bluuebunny/crossref_metadata_2025_split huggingface dataset:
Emergence of an Outbreak-Associated Clostridium difficile Variant with Increased Virulence
Authors: Quesada-Gómez Carlos , López-Ureña Diana , Acuña-Amador Luis , Villalobos-Zúñiga Manuel , Du Tim , Freire Rosemayre , Guzmán-Verri Caterina , Gamboa-Coronado María del Mar , Lawley Trevor D. , Moreno Edgardo , Mulvey Michael R. , Brito Gerly Anne de Castro , Rodríguez-Cavallini Evelyn , Rodríguez César , Chaves-Olarte Esteban 
Published: February 2015

ABSTRACT

The prevalence of
Clostridium difficile
infections has increased due to the emergence of epidemic variants from diverse genetic lineages. Here we describe the emergence of a novel variant during an outbreak in a Costa Rican hospital that was associated with severe clinical presentations. This
C. difficile
variant elicited higher white blood cell counts and caused disease in younger patients than did other strains isolated during the outbreak. Furthermore, it had a recurrence rate, a 30-day attributable disease rate, and disease severity as great as those of the epidemic strain NAP1. Pulsed-field gel electrophoresis genotyping indicated that the outbreak strains belong to a previously undescribed variant, designated NAP
CR1
. Whole-genome sequencing and ribotyping indicated that the NAP
CR1
variant belongs to
C. difficile
ribotype 012 and sequence type 54, as does the reference strain 630. NAP
CR1
strains are resistant to fluoroquinolones due to a mutation in
gyrA
, and they possess an 18-bp deletion in
tcdC
that is characteristic of the epidemic, evolutionarily distinct,
C. difficile
NAP1 variant. NAP
CR1
genomes contain 10% more predicted genes than strain 630, most of which are of hypothetical function and are present on phages and other mobile genetic elements. The increased virulence of NAP
CR1
was confirmed by mortality rates in the hamster model and strong inflammatory responses induced by bacteria-free supernatants in the murine ligated loop model. However, NAP
CR1
strains do not synthesize toxin A and toxin B at levels comparable to those in NAP1 strains. Our results suggest that the pathogenic potential of this emerging
C. difficile
variant is due to the acquisition of hypothetical functions associated with laterally acquired DNA.
Development of a Novel Liquid Bead Array Human Papillomavirus Genotyping Assay (PGMY-LX) and Comparison with Linear Array for Continuity in Longitudinal Cohort Studies
Authors: Farhat Sepideh , Scott Mark E. , Ma Yifei , Moscicki Anna-Barbara 
Published: February 2015

ABSTRACT
Studies of the natural history of human papillomavirus (HPV) infection require reproducible, type-specific testing of the viral types that infect cervical tissue; Linear Array (LA) is one method that has been widely used. We sought to develop a cost-effective, high-throughput alternative using the same PGMY09/11 primer/probe system and offering sensitivity and specificity comparable to those with LA to ensure continuity in longitudinal studies. We report here on a Luminex-based approach, PGMY-LX, that offers type-specific detection of 33 oncogenic and nononcogenic types. Detection of HPV type-specific plasmid DNA was highly specific, with high signal-to-noise ratios for all types except nononcogenic type 40 and no cross-reactivity between types. Cohen's unweighted κ values for 378 clinical samples tested by both LA and PGMY-LX were ≥0.8 (range, 0.80 to 1.0) for 25 types, including oncogenic HPV types 16, 31, 33, 39, 45, 58, and 59 and possibly oncogenic types 53, 66, 73, and 82) and >0.7 (range, 0.74 to 0.79) for oncogenic types 18, 35, 51, and 56 and probable oncogenic type 68b, indicating substantial or better type-specific agreement between the two methods. The reproducibility by PGMY-LX of the types detected by LA varied from 94% when a single HPV type was present to 66% when multiple types were present. The interrun reproducibility for PGMY-LX varied from 98% for single-type infections to 85% for multiple-type infections. The high reproducibility of PGMY-LX and the type-specific agreement with LA allows PGMY-LX to be incorporated into longitudinal, cohort studies that have historically relied on LA.
Recurrence of Disseminated Mycobacterium avium Complex Disease in a Patient with Anti-Gamma Interferon Autoantibodies by Reinfection
Authors: Nishimura Tomoyasu , Fujita-Suzuki Yukiko , Yonemaru Makoto , Ohkusu Kiyofumi , Sakagami Takuro , Carpenter Stephen M. , Otsuka Yoshihito , Namkoong Ho , Yano Ikuya , Hasegawa Naoki 
Published: February 2015

ABSTRACT

We report a case of recurrent disseminated
Mycobacterium avium
complex (DMAC) disease with anti-gamma interferon autoantibodies. To our knowledge, this is the first reported case caused by reinfection with a separate isolate of
M. avium
. DMAC disease activity was monitored using serum IgG antibody titers against lipid antigens extracted from a MAC strain.
Improving the Sensitivity of the Xpert MTB/RIF Assay on Sputum Pellets by Decreasing the Amount of Added Sample Reagent: a Laboratory and Clinical Evaluation
Authors: Dharan Nila J. , Amisano Danielle , Mboowa Gerald , Ssengooba Willy , Blakemore Robert , Kubiak Rachel W. , Armstrong Derek T. , Jones Martin , Manabe Yukari C. , Joloba Moses L. , Ellner Jerrold J. , Dorman Susan E. , Alland David 
Published: February 2015

ABSTRACT

The Xpert MTB/RIF (Xpert) assay permits rapid near-patient detection of
Mycobacterium tuberculosis
in sputum; however, the test sensitivity remains suboptimal in paucibacillary specimens that are negative for acid-fast bacilli using smear microscopy. Xpert testing includes dilution with sample reagent, and when processed sputum pellets are tested, the recommended sample reagent/pellet ratio is 3:1. We evaluated whether a decreased sample reagent/pellet ratio of 2:1 increased Xpert sensitivity compared to the recommended 3:1. The limit of detection was determined by inoculating serial dilutions of
M. tuberculosis
into sputum samples, preparing sputum pellets, and testing each pellet by Xpert at both sample reagent ratios. Processed sputum pellets obtained from
M. tuberculosis
culture-positive clinical specimens were also tested by Xpert at both ratios. Among spiked sputum pellets, the limit of detection was 1,478 CFU/ml (95% confidence interval [CI], 1,211 to 1,943) at a 3:1 ratio and decreased to 832 CFU/ml (95% CI, 671 to 1,134) at 2:1. The proportion of specimens in which
M. tuberculosis
was detected was greater at 2:1 than at 3:1 for almost all numbers of CFU/ml; this difference was most prominent at lower numbers of CFU/ml. Among 134 concentrated sputum pellets from the clinical study, the sensitivity of Xpert at 2:1 was greater than at 3:1 overall (80% versus 72%;
P
= 0.03) and for smear-negative specimens (67% versus 58%;
P
= 0.12). For Xpert testing of sputum pellets, using a lower sample reagent/pellet ratio increased
M. tuberculosis
detection, especially for paucibacillary specimens. Our study supports use of a 2:1 sample reagent/pellet dilution for Xpert testing of sputum pellets.
Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein
Authors: Viktorova Ekaterina G. , Nchoutmboube Jules , Ford-Siltz Lauren A. , Belov George A. 
Published: February 2015

ABSTRACT
It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses.

IMPORTANCE
Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can easily generate mutants resistant to practically any compounds targeting viral proteins. An alternative approach is to target stable cellular factors recruited for the virus-specific functions. In the present study, we analyzed the factors permitting and restricting the establishment of the resistance of poliovirus, a small (+)RNA virus, to brefeldin A (BFA), a drug targeting a cellular component of the viral replication complex. We found that the emergence and replication potential of resistant mutants is cell type dependent and that BFA resistance reduces virus fitness. Our data provide a rational approach to the development of antiviral therapeutics targeting host factors.
Influenza Hemagglutinin (HA) Stem Region Mutations That Stabilize or Destabilize the Structure of Multiple HA Subtypes
Authors: Byrd-Leotis Lauren , Galloway Summer E. , Agbogu Evangeline , Steinhauer David A. 
Published: February 2015

ABSTRACT
Influenza A viruses enter host cells through endosomes, where acidification induces irreversible conformational changes of the viral hemagglutinin (HA) that drive the membrane fusion process. The prefusion conformation of the HA is metastable, and the pH of fusion can vary significantly among HA strains and subtypes. Furthermore, an accumulating body of evidence implicates HA stability properties as partial determinants of influenza host range, transmission phenotype, and pathogenic potential. Although previous studies have identified HA mutations that can affect HA stability, these have been limited to a small selection of HA strains and subtypes. Here we report a mutational analysis of HA stability utilizing a panel of expressed HAs representing a broad range of HA subtypes and strains, including avian representatives across the phylogenetic spectrum and several human strains. We focused on two highly conserved residues in the HA stem region: HA2 position 58, located at the membrane distal tip of the short helix of the hairpin loop structure, and HA2 position 112, located in the long helix in proximity to the fusion peptide. We demonstrate that a K58I mutation confers an acid-stable phenotype for nearly all HAs examined, whereas a D112G mutation consistently leads to elevated fusion pH. The results enhance our understanding of HA stability across multiple subtypes and provide an additional tool for risk assessment for circulating strains that may have other hallmarks of human adaptation. Furthermore, the K58I mutants, in particular, may be of interest for potential use in the development of vaccines with improved stability profiles.

IMPORTANCE
The influenza A hemagglutinin glycoprotein (HA) mediates the receptor binding and membrane fusion functions that are essential for virus entry into host cells. While receptor binding has long been recognized for its role in host species specificity and transmission, membrane fusion and associated properties of HA stability have only recently been appreciated as potential determinants. We show here that mutations can be introduced at highly conserved positions to stabilize or destabilize the HA structure of multiple HA subtypes, expanding our knowledge base for this important phenotype. The practical implications of these findings extend to the field of vaccine design, since the HA mutations characterized here could potentially be utilized across a broad spectrum of influenza virus subtypes to improve the stability of vaccine strains or components.
AP-2-Associated Protein Kinase 1 and Cyclin G-Associated Kinase Regulate Hepatitis C Virus Entry and Are Potential Drug Targets
Authors: Neveu Gregory , Ziv-Av Amotz , Barouch-Bentov Rina , Berkerman Elena , Mulholland Jon , Einav Shirit 
Published: February 2015

ABSTRACT
Hepatitis C virus (HCV) enters its target cell via clathrin-mediated endocytosis. AP-2-associated protein kinase 1 (AAK1) and cyclin G-associated kinase (GAK) are host kinases that regulate clathrin adaptor protein (AP)-mediated trafficking in the endocytic and secretory pathways. We previously reported that AAK1 and GAK regulate HCV assembly by stimulating binding of the μ subunit of AP-2, AP2M1, to HCV core protein. We also discovered that AAK1 and GAK inhibitors, including the approved anticancer drugs sunitinib and erlotinib, could block HCV assembly. Here, we hypothesized that AAK1 and GAK regulate HCV entry independently of their effect on HCV assembly. Indeed, silencing AAK1 and GAK expression inhibited entry of pseudoparticles and cell culture grown-HCV and internalization of Dil-labeled HCV particles with no effect on HCV attachment or RNA replication. AAK1 or GAK depletion impaired epidermal growth factor (EGF)-mediated enhanced HCV entry and endocytosis of EGF receptor (EGFR), an HCV entry cofactor and erlotinib's cancer target. Moreover, either RNA interference-mediated depletion of AP2M1 or NUMB, each a substrate of AAK1 and/or GAK, or overexpression of either an AP2M1 or NUMB phosphorylation site mutant inhibited HCV entry. Last, in addition to affecting assembly, sunitinib and erlotinib inhibited HCV entry at a postbinding step, their combination was synergistic, and their antiviral effect was reversed by either AAK1 or GAK overexpression. Together, these results validate AAK1 and GAK as critical regulators of HCV entry that function in part by activating EGFR, AP2M1, and NUMB and as the molecular targets underlying the antiviral effect of sunitinib and erlotinib (in addition to EGFR), respectively.

IMPORTANCE
Understanding the host pathways hijacked by HCV is critical for developing host-centered anti-HCV approaches. Entry represents a potential target for antiviral strategies; however, no FDA-approved HCV entry inhibitors are currently available. We reported that two host kinases, AAK1 and GAK, regulate HCV assembly. Here, we provide evidence that AAK1 and GAK regulate HCV entry independently of their role in HCV assembly and define the mechanisms underlying AAK1- and GAK-mediated HCV entry. By regulating temporally distinct steps in the HCV life cycle, AAK1 and GAK represent “master regulators” of HCV infection and potential targets for antiviral strategies. Indeed, approved anticancer drugs that potently inhibit AAK1 or GAK inhibit HCV entry in addition to assembly. These results contribute to an understanding of the mechanisms of HCV entry and reveal attractive host targets for antiviral strategies as well as approved candidate inhibitors of these targets, with potential implications for other viruses that hijack clathrin-mediated pathways.
Scanning Mutagenesis Studies Reveal a Potential Intramolecular Interaction within the C-Terminal Half of Dengue Virus NS2A Involved in Viral RNA Replication and Virus Assembly and Secretion
Authors: Wu Ren-Huang , Tsai Ming-Han , Chao Day-Yu , Yueh Andrew 
Published: February 2015

ABSTRACT

The NS2A protein of dengue virus (DENV) has eight predicted transmembrane segments (pTMSs; pTMS1 to pTMS8). NS2A has been shown to participate in RNA replication, virion assembly, and the host antiviral response. However, the role of the amino acid residues within the pTMS regions of NS2A during the virus life cycle is poorly understood. In the study described here, we explored the function of DENV NS2A by introducing a series of double or triple alanine substitutions into the C-terminal half (pTMS4 to pTMS8) of NS2A in the context of a DENV infectious clone or subgenomic replicon. Fourteen (8 within pTMS8) of 35 NS2A mutants displayed a lethal phenotype due to impairment of RNA replication by a replicon assay. Three NS2A mutants with mutations within pTMS7, the CM20, CM25, and CM27 mutants, displayed similar phenotypes, low virus yields (>100-fold reduction), wild-type-like replicon activity, and low infectious virus-like particle yields by transient
trans
-packaging experiments, suggesting a defect in virus assembly and secretion. The sequencing of revertant viruses derived from CM20, CM25, and CM27 mutant viruses revealed a consensus reversion mutation, leucine (L) to phenylalanine (F), at codon 181 within pTMS7. The introduction of an L181F mutation into a full-length NS2A mutant, i.e., the CM20, CM25, and CM27 constructs, completely restored wild-type infectivity. Notably, L181F also substantially rescued the other severely RNA replication-defective mutants with mutations within pTMS4, pTMS6, and pTMS8, i.e., the CM2, CM3, CM13, CM31, and CM32 mutants. In conclusion, the results revealed the essential roles of pTMS4 to pTMS8 of NS2A in RNA replication and/or virus assembly and secretion. The intramolecular interaction between pTMS7 and pTMS4, pTMS6, or pTMS8 of the NS2A protein was also implicated.

IMPORTANCE
The reported characterization of the C-terminal half of dengue virus NS2A is the first comprehensive mutagenesis study to investigate the function of flavivirus NS2A involved in the steps of the virus life cycle. In particular, detailed mapping of the amino acid residues within the predicted transmembrane segments (pTMSs) of NS2A involved in RNA replication and/or virus assembly and secretion was performed. A revertant genetics study also revealed that L181F within pTMS7 is a consensus reversion mutation that rescues both RNA replication-defective and virus assembly- and secretion-defective mutants with mutations within the other three pTMSs of NS2A. Collectively, these findings elucidate the role played by NS2A during the virus life cycle, possibly through the intricate intramolecular interaction between pTMS7 and other pTMSs within the NS2A protein.
Isolation, Genetic Characterization, and Seroprevalence of Adana Virus, a Novel Phlebovirus Belonging to the Salehabad Virus Complex, in Turkey
Authors: Alkan Cigdem , Alwassouf Sulaf , Piorkowski Géraldine , Bichaud Laurence , Tezcan Seda , Dincer Ender , Ergunay Koray , Ozbel Yusuf , Alten Bulent , de Lamballerie Xavier , Charrel Rémi N. 
Published: February 2015

ABSTRACT

A new phlebovirus, Adana virus, was isolated from a pool of
Phlebotomus
spp. (
Diptera
;
Psychodidae
) in the province of Adana, in the Mediterranean region of Turkey. Genetic analysis based on complete coding of genomic sequences indicated that Adana virus belongs to the
Salehabad virus
species of the genus
Phlebovirus
in the family
Bunyaviridae
. Adana virus is the third virus of the
Salehabad virus
species for which the complete sequence has been determined. To understand the epidemiology of Adana virus, a seroprevalence study using microneutralization assay was performed to detect the presence of specific antibodies in human and domestic animal sera collected in Adana as well as Mersin province, located 147 km west of Adana. The results demonstrate that the virus is present in both provinces. High seroprevalence rates in goats, sheep, and dogs support intensive exposure to Adana virus in the region, which has not been previously reported for any virus included in the Salehabad serocomplex; however, low seroprevalence rates in humans suggest that Adana virus is not likely to constitute an important public health problem in exposed human populations, but this deserves further studies.

IMPORTANCE
Until recently, in the genus
Phlebovirus
, the
Salehabad virus
species consisted of two viruses: Salehabad virus, isolated from sand flies in Iran, and Arbia virus, isolated from sand flies in Italy. Here we present the isolation and complete genome characterization of the Adana virus, which we propose to be included in the
Salehabad virus
species. To our knowledge, this is the first report of the isolation and complete genome characterization, from sand flies in Turkey, of a Salehabad virus-related phlebovirus with supporting seropositivity in the Mediterranean, Aegean, and Central Anatolia regions, where phleboviruses have been circulating and causing outbreaks. Salehabad species viruses have generally been considered to be a group of viruses with little medical or veterinary interest. This view deserves to be revisited according to our results, which indicate a high animal infection rate of Adana virus and recent evidence of human infection with Adria virus in Greece.
Rice Stripe Tenuivirus Nonstructural Protein 3 Hijacks the 26S Proteasome of the Small Brown Planthopper via Direct Interaction with Regulatory Particle Non-ATPase Subunit 3
Authors: Xu Yi , Wu Jianxiang , Fu Shuai , Li Chenyang , Zhu Zeng-Rong , Zhou Xueping 
Published: February 2015

ABSTRACT

The ubiquitin/26S proteasome system plays a vital role in regulating host defenses against pathogens. Previous studies have highlighted different roles for the ubiquitin/26S proteasome in defense during virus infection in both mammals and plants, but their role in the vectors that transmit those viruses is still unclear. In this study, we determined that the 26S proteasome is present in the small brown planthopper (SBPH) (
Laodelphgax striatellus
) and has components similar to those in plants and mammals. There was an increase in the accumulation of
Rice stripe virus
(RSV) in the transmitting vector SBPH after disrupting the 26S proteasome, indicating that the SBPH 26S proteasome plays a role in defense against RSV infection by regulating RSV accumulation. Yeast two-hybrid analysis determined that a subunit of the 26S proteasome, named RPN3, could interact with RSV NS3. Transient overexpression of RPN3 had no effect on the RNA silencing suppressor activity of RSV NS3. However, NS3 could inhibit the ability of SBPH
rpn
3 to complement an
rpn
3 mutation in yeast. Our findings also indicate that the direct interaction between RPN3 and NS3 was responsible for inhibiting the complementation ability of RPN3.
In vivo
, we found an accumulation of ubiquitinated protein in SBPH tissues where the RSV titer was high, and silencing of
rpn
3 resulted in malfunction of the SBPH proteasome-mediated proteolysis. Consequently, viruliferous SBPH in which RPN3 was repressed transmitted the virus more effectively as a result of higher accumulation of RSV. Our results suggest that the RSV NS3 protein is able to hijack the 26S proteasome in SBPH via a direct interaction with the RPN3 subunit to attenuate the host defense response.

IMPORTANCE
We show, for the first time, that the 26S proteasome components are present in the small brown planthopper and play a role in defense against its vectored plant virus (RSV). In turn, RSV encodes a protein that subverts the SBPH 26S proteasome via direct interaction with the 26S proteasome subunit RPN3. Our results imply that the molecular arms race observed in plant hosts can be extended to the insect vector that transmits those viruses.