of wt MCMV from latency occurred with similar incidences in lung explant cultures at 4, 7, and 12 months postinfection. The increase in the frequency of a subset of MCMV-specific memory T cells, a possible indicator of frequent transcriptional reactivation events during latency, was in a comparable range for both viruses. Recurrence of the Δie1 virus infection in vivo could also be induced by hematoablative treatment of latently infected mice. We conclude that the ie1 gene is not essential for the establishment of latency or for the reactivation of MCMV.
Molecular Characterization of Infectious Clones of the Minute Virus of Canines Reveals Unique Features of Bocaviruses
Authors: Sun Yuning , Chen Aaron Yun , Cheng Fang , Guan Wuxiang , Johnson F. Brent , Qiu Jianming 
Published: February 2009

ABSTRACTMinute virus of canines (MVC) is a member of the genusBocavirusin the familyParvoviridae. We have molecularly cloned and sequenced the 5′- and 3′-terminal palindromes of MVC. The MVC genome, 5,404 nucleotides (nt) in length, shared an identity of 52.6% and 52.1% with that of human bocavirus and bovine parvovirus, respectively. It had distinct palindromic hairpins of 183 nt and 198 nt at the left-end and right-end termini of the genome, respectively. The left-end terminus was also found in two alternative orientations (flip or flop). Both termini shared extensive similarities with those of bovine parvovirus. Four full-length molecular clones constructed with different orientations of the left-end terminus proved to be infectious in Walter Reed canine cell/3873D (WRD) canine cells. Both MVC infection and transfection of the infectious clone in WRD cells revealed an identical RNA transcription profile that was similar to that of bovine parvovirus. Mutagenesis of the infectious clone demonstrated that the middle open reading frame encodes the NP1 protein. This protein, unique to the genusBocavirus, was essential for MVC DNA replication. Moreover, the phospholipase A2 motif in the VP1 unique region was also critical for MVC infection. Thus, our studies revealed important information about the genusBocavirusthat may eventually help us to clone the human bocavirus and study its pathogenesis.
Adenovirus Induction of IRF3 Occurs through a Binary Trigger Targeting Jun N-Terminal Kinase and TBK1 Kinase Cascades and Type I Interferon Autocrine Signaling
Authors: Nociari Marcelo , Ocheretina Oksana , Murphy Mary , Falck-Pedersen Erik 
Published: February 2009

ABSTRACT
Pathogen recognition is a critical function of immune sentinel cells. Naïve macrophages or dendritic cells (DCs) undergo pathogen-directed activation and maturation, and as mature antigen-presenting cells (APCs), they contribute essential functions to both innate and adaptive immunity. Using recombinant adenovirus (rAdV) as a model for murine APC activation by DNA viruses, we demonstrate a critical role for stress kinase activation in cell intrinsic and extrinsic antiviral signaling cascades. We propose two viral triggers, viral capsid and viral DNA, are required for APC activation. Endosomal escape and presentation of cytosolic rAdV DNA induces phosphorylation of TANK-binding kinase 1 (TBK1) at serine 172 but does not induce IκB kinase ε activity as determined by in vitro kinase assays. However, induction of TBK1 alone is not sufficient for interferon regulatory factor 3 (IRF3) phosphorylation. We show that capsid-dependent activation of Jun N-terminal kinase (JNK) stress kinase is a necessary step, licensing TBK1 phosphorylation of IRF3 at Ser 396. A second later phase of JNK activity is required to coordinate phosphorylation of JNK-dependent transcription factors (c-Jun/ATF2) with activated IRF3 in the induction of primary IRF3-responsive transcripts. Finally, we demonstrate that maximal JNK/TBK1/IRF3 stimulation by rAdV depends on an intact type I interferon (IFN) signaling cascade. By requiring multiple viral triggers and type I IFN autocrine regulation, APCs have an inherent fail-safe mechanism against inappropriate activation and maturation.