bluuebunny/crossref_metadata_2025_split huggingface dataset:
EuroCareCF Quality Assessment of Diagnostic Microbiology of Cystic Fibrosis Isolates
Authors: Hogardt Michael , Ulrich Jutta , Riehn-Kopp Helga , Tümmler Burkhard 
Published: September 2009

ABSTRACT

The identification of microbial species from respiratory specimens and their susceptibility to antimicrobial agents are among the most important diagnostic measures of care for patients with cystic fibrosis (CF). Under the umbrella of EuroCareCF, two quality assurance trials of CF microbiology were performed in 2007 and 2008. Nine formulations with CF bacterial isolates were dispatched. A total of 31/37 laboratories from 18/21 European countries participated in the 2007 and 2008 trials. The common CF pathogens
Pseudomonas aeruginosa
and
Staphylococcus aureus
were correctly identified by almost all participants in both trials, even if the strains presented uncommon phenotypes.
Burkholderia cenocepacia
IIIB and
Burkholderia vietnamensis
CF isolates, however, were correctly assigned to the species level by only 26% and 27% of the laboratories, respectively. Emerging pathogens such as
Achromobacter xylosoxidans
,
Inquilinus limosus
, and
Pandoraea pnomenusa
were also not detected or were misclassified by many laboratories. One participant correctly identified all CF isolates in both trials. The percentages of correct classifications (susceptible, intermediate, resistant) by antimicrobial susceptibility testing ranged from 55 to 100% (median, 96%) per isolate and drug. The shortcomings in the diagnostics of rare and emerging pathogens point to the need for continuing education in CF microbiology and suggest the establishment of CF microbiology reference laboratories.
Multicenter Evaluation of a Transcription-Reverse Transcription Concerted Assay for Rapid Detection of
Mycobacterium tuberculosis
Complex in Clinical Specimens
Authors: Drouillon V. , Delogu G. , Dettori G. , Lagrange P. H. , Benecchi M. , Houriez F. , Baroli K. , Ardito F. , Torelli R. , Chezzi C. , Fadda G. , Herrmann J.-L. 
Published: September 2009

ABSTRACT

A European multicenter study was performed to evaluate the performance of a new method, based on the transcription-reverse transcription concerted reaction (TRC-2), which enabled one-step amplification and real-time detection of the
Mycobacterium tuberculosis
16S rRNA target directly in clinical specimens. A total of 633 respiratory and nonrespiratory specimens were tested, and the results were compared with those from smears and cultures. A total of 129 patients (Paris center) were followed up in order to evaluate the clinical performance of TRC-2. By using
M. tuberculosis
complex strains to inoculate sterile sputa, the detection limit of TRC-2 was found to be 30 to 50 CFU/ml. A total of 548 respiratory specimens and 59 extrapulmonary specimens were assessable. For pulmonary specimens, the sensitivities of TRC-2 and acid-fast smear were 86.8% and 50.4%, respectively (
P
= 0.002). The specificities were 97.5% and 100%, respectively. For extrapulmonary specimens, the sensitivities of TRC-2 and acid-fast smear were 83.3% and 8.3% (
P
< 0.0001), and the specificities were 95.8% and 100%, respectively. Fifteen of 129 patients were diagnosed with pulmonary tuberculosis (TB). The sensitivities of culture and TRC-2 were 80% (12/15) and 86.7% (13/15) (
P
= 0.16), and the specificities were 100% and 93.9%, respectively. Based on an 11.6% incidence of TB in our population, the positive predictive values of TRC-2 and culture were 81.3% and 100%, respectively, and the negative predictive values were 98.2% and 97.4%, respectively. These results demonstrated that detection of
M. tuberculosis
complex in clinical specimens by TRC-2 with ready-to-use reagents was an efficient and rapid method for the diagnosis of pulmonary and extrapulmonary TB.
Clinical Relevance of the Recently Described SpeciesAeromonas aquariorum
Authors: Figueras M. José , Alperi Anabel , Saavedra M. José , Ko Wen-Chien , Gonzalo Nieves , Navarro Maria , Martínez-Murcia Antonio J. 
Published: September 2009

ABSTRACTTwenty-two human extraintestinal isolates (11 from blood) and three isolates recovered from patients with diarrhea were genetically characterized asAeromonas aquariorum, a novel species known only from ornamental fish. The isolates proved to bear a considerable number of virulence genes, and all were resistant to amoxicillin (amoxicilline), cephalothin (cefalotin), and cefoxitin. Biochemical differentiation from the most relevant clinical species is provided.
Full-Length Characterization of Hepatitis C Virus Subtype 3a Reveals Novel Hypervariable Regions under Positive Selection during Acute Infection
Authors: Humphreys Isla , Fleming Vicki , Fabris Paolo , Parker Joe , Schulenberg Bodo , Brown Anthony , Demetriou Charis , Gaudieri Silvana , Pfafferott Katja , Lucas Michaela , Collier Jane , Huang Kuan-Hsiang Gary , Pybus Oliver G. , Klenerman Paul , Barnes Eleanor 
Published: September 2009

ABSTRACT
Hepatitis C virus subtype 3a is a highly prevalent and globally distributed strain that is often associated with infection via injection drug use. This subtype exhibits particular phenotypic characteristics. In spite of this, detailed genetic analysis of this subtype has rarely been performed. We performed full-length viral sequence analysis in 18 patients with chronic HCV subtype 3a infection and assessed genomic viral variability in comparison to other HCV subtypes. Two novel regions of intragenotypic hypervariability within the envelope protein E2, of HCV genotype 3a, were identified. We named these regions HVR495 and HVR575. They consisted of flanking conserved hydrophobic amino acids and central variable residues. A 5-amino-acid insertion found only in genotype 3a and a putative glycosylation site is contained within HVR575. Evolutionary analysis of E2 showed that positively selected sites within genotype 3a infection were largely restricted to HVR1, HVR495, and HVR575. Further analysis of clonal viral populations within single hosts showed that viral variation within HVR495 and HVR575 were subject to intrahost positive selecting forces. Longitudinal analysis of four patients with acute HCV subtype 3a infection sampled at multiple time points showed that positively selected mutations within HVR495 and HVR575 arose early during primary infection. HVR495 and HVR575 were not present in HCV subtypes 1a, 1b, 2a, or 6a. Some variability that was not subject to positive selection was present in subtype 4a HVR575. Further defining the functional significance of these regions may have important implications for genotype 3a E2 virus-receptor interactions and for vaccine studies that aim to induce cross-reactive anti-E2 antibodies.
Protective Role of Fas-FasL Signaling in Lethal Infection with Herpes Simplex Virus Type 2 in Mice
Authors: Ishikawa Takahiro , Yamada Hisakata , Oyamada Akiko , Goshima Fumi , Nishiyama Yukihiro , Yoshikai Yasunobu 
Published: September 2009

ABSTRACTHerpes simplex virus type 2 (HSV-2) induces acute local infection followed by latent infection in the nervous system and often leads to the development of lethal encephalitis in immunocompromised hosts. The mechanisms of immune protection against lethal HSV-2 infection, however, have not been clarified. In this study, we examined the roles of Fas-Fas ligand (FasL) signaling in lethal infection with HSV-2 by using mice with mutated Fas (lpr) or FasL (gld) in C57BL/6 background. Bothlprandgldmice exhibited higher mortality than wild-type (WT) C57BL/6 mice after infection with virulent HSV-2 strain 186 and showed significantly increased viral titers in the spinal cord compared with WT mice 9 days after infection, just before the mice started to die. There were no differences in the numbers of CD4+and CD8+T cells infiltrated in the spinal cord or in the levels of HSV-2-specific gamma interferon produced by those cells in a comparison oflprand WT mice 9 days after infection. Adoptive transfer studies demonstrated that CD4+T cells from WT mice protectedgldmice from lethal infection by HSV-2. Furthermore, CD4+T cells infiltrated in the spinal cord of HSV-2-infected WT mice expressed functional FasL that induced apoptosis of Fas-expressing target cells in vitro. These results suggest that FasL-mediated cytotoxic activity of CD4+T cells plays an important role in host defense against lethal infection with HSV-2.
Structural and Functional Analysis of Hepatitis C Virus Strain JFH1 Polymerase
Authors: Simister Philip , Schmitt Melanie , Geitmann Matthis , Wicht Oliver , Danielson U. Helena , Klein Rahel , Bressanelli Stéphane , Lohmann Volker 
Published: September 2009

ABSTRACT
The hepatitis C virus (HCV) isolate JFH1 represents the only cloned wild-type sequence capable of efficient replication in cell culture, as well as in chimpanzees. Previous reports have pointed to the viral polymerase NS5B as a major determinant for efficient replication of this isolate. To understand the underlying mechanisms, we expressed and purified NS5B of JFH1 and of the closely related isolate J6, which replicates below the limit of detection in cell culture. The JFH1 enzyme exhibited a 5- to 10-fold-higher specific activity in vitro, consistent with the polymerase activity itself contributing to efficient replication of JFH1. The higher in vitro activity of the JFH1 enzyme was not due to increased RNA binding, elongation rate, or processivity of the polymerase but to higher initiation efficiency. By using homopolymeric and heteropolymeric templates, we found that purified JFH1 NS5B was significantly more efficient in de novo initiation of RNA synthesis than the J6 counterpart, particularly at low GTP concentrations, probably representing an important prerequisite for the rapid replication kinetics of JFH1. Furthermore, we solved the crystal structure of JFH1 NS5B, which displays a very closed conformation that is expected to facilitate de novo initiation. Structural analysis shows that this closed conformation is stabilized by a sprinkle of substitutions that together promote extra hydrophobic interactions between the subdomains “thumb” and “fingers.” These analyses provide deeper insights into the initiation of HCV RNA synthesis and might help to establish more efficient cell culture models for HCV using alternative isolates.
Why Do Phage Play Dice?
Authors: Avlund Mikkel , Dodd Ian B. , Semsey Szabolcs , Sneppen Kim , Krishna Sandeep 
Published: September 2009

ABSTRACT
Phage lambda is among the simplest organisms that make a developmental decision. An infected bacterium goes either into the lytic state, where the phage particles rapidly replicate and eventually lyse the cell, or into a lysogenic state, where the phage goes dormant and replicates along with the cell. Experimental observations by P. Kourilsky are consistent with a single phage infection deterministically choosing lysis and double infection resulting in a stochastic choice. We argue that the phage are playing a “game” of minimizing the chance of extinction and that the shift from determinism to stochasticity is due to a shift from a single-player to a multiplayer game. Crucial to the argument is the clonal identity of the phage.
GBF1, a Guanine Nucleotide Exchange Factor for Arf, Is Crucial for Coxsackievirus B3 RNA Replication
Authors: Lanke Kjerstin H. W. , van der Schaar Hilde M. , Belov George A. , Feng Qian , Duijsings Daniël , Jackson Catherine L. , Ehrenfeld Ellie , van Kuppeveld Frank J. M. 
Published: September 2009

ABSTRACT
The replication of enteroviruses is sensitive to brefeldin A (BFA), an inhibitor of endoplasmic reticulum-to-Golgi network transport that blocks activation of guanine exchange factors (GEFs) of the Arf GTPases. Mammalian cells contain three BFA-sensitive Arf GEFs: GBF1, BIG1, and BIG2. Here, we show that coxsackievirus B3 (CVB3) RNA replication is insensitive to BFA in MDCK cells, which contain a BFA-resistant GBF1 due to mutation M832L. Further evidence for a critical role of GBF1 stems from the observations that viral RNA replication is inhibited upon knockdown of GBF1 by RNA interference and that replication in the presence of BFA is rescued upon overexpression of active, but not inactive, GBF1. Overexpression of Arf proteins or Rab1B, a GTPase that induces GBF1 recruitment to membranes, failed to rescue RNA replication in the presence of BFA. Additionally, the importance of the interaction between enterovirus protein 3A and GBF1 for viral RNA replication was investigated. For this, the rescue from BFA inhibition of wild-type (wt) replicons and that of mutant replicons of both CVB3 and poliovirus (PV) carrying a 3A protein that is impaired in binding GBF1 were compared. The BFA-resistant GBF1-M832L protein efficiently rescued RNA replication of both wt and mutant CVB3 and PV replicons in the presence of BFA. However, another BFA-resistant GBF1 protein, GBF1-A795E, also efficiently rescued RNA replication of the wt replicons, but not that of mutant replicons, in the presence of BFA. In conclusion, this study identifies a critical role for GBF1 in CVB3 RNA replication, but the importance of the 3A-GBF1 interaction requires further study.
Histone Deacetylases 1 and 2 Are Phosphorylated at Novel Sites during Varicella-Zoster Virus Infection
Authors: Walters Matthew S. , Erazo Angela , Kinchington Paul R. , Silverstein Saul 
Published: September 2009

ABSTRACT

ORF66p, a virion-associated varicella-zoster virus (VZV) protein, is a member of a conserved
A
lphaherpesvirinae
kinase family with homology to herpes simplex virus U
S
3 kinase. Expression of ORF66p in cells infected with VZV or an adenovirus expressing only ORF66p results in hyperphosphorylation of histone deacetylase 1 (HDAC1) and HDAC2. Mapping studies reveal that phosphorylation is at a unique conserved Ser residue in the C terminus of both HDACs. This modification requires an active kinase domain in ORF66p, as neither protein is phosphorylated in cells infected with VZV lacking kinase activity. However, hyperphosphorylation appears to occur indirectly, as within the context of in vitro kinase reactions, purified ORF66p phosphorylates a peptide derived from ORF62p, a known substrate, but does not phosphorylate HDAC. These results support a model where ORF66p is necessary but not sufficient to effect hyperphosphorylation of HDAC1 and HDAC2.
Inhibition of Human Cytomegalovirus Replication via Peptide Aptamers Directed against the Nonconventional Nuclear Localization Signal of the Essential Viral Replication Factor pUL84
Authors: Kaiser Nina , Lischka Peter , Wagenknecht Nadine , Stamminger Thomas 
Published: September 2009

ABSTRACT
The UL84 open reading frame of human cytomegalovirus encodes an essential multifunctional regulatory protein that is thought to act in the nucleus as an initiator of lytic viral replication. Nuclear trafficking of pUL84 is facilitated by a complex nonconventional nuclear localization signal (NLS) that mediates its interaction with the cellular importin-α/β pathway. Since binding of pUL84 to importin-α proteins mechanistically differs from that of cellular proteins containing a classical NLS, we assumed that specific interference with the nuclear import of pUL84 might be possible and that this could constitute a novel principle for antiviral therapy. In order to test this hypothesis, we employed peptide aptamer technology and isolated several peptide aptamers from a randomized peptide expression library that specifically bind with high affinity to the unconventional pUL84 NLS under intracellular conditions. Coimmunoprecipitation experiments confirmed these interactions in mammalian cells, and the antiviral potential of the identified peptide aptamers was determined using three independent experimental approaches. (i) Infection experiments with a recombinant human cytomegalovirus expressing green fluorescent protein demonstrated 50 to 60% decreased viral replication in primary human fibroblasts stably expressing pUL84-specific aptamers. (ii) A 50 to 70% reduction of viral plaque formation, as well as a 70 to 90% inhibition of virus release in the presence of pUL84-specific aptamers, was observed. (iii) Immunofluorescence analyses revealed a shift from an almost exclusively nuclear pUL84 staining pattern to a nucleocytoplasmic distribution upon coexpression of the identified molecules, indicating that interference with the nuclear import of pUL84 contributes to the observed antiviral activity of the identified pUL84-binding aptamer molecules.
Adult AIDS-Like Disease in a Novel Inducible Human Immunodeficiency Virus Type 1 Nef Transgenic Mouse Model: CD4
+
T-Cell Activation Is Nef Dependent and Can Occur in the Absence of Lymphophenia
Authors: Rahim Mir Munir Ahmed , Chrobak Pavel , Hu Chunyan , Hanna Zaher , Jolicoeur Paul 
Published: September 2009

ABSTRACT

CD4C/HIV
nef
transgenic (Tg) mice express Nef in CD4
+
T cells and in the cells of the macrophage/monocyte/dendritic lineage, and they develop an AIDS-like disease similar to human AIDS. In these mice, Nef is constitutively expressed throughout life. To rule out the contribution of any developmental defects caused by early expression of Nef, we generated inducible human immunodeficiency virus type 1 (HIV-1) Nef Tg mice by using the tetracycline-inducible system. Faithful expression of the Nef transgene was induced in (CD4C/rtTA × TRE/HIV
Nef
) or (CD4C/rtTA2
S
-M2 × TRE/HIV
Nef
) double-Tg mice upon doxycycline (DOX) treatment in drinking water. Long-term treatment of these mice with DOX also led to loss, apoptosis, and activation of CD4
+
T cells, this latter phenotype being observed even with low levels of Nef. These phenotypes could be transferred by bone marrow (BM) transplantation, indicating a hematopoietic cell autonomous effect. In addition, in mixed Tg:non-Tg BM chimeras, only Tg and not non-Tg CD4
+
T cells exhibited an effector/memory phenotype in the absence of lymphopenia. Finally, the DOX-induced double-Tg mice developed nonlymphoid organ diseases similar to those of CD4C/HIV
Nef
Tg mice and of humans infected with HIV-1. These results show for the first time that adult mice are susceptible to the detrimental action of Nef and that Nef-mediated T-cell activation can be independent of lymphopenia. These Tg mice represent a unique model which is likely to be instrumental for understanding the cellular and molecular pathways of Nef action as well as the main characteristics of immune reconstitution following DOX withdrawal.
Semliki Forest Virus Expressing Interleukin-12 Induces Antiviral and Antitumoral Responses in Woodchucks with Chronic Viral Hepatitis and Hepatocellular Carcinoma
Authors: Rodriguez-Madoz Juan R. , Liu Katherine H. , Quetglas Jose I. , Ruiz-Guillen Marta , Otano Itziar , Crettaz Julien , Butler Scott D. , Bellezza Christine A. , Dykes Nathan L. , Tennant Bud C. , Prieto Jesus , González-Aseguinolaza Gloria , Smerdou Cristian , Menne Stephan 
Published: September 2009

ABSTRACT

A vector based on Semliki Forest virus (SFV) expressing high levels of interleukin-12 (SFV-enhIL-12) has previously demonstrated potent antitumoral efficacy in small rodents with hepatocellular carcinoma (HCC) induced by transplantation of tumor cells. In the present study, the infectivity and antitumoral/antiviral effects of SFV vectors were evaluated in the clinically more relevant woodchuck model, in which primary HCC is induced by chronic infection with woodchuck hepatitis virus (WHV). Intratumoral injection of SFV vectors expressing luciferase or IL-12 resulted in high reporter gene activity within tumors and cytokine secretion into serum, respectively, demonstrating that SFV vectors infect woodchuck tumor cells. For evaluating antitumoral efficacy, woodchuck tumors were injected with increasing doses of SFV-enhIL-12, and tumor size was measured by ultrasonography following treatment. In five (83%) of six woodchucks, a dose-dependent, partial tumor remission was observed, with reductions in tumor volume of up to 80%, but tumor growth was restored thereafter. Intratumoral treatment further produced transient changes in WHV viremia and antigenemia, with ≥1.5-log
10
reductions in serum WHV DNA in half of the woodchucks. Antitumoral and antiviral effects were associated with T-cell responses to tumor and WHV antigens and with expression of CD4 and CD8 markers, gamma interferon, and tumor necrosis factor alpha in peripheral blood mononuclear cells, suggesting that immune responses against WHV and HCC had been induced. These experimental observations suggest that intratumoral administration of SFV-enhIL-12 may represent a strategy for treatment of chronic HBV infection and associated HCC in humans but indicate that this approach could benefit from further improvements.
Crystallographic Definition of the Epitope Promiscuity of the Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2F5: Vaccine Design Implications
Authors: Bryson Steve , Julien Jean-Philippe , Hynes Rosemary C. , Pai Emil F. 
Published: September 2009

ABSTRACT

The quest to create a human immunodeficiency virus type 1 (HIV-1) vaccine capable of eliciting broadly neutralizing antibodies against Env has been challenging. Among other problems, one difficulty in creating a potent immunogen resides in the substantial overall sequence variability of the HIV envelope protein. The membrane-proximal region (MPER) of gp41 is a particularly conserved tryptophan-rich region spanning residues 659 to 683, which is recognized by three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, Z13, and 4E10. In this study, we first describe the variability of residues in the gp41 MPER and report on the invariant nature of 15 out of 25 amino acids comprising this region. Subsequently, we evaluate the ability of the bnMAb 2F5 to recognize 31 varying sequences of the gp41 MPER at a molecular level. In 19 cases, resulting crystal structures show the various MPER peptides bound to the 2F5 Fab′. A variety of amino acid substitutions outside the
664
DKW
666
core epitope are tolerated. However, changes at the
664
DKW
666
motif itself are restricted to those residues that preserve the aspartate's negative charge, the hydrophobic alkyl-π stacking arrangement between the β-turn lysine and tryptophan, and the positive charge of the former. We also characterize a possible molecular mechanism of 2F5 escape by sequence variability at position 667, which is often observed in HIV-1 clade C isolates. Based on our results, we propose a somewhat more flexible molecular model of epitope recognition by bnMAb 2F5, which could guide future attempts at designing small-molecule MPER-like vaccines capable of eliciting 2F5-like antibodies.