bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of Commercial Extraction Systems and PCR Assays for Quantification of Epstein-Barr Virus DNA Load in Whole Blood
Authors: Germi Raphaële , Lupo Julien , Semenova Touyana , Larrat Sylvie , Magnat Nelly , Grossi Laurence , Seigneurin Jean-Marie , Morand Patrice 
Published: January 2012

ABSTRACT

The automation of DNA extraction and the use of commercial quantitative real-time PCR assays could help obtain more reliable results for the quantification of Epstein-Barr virus DNA loads (EBV VL). This study compared two automated extraction platforms and two commercial PCRs for measurement of EBV VL in 10 EBV specimens from Quality Control for Molecular Diagnostics (QCMD) and in 200 whole-blood (WB) specimens from transplant (
n
= 137) and nontransplant (
n
= 63) patients. The WB specimens were extracted using the QIAcube or MagNA Pure instrument; VL were quantified with the EBV R-gene quantification kit (Argene) or the artus EBV RG PCR kit (Qiagen) on the Rotor-Gene 6000 real-time analyzer; and the results were compared with those of a laboratory-developed PCR. DNA was extracted from the QCMD specimens by use of the QIAamp DNA minikit and was quantified by the three PCR assays. The extraction platforms and the PCR assays showed good correlation (
R
, >0.9;
P
, <0.0001), but as many as 10% discordant results were observed, mostly for low viral loads (<3 log
10
copies/ml), and standard deviations reached as high as 0.49 log
10
copy/ml. In WB but not in QCMD samples, Argene PCR tended to give higher VL values than artus PCR or the laboratory-developed PCR (mean difference for the 200 WB VL, −0.42 or −0.36, respectively). In conclusion, the two automated extraction platforms and the two PCRs provided reliable and comparable VL results, but differences greater than 0.5 log
10
copy/ml remained between the two commercial PCRs after common DNA extraction.
Genotype MTBDRplus for Direct Detection of Mycobacterium tuberculosis and Drug Resistance in Strains from Gold Miners in South Africa
Authors: Dorman Susan E. , Chihota Violet N. , Lewis James J. , van der Meulen Minty , Mathema Barun , Beylis Natalie , Fielding Katherine L. , Grant Alison D. , Churchyard Gavin J. 
Published: January 2012

ABSTRACT

GenoType MTBDRplus is a molecular assay for detection of
Mycobacterium tuberculosis
and drug resistance. Assay performance as applied directly to consecutive unselected sputum samples has not been established. The objective of this study was to determine the accuracy of the MTBDRplus test for direct detection of
M. tuberculosis
(in sputum) and for drug resistance in consecutively submitted sputum samples. In this cross-sectional study in South Africa, one sputum specimen from each person suspected of having pulmonary tuberculosis was tested by smear microscopy, direct MTBDRplus, and Mycobacterial Growth Indicator Tube (MGIT) culture with MGIT drug susceptibility testing. MGIT results were the reference standard. We tested 2,510 sputum samples, and 529 (21.1%) were positive for
M. tuberculosis
by MGIT. Direct MTBDRplus identified
M. tuberculosis
in 256 of 529 specimens (sensitivity, 48.4%; 95% confidence interval [CI], 44.1, 52.7). The sensitivity of MTBDRplus for
M. tuberculosis
detection by sputum smear status was as follows: smear negative, 13.7% (95% CI, 9.8, 18.4); smear scanty, 46.2% (95% CI, 19.2, 74.9); smear 1+, 69.1% (95% CI, 55.2, 80.9); smear 2+, 86.3% (95% CI, 73.7, 94.3); smear 3+, 89.8% (95% CI, 83.7, 94.2). Direct MTBDRplus testing was negative for 1,594/1,612 sputum samples that were culture negative for
M. tuberculosis
(specificity, 98.9%; 95% CI, 98.2, 99.3). For specimens positive for
M. tuberculosis
by MTBDRplus, this assay's sensitivity and specificity for rifampin resistance were 85.7% (95% CI, 57.2, 98.2) and 96.6% (95% CI, 93.2, 98.6) and for isoniazid resistance they were 62.1% (95% CI, 42.3, 79.3) and 97.9% (95% CI, 94.8, 99.4). For sputum testing, the sensitivity of MTBDRplus is directly related to the specimen's bacillary burden. Our results support recommendations that the MTBDRplus test not be used for direct testing of smear-negative or paucibacillary sputum samples.
Bacteriuria Screening by Automated Whole-Field-Image-Based Microscopy Reduces the Number of Necessary Urine Cultures
Authors: Falbo Rosanna , Sala M. Roberta , Signorelli Silvia , Venturi Natascia , Signorini Stefano , Brambilla Paolo 
Published: January 2012

ABSTRACT
We evaluated a new automated urine sediment analyzer that provides whole-field images for the screening of urine samples prior to bacterial culture. Sterile urine samples from 1,011 male and female outpatients and inpatients (mean age 54.7) with a urinary tract infection prevalence of 18.3% were studied. Screening rapidly provides negative results.
Multilocus Sequence Typing of Total-Genome-Sequenced Bacteria
Authors: Larsen Mette V. , Cosentino Salvatore , Rasmussen Simon , Friis Carsten , Hasman Henrik , Marvig Rasmus Lykke , Jelsbak Lars , Sicheritz-Pontén Thomas , Ussery David W. , Aarestrup Frank M. , Lund Ole 
Published: January 2012

ABSTRACT

Accurate strain identification is essential for anyone working with bacteria. For many species, multilocus sequence typing (MLST) is considered the “gold standard” of typing, but it is traditionally performed in an expensive and time-consuming manner. As the costs of whole-genome sequencing (WGS) continue to decline, it becomes increasingly available to scientists and routine diagnostic laboratories. Currently, the cost is below that of traditional MLST. The new challenges will be how to extract the relevant information from the large amount of data so as to allow for comparison over time and between laboratories. Ideally, this information should also allow for comparison to historical data. We developed a Web-based method for MLST of 66 bacterial species based on WGS data. As input, the method uses short sequence reads from four sequencing platforms or preassembled genomes. Updates from the MLST databases are downloaded monthly, and the best-matching MLST alleles of the specified MLST scheme are found using a BLAST-based ranking method. The sequence type is then determined by the combination of alleles identified. The method was tested on preassembled genomes from 336 isolates covering 56 MLST schemes, on short sequence reads from 387 isolates covering 10 schemes, and on a small test set of short sequence reads from 29 isolates for which the sequence type had been determined by traditional methods. The method presented here enables investigators to determine the sequence types of their isolates on the basis of WGS data. This method is publicly available at
www.cbs.dtu.dk/services/MLST
.
A Simple, Semiselective Medium for Anaerobic Isolation of Anginosus Group Streptococci from Patients with Chronic Lung Disease
Authors: Waite Richard D. , Wareham David W. , Gardiner Samuel , Whiley Robert A. 
Published: January 2012

ABSTRACT
The anaerobic isolation of anginosus group streptococci (AGS) from respiratory specimens containing diverse microbiota using a semiselective blood agar medium incorporating nalidixic acid and sulfamethazine (NAS) is described. AGS were detected in 60% of tested sputa from patients with cystic fibrosis, chronic obstructive pulmonary disease, and bronchiectasis. This demonstrates NAS to be a diagnostic tool for detecting AGS within the complex microbial communities associated with chronic lung disorders.
Biological and Phylogenetic Characterization of a Genotype VII Newcastle Disease Virus from Venezuela: Efficacy of Field Vaccination
Authors: Perozo Francisco , Marcano Rosmar , Afonso Claudio L. 
Published: January 2012

ABSTRACT

Here we report the biological and molecular characterization of a virulent genotype VII Newcastle disease virus (NDV) circulating in Venezuela and the assessment of the vaccination efficacy under field conditions compared to controlled rearing conditions. Biological pathotyping showed a mean embryo dead time of 50 h and an intracerebral pathogenicity index of 1.86. Sequence-based phylogenetic analysis demonstrated that the virus belongs to genotype VII in class II (a genotype often found in Asia and Africa), representing the first report of the presence of this genotype in the continent of South America. A vaccine-challenge trial in commercial broilers reared in fields or in a experimental setting included dual (live/killed) priming of 1-day-old chicks plus two live NDV and infectious bursal disease virus (IBDV) field vaccinations at days 7 and 17, followed by a very stringent genotype VII NDV challenge at day 28. Serology for NDV and IBDV, bursal integrity, and protection against NDV lethal challenge were assessed. At 28 days, field vaccinates showed significantly lower NDV (1,356 versus 2,384) and higher IBD (7,295 versus 1,489) enzyme-linked immunosorbent assay (ELISA) antibody titers than the experimentally reared birds. A lower bursal size and bursa-body weight ratio (
P
< 0.05) and higher bursa lesion score were also detected in the field set. Only 57.1% of field vaccinates survived the lethal challenge, differing (
P
< 0.05) from 90.5% survival in the experimental farm. Overall, results confirmed the presence of the genotype VII viruses in South America and suggest that field-associated factors such as immunosuppression compromise the efficacy of the vaccination protocols implemented.
Mast Cell-Induced Lung Injury in Mice Infected with H5N1 Influenza Virus
Authors: Hu Yanxin , Jin Yi , Han Deping , Zhang Guozhong , Cao Shanping , Xie Jingjing , Xue Jia , Li Yi , Meng Di , Fan Xiaoxu , Sun Lun-Quan , Wang Ming 
Published: January 2012

ABSTRACTAlthough an important role for mast cells in several viral infections has been demonstrated, its role in the invasion of highly pathogenic H5N1 influenza virus is unknown. In the present study, we demonstrate that mast cells were activated significantly by H5N1 virus (A/chicken/Henan/1/2004) infection bothin vivoandin vitro. Mast cells could possibly intensify the lung injury that results from H5N1 infection by releasing proinflammatory mediators, including histamine, tryptase, and gamma interferon (IFN-γ). Lung lesions and apoptosis induced by H5N1 infection were reduced dramatically by treatment with ketotifen, which is a mast cell degranulation inhibitor. A combination of ketotifen and the neuraminidase inhibitor oseltamivir protected 100% of the mice from death postinfection. In conclusion, our data suggest that mast cells play a crucial role in the early stages of H5N1 influenza virus infection and provide a new approach to combat highly pathogenic influenza virus infection.
Simian Virus 40 Strains with Novel Properties Generated by Replacing the Viral Enhancer with Synthetic Oligonucleotides
Authors: Günther Viola , Strassen Till , Lindert Uschi , Dagani Patrizia , Waldvogel Dominique , Georgiev Oleg , Schaffner Walter , Bethge Tobias 
Published: January 2012

ABSTRACT
Typical enhancers of viral or cellular genes are approximately 100 to 400 bp long and contain several transcription factor binding sites. Previously, we have shown that simian virus 40 (SV40) genomic DNA that lacks its own enhancer can be used as an “enhancer trap” since it reacquires infectivity upon incorporation of heterologous enhancers. Here, we show that SV40 infectivity can be restored with synthetic enhancers that are assembled by the host cell. We found that several oligonucleotides, cotransfected with enhancerless SV40 DNA into host cells, were incorporated into the viral genome via cellular DNA end joining. The oligonucleotides tested included metal response elements (MREs), the binding sites for the transcription factor MTF-1, which induces gene activity in response to heavy metals. These recombinant SV40 strains showed preferential growth on cells overloaded with zinc or cadmium. We also cotransfected enhancerless SV40 DNA with oligonucleotides corresponding to enhancer motifs of human and mouse cytomegalovirus (HCMV and MCMV, respectively). In contrast to SV40 wild type, the viruses with cytomegalovirus-derived patchwork enhancers strongly expressed T-antigen in human HEK293 cells, accompanied by viral DNA replication. Occasionally, we also observed the assembly of functional viral genomes by incorporation of fragments of bovine DNA, an ingredient of the fetal calf serum in the medium. These fragments contained, among other sites, binding sites for AP-1 and CREB transcription factors. Taken together, our studies show that viruses with novel properties can be generated by intracellular incorporation of synthetic enhancer DNA motifs.
Filoviruses Require Endosomal Cysteine Proteases for Entry but Exhibit Distinct Protease Preferences
Authors: Misasi John , Chandran Kartik , Yang Jin-Yi , Considine Bryden , Filone Claire Marie , Côté Marceline , Sullivan Nancy , Fabozzi Giulia , Hensley Lisa , Cunningham James 
Published: January 2012

ABSTRACT

Filoviruses are enveloped viruses that cause sporadic outbreaks of severe hemorrhagic fever [CDC, MMWR Morb. Mortal. Wkly. Rep. 50:73–77, 2001; Colebunders and Borchert, J. Infect. 40:16–20, 2000; Colebunders et al., J. Infect. Dis. 196(Suppl. 2):S148–S153, 2007; Geisbert and Jahrling, Nat. Med. 10:S110-S121, 2004]. Previous studies revealed that endosomal cysteine proteases are host factors for ebolavirus Zaire (Chandran et al., Science 308:1643–1645, 2005; Schornberg et al., J. Virol. 80:4174–4178, 2006). In this report, we show that infection mediated by glycoproteins from other phylogenetically diverse filoviruses are also dependent on these proteases and provide additional evidence indicating that they cleave GP1 and expose the binding domain for the critical host factor Niemann-Pick C1. Using selective inhibitors and knockout-derived cell lines, we show that the ebolaviruses Zaire and Cote d'Ivoire are strongly dependent on cathepsin B, while the ebolaviruses Sudan and Reston and Marburg virus are not. Taking advantage of previous studies of cathepsin B inhibitor-resistant viruses (Wong et al., J. Virol. 84:163–175, 2010), we found that virus-specific differences in the requirement for cathepsin B are correlated with sequence polymorphisms at residues 47 in GP1 and 584 in GP2. We applied these findings to the analysis of additional ebolavirus isolates and correctly predicted that the newly identified ebolavirus species Bundibugyo, containing D47 and I584, is cathepsin B dependent and that ebolavirus Zaire-1995, the single known isolate of ebolavirus Zaire that lacks D47, is not. We also obtained evidence for virus-specific differences in the role of cathepsin L, including cooperation with cathepsin B. These studies strongly suggest that the use of endosomal cysteine proteases as host factors for entry is a general property of members of the family
Filoviridae
.
Deep Sequencing Identifies Viral and Wasp Genes with Potential Roles in Replication of Microplitis demolitor Bracovirus
Authors: Burke Gaelen R. , Strand Michael R. 
Published: January 2012

ABSTRACT

Viruses in the genus
Bracovirus
(BV) (
Polydnaviridae
) are symbionts of parasitoid wasps that specifically replicate in the ovaries of females. Recent analysis of expressed sequence tags from two wasp species,
Cotesia congregata
and
Chelonus inanitus
, identified transcripts related to 24 different nudivirus genes. These results together with other data strongly indicate that BVs evolved from a nudivirus ancestor. However, it remains unclear whether BV-carrying wasps contain other nudivirus-like genes and what types of wasp genes may also be required for BV replication.
Microplitis demolitor
carries
Microplitis demolitor
bracovirus (
Md
BV). Here we characterized
Md
BV replication and performed massively parallel sequencing of
M. demolitor
ovary transcripts. Our results indicated that
Md
BV replication begins in stage 2 pupae and continues in adults. Analysis of prereplication- and active-replication-stage ovary RNAs yielded 22 Gb of sequence that assembled into 66,425 transcripts. This breadth of sampling indicated that a large percentage of genes in the
M. demolitor
genome were sequenced. A total of 41 nudivirus-like transcripts were identified, of which a majority were highly expressed during
Md
BV replication. Our results also identified a suite of wasp genes that were highly expressed during
Md
BV replication. Among these products were several transcripts with conserved roles in regulating locus-specific DNA amplification by eukaryotes. Overall, our data set together with prior results likely identify the majority of nudivirus-related genes that are transcriptionally functional during BV replication. Our results also suggest that amplification of proviral DNAs for packaging into BV virions may depend upon the replication machinery of wasps.
The E1 Protein of Human Papillomavirus Type 16 Is Dispensable for Maintenance Replication of the Viral Genome
Authors: Egawa Nagayasu , Nakahara Tomomi , Ohno Shin-ichi , Narisawa-Saito Mako , Yugawa Takashi , Fujita Masatoshi , Yamato Kenji , Natori Yukikazu , Kiyono Tohru 
Published: January 2012

ABSTRACT
Papillomavirus genomes are thought to be amplified to about 100 copies per cell soon after infection, maintained constant at this level in basal cells, and amplified for viral production upon keratinocyte differentiation. To determine the requirement for E1 in viral DNA replication at different stages, an E1-defective mutant of the human papillomavirus 16 (HPV16) genome featuring a translation termination mutation in the E1 gene was used. The ability of the mutant HPV16 genome to replicate as nuclear episomes was monitored with or without exogenous expression of E1. Unlike the wild-type genome, the E1-defective HPV16 genome became established in human keratinocytes only as episomes in the presence of exogenous E1 expression. Once established, it could replicate with the same efficiency as the wild-type genome, even after the exogenous E1 was removed. However, upon calcium-induced keratinocyte differentiation, once again amplification was dependent on exogenous E1. These results demonstrate that the E1 protein is dispensable for maintenance replication but not for initial and productive replication of HPV16.
Mutating Conserved Cysteines in the Alphavirus E2 Glycoprotein Causes Virus-Specific Assembly Defects
Authors: Snyder Anthony J. , Sokoloski Kevin J. , Mukhopadhyay Suchetana 
Published: January 2012

ABSTRACT

There are 80 trimeric, glycoprotein spikes that cover the surface of an alphavirus particle. The spikes, which are composed of three E2 and E1 glycoprotein heterodimers, are responsible for receptor binding and mediating fusion between the viral and host-cell membranes during entry. In addition, the cytoplasmic domain of E2 interacts with the nucleocapsid core during the last stages of particle assembly, possibly to aid in particle stability. During assembly, the spikes are nonfusogenic until the E3 glycoprotein is cleaved from E2 in the
trans
-Golgi network. Thus, a mutation in E2 potentially has effects on virus entry, spike assembly, or spike maturation. E2 is a highly conserved, cysteine-rich transmembrane glycoprotein. We made single cysteine-to-serine mutations within two distinct regions of the E2 ectodomain in both Sindbis virus and Ross River virus. Each of the E2 Cys mutants produced fewer infectious particles than wild-type virus. Further characterization of the mutant viruses revealed differences in particle morphology, fusion activity, and polyprotein cleavage between Sindbis and Ross River virus mutants, despite the mutations being made at corresponding positions in E2. The nonconserved assembly defects suggest that E2 folding and function is species dependent, possibly due to interactions with a virus-specific chaperone.
Endogenous Lentiviruses in the Ferret Genome
Authors: Cui Jie , Holmes Edward C. 
Published: January 2012

ABSTRACT

By screening 74 chordate genomes for endogenous lentiviruses using Pol sequences of exogenous lentiviruses as a reference, we identified a novel endogenous lentivirus in the genome of the ferret (
Mustela putorius furo
). Phylogenetic analysis suggested that the ferret endogenous lentivirus, denoted ELVmpf, diverged early in the evolution of the mammalian lentiviruses, although with a lack of resolution at key nodes. These data support the notion that lentiviruses have evolved on timescales of millions of years.
Postexposure Treatment with the Live-Attenuated Rabies Virus (RV) Vaccine TriGAS Triggers the Clearance of Wild-Type RV from the Central Nervous System (CNS) through the Rapid Induction of Genes Relevant to Adaptive Immunity in CNS Tissues
Authors: Li Jianwei , Ertel Adam , Portocarrero Carla , Barkhouse Darryll A. , Dietzschold Bernhard , Hooper D. Craig , Faber Milosz 
Published: January 2012

ABSTRACTPostexposure treatment (PET) of wild-type rabies virus (RV)-infected mice with a live-attenuated triple-glycoprotein RV variant (TriGAS) promotes survival but does not prevent the pathogenic RV from invading and replicating in the brain. Successful PET is associated with the induction of a robust virus-neutralizing antibody response and clearance of the wild-type RV from brain tissues. Comparison of the transcriptomes of normal mouse brain with those of wild-type-RV-infected mice that had received either mock or TriGAS PET treatment revealed that many of the host genes activated in the mock-treated mice represent type I interferon (IFN) response genes. This indicates that RV infection induces an early type I IFN response that is unable to control the infection. In contrast, most of the activated genes in the brain of the RV-infected, TriGAS-treated mouse play a role in adaptive immunity, including the regulation of T cell activation, T cell differentiation, and the regulation of lymphocyte and mononuclear cell proliferation. These findings were confirmed by quantitative PCR (qPCR) array studies, which showed that 3 genes in particular, encoding chemokine ligand 3 (Ccl3), natural killer cell activator 2 (interleukin 12B [IL-12B]), and granzyme A (GzmA), were activated earlier and to a greater extent in the brains of RV-infected mice treated with TriGAS than in the brains of mock-treated mice. The activation of these genes, known to play key roles in the regulation of lymphocyte and mononuclear cell proliferation, is likely an important part of the mechanism by which TriGAS mediates its PET activity.
Varicella-Zoster Virus Infects Human Embryonic Stem Cell-Derived Neurons and Neurospheres but Not Pluripotent Embryonic Stem Cells or Early Progenitors
Authors: Dukhovny Anna , Sloutskin Anna , Markus Amos , Yee Michael B. , Kinchington Paul R. , Goldstein Ronald S. 
Published: January 2012

ABSTRACT

Pluripotent human stem cells are a powerful tool for the generation of differentiated cells that can be used for the study of human disease. We recently demonstrated that neurons derived from pluripotent human embryonic stem cells (hESC) can be infected by the highly host-restricted human alphaherpesvirus varicella-zoster virus (VZV), permitting the interaction of VZV with neurons to be readily evaluated in culture. In the present study, we examine whether pluripotent hESC and neural progenitors at intermediate stages of differentiation are permissive for VZV infection. We demonstrate here that VZV infection is blocked in naïve hESC. A block to VZV replication is also seen when a bacterial artificial chromosome (BAC) containing the VZV genome is transfected into hESC. In contrast, related alphaherpesviruses herpes simplex virus 1 (HSV-1) and pseudorabies virus (PrV) productively infect naïve hESC in a cell-free manner, and PrV replicates from a BAC transfected into hESC. Neurons differentiate from hESC via neural progenitor intermediates, as is the case in the embryo. The first
in vitro
stage at which permissiveness of hESC-derived neural precursors to VZV replication is observed is upon formation of “neurospheres,” immediately after detachment from the inductive stromal feeder layer. These findings suggest that hESC may be useful in deciphering the yet enigmatic mechanisms of specificity of VZV infection and replication.