bluuebunny/crossref_metadata_2025_split huggingface dataset:
Exploring the Potential of Next-Generation Sequencing in Detection of Respiratory Viruses
Authors: Prachayangprecha Slinporn , Schapendonk Claudia M. E. , Koopmans Marion P. , Osterhaus Albert D. M. E. , Schürch Anita C. , Pas Suzan D. , van der Eijk Annemiek A. , Poovorawan Yong , Haagmans Bart L. , Smits Saskia L. 
Published: August 2014

ABSTRACT
Efficient detection of human respiratory viral pathogens is crucial in the management of patients with acute respiratory tract infection. Sequence-independent amplification of nucleic acids combined with next-generation sequencing technology and bioinformatics analyses is a promising strategy for identifying pathogens in clinical and public health settings. It allows the characterization of hundreds of different known pathogens simultaneously and of novel pathogens that elude conventional testing. However, major hurdles for its routine use exist, including cost, turnaround time, and especially sensitivity of the assay, as the detection limit is dependent on viral load, host genetic material, and sequencing depth. To obtain insights into these aspects, we analyzed nasopharyngeal aspirates from a cohort of 81 Thai children with respiratory disease for the presence of respiratory viruses using a sequence-independent next-generation sequencing approach and routinely used diagnostic real-time reverse transcriptase PCR (real-time RT-PCR) assays. With respect to the detection of rhinovirus and human metapneumovirus, the next-generation sequencing approach was at least as sensitive as diagnostic real-time RT-PCR in this small cohort, whereas for bocavirus and enterovirus, next-generation sequencing was less sensitive than real-time RT-PCR. The advantage of the sequencing approach over real-time RT-PCR was the immediate availability of virus-typing information. Considering the development of platforms capable of generating more output data at declining costs, next-generation sequencing remains of interest for future virus diagnosis in clinical and public health settings and certainly as an additional tool when screening results from real-time RT-PCR are negative.
Integrating Forecast Probabilities in Antibiograms: a Way To Guide Antimicrobial Prescriptions More Reliably?
Authors: Maurer Florian P. , Courvalin Patrice , Böttger Erik C. , Hombach Michael 
Published: August 2014

ABSTRACT

Antimicrobial susceptibility testing (AST) assigns pathogens to “susceptible” or “resistant” clinical categories based on clinical breakpoints (CBPs) derived from MICs or inhibition zone diameters and indicates the likelihood for therapeutic success. AST reports do not provide quantitative measures for the reliability of such categorization. Thus, it is currently impossible for clinicians to estimate the technical forecast uncertainty of an AST result regarding clinical categorization. AST error rates depend on the localization of pathogen populations in relation to CBPs. Bacterial species are, however, not homogeneous, and subpopulations behave differently with respect to AST results. We addressed how AST reporting errors differ between isolates with and without acquired drug resistance determinants. Using as an example the beta-lactams and their most important resistance mechanisms, we analyzed different pathogen populations for their individual reporting error probabilities. Categorization error rates were significantly higher for bacterial populations harboring resistance mechanisms than for the wild-type population. Reporting errors for amoxicillin-clavulanic acid and piperacillin-tazobactam in
Escherichia coli
infection cases were almost exclusively due to the presence of broad-spectrum- and extended-spectrum-beta-lactamase (ESBL)-producing microorganisms (79% and 20% of all errors, respectively). Clinicians should be aware of the significantly increased risk of erroneous AST reports for isolates producing beta-lactamases, particularly ESBL and AmpC. Including probability indicators for interpretation would improve AST reports.
Isolation of Dengue Virus-Specific Memory B Cells with Live Virus Antigen from Human Subjects following Natural Infection Reveals the Presence of Diverse Novel Functional Groups of Antibody Clones
Authors: Smith Scott A. , de Alwis A. Ruklanthi , Kose Nurgun , Jadi Ramesh S. , de Silva Aravinda M. , Crowe James E. 
Published: August 2014

ABSTRACT
Natural dengue virus (DENV) infection in humans induces antibodies (Abs) that neutralize the serotype of infection in a potent and type-specific manner; however, most Abs generated in response to infection are serotype cross-reactive and poorly neutralizing. Such cross-reactive Abs may enhance disease during subsequent infection with a virus of a different DENV serotype. Previous screening assays for DENV-specific human B cells and antibodies, using viral and recombinant antigens, mainly led to the isolation of dominant nonneutralizing B cell clones. To improve upon our ability to recover and study rare but durable and potently neutralizing DENV-specific Abs, we isolated human DENV-specific B cells by using a primary screen of binding to live virus, followed by a secondary screen with a high-throughput, flow cytometry-based neutralization assay to identify DENV-specific B cell lines prior to generation of hybridomas. Using this strategy, we identified several new classes of serotype-specific and serotype-cross-neutralizing anti-DENV monoclonal Abs (MAbs), including ultrapotent inhibitory antibodies with neutralizing activity concentrations of <10 ng/ml. We isolated serotype-specific neutralizing Abs that target diverse regions of the E protein, including epitopes present only on the intact, fully assembled viral particle. We also isolated a number of serotype-cross-neutralizing MAbs, most of which recognized a region in E protein domain I/II containing the fusion loop. These data provide insights into targets of the protective Ab-mediated immune response to natural DENV infection, which will prove valuable in the design and testing of new experimental DENV vaccines.

IMPORTANCE
Dengue virus infection is one of the most common mosquito-borne diseases and occurs in most countries of the world. Infection of humans with dengue virus induces a small number of antibodies that inhibit the infecting strain but also induces a large number of antibodies that can bind but do not inhibit dengue virus strains of other serotypes. We used a focused screening strategy to discover a large number of rare potently inhibiting antibodies, and we mapped the regions on the virus that were recognized by such antibodies. Our studies revealed that humans have the potential to generate very potent antibodies directed to diverse regions of the dengue virus surface protein. These studies provide important new information about protection from dengue virus infection that will be useful in the design and testing of new experimental dengue vaccines for humans.
Inhibition of Enterovirus 71 by Adenosine Analog NITD008
Authors: Deng Cheng-Lin , Yeo Huimin , Ye Han-Qing , Liu Si-Qing , Shang Bao-Di , Gong Peng , Alonso Sylvie , Shi Pei-Yong , Zhang Bo 
Published: August 2014

ABSTRACT
Enterovirus 71 (EV71) is a major viral pathogen in China and Southeast Asia. There is no clinically approved vaccine or antiviral therapy for EV71 infection. NITD008, an adenosine analog, is an inhibitor of flavivirus that blocks viral RNA synthesis. Here we report that NITD008 has potent antiviral activity against EV71. In cell culture, the compound inhibits EV71 at a 50% effective concentration of 0.67 μM and a 50% cytotoxic concentration of 119.97 μM. When administered at 5 mg/kg in an EV71 mouse model, the compound reduced viral loads in various organs and completely prevented clinical symptoms and death. To study the antiviral mechanism and drug resistance, we selected escape mutant viruses by culturing EV71 with increasing concentrations of NITD008. Resistance mutations were reproducibly mapped to the viral 3A and 3D polymerase regions. Resistance analysis with recombinant viruses demonstrated that either a 3A or a 3D mutation alone could lead to resistance to NITD008. A combination of both 3A and 3D mutations conferred higher resistance, suggesting a collaborative interplay between the 3A and 3D proteins during viral replication. The resistance results underline the importance of combination therapy required for EV71 treatment.

IMPORTANCE
Human enterovirus 71 (EV71) has emerged as a major cause of viral encephalitis in children worldwide, especially in the Asia-Pacific region. Vaccines and antivirals are urgently needed to prevent and treat EV71 infections. In this study, we report the
in vitro
and
in vivo
efficacy of NITD008 (an adenosine analog) as an inhibitor of EV71. The efficacy results validated the potential of nucleoside analogs as antiviral drugs for EV71 infections. Mechanistically, we showed that mutations in the viral 3A and 3D polymerases alone or in combination could confer resistance to NITD008. The resistance results suggest an intrinsic interaction between viral proteins 3A and 3D during replication, as well as the importance of combination therapy for the treatment of EV71 infections.
The 3′ Untranslated Region of Pea Enation Mosaic Virus Contains Two T-Shaped, Ribosome-Binding, Cap-Independent Translation Enhancers
Authors: Gao Feng , Kasprzak Wojciech K. , Szarko Christine , Shapiro Bruce A. , Simon Anne E. 
Published: August 2014

ABSTRACT

Many plant viruses without 5′caps or 3′ poly(A) tails contain 3′ proximal, cap-independent translation enhancers (3′CITEs) that bind to ribosomal subunits or translation factors thought to assist in ribosome recruitment. Most 3′CITEs participate in a long-distance kissing-loop interaction with a 5′ proximal hairpin to deliver ribosomal subunits to the 5′ end for translation initiation.
Pea Enation Mosaic Virus
(PEMV) contains two adjacent 3′CITEs in the center of its 703-nucleotide 3′ untranslated region (3′UTR), the ribosome-binding, kissing-loop T-shaped structure (kl-TSS) and eukaryotic translation initiation factor 4E-binding Panicum mosaic virus-like translation enhance (PTE). We now report that PEMV contains a third, independent 3′CITE located near the 3′ terminus. This 3′CITE is composed of three hairpins and two pseudoknots, similar to the TSS 3′CITE of the carmovirus
Turnip crinkle virus
(TCV). As with the TCV TSS, the PEMV 3′TSS is predicted to fold into a T-shaped structure that binds to 80S ribosomes and 60S ribosomal subunits. A small hairpin (kl-H) upstream of the 3′TSS contains an apical loop capable of forming a kissing-loop interaction with a 5′ proximal hairpin and is critical for the accumulation of full-length PEMV in protoplasts. Although the kl-H and 3′TSS are dispensable for the translation of a reporter construct containing the complete PEMV 3′UTR
in vitro
, deleting the normally required kl-TSS and PTE 3′CITEs and placing the kl-H and 3′TSS proximal to the reporter termination codon restores translation to near wild-type levels. This suggests that PEMV requires three 3′CITEs for proper translation and that additional translation enhancers may have been missed if reporter constructs were used in 3′CITE identification.

IMPORTANCE
The rapid life cycle of viruses requires efficient translation of viral-encoded proteins. Many plant RNA viruses contain 3′ cap-independent translation enhancers (3′CITEs) to effectively compete with ongoing host translation. Since only single 3′CITEs have been identified for the vast majority of individual viruses, it is widely accepted that this is sufficient for a virus's translational needs.
Pea Enation Mosaic Virus
possesses a ribosome-binding 3′CITE that can connect to the 5′ end through an RNA-RNA interaction and an adjacent eukaryotic translation initiation factor 4E-binding 3′CITE. We report the identification of a third 3′CITE that binds weakly to ribosomes and requires an upstream hairpin to form a bridge between the 3′ and 5′ ends. Although both ribosome-binding 3′CITEs are critical for virus accumulation
in vivo
, only the CITE closest to the termination codon of a reporter open reading frame is active, suggesting that artificial constructs used for 3′CITE identification may underestimate the number of CITEs that participate in translation.
Coronaviruses Resistant to a 3C-Like Protease Inhibitor Are Attenuated for Replication and Pathogenesis, Revealing a Low Genetic Barrier but High Fitness Cost of Resistance
Authors: Deng Xufang , StJohn Sarah E. , Osswald Heather L. , O'Brien Amornrat , Banach Bridget S. , Sleeman Katrina , Ghosh Arun K. , Mesecar Andrew D. , Baker Susan C. 
Published: August 2014

ABSTRACT
Viral protease inhibitors are remarkably effective at blocking the replication of viruses such as human immunodeficiency virus and hepatitis C virus, but they inevitably lead to the selection of inhibitor-resistant mutants, which may contribute to ongoing disease. Protease inhibitors blocking the replication of coronavirus (CoV), including the causative agents of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), provide a promising foundation for the development of anticoronaviral therapeutics. However, the selection and consequences of inhibitor-resistant CoVs are unknown. In this study, we exploited the model coronavirus, mouse hepatitis virus (MHV), to investigate the genotype and phenotype of MHV quasispecies selected for resistance to a broad-spectrum CoV 3C-like protease (3CLpro) inhibitor. Clonal sequencing identified single or double mutations within the 3CLpro coding sequence of inhibitor-resistant virus. Using reverse genetics to generate isogenic viruses with mutant 3CLpros, we found that viruses encoding double-mutant 3CLpros are fully resistant to the inhibitor and exhibit a significant delay in proteolytic processing of the viral replicase polyprotein. The inhibitor-resistant viruses also exhibited postponed and reduced production of infectious virus particles. Biochemical analysis verified double-mutant 3CLpro enzyme as impaired for protease activity and exhibiting reduced sensitivity to the inhibitor and revealed a delayed kinetics of inhibitor hydrolysis and activity restoration. Furthermore, the inhibitor-resistant virus was shown to be highly attenuated in mice. Our study provides the first insight into the pathogenicity and mechanism of 3CLpro inhibitor-resistant CoV mutants, revealing a low genetic barrier but high fitness cost of resistance.

IMPORTANCE
RNA viruses are infamous for their ability to evolve in response to selective pressure, such as the presence of antiviral drugs. For coronaviruses such as the causative agent of Middle East respiratory syndrome (MERS), protease inhibitors have been developed and shown to block virus replication, but the consequences of selection of inhibitor-resistant mutants have not been studied. Here, we report the low genetic barrier and relatively high deleterious consequences of CoV resistance to a 3CLpro protease inhibitor in a coronavirus model system, mouse hepatitis virus (MHV). We found that although mutations that confer resistance arise quickly, the resistant viruses replicate slowly and do not cause lethal disease in mice. Overall, our study provides the first analysis of the low barrier but high cost of resistance to a CoV 3CLpro inhibitor, which will facilitate the further development of protease inhibitors as anti-coronavirus therapeutics.
Epstein-Barr Virus-Encoded Latent Membrane Protein 2A Promotes the Epithelial-Mesenchymal Transition in Nasopharyngeal Carcinoma via Metastatic Tumor Antigen 1 and Mechanistic Target of Rapamycin Signaling Induction
Authors: Lin Zhe , Wan Xin , Jiang Runqiu , Deng Lei , Gao Yun , Tang Junwei , Yang Yu , Zhao Wei , Yan Xin , Yao Kun , Sun Beicheng , Chen Yun 
Published: August 2014

ABSTRACT
Epstein-Barr virus-encoded latent membrane protein 2A (LMP2A) promotes the epithelial-mesenchymal transition (EMT) of nasopharyngeal carcinoma (NPC), thereby increasing tumor invasion. Recently, the dysregulation of metastatic tumor antigen 1 (MTA1) was found to enhance tumor metastasis in a variety of cancers. A molecular connection between these two proteins has been proposed but not firmly established. In this study, we reported the overexpression of MTA1 in 29/60 (48.3%) NPC patients, and the overexpression of MTA1 significantly correlated with tumor metastasis. The overexpression of MTA1 promoted EMT via the Wnt1 pathway and β-catenin activation. We demonstrated that LMP2A reinforces the expression of MTA1 via the mechanistic target of rapamycin (mTOR) pathway to promote EMT in NPC. Furthermore, by knocking down 4EBP1 in combination with the new mTOR inhibitor INK-128 treatment, we discovered that LMP2A expression activates the 4EBP1-eIF4E axis and increases the expression of MTA1 at the translational level partially independent of c-myc. These findings provided novel insights into the correlation between the LMP2A and MTA1 proteins and reveal a novel function of the 4EBP1-eIF4E axis in EMT of nasopharyngeal carcinoma.

IMPORTANCE
Prevention of the recurrence and metastasis of NPC is critical to achieving a successful NPC treatment. As we all know, EMT has a vital role in metastasis of malignancies. LMP2A, an oncoprotein of Epstein-Barr virus, a well-known NPC activator, induces EMT and has been proved to exert a promoting effect in tumor metastasis. Our study demonstrated that LMP2A could induce EMT by activating MTA1 at the translational level via activating mTOR signaling and the 4EBP1-eIF4E axis. Taken together, our findings bridge the gap between the NPC-specific cell surface molecule and the final phenotype of the NPC cells. Additionally, our findings indicate that LMP2A and mTOR will serve as targets for NPC therapy in the future.
An Eriophyid Mite-Transmitted Plant Virus Contains Eight Genomic RNA Segments with Unusual Heterogeneity in the Nucleocapsid Protein
Authors: Tatineni Satyanarayana , McMechan Anthony J. , Wosula Everlyne N. , Wegulo Stephen N. , Graybosch Robert A. , French Roy , Hein Gary L. 
Published: August 2014

ABSTRACT

Eriophyid mite-transmitted, multipartite, negative-sense RNA plant viruses with membrane-bound spherical virions are classified in the genus
Emaravirus
. We report here that the eriophyid mite-transmitted
Wheat mosaic virus
(WMoV), an
Emaravirus
, contains eight genomic RNA segments, the most in a known negative-sense RNA plant virus. Remarkably, two RNA 3 consensus sequences, encoding the nucleocapsid protein, were found with 12.5% sequence divergence, while no heterogeneity was observed in the consensus sequences of additional genomic RNA segments. The RNA-dependent RNA polymerase, glycoprotein precursor, nucleocapsid, and P4 proteins of WMoV exhibited limited sequence homology with the orthologous proteins of other emaraviruses, while proteins encoded by additional genomic RNA segments displayed no significant homology with proteins reported in GenBank, suggesting that the genus
Emaravirus
evolved further with a divergent octapartite genome. Phylogenetic analyses revealed that WMoV formed an evolutionary link between members of the
Emaravirus
genus and the family
Bunyaviridae
. Furthermore, genomic-length virus- and virus-complementary (vc)-sense strands of all WMoV genomic RNAs accumulated asymmetrically in infected wheat, with 10- to 20-fold more virus-sense genomic RNAs than vc-sense RNAs. These data further confirm the octapartite negative-sense polarity of the WMoV genome. In WMoV-infected wheat, subgenomic-length mRNAs of vc sense were detected for genomic RNAs 3, 4, 7, and 8 but not for other RNA species, suggesting that the open reading frames present in the complementary sense of genomic RNAs are expressed through subgenomic- or near-genomic-length vc-sense mRNAs.

IMPORTANCE
Wheat mosaic virus
(WMoV), an
Emaravirus
, is the causal agent of High Plains disease of wheat and maize. In this study, we demonstrated that the genome of WMoV comprises eight negative-sense RNA segments with an unusual sequence polymorphism in an RNA encoding the nucleocapsid protein but not in the additional genomic RNA segments. WMoV proteins displayed weak or no homology with reported emaraviruses, suggesting that the genus
Emaravirus
further evolved with a divergent octapartite genome. The current study also examined the profile of WMoV RNA accumulation in wheat and provided evidence for the synthesis of subgenomic-length mRNAs of virus complementary sense. This is the first report to demonstrate that emaraviruses produce subgenomic-length mRNAs that are most likely utilized for genome expression. Importantly, this study facilitates the examination of gene functions and virus diversity and the development of effective diagnostic methods and management strategies for an economically important but poorly understood virus.
Cellular Visualization of Macrophage Pyroptosis and Interleukin-1β Release in a Viral Hemorrhagic Infection in Zebrafish Larvae
Authors: Varela Mónica , Romero Alejandro , Dios Sonia , van der Vaart Michiel , Figueras Antonio , Meijer Annemarie H. , Novoa Beatriz 
Published: August 2014

ABSTRACTHemorrhagic viral diseases are distributed worldwide with important pathogens, such as dengue virus or hantaviruses. The lack of adequatein vivoinfection models has limited the research on viral pathogenesis and the current understanding of the underlying infection mechanisms. Although hemorrhages have been associated with the infection of endothelial cells, other cellular types could be the main targets for hemorrhagic viruses. Our objective was to take advantage of the use of zebrafish larvae in the study of viral hemorrhagic diseases, focusing on the interaction between viruses and host cells. Cellular processes, such as transendothelial migration of leukocytes, virus-induced pyroptosis of macrophages. and interleukin-1β (Il-1β) release, could be observed in individual cells, providing a deeper knowledge of the immune mechanisms implicated in the disease. Furthermore, the application of these techniques to other pathogens will improve the current knowledge of host-pathogen interactions and increase the potential for the discovery of new therapeutic targets.IMPORTANCEPathogenic mechanisms of hemorrhagic viruses are diverse, and most of the research regarding interactions between viruses and host cells has been performed in cell lines that might not be major targets during natural infections. Thus, viral pathogenesis research has been limited because of the lack of adequatein vivoinfection models. The understanding of the relative pathogenic roles of the viral agent and the host response to the infection is crucial. This will be facilitated by the establishment ofin vivoinfection models using organisms such as zebrafish, which allows the study of the diseases in the context of a complete individual. The use of this animal model with other pathogens could improve the current knowledge on host-pathogen interactions and increase the potential for the discovery of new therapeutic targets against diverse viral diseases.