bluuebunny/crossref_metadata_2025_split huggingface dataset:
Targeted Enrichment for Pathogen Detection and Characterization in Three Felid Species
Authors: Lee Justin S. , Mackie Ryan S. , Harrison Thomas , Shariat Basir , Kind Trey , Kehl Timo , Löchelt Martin , Boucher Christina , VandeWoude Sue 
Published: March 2017

ABSTRACT
Traditional diagnostic assays often lack sensitivity and can be difficult to multiplex across many pathogens. Next-generation sequencing (NGS) can overcome some of these problems but has limited application in the detection of low-copy-number pathogens in complex samples. Targeted genome capture (TGC) utilizes oligonucleotide probes to enrich specific nucleic acids in heterogeneous extracts and can therefore increase the proportion of NGS reads for low-abundance targets. While earlier studies have demonstrated the utility of this technology for detection of novel pathogens in human clinical samples, the capacity and practicality of TGC-NGS in a veterinary diagnostic setting have not yet been evaluated. Here we report the use of TGC-NGS assays for the detection and characterization of diverse feline pathogen taxa. We detected 31 pathogens comprising nine pathogen taxa in 28 felid samples analyzed. This included 20 pathogens detected via traditional PCR and 11 additional pathogens that had not been previously detected in the same samples. Most of the pathogens detected were sequenced at sufficient breadth and depth to confidently classify them at the species or subspecies level. Target nucleic acids were enriched from a low of 58-fold to 56 million-fold relative to host nucleic acids. Despite the promising performance of these assays, a number of pathogens detected by conventional PCR or serology were not isolated by TGC-NGS, suggesting that further validation is required before this technology can be used in lieu of quality-controlled standard assays. We conclude that TGC-NGS offers great potential as a broad multiplex pathogen characterization assay in veterinary diagnostic and research settings.
Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis
Authors: Anthwal Divya , Gupta Rakesh Kumar , Bhalla Manpreet , Bhatnagar Shinjini , Tyagi Jaya Sivaswami , Haldar Sagarika 
Published: March 2017

ABSTRACT

Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in
Mycobacterium tuberculosis
in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of
rpoB
,
katG
, and an
inhA
promoter and used as positive controls in HRM. The assay was first validated in 25 MDR
M. tuberculosis
clinical isolates. The assay was evaluated on DNA isolated from 99
M. tuberculosis
culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH-
katG
, and INH-
inhA
assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% (κ value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR]400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases.
Species Identification and Strain Typing of Staphylococcus agnetis and Staphylococcus hyicus Isolates from Bovine Milk by Use of a Novel Multiplex PCR Assay and Pulsed-Field Gel Electrophoresis
Authors: Adkins P. R. F. , Middleton J. R. , Calcutt M. J. , Stewart G. C. , Fox L. K. 
Published: March 2017

ABSTRACTStaphylococcus hyicusandStaphylococcus agnetisare two coagulase-variable staphylococcal species that can be isolated from bovine milk and are difficult to differentiate. The objectives of this study were to characterize isolates of bovine milk origin from a collection that had previously been characterized as coagulase-positiveS. hyicusbased on phenotypic species identification methods and to develop a PCR-based method for differentiatingS. hyicus,S. agnetis, andStaphylococcus aureus. Isolates (n= 62) were selected from a previous study in which milk samples were collected from cows on 15 dairy herds. Isolates were coagulase tested and identified to the species level using housekeeping gene sequencing. A multiplex PCR to differentiateS. hyicus,S. agnetis, andS. aureuswas developed. Pulsed-field gel electrophoresis was conducted to strain type the isolates. Based on gene sequencing, 44/62 of the isolates were determined to be eitherS. agnetis(n= 43) orS. hyicus(n= 1). Overall, 88% (37/42) of coagulase-positiveS. agnetisisolates were found to be coagulase positive at 4 h. The herd-level prevalence of coagulase-positiveS. agnetisranged from 0 to 2.17%. Strain typing identified 23 different strains. Six strains were identified more than once and from multiple cows within the herd. Three strains were isolated from cows at more than one time point, with 41 to 264 days between samplings. These data suggest thatS. agnetisis likely more prevalent on dairy farms thanS. hyicus. Also, someS. agnetisisolates in this study appeared to be contagious and associated with persistent infections.
Usefulness of Two Aspergillus PCR Assays and Aspergillus Galactomannan and β-
d
-Glucan Testing of Bronchoalveolar Lavage Fluid for Diagnosis of Chronic Pulmonary Aspergillosis
Authors: Urabe Naohisa , Sakamoto Susumu , Sano Go , Suzuki Junko , Hebisawa Akira , Nakamura Yasuhiko , Koyama Kazuya , Ishii Yoshikazu , Tateda Kazuhiro , Homma Sakae 
Published: March 2017

ABSTRACT

We evaluated the usefulness of an
Aspergillus
galactomannan (GM) test, a β-
d
-glucan (βDG) test, and two different
Aspergillus
PCR assays of bronchoalveolar lavage fluid (BALF) samples for the diagnosis of chronic pulmonary aspergillosis (CPA). BALF samples from 30 patients with and 120 patients without CPA were collected. We calculated the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio for each test individually and in combination with other tests. The optical density index values, as determined by receiver operating characteristic analysis, for the diagnosis of CPA were 0.5 and 100 for GM and βDG testing of BALF, respectively. The sensitivity and specificity of the GM test, βDG test, and PCR assays 1 and 2 were 77.8% and 90.0%, 77.8% and 72.5%, 86.7% and 84.2%, and 66.7% and 94.2%, respectively. A comparison of the PCR assays showed that PCR assay 1 had a better sensitivity, a better negative predictive value, and a better negative likelihood ratio and PCR assay 2 had a better specificity, a better positive predictive value, and a better positive likelihood ratio. The combination of the GM and βDG tests had the highest diagnostic odds ratio. The combination of the GM and βDG tests on BALF was more useful than any single test for diagnosing CPA.
Heterologous Packaging Signals on Segment 4, but Not Segment 6 or Segment 8, Limit Influenza A Virus Reassortment
Authors: White Maria C. , Steel John , Lowen Anice C. 
Published: March 2017

ABSTRACT
Influenza A virus (IAV) RNA packaging signals serve to direct the incorporation of IAV gene segments into virus particles, and this process is thought to be mediated by segment-segment interactions. These packaging signals are segment and strain specific, and as such, they have the potential to impact reassortment outcomes between different IAV strains. Our study aimed to quantify the impact of packaging signal mismatch on IAV reassortment using the human seasonal influenza A/Panama/2007/99 (H3N2) and pandemic influenza A/Netherlands/602/2009 (H1N1) viruses. Focusing on the three most divergent segments, we constructed pairs of viruses that encoded identical proteins but differed in the packaging signal regions on a single segment. We then evaluated the frequency with which segments carrying homologous versus heterologous packaging signals were incorporated into reassortant progeny viruses. We found that, when segment 4 (HA) of coinfecting parental viruses was modified, there was a significant preference for the segment containing matched packaging signals relative to the background of the virus. This preference was apparent even when the homologous HA constituted a minority of the HA segment population available in the cell for packaging. Conversely, when segment 6 (NA) or segment 8 (NS) carried modified packaging signals, there was no significant preference for homologous packaging signals. These data suggest that movement of NA and NS segments between the human H3N2 and H1N1 lineages is unlikely to be restricted by packaging signal mismatch, while movement of the HA segment would be more constrained. Our results indicate that the importance of packaging signals in IAV reassortment is segment dependent.

IMPORTANCE
Influenza A viruses (IAVs) can exchange genes through reassortment. This process contributes to both the highly diverse population of IAVs found in nature and the formation of novel epidemic and pandemic IAV strains. Our study sought to determine the extent to which IAV packaging signal divergence impacts reassortment between seasonal IAVs. Our knowledge in this area is lacking, and insight into the factors that influence IAV reassortment will inform and strengthen ongoing public health efforts to anticipate the emergence of new viruses. We found that the packaging signals on the HA segment, but not the NA or NS segments, restricted IAV reassortment. Thus, the packaging signals of the HA segment could be an important factor in determining the likelihood that two IAV strains of public health interest will undergo reassortment.
Evaluation of the Immune Responses to and Cross-Protective Efficacy of Eurasian H7 Avian Influenza Viruses
Authors: Kwon Hyeok-Il , Kim Young-Il , Park Su-Jin , Song Min-Suk , Kim Eun-Ha , Kim Se Mi , Si Young-Jae , Lee In-Won , Song Byung-Min , Lee Youn-Jeong , Yun Seok Joong , Kim Wun-Jae , Choi Young Ki 
Published: March 2017

ABSTRACT

Due to increasing concerns about human infection by various H7 influenza viruses, including recent H7N9 viruses, we evaluated the genetic relationships and cross-protective efficacies of three different Eurasian H7 avian influenza viruses. Phylogenic and molecular analyses revealed that recent Eurasian H7 viruses can be separated into two different lineages, with relatively high amino acid identities within groups (94.8 to 98.8%) and low amino acid identities between groups (90.3 to 92.6%).
In vivo
immunization with representatives of each group revealed that while group-specific cross-reactivity was induced, cross-reactive hemagglutination inhibition (HI) titers were approximately 4-fold lower against heterologous group viruses than against homologous group viruses. Moreover, the group I (RgW109/06) vaccine protected 100% of immunized mice from various group I viruses, while only 20 to 40% of immunized mice survived lethal challenge with heterologous group II viruses and exhibited high viral titers in the lung. Moreover, while the group II (RgW478/14) vaccine also protected mice from lethal challenge with group II viruses, it failed to elicit cross-protection against group I viruses. However, it is noteworthy that vaccination with RgAnhui1/13, a virus of a sublineage of group I, cross-protected immunized mice against lethal challenge with both group I and II viruses and significantly attenuated lung viral titers. Interestingly, immune sera from RgAnhui1/13-vaccinated mice showed a broad neutralizing spectrum rather than the group-specific pattern observed with the other viruses. These results suggest that the recent human-infective H7N9 strain may be a candidate broad cross-protective vaccine for Eurasian H7 viruses.

IMPORTANCE
Genetic and phylogenic analyses have demonstrated that the Eurasian H7 viruses can be separated into at least two different lineages, both of which contain human-infective fatal H7 viruses, including the recent novel H7N9 viruses isolated in China since 2013. Due to the increasing concerns regarding the global public health risk posed by H7 viruses, we evaluated the genetic relationships between Eurasian H7 avian influenza viruses and the cross-protective efficacies of three different H7 viruses: W109/06 (group I), W478/14 (group II), and Anhui1/13 (a sublineage of group I). While each vaccine induced group-specific antibody responses and cross-protective efficacy, only Anhui1/13 was able to cross-protect immunized hosts against lethal challenge across groups. In fact, the Anhui1/13 virus induced not only cross-protection but also broad serum neutralizing antibody responses against both groups of viruses. This suggests that Anhui1/13-like H7N9 viruses may be viable vaccine candidates for broad protection against Eurasian H7 viruses.
Kaposi's Sarcoma-Associated Herpesvirus Hijacks RNA Polymerase II To Create a Viral Transcriptional Factory
Authors: Chen Christopher Phillip , Lyu Yuanzhi , Chuang Frank , Nakano Kazushi , Izumiya Chie , Jin Di , Campbell Mel , Izumiya Yoshihiro 
Published: March 2017

ABSTRACT

Locally concentrated nuclear factors ensure efficient binding to DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable preinitiation complexes. We have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around Kaposi's sarcoma-associated herpesvirus (KSHV) genomes in the host cell nucleus. Using immunofluorescence labeling of latent nuclear antigen (LANA) protein, together with fluorescence
in situ
RNA hybridization (RNA-FISH) of the intron region of immediate early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At the single-cell level, we found that not all episomes were uniformly transcribed following reactivation stimuli. However, those episomes that were being transcribed would spontaneously aggregate to form transcriptional “factories,” which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of “viral transcriptional factories” decreased the pool of cellular RNA polymerase II available for cellular gene transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an “all-in-one” factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells.

IMPORTANCE
B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. Three-dimensional imaging of viral gene expression in the nucleus allows us to study interactions and changes in the physical distribution of these episomes following stimulation. The results showed heterogeneity in the responses of individual KSHV episomes to stimuli within a single reactivating cell; those episomes that did respond to stimulation, aggregated within large domains that appear to function as viral transcription factories. A significant portion of cellular RNA polymerase II was trapped in these factories and served to transcribe viral genomes, which coincided with an overall decrease in cellular gene expression. Our findings uncover a strategy of KSHV gene regulation through focal assembly of KSHV episomes and a molecular mechanism of late gene expression.
Structural Characterization of Human Coronavirus NL63 N Protein
Authors: Szelazek Bozena , Kabala Wojciech , Kus Krzysztof , Zdzalik Michal , Twarda-Clapa Aleksandra , Golik Przemyslaw , Burmistrz Michal , Florek Dominik , Wladyka Benedykt , Pyrc Krzysztof , Dubin Grzegorz 
Published: March 2017

ABSTRACT
Coronaviruses are responsible for upper and lower respiratory tract infections in humans. It is estimated that 1 to 10% of the population suffers annually from cold-like symptoms related to infection with human coronavirus NL63 (HCoV-NL63), an alphacoronavirus. The nucleocapsid (N) protein, the major structural component of the capsid, facilitates RNA packing, links the capsid to the envelope, and is also involved in multiple other processes, including viral replication and evasion of the immune system. Although the role of N protein in viral replication is relatively well described, no structural data are currently available regarding the N proteins of alphacoronaviruses. Moreover, our understanding of the mechanisms of RNA binding and nucleocapsid formation remains incomplete. In this study, we solved the crystal structures of the N- and C-terminal domains (NTD, residues 10 to 140, and CTD, residues 221 to 340, respectively) of the N protein of HCoV-NL63, both at a 1.5-Å resolution. Based on our structure of NTD solved here, we proposed and experimentally evaluated a model of RNA binding. The structure of the CTD reveals the mode of N protein dimerization. Overall, this study expands our understanding of the initial steps of N protein-nucleic acid interaction and may facilitate future efforts to control the associated infections.

IMPORTANCE
Coronaviruses are responsible for the common cold and other respiratory tract infections in humans. According to multiple studies, 1 to 10% of the population is infected each year with HCoV-NL63. Viruses are relatively simple organisms composed of a few proteins and the nucleic acids that carry the information determining their composition. The nucleocapsid (N) protein studied in this work protects the nucleic acid from the environmental factors during virus transmission. This study investigated the structural arrangement of N protein, explaining the first steps of its interaction with nucleic acid at the initial stages of virus structure assembly. The results expand our understanding of coronavirus physiology and may facilitate future efforts to control the associated infections.
Estimated disability-adjusted life years averted by free-of-charge provision of the levonorgestrel-releasing intrauterine system over a 9-year period in Brazil
Authors: Ferreira Jessica M , Monteiro Ilza , Fernandes Arlete , Bahamondes Maria V , Pitoli Ana , Bahamondes Luis 
Published: March 2017

BackgroundThe objective was to analyse the contribution of the provision at no cost to users of the 20 µg/day levonorgestrel-releasing intrauterine system (LNG-IUS) towards disability-adjusted life years (DALY) averted over a 9-year period.MethodsWe analysed data from 15 030 new users of the LNG-IUS who had the device inserted at 26 Brazilian teaching hospitals between January 2007 and December 2015. The devices came from the International Contraceptive Access Foundation (ICA), a not-for-profit foundation that donates the devices to developing countries for use by low-income women who desire long-term contraception and who freely choose to use this device. Estimation of the DALY averted included live births averted, maternal morbidity and mortality, child mortality and unsafe abortions averted.ResultsA total of 15 030 women chose the LNG-IUS as a contraceptive method during the study period. Over the 9 years of evaluation, the estimated cumulative contribution of the Brazilian program in terms of DALY averted consisted of 486 live births, 14 cases of combined maternal mortality and morbidity, 143 cases of child mortality and 410 unsafe abortions.ConclusionsProvision of the LNG-IUS at no cost to low-income Brazilian women reduced unwanted pregnancies and probably averted maternal mortality and morbidity, child mortality and unsafe abortions. Family planning programs, policymakers and stakeholders based in low-resource settings could take advantage of the information that the provision of this contraceptive at no cost, or at affordable cost to a publicly-insured population, is an effective policy to help promote women’s health.
Mechanisms of ECG signs in chronic obstructive pulmonary disease
Authors: Larssen Marte Strømsnes , Steine Kjetil , Hilde Janne Mykland , Skjørten Ingunn , Hodnesdal Christian , Liestøl Knut , Gjesdal Knut 
Published: March 2017

ObjectivePatients with chronic obstructive pulmonary disease (COPD) often have abnormal ECGs. Our aim was to separate the effects on ECG by airway obstruction, emphysema and right ventricular (RV) afterload in patients with COPD.MethodsA cross-sectional study was performed on 101 patients with COPD without left heart disease and 32 healthy age-matched controls. Body mass index (BMI) was measured, and pulmonary function tests, ECG, echocardiography and right heart catheterisation (only patients) were performed. Variables were grouped into (1) airway obstruction by FEV% (percentage of forced expiratory volume)_predicted, (2) emphysema by residual volume/total lung capacity and residual volume (percent of predicted) and (3) RV afterload by mean pulmonary pressure, artery compliance, vascular resistance and RV wall thickness.ResultsIn multivariate regression analysis, emphysema correlated negatively to R+S amplitudes in horizontal and frontal leads, RV/left ventricle (LV) end-diastolic volume ratio to horizontal amplitudes and BMI negatively to frontal amplitudes. Increased airway obstruction, RV afterload and BMI correlated with horizontal QRS-axis clockwise rotation. Airway obstruction, RV afterload, RV/LV end-diastolic volume ratio and BMI correlated to the Sokolow-Lyon Index for RV, and RV afterload negatively to Sokolow-LyonIndex for LV. Several classical ECG changes could, however, not be ascribed to specific mechanisms.ConclusionsIn COPD, the various pathophysiological mechanisms modify the ECG differently. Increased airway obstruction and RV afterload mainly increase the Sokolow-Lyon Index for RV mass and associate with clockwise rotation of the horizontal QRS-axis, whereas emphysema reduces the QRS amplitudes. BMI is an equally important determinant for the majority of the ECG changes.