bluuebunny/crossref_metadata_2025_split huggingface dataset:
A 10-Year Retrospective Comparison of Two Target Sequences, REP-529 and B1, for Toxoplasma gondii Detection by Quantitative PCR
Authors: Belaz Sorya , Gangneux Jean-Pierre , Dupretz Peggy , Guiguen Claude , Robert-Gangneux Florence 
Published: February 2015

ABSTRACT

This study aimed to evaluate the repeated sequence REP-529 compared to that of the B1 gene in the molecular diagnosis of toxoplasmosis by quantitative PCR (qPCR) in routine diagnosis. Over a 10-year period (2003 to 2013), all patients prospectively diagnosed with a positive REP-529 qPCR result for toxoplasmosis were included. All DNA samples (76 samples from 56 patients) were simultaneously tested using the two qPCR methods (REP-529 and B1). The mean cycle threshold (

C
T

) obtained with the B1 qPCR was significantly higher (+4.71 cycles) than that obtained with REP-529 qPCR (
P
< 0.0001). Thirty-one out of 69 extracts (45.6%) positive with REP-529 qPCR were not amplified with the B1 qPCR (relative sensitivity of 54.4% compared to that with REP-529), yielding false-negative results with 15/28 placenta, 5 cord blood, 2 amniotic fluid, 4 cerebrospinal fluid, 1 aqueous humor, 2 lymph node puncture, and 1 abortion product sample. This defect in sensitivity would have left 20/56 patients undiagnosed, distributed as follows: 12/40 congenital toxoplasmosis, 4/5 cerebral toxoplasmosis, 2/8 patients with retinochoroiditis, and 2 patients with chronic lymphadenopathy. This poor performance of B1 qPCR might be related to low parasite loads, since the mean
Toxoplasma
quantification in extracts with B1 false-negative results was 0.4 parasite/reaction. These results clearly show the superiority of the REP-529 sequence in the diagnosis of toxoplasmosis by PCR and suggest that this target should be adopted as part of the standardization of the PCR assay.
Community-Onset Pyomyositis Caused by a Salmonella enterica Serovar Enteritidis Sequence Type 11 Strain Producing CTX-M-15 Extended-Spectrum β-Lactamase
Authors: Hwang Jeong-Hwan , Shin Gee-Wook , Lee Chang-Seop 
Published: February 2015

ABSTRACT

We report a case of community-onset pyomyositis due to
Salmonella enterica
serovar Enteritidis in South Korea. The isolated strain was resistant to extended-spectrum cephalosporins and harbored sequence type 11 coproducing CTX-M-15 extended-spectrum β-lactamase (ESBL). Physicians should be alert for early diagnosis and appropriate treatment since ESBL-producing nontyphoidal
Salmonella
infections are difficult to treat without initiation of appropriate empirical antibiotics.
First Report of Chronic Pulmonary Infection by KPC-3-Producing and Colistin-Resistant Klebsiella pneumoniae Sequence Type 258 (ST258) in an Adult Patient with Cystic Fibrosis
Authors: Delfino Emanuele , Giacobbe Daniele Roberto , Del Bono Valerio , Coppo Erika , Marchese Anna , Manno Graziana , Morelli Patrizia , Minicucci Laura , Viscoli Claudio 
Published: February 2015

ABSTRACT

The spread of
Klebsiella pneumoniae
carbapenemase (KPC)-producing
K. pneumoniae
continues to increase, and the possible development of KPC-producing
K. pneumoniae
infections in cystic fibrosis (CF) patients is a matter of concern. Here, we describe the establishment of a chronic lung infection due to a colistin-resistant KPC-producing
K. pneumoniae
isolate in an Italian CF patient.
Evaluation of a New Phenotypic OXA-48 Disk Test for Differentiation of OXA-48 Carbapenemase-Producing Enterobacteriaceae Clinical Isolates
Authors: Tsakris Athanassios , Poulou Aggeliki , Bogaerts Pierre , Dimitroulia Evangelia , Pournaras Spyros , Glupczynski Youri 
Published: February 2015

ABSTRACT

The current phenotypic methods for detecting carbapenemase-producing
Enterobacteriaceae
(CPE) allow differentiation between class A and B carbapenemases, but they cannot confirm in a single test class D OXA-48 carbapenemase producers. In this study, we evaluated a new phenotypic test, the OXA-48 disk test, which is based on an imipenem disk and two blank disks adjacent to the imipenem disk, loaded with the tested strain and impregnated with EDTA and EDTA plus phenyl boronic acid (PBA), respectively. The evaluation of the OXA-48 disk test was performed with 81 genotypically confirmed OXA-48-type-producing
Enterobacteriaceae
isolates (41 extended-spectrum β-lactamase [ESBL] producers, 3 AmpC producers, and 37 non-ESBL, non-AmpC producers). To measure the specificity of the test, 173 genotypically confirmed OXA-48-negative
Enterobacteriaceae
isolates (57
Klebsiella pneumoniae
carbapenemase [KPC] producers, 34 VIM producers, 23 KPC/VIM producers, 22 NDM producers, and 37 AmpC or ESBL producers and porin deficient) that were nonsusceptible to at least one carbapenem were chosen for testing. Using the imipenem disk and the distortion of the inhibition halo around both blank disks containing EDTA and EDTA/PBA, the test differentiated all but 3 of the 81 OXA-48 producers (sensitivity of 96.3%). The test was negative for OXA-48 production in all but 4 of the 173 carbapenem-nonsusceptible isolates producing other carbapenemases, AmpCs, or ESBLs (specificity of 97.7%). This evaluation shows that the OXA-48 disk test is an accurate phenotypic method for the direct differentiation of OXA-48-producing
Enterobacteriaceae
. Its use along with combined disk tests employing inhibitor-supplemented carbapenem disks might allow the differentiation of the currently known carbapenemase types in
Enterobacteriaceae
species and provide important infection control information.
Monitoring of Cytomegalovirus Viral Loads by Two Molecular Assays in Whole-Blood and Plasma Samples from Hematopoietic Stem Cell Transplant Recipients
Authors: Babady N. Esther , Cheng Cindy , Cumberbatch Evelyn , Stiles Jeffrey , Papanicolaou Genovefa , Tang Yi-Wei 
Published: February 2015

ABSTRACT
Cytomegalovirus (CMV) viral loads in hematopoietic stem cell transplant (HSCT) recipients are typically monitored using quantitative molecular assays. The Roche Cobas AmpliPrep/Cobas TaqMan CMV test (Cobas CMV) has recently been cleared by the FDA for the monitoring of CMV viral loads in plasma samples from transplant patients. In this study, we compare and correlate the viral loads obtained by a laboratory-developed test (LC CMV) (using Roche analyte-specific reagents [ASR] on the LightCycler 2.0) on whole-blood specimens with those obtained on corresponding plasma and whole-blood specimens by the Cobas CMV assay. Testing was performed on 773 archived patient specimens. The strength of the agreement was good for the two assays performed on whole blood (κ = 0.6; 95% confidence interval [CI], 0.51 to 0.7) and moderate when the tests were performed on different sample types (κ = 0.54; 95% CI, 0.47 to 0.62 for the LC CMV whole blood [WB] assay versus Cobas plasma [PL], and κ = 0.57; 95% CI, 0.5 to 0.65 for the Cobas WB assay versus Cobas PL), although the difference was not statistically significant. Using a combination gold standard (i.e., a true positive was a specimen that was positive by two or more methods), the sensitivity and specificity of the assays were 78.8% and 99.3% for the LC CMV assay, 85.2% and 98.1% for the Cobas CMV WB assay, and 100% and 90.5% for Cobas CMV PL assay, respectively. A comparison of the CMV viral load trends in both plasma and whole blood from a few patients with multiple positive successive samples showed similar slopes, with differences in the slope ranging from 0.01 to 0.22. However, the absolute value for individual viral load differed markedly with whole-blood viral loads, being on average 0.5- to 1.22-log higher than those in plasma. The Cobas CMV assay provides a valid option for the monitoring of viral loads in transplant patients. Due to its increased sensitivity, the detection of CMV DNA in patients with low viral loads (i.e., those below limit of quantification [LOQ]) is increased with the Cobas CMV assay in plasma specimens. Longitudinal prospective studies will be needed to examine the clinical significance of these low-level viral loads.
MicroRNA miR-24-3p Promotes Porcine Reproductive and Respiratory Syndrome Virus Replication through Suppression of Heme Oxygenase-1 Expression
Authors: Xiao Shuqi , Wang Xue , Ni Huaibao , Li Na , Zhang Angke , Liu Hongliang , Pu Fengxing , Xu Lele , Gao Jiming , Zhao Qin , Mu Yang , Wang Chengbao , Sun Yani , Du Taofeng , Xu Xingang , Zhang Gaiping , Hiscox Julian A. , Goodfellow Ian G. , Zhou En-Min 
Published: February 2015

ABSTRACT
Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important viruses affecting the swine industry worldwide. Our previous research showed that PRRSV downregulates the expression of heme oxygenase-1 (HO-1), a pivotal cytoprotective enzyme, postinfection and that overexpression of HO-1 inhibits PRRSV replication. MicroRNAs regulate gene expression at the posttranscriptional level and have recently been demonstrated to play vital roles in pathogen-host interactions. The present study sought to determine whether microRNAs modulate HO-1 expression and, by doing so, regulate PRRSV replication. Using bioinformatic prediction and experimental verification, we demonstrate that HO-1 expression is regulated by miR-24-3p. A direct interaction between miR-24-3p and HO-1 mRNA was confirmed using a number of approaches. Overexpression of miR-24-3p significantly decreased HO-1 mRNA and protein levels. PRRSV infection induced miR-24-3p expression to facilitate viral replication. The suppressive effect of HO-1 induction by protoporphyrin IX cobalt chloride (CoPP; a classical inducer of HO-1 expression) on PRRSV replication in MARC-145 cells and primary porcine alveolar macrophages could also be reversed by overexpression of miR-24-3p. Collectively, these results suggested that miR-24-3p promotes PRRSV replication through suppression of HO-1 expression, which not only provides new insights into virus-host interactions during PRRSV infection but also suggests potential new antiviral strategies against PRRSV infection.

IMPORTANCE
MicroRNAs (miRNAs) play vital roles in viral infections by regulating the expression of viral or host genes at the posttranscriptional level. Heme oxygenase-1 (HO-1), a pivotal cytoprotective enzyme, has antiviral activity for a number of viruses, such as Ebola virus, hepatitis C virus, human immunodeficiency virus, and our focus, PRRSV, which causes great economic losses each year in the swine industry worldwide. Here, we show that PRRSV infection induces host miRNA miR-24-3p expression and that miR-24-3p regulates HO-1 expression through both mRNA degradation and translation repression. Suppression of HO-1 expression by miR-24-3p facilitates PRRSV replication. This work lends credibility to the hypothesis that an arterivirus can manipulate cellular miRNAs to enhance virus replication by regulating antiviral responses following viral infection. Therefore, our findings provide new insights into the pathogenesis of PRRSV.
Inhibition of Hepatitis B Virus Gene Expression and Replication by Hepatocyte Nuclear Factor 6
Authors: Hao Ruidong , He Jing , Liu Xing , Gao Guozhen , Liu Dan , Cui Lei , Yu Guojun , Yu Wenhui , Chen Yu , Guo Deyin 
Published: February 2015

ABSTRACT

Hepatitis B virus (HBV), a small enveloped DNA virus, chronically infects more than 350 million people worldwide and causes liver diseases from hepatitis to cirrhosis and liver cancer. Here, we report that hepatocyte nuclear factor 6 (HNF6), a liver-enriched transcription factor, can inhibit HBV gene expression and DNA replication. Overexpression of HNF6 inhibited, while knockdown of HNF6 expression enhanced, HBV gene expression and replication in hepatoma cells. Mechanistically, the SP2 promoter was inhibited by HNF6, which partly accounts for the inhibition on S mRNA. Detailed analysis showed that a
cis
element on the HBV genome (nucleotides [nt] 3009 to 3019) was responsible for the inhibition of the SP2 promoter by HNF6. Moreover, further analysis showed that HNF6 reduced viral pregenomic RNA (pgRNA) posttranscriptionally via accelerating the degradation of HBV pgRNA independent of La protein. Furthermore, by using truncated mutation experiments, we demonstrated that the N-terminal region of HNF6 was responsible for its inhibitory effects. Importantly, introduction of an HNF6 expression construct with the HBV genome into the mouse liver using hydrodynamic injection resulted in a significant reduction in viral gene expression and DNA replication. Overall, our data demonstrated that HNF6 is a novel host factor that can restrict HBV replication via both transcriptional and posttranscriptional mechanisms.

IMPORTANCE
HBV is a major human pathogen whose replication is regulated by host factors. Liver-enriched transcription factors are critical for many liver functions, including metabolism, development, and cell proliferation, and some of them have been shown to regulate HBV gene expression or replication in different manners. In this study, we showed that HNF6 could inhibit the gene expression and DNA replication of HBV via both transcriptional and posttranscriptional mechanisms. As HNF6 is differentially expressed in men and women, the current results may suggest a role of HNF6 in the gender dimorphism of HBV infection.
X-Ray Structure of the Pestivirus NS3 Helicase and Its Conformation in Solution
Authors: Tortorici M. Alejandra , Duquerroy Stéphane , Kwok Jane , Vonrhein Clemens , Perez Javier , Lamp Benjamin , Bricogne Gerard , Rümenapf Till , Vachette Patrice , Rey Félix A. 
Published: February 2015

ABSTRACT

Pestiviruses form a genus in the
Flaviviridae
family of small enveloped viruses with a positive-sense single-stranded RNA genome. Viral replication in this family requires the activity of a superfamily 2 RNA helicase contained in the C-terminal domain of nonstructural protein 3 (NS3). NS3 features two conserved RecA-like domains (D1 and D2) with ATPase activity, plus a third domain (D3) that is important for unwinding nucleic acid duplexes. We report here the X-ray structure of the pestivirus NS3 helicase domain (pNS3h) at a 2.5-Å resolution. The structure deviates significantly from that of NS3 of other genera in the
Flaviviridae
family in D3, as it contains two important insertions that result in a narrower nucleic acid binding groove. We also show that mutations in pNS3h that rescue viruses from which the core protein is deleted map to D3, suggesting that this domain may be involved in interactions that facilitate particle assembly. Finally, structural comparisons of the enzyme in different crystalline environments, together with the findings of small-angle X-ray-scattering studies in solution, show that D2 is mobile with respect to the rest of the enzyme, oscillating between closed and open conformations. Binding of a nonhydrolyzable ATP analog locks pNS3h in a conformation that is more compact than the closest apo-form in our crystals. Together, our results provide new insight and bring up new questions about pNS3h function during pestivirus replication.

IMPORTANCE
Although pestivirus infections impose an important toll on the livestock industry worldwide, little information is available about the nonstructural proteins essential for viral replication, such as the NS3 helicase. We provide here a comparative structural and functional analysis of pNS3h with respect to its orthologs in other viruses of the same family, the flaviviruses and hepatitis C virus. Our studies reveal differences in the nucleic acid binding groove that could have implications for understanding the unwinding specificity of pNS3h, which is active only on RNA duplexes. We also show that pNS3h has a highly dynamic behavior—a characteristic probably shared with NS3 helicases from all
Flaviviridae
members—that could be targeted for drug design by using recent algorithms to specifically block molecular motion. Compounds that lock the enzyme in a single conformation or limit its dynamic range of conformations are indeed likely to block its helicase function.
ATP1A1-Mediated Src Signaling Inhibits Coronavirus Entry into Host Cells
Authors: Burkard Christine , Verheije Monique H. , Haagmans Bart L. , van Kuppeveld Frank J. , Rottier Peter J. M. , Bosch Berend-Jan , de Haan Cornelis A. M. 
Published: February 2015

ABSTRACT

In addition to transporting ions, the multisubunit Na
+
,K
+
-ATPase also functions by relaying cardiotonic steroid (CTS)-binding-induced signals into cells. In this study, we analyzed the role of Na
+
,K
+
-ATPase and, in particular, of its ATP1A1 α subunit during coronavirus (CoV) infection. As controls, the vesicular stomatitis virus (VSV) and influenza A virus (IAV) were included. Using gene silencing, the ATP1A1 protein was shown to be critical for infection of cells with murine hepatitis virus (MHV), feline infectious peritonitis virus (FIPV), and VSV but not with IAV. Lack of ATP1A1 did not affect virus binding to host cells but resulted in inhibited entry of MHV and VSV. Consistently, nanomolar concentrations of the cardiotonic steroids ouabain and bufalin, which are known not to affect the transport function of Na
+
,K
+
-ATPase, inhibited infection of cells with MHV, FIPV, Middle East respiratory syndrome (MERS)-CoV, and VSV, but not IAV, when the compounds were present during virus inoculation. Cardiotonic steroids were shown to inhibit entry of MHV at an early stage, resulting in accumulation of virions close to the cell surface and, as a consequence, in reduced fusion. In agreement with an early block in infection, the inhibition of VSV by CTSs could be bypassed by low-pH shock. Viral RNA replication was not affected when these compounds were added after virus entry. The antiviral effect of ouabain could be relieved by the addition of different Src kinase inhibitors, indicating that Src signaling mediated via ATP1A1 plays a crucial role in the inhibition of CoV and VSV infections.

IMPORTANCE
Coronaviruses (CoVs) are important pathogens of animals and humans, as demonstrated by the recent emergence of new human CoVs of zoonotic origin. Antiviral drugs targeting CoV infections are lacking. In the present study, we show that the ATP1A1 subunit of Na
+
,K
+
-ATPase, an ion transporter and signaling transducer, supports CoV infection. Targeting ATP1A1 either by gene silencing or by low concentrations of the ATP1A1-binding cardiotonic steroids ouabain and bufalin resulted in inhibition of infection with murine, feline, and MERS-CoVs at an early entry stage. Infection with the control virus VSV was also inhibited. Src signaling mediated by ATP1A1 was shown to play a crucial role in the inhibition of virus entry by ouabain and bufalin. These results suggest that targeting the Na
+
,K
+
-ATPase using cardiotonic steroids, several of which are FDA-approved compounds, may be an attractive therapeutic approach against CoV and VSV infections.
Analysis by Live Imaging of Effects of the Adenovirus E4orf4 Protein on Passage through Mitosis of H1299 Tumor Cells
Authors: Sriskandarajah Neera , Blanchette Paola , Kucharski Thomas J. , Teodoro Jose G. , Branton Philip E. 
Published: February 2015

ABSTRACT

The adenovirus E4orf4 protein expressed at high levels kills cancer cells but not normal human primary cells. Previous studies suggested that disruption of processes that regulate mitosis may underlie E4orf4 toxicity. Here we have used live imaging to show that E4orf4 induces a slowed defective transit through mitosis, exhibiting a delay or often failure in cytokinesis that may account for an accumulation of G
1
tetraploids in the population of dying E4orf4-expressing cells.
Dolutegravir Resistance Mutation R263K Cannot Coexist in Combination with Many Classical Integrase Inhibitor Resistance Substitutions
Authors: Anstett Kaitlin , Mesplede Thibault , Oliveira Maureen , Cutillas Vincent , Wainberg Mark A. 
Published: February 2015

ABSTRACT
The new integrase strand transfer inhibitor (INSTI) dolutegravir (DTG) displays limited cross-resistance with older drugs of this class and selects for the R263K substitution in treatment-experienced patients. We performed tissue culture selections with DTG, using viruses resistant to older INSTIs and infectivity and resistance assays, and showed that the presence of the E92Q or N155H substitution was compatible with the emergence of R263K, whereas the G140S Q148R, E92Q N155H, G140S, Y143R, and Q148R substitutions were not.
A Nucleotide Exchange Factor Promotes Endoplasmic Reticulum-to-Cytosol Membrane Penetration of the Nonenveloped Virus Simian Virus 40
Authors: Inoue Takamasa , Tsai Billy 
Published: February 2015

ABSTRACT
The nonenveloped simian polyomavirus (PyV) simian virus 40 (SV40) hijacks the endoplasmic reticulum (ER) quality control machinery to penetrate the ER membrane and reach the cytosol, a critical infection step. During entry, SV40 traffics to the ER, where host-induced conformational changes render the virus hydrophobic. The hydrophobic virus binds and integrates into the ER lipid bilayer to initiate membrane penetration. However, prior to membrane transport, the hydrophobic SV40 recruits the ER-resident Hsp70 BiP, which holds the virus in a transport-competent state until it is ready to cross the ER membrane. Here we probed how BiP disengages from SV40 to enable the virus to penetrate the ER membrane. We found that nucleotide exchange factor (NEF) Grp170 induces nucleotide exchange of BiP and releases SV40 from BiP. Importantly, this reaction promotes SV40 ER-to-cytosol transport and infection. The human BK PyV also relies on Grp170 for successful infection. Interestingly, SV40 mobilizes a pool of Grp170 into discrete puncta in the ER called foci. These foci, postulated to represent the ER membrane penetration site, harbor ER components, including BiP, known to facilitate viral ER-to-cytosol transport. Our results thus identify a nucleotide exchange activity essential for catalyzing the most proximal event before ER membrane penetration of PyVs.

IMPORTANCE
PyVs are known to cause debilitating human diseases. During entry, this virus family, including monkey SV40 and human BK PyV, hijacks ER protein quality control machinery to breach the ER membrane and access the cytosol, a decisive infection step. In this study, we pinpointed an ER-resident factor that executes a crucial role in promoting ER-to-cytosol membrane penetration of PyVs. Identifying a host factor that facilitates entry of the PyV family thus provides additional therapeutic targets to combat PyV-induced diseases.