bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of Test Methodologies for Foot-and-Mouth Disease Virus Serotype A Vaccine Matching
Authors: Tekleghiorghis Tesfaalem , Weerdmeester Klaas , van Hemert-Kluitenberg Froukje , Moormann Rob J. M. , Dekker Aldo 
Published: March 2014

ABSTRACTVaccination has been one of the most important interventions in disease prevention and control. The impact of vaccination largely depends on the quality and suitability of the chosen vaccine. To determine the suitability of a vaccine strain, antigenic matching is usually studied byin vitroanalysis. In this study, we performed threein vitrotest methods to determine which one gives the lowest variability and the highest discriminatory capacity. Binary ethylenimine inactivated vaccines, prepared from 10 different foot-and-mouth disease (FMD) virus serotype A strains, were used to vaccinate cattle (5 animals for each strain). The antibody titers in blood serum samples 3 weeks postvaccination (w.p.v.) were determined by a virus neutralization test, neutralization index test, and liquid-phase blocking enzyme-linked immunosorbent assay (ELISA). The titers were then used to calculate relationship coefficient (r1) values. Theser1values were compared to the genetic lineage using receiver operating characteristic (ROC) analysis. In the two neutralization test methods, the median titers observed against the test strains differed considerably, and the sera of the vaccinated animals did not always show the highest titers against their respective homologous virus strains. When the titers were corrected for test strain effect (scaling), the variability (standard error of the mean per vaccinated group) increased because the results were on a different scale, but the discriminatory capacity improved. An ROC analysis of ther1value calculated on both observed and scaled titers showed that onlyr1values of the liquid-phase blocking ELISA gave a consistent statistically significant result. Under the conditions of the present study, the liquid-phase blocking ELISA showed less variation and still had a higher discriminatory capacity than the other tests.
Development and Optimization of a High-Throughput Assay To Measure Neutralizing Antibodies against Clostridium difficile Binary Toxin
Authors: Xie Jinfu , Horton Melanie , Zorman Julie , Antonello Joseph M. , Zhang Yuhua , Arnold Beth A. , Secore Susan , Xoconostle Rachel , Miezeiewski Matthew , Wang Su , Price Colleen E. , Thiriot David , Goerke Aaron , Gentile Marie-Pierre , Skinner Julie M. , Heinrichs Jon H. 
Published: March 2014

ABSTRACTClostridium difficilestrains producing binary toxin, in addition to toxin A (TcdA) and toxin B (TcdB), have been associated with more severe disease and increased recurrence ofC. difficileinfection in recent outbreaks. Binary toxin comprises two subunits (CDTa and CDTb) and catalyzes the ADP-ribosylation of globular actin (G-actin), which leads to the depolymerization of filamentous actin (F-actin) filaments. A robust assay is highly desirable for detecting the cytotoxic effect of the toxin and the presence of neutralizing antibodies in animal and human sera to evaluate vaccine efficacy. We describe here the optimization, using design-of-experiment (DOE) methodology, of a high-throughput assay to measure the toxin potency and neutralizing antibodies (NAb) against binary toxin. Vero cells were chosen from a panel of cells screened for sensitivity and specificity. We have successfully optimized the CDTa-to-CDTb molar ratio, toxin concentration, cell-seeding density, and sera-toxin preincubation time in the NAb assay using DOE methodology. This assay is robust, produces linear results across serial dilutions of hyperimmune serum, and can be used to quantify neutralizing antibodies in sera from hamsters and monkeys immunized withC. difficilebinary toxin-containing vaccines. The assay will be useful forC. difficilediagnosis, for epidemiology studies, and for selecting and optimizing vaccine candidates.
Evaluation of the Architect Epstein-Barr Virus (EBV) Viral Capsid Antigen (VCA) IgG, VCA IgM, and EBV Nuclear Antigen 1 IgG Chemiluminescent Immunoassays for Detection of EBV Antibodies and Categorization of EBV Infection Status Using Immunofluorescence Assays as the Reference Method
Authors: Corrales Isabel , Giménez Estela , Navarro David 
Published: March 2014

ABSTRACTCommercial immunoassays for detecting IgG and IgM antibodies against Epstein-Barr virus (EBV), viral capsid antigens (VCA), and IgGs toward EBV nuclear antigen-1 (EBNA-1) are routinely used in combination to categorize EBV infection status. In this study, we evaluated the performances of the Architect EBV VCA IgG, VCA IgM, and EBNA-1 IgG chemiluminescent microparticle assays (CMIAs) in EBV serological analyses using indirect immunofluorescence assays and anticomplement immunofluorescence assays as the reference methods for VCA IgG, VCA IgM, and EBNA-1 IgG antibody detection, respectively. A total of 365 serum samples representing different EBV serological profiles were included in this study. The κ values (concordances between the results) obtained in the Architect CMIA and those in the reference assays were 0.905 (P< 0.0001) for VCA IgM, 0.889 (P< 0.0001) for VCA IgG, and 0.961 (P< 0.0001) for EBNA-1 IgG. The sensitivities and specificities were, respectively, 91.08% and 99.48% for VCA IgM, 99.23% and 86.27% for VCA IgG, and 96.77% and 99.16% for EBNA-1 IgG. The sensitivities and specificities of the Architect CMIA panel were, respectively, 99.15% and 98.6% for diagnosing a primary infection, 97.62% and 93.39% for diagnosing a past EBV infection, and 92.42% and 97.82% for diagnosing the absence of an EBV infection. In summary, we demonstrated that the Architect EBV antibody panel performs very well for EBV antibody detection and correctly categorizes clinically relevant EBV infection states.
Epidemiology of Candida kefyr in Patients with Hematologic Malignancies
Authors: Dufresne Simon F. , Marr Kieren A. , Sydnor Emily , Staab Janet F. , Karp Judith E. , Lu Kit , Zhang Sean X. , Lavallée Christian , Perl Trish M. , Neofytos Dionysios 
Published: March 2014

ABSTRACT

Candida kefyr
is an emerging pathogen among patients with hematologic malignancies (HM). We performed a retrospective study at Johns Hopkins Hospital to evaluate the epidemiology of
C. kefyr
colonization and infection in HM patients between 2004 and 2010. Eighty-three patients were colonized and/or infected with
C. kefyr
, with 8 (9.6%) having invasive candidiasis (IC). The yearly incidence of
C. kefyr
colonization and candidemia increased over the study period (
P
< 0.01), particularly after 2009. In 2010,
C. kefyr
caused 16.7% of candidemia episodes. The monthly incidence of
C. kefyr
was higher during the summer throughout the study. In a cohort of patients with acute myelogenic leukemia receiving induction chemotherapy, risks for
C. kefyr
colonization included the summer season (odds ratio [OR], 3.1;
P
= 0.03); administration of an azole (OR, 0.06;
P
< 0.001) or amphotericin B (OR, 0.35;
P
= 0.05) was protective. Fingerprinting of 16 isolates by repetitive sequence-based PCR showed that all were different genotypes. The epidemiology of
C. kefyr
candidemia was evaluated in another hospital in Montreal, Canada; data confirmed higher rates of
C. kefyr
infection in the summer.
C. kefyr
appears to be increasing in HM patients, with prominent summer seasonality. These findings raise questions about the effect of antifungal agents and health care exposures (e.g., yogurt) on the epidemiology of this yeast.
Characterization of Multidrug-Resistant Group B Streptococci with Reduced Penicillin Susceptibility Forming Small Non-Beta-Hemolytic Colonies on Sheep Blood Agar Plates
Authors: Banno Hirotsugu , Kimura Kouji , Tanaka Yosuke , Kitanaka Hiromitsu , Jin Wanchun , Wachino Jun-ichi , Yamada Keiko , Shibayama Keigo , Arakawa Yoshichika 
Published: March 2014

ABSTRACT
We isolated and characterized three multidrug-resistant clinical isolates of group B streptococci with reduced penicillin susceptibility (PRGBS) that formed small non-beta-hemolytic colonies on sheep blood agar plates but grew well on chocolate agar plates. They can be overlooked in the bacterial identification step, leading to clinical misdiagnosis and treatment failure.
Anaplasma platys in Bone Marrow Megakaryocytes of Young Dogs
Authors: De Tommasi A. Sara , Baneth Gad , Breitschwerdt Edward B. , Stanneck Dorothee , Dantas-Torres Filipe , Otranto Domenico , de Caprariis Donato 
Published: March 2014

ABSTRACTAnaplasma platysis an obligate intracellular rickettsial pathogen that infects platelets of dogs, forming basophilic intracellular morulae. In the present report, cellular inclusions were documented in bone marrow thrombocyte precursors of two young naturally infected dogs, indicating thatA. platyscan infect megakaryocytes and promegakaryocytes.
Coronavirus Replicase-Reporter Fusions Provide Quantitative Analysis of Replication and Replication Complex Formation
Authors: Freeman Megan Culler , Graham Rachel L. , Lu Xiaotao , Peek Christopher T. , Denison Mark R. 
Published: March 2014

ABSTRACT
The replication of coronaviruses occurs in association with multiple virus-induced membrane structures that evolve during the course of infection; however, the dynamics of this process remain poorly understood. Previous studies of coronavirus replication complex organization and protein interactions have utilized protein overexpression studies and immunofluorescence of fixed cells. Additionally, live-imaging studies of coronavirus replicase proteins have used fluorescent reporter molecules fused to replicase proteins, but expressed from nonnative locations, mostly late-transcribed subgenomic mRNAs, in the presence or absence of the native protein. Thus, the timing and targeting of native replicase proteins expressed in real time from native locations in the genome remain unknown. In this study, we tested whether reporter molecules could be expressed from the replicase polyprotein of murine hepatitis virus as fusions with nonstructural protein 2 or 3 and whether such reporters could define the targeting and activity of replicase proteins during infection. We demonstrate that the fusion of green fluorescent protein and firefly luciferase with either nonstructural protein 2 or 3 is tolerated and that these reporter-replicase fusions can be used to quantitate replication complex formation and virus replication. The results show that the replicase gene has flexibility to accommodate a foreign gene addition and can be used directly to study replicase complex formation and evolution during infection as well as to provide highly sensitive and specific markers for protein translation and genome replication.

IMPORTANCE
Coronaviruses are a family of enveloped, positive-sense RNA viruses that are important agents of disease, including severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus. Replication is associated with multiple virus-induced membrane structures that evolve during infection; however, the dynamics of this process remain poorly understood. In this study, we tested whether reporter molecules expressed from native locations within the replicase polyprotein of murine hepatitis virus as fusions with nonstructural proteins could define the expression and targeting of replicase proteins during infection in live cells. We demonstrate that the replicase gene tolerates the introduction of green fluorescent protein or firefly luciferase as fusions with replicase proteins. These viruses allow early quantitation of virus replication as well as real-time measurement of replication complexes.
Identification of Cellular Proteins Interacting with the Retroviral Restriction Factor SAMHD1
Authors: St. Gelais Corine , de Silva Suresh , Hach Jocelyn C. , White Tommy E. , Diaz-Griffero Felipe , Yount Jacob S. , Wu Li 
Published: March 2014

ABSTRACT

Human and mouse SAMHD1 proteins block human immunodeficiency virus type 1 (HIV-1) infection in noncycling human monocytic cells by reducing the intracellular deoxynucleoside triphosphate (dNTP) concentrations. Phosphorylation of human SAMHD1 at threonine 592 (T592) by cyclin-dependent kinase 1 (CDK1) and cyclin A2 impairs its HIV-1 restriction activity, but not the dNTP hydrolase activity, suggesting that dNTP depletion is not the sole mechanism of SAMHD1-mediated HIV-1 restriction. Using coimmunoprecipitation and mass spectrometry, we identified and validated two additional host proteins interacting with human SAMHD1, namely, cyclin-dependent kinase 2 (CDK2) and S-phase kinase-associated protein 2 (SKP2). We observed that mouse SAMHD1 specifically interacted with cyclin A2, cyclin B1, CDK1, and CDK2. Given the role of these SAMHD1-interacting proteins in cell cycle progression, we investigated the regulation of these host proteins by monocyte differentiation and activation of CD4
+
T cells and examined their effect on the phosphorylation of human SAMHD1 at T592. Our results indicate that primary monocyte differentiation and CD4
+
T-cell activation regulate the expression of these SAMHD1-interacting proteins. Furthermore, our results suggest that, in addition to CDK1 and cyclin A2, CDK2 phosphorylates T592 of human SAMHD1 and thereby regulates its HIV-1 restriction function.

IMPORTANCE
SAMHD1 is the first dNTP triphosphohydrolase found in mammalian cells. Human and mouse SAMHD1 proteins block HIV-1 infection in noncycling cells. Previous studies suggested that phosphorylation of human SAMHD1 at threonine 592 by CDK1 and cyclin A2 negatively regulates its HIV-1 restriction activity. However, it is unclear whether human SAMHD1 interacts with other host proteins in the cyclin A2 and CDK1 complex and whether mouse SAMHD1 shares similar cellular interacting partners. Here, we identify five cell cycle-related host proteins that interact with human and mouse SAMHD1, including three previously unknown cellular proteins (CDK2, cyclin B1, and SKP2). Our results demonstrate that several SAMHD1-interacting cellular proteins regulate phosphorylation of SAMHD1 and play an important role in HIV-1 restriction function. Our findings help define the role of these cellular interacting partners of SAMHD1 that regulate its HIV-1 restriction function.
The 5′ Untranslated Region of the Human T-Cell Lymphotropic Virus Type 1 mRNA Enables Cap-Independent Translation Initiation
Authors: Olivares Eduardo , Landry Dori M. , Cáceres C. Joaquín , Pino Karla , Rossi Federico , Navarrete Camilo , Huidobro-Toro Juan Pablo , Thompson Sunnie R. , López-Lastra Marcelo 
Published: March 2014

ABSTRACT

The human T-cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes adult T cell leukemia and of HTLV-associated myelopathy/tropical spastic paraparesis. The mRNA of some complex retroviruses, including the human and simian immunodeficiency viruses (HIV and SIV), can initiate translation using a canonical cap-dependent mechanism or through an internal ribosome entry site (IRES). In this study, we present strong evidence showing that like HIV-1 and SIV, the 5′-untranslated region (5′UTR) of the HTLV-1 full-length mRNA harbors an IRES. Cap-independent translational activity was evaluated and demonstrated using dual luciferase bicistronic mRNAs in rabbit reticulocyte lysate, in mammalian cell culture, and in
Xenopus laevis
oocytes. Characterization of the HTLV-1 IRES shows that its activity is dependent on the ribosomal protein S25 (RPS25) and that its function is highly sensitive to the drug edeine. Together, these findings suggest that the 5′UTR of the HTLV-1 full-length mRNA enables internal recruitment of the eukaryotic translation initiation complex. However, the recognition of the initiation codon requires ribosome scanning. These results suggest that, after internal recruitment by the HTLV-1 IRES, a scanning step takes place for the 40S ribosomal subunit to be positioned at the translation initiation codon.

IMPORTANCE
The mechanism by which retroviral mRNAs recruit the 40S ribosomal subunit internally is not understood. This study provides new insights into the mechanism of translation initiation used by the human T-cell lymphotropic virus type 1 (HTLV-1). The results show that the HTLV-1 mRNA can initiate translation via a noncanonical mechanism mediated by an internal ribosome entry site (IRES). This study also provides evidence showing the involvement of cellular proteins in HTLV-1 IRES-mediated translation initiation. Together, the data presented in this report significantly contribute to the understanding of HTLV-1 gene expression.
Conserved Features of the PB2 627 Domain Impact Influenza Virus Polymerase Function and Replication
Authors: Kirui James , Bucci Michael D. , Poole Daniel S. , Mehle Andrew 
Published: March 2014

ABSTRACT
Successful replication of influenza virus requires the coordinated expression of viral genes and replication of the genome by the viral polymerase, composed of the subunits PA, PB1, and PB2. Polymerase activity is regulated by both viral and host factors, yet the mechanisms of regulation and how they contribute to viral pathogenicity and tropism are poorly understood. To characterize these processes, we created a series of mutants in the 627 domain of the PB2 subunit. This domain contains a conserved “P[F/P]AAAPP” sequence motif and the well-described amino acid 627, whose identity regulates host range. A lysine present at position 627 in most mammalian viral isolates creates a basic face on the domain surface and confers high-level activity in humans compared to the glutamic acid found at this position in avian isolates. Mutation of the basic face or the P[F/P]AAAPP motif impaired polymerase activity, assembly of replication complexes, and viral replication. Most of these residues are required for general polymerase activity, whereas PB2 K586 and R589 were preferentially required for function in human versus avian cells. Thus, these data identify residues in the 627 domain and other viral proteins that regulate polymerase activity, highlighting the importance of the surface charge and structure of this domain for virus replication and host adaptation.

IMPORTANCE
Influenza virus faces barriers to transmission across species as it emerges from its natural reservoir in birds to infect mammals. The viral polymerase is an important regulator of this process and undergoes discrete changes to adapt to replication in mammals. Many of these changes occur in the polymerase subunit PB2. Here we describe the systematic analysis of a key region in PB2 that controls species-specific polymerase activity. We report the importance of conserved residues that contribute to the overall charge of the protein as well as those that likely affect protein structure. These findings provide further insight into the molecular events dictating species-specific polymerase function and viral replication.
Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Matrix Proteins Specify Different Capabilities To Modulate B Cell Growth
Authors: Caccuri Francesca , Giagulli Cinzia , Reichelt Joachim , Martorelli Debora , Marsico Stefania , Bugatti Antonella , Barone Ines , Rusnati Marco , Guzman Carlos A. , Dolcetti Riccardo , Caruso Arnaldo 
Published: March 2014

ABSTRACTExogenous HIV-1 matrix protein p17 (p17) deregulates the function of different cells after its N-terminal loop (AT20) binding to the chemokine receptors CXCR1 and CXCR2. One site within AT20 has been recently found to be the major determinant of viral fitness following transmission of simian immunodeficiency virus (SIV) to the human host. Therefore, we sought to determine whether SIV matrix protein (MA) was already capable of interacting with CXCR1 and CXCR2 and mimic p17 biological activities rather than this being a newly acquired function during host adaptation. We show here that SIV MA binds with the same affinity of p17 to CXCR1 and CXCR2 and displays both p17 proangiogenic on human primary endothelial cells and chemotactic activity on human primary monocytes and B cells. However, SIV MA exhibited a higher degree of plasticity than p17 in the C terminus, a region known to play a role in modulating B cell growth. Indeed, in contrast to p17, SIV MA was found to activate the phosphatidylinositol 3-kinase/Akt signaling pathway and strongly promote B cell proliferation and clonogenic activity. Interestingly, we have recently highlighted the existence of a Ugandan HIV-1 strain-derived p17 variant (S75X) with the same B cell growth-promoting activity of SIV MA. Computational modeling allowed us to hypothesize an altered C terminus/core region interaction behind SIV MA and S75X activity. Our findings suggest the appearance of a structural constraint in the p17 C terminus that controls B cell growth, which may help to elucidate the evolutionary trajectory of HIV-1.IMPORTANCEThe HIV-1 matrix protein p17 (p17) deregulates the biological activities of different cells after binding to the chemokine receptors CXCR1 and CXCR2. The p17 functional domain responsible for receptors interaction includes an amino acid which is considered the major determinant of SIV replication in humans. Therefore, we sought to determine whether SIV matrix protein (SIV MA) already had the ability to bind to both chemokine receptors rather than being a function newly acquired during host adaptation. We show here that SIV MA binds to CXCR1 and CXCR2 and fully mimics the p17 proangiogenic and chemokine activity. However, it differs from p17 in its ability to signal into B cells and promote B cell growth and clonogenicity. Computational analysis suggests that the accumulation of mutations in the C-terminal region may have led to a further SIV MA adaptation to the human host. This finding in turn sheds light on the evolutionary trajectory of HIV-1.
Dengue Virus- and Hepatitis C Virus-Induced Replication and Assembly Compartments: the Enemy Inside—Caught in the Web
Authors: Chatel-Chaix Laurent , Bartenschlager Ralf 
Published: March 2014

ABSTRACT

Dengue virus (DENV) and hepatitis C virus (HCV), members of the family
Flaviviridae
, are global human health concerns. As positive-strand RNA viruses, they each replicate in the cytoplasm of infected cells and induce distinct membranous replication compartments where most, if not all, steps of the viral life cycle occur. This Gem briefly reviews the most recent insights into the architecture and functional properties of membranous replication and assembly sites induced by DENV and HCV.