bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of the Bruker MALDI Biotyper for Identification of Fastidious Gram-Negative Rods
Authors: Schulthess Bettina , Bloemberg Guido V. , Zbinden Andrea , Mouttet Forouhar , Zbinden Reinhard , Böttger Erik C. , Hombach Michael 
Published: December 2015

ABSTRACTMatrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS) has entered clinical laboratories, facilitating identification of bacteria. Here, we evaluated the MALDI Biotyper (Bruker Daltonics) for the identification of fastidious Gram-negative rods (GNR). Three sample preparation methods, direct colony transfer, direct transfer plus on-target formic acid preparation, and ethanol-formic acid extraction, were analyzed for 151 clinical isolates. Direct colony transfer applied with the manufacturer's interpretation criteria resulted in overall species and genus identification rates of 43.0% and 32.5%, respectively; 23.2% of the isolates were not identified, and two misidentifications (1.3%) were observed. The species identification rates increased to 46.4% and 53.7% for direct transfer plus formic acid preparation and ethanol-formic acid extraction, respectively. In addition, we evaluated score value cutoff alterations. The identification rates hardly increased by reducing the genus cutoff, while reducing the 2.0 species cutoff to 1.9 and to 1.8 increased the identification rates to up to 66.2% without increasing the rate of misidentifications. This study shows that fastidious GNR can reliably be identified using the MALDI Biotyper. However, the identification rates do not reach those of nonfastidious GNR such as theEnterobacteriaceae. In addition, two approaches optimizing the identification of fastidious GNR by the MALDI Biotyper were demonstrated: formic acid-based on-target sample treatment and reductions in cutoff scores to increase the species identification rates.
Laboratory Evaluation of a Dual-Path Platform Assay for Rapid Point-of-Care HIV and Syphilis Testing
Authors: Leon S. R. , Ramos L. B. , Vargas S. K. , Kojima N. , Perez D. G. , Caceres C. F. , Klausner J. D. 
Published: December 2015

ABSTRACT

We assessed the laboratory performance of the Chembio dual-path platform HIV-syphilis rapid immunodiagnostic test and electronic reader for detection of HIV and
Treponema pallidum
antibodies in 450 previously characterized serum specimens. For visual or electronic reader HIV antibody detection, the sensitivity was 100% and the specificity was 98.7%. For visual
T. pallidum
antibody detection, the test sensitivity was 94.7% and the specificity was 100.0%; with the electronic reader, the sensitivity was 94.7% and the specificity was 99.7%.
Crystal Structure of Marburg Virus VP40 Reveals a Broad, Basic Patch for Matrix Assembly and a Requirement of the N-Terminal Domain for Immunosuppression
Authors: Oda Shun-ichiro , Noda Takeshi , Wijesinghe Kaveesha J. , Halfmann Peter , Bornholdt Zachary A. , Abelson Dafna M. , Armbrust Tammy , Stahelin Robert V. , Kawaoka Yoshihiro , Saphire Erica Ollmann 
Published: December 2015

ABSTRACT
Marburg virus (MARV), a member of the filovirus family, causes severe hemorrhagic fever with up to 90% lethality. MARV matrix protein VP40 is essential for assembly and release of newly copied viruses and also suppresses immune signaling in the infected cell. Here we report the crystal structure of MARV VP40. We found that MARV VP40 forms a dimer in solution, mediated by N-terminal domains, and that formation of this dimer is essential for budding of virus-like particles. We also found the N-terminal domain to be necessary and sufficient for immune antagonism. The C-terminal domains of MARV VP40 are dispensable for immunosuppression but are required for virus assembly. The C-terminal domains are only 16% identical to those of Ebola virus, differ in structure from those of Ebola virus, and form a distinct broad and flat cationic surface that likely interacts with the cell membrane during virus assembly.

IMPORTANCE
Marburg virus, a cousin of Ebola virus, causes severe hemorrhagic fever, with up to 90% lethality seen in recent outbreaks. Molecular structures and visual images of the proteins of Marburg virus are essential for the development of antiviral drugs. One key protein in the Marburg virus life cycle is VP40, which both assembles the virus and suppresses the immune system. Here we provide the molecular structure of Marburg virus VP40, illustrate differences from VP40 of Ebola virus, and reveal surfaces by which Marburg VP40 assembles progeny and suppresses immune function.
Bovine Herpesvirus 4 Modulates Its β-1,6-
N
-Acetylglucosaminyltransferase Activity through Alternative Splicing
Authors: Lété Céline , Markine-Goriaynoff Nicolas , Machiels Bénédicte , Pang Poh-Choo , Xiao Xue , Canis Kevin , Suzuki Masami , Fukuda Minoru , Dell Anne , Haslam Stuart M. , Vanderplasschen Alain , Gillet Laurent 
Published: December 2015

ABSTRACT

Carbohydrates play major roles in host-virus interactions. It is therefore not surprising that, during coevolution with their hosts, viruses have developed sophisticated mechanisms to hijack for their profit different pathways of glycan synthesis. Thus, the Bo17 gene of Bovine herpesvirus 4 (BoHV-4) encodes a homologue of the cellular core 2 protein β-1,6-
N
-acetylglucosaminyltransferase-mucin type (C2GnT-M), which is a key player for the synthesis of complex
O
-glycans. Surprisingly, we show in this study that, as opposed to what is observed for the cellular enzyme, two different mRNAs are encoded by the Bo17 gene of all available BoHV-4 strains. While the first one corresponds to the entire coding sequence of the Bo17 gene, the second results from the splicing of a 138-bp intron encoding critical residues of the enzyme. Antibodies generated against the Bo17 C terminus showed that the two forms of Bo17 are expressed in BoHV-4 infected cells, but enzymatic assays revealed that the spliced form is not active. In order to reveal the function of these two forms, we then generated recombinant strains expressing only the long or the short form of Bo17. Although we did not highlight replication differences between these strains, glycomic analyses and lectin neutralization assays confirmed that the splicing of the Bo17 gene gives the potential to BoHV-4 to fine-tune the global level of core 2 branching activity in the infected cell. Altogether, these results suggest the existence of new mechanisms to regulate the activity of glycosyltransferases from the Golgi apparatus.

IMPORTANCE
Viruses are masters of adaptation that hijack cellular pathways to allow their growth. Glycans play a central role in many biological processes, and several studies have highlighted mechanisms by which viruses can affect glycosylation. Glycan synthesis is a nontemplate process regulated by the availability of key glycosyltransferases. Interestingly, bovine herpesvirus 4 encodes one such enzyme which is a key enzyme for the synthesis of complex
O
-glycans. In this study, we show that, in contrast to cellular homologues, this virus has evolved to alternatively express two proteins from this gene. While the first one is enzymatically active, the second results from the alternative splicing of the region encoding the catalytic site of the enzyme. We postulate that this regulatory mechanism could allow the virus to modulate the synthesis of some particular glycans for function at the location and/or the moment of infection.
Pan-genotypic Hepatitis C Virus Inhibition by Natural Products Derived from the Wild Egyptian Artichoke
Authors: Elsebai Mahmoud Fahmi , Koutsoudakis George , Saludes Verónica , Pérez-Vilaró Gemma , Turpeinen Ari , Mattila Sampo , Pirttilä Anna Maria , Fontaine-Vive Fabien , Mehiri Mohamed , Meyerhans Andreas , Diez Juana 
Published: December 2015

ABSTRACT

Hepatitis C virus (HCV) infection is the leading cause of chronic liver diseases. Water extracts of the leaves of the wild Egyptian artichoke (WEA) [
Cynara cardunculus
L. var.
sylvestris
(Lam.) Fiori] have been used for centuries in the Sinai Peninsula to treat hepatitis symptoms. Here we isolated and characterized six compounds from the water extracts of WEA and evaluated their HCV inhibition capacities
in vitro
. Importantly, two of these compounds, grosheimol and cynaropicrin, inhibited HCV with half-maximal effective concentrations (EC
50
s) in the low micromolar range. They inhibited HCV entry into target cells and were active against both cell-free infection as well as cell-cell transmission. Furthermore, the antiviral activity of both compounds was pan-genotypic as HCV genotypes 1a, 1b, 2b, 3a, 4a, 5a, 6a, and 7a were inhibited. Thus, grosheimol and cynaropicrin are promising candidates for the development of new pan-genotypic entry inhibitors of HCV infection.

IMPORTANCE
Because there is no preventive HCV vaccine available today, the discovery of novel anti-HCV cell entry inhibitors could help develop preventive measures against infection. The present study describes two compounds isolated from the wild Egyptian artichoke (WEA) with respect to their structural elucidation, absolute configuration, and quantitative determination. Importantly, both compounds inhibited HCV infection
in vitro
. The first compound was an unknown molecule, and it was designated “grosheimol,” while the second compound is the known molecule cynaropicrin. Both compounds belong to the group of sesquiterpene lactones. The mode of action of these compounds occurred during the early steps of the HCV life cycle, including cell-free and cell-cell infection inhibition. These natural compounds present promising candidates for further development into anti-HCV therapeutics.
Interaction of Human Parainfluenza Virus Type 3 Nucleoprotein with Matrix Protein Mediates Internal Viral Protein Assembly
Authors: Zhang Guangyuan , Zhong Yi , Qin Yali , Chen Mingzhou 
Published: December 2015

ABSTRACT

Human parainfluenza virus type 3 (HPIV3) belongs to the
Paramyxoviridae
family. Its three internal viral proteins, the nucleoprotein (N), the phosphoprotein (P), and the polymerase (L), form the ribonucleoprotein (RNP) complex, which encapsidates the viral genome and associates with the matrix protein (M) for virion assembly. We previously showed that the M protein expressed alone is sufficient to assemble and release virus-like particles (VLPs) and a mutant with the L305A point mutation in the M protein (M
L305A
) has a VLP formation ability similar to that of wild-type M protein. In addition, recombinant HPIV3 (rHPIV3) containing the M
L305A
mutation (rHPIV3-M
L305A
) could be successfully recovered. In the present study, we found that the titer of rHPIV3-M
L305A
was at least 10-fold lower than the titer of rHPIV3. Using VLP incorporation and coimmunoprecipitation assays, we found that VLPs expressing the M protein (M-VLPs) can efficiently incorporate N and P via an N-M or P-M interaction and M
L305A
-VLPs had an ability to incorporate P via a P-M interaction similar to that of M-VLPs but were unable to incorporate N and no longer interacted with N. Furthermore, we found that the incorporation of P into M
L305A
-VLPs but not M-VLPs was inhibited in the presence of N. In addition, we provide evidence that the C-terminal region of P is involved in its interaction with both N and M and N binding to the C-terminal region of P inhibits the incorporation of P into M
L305A
-VLPs. Our findings provide new molecular details to support the idea that the N-M interaction and not the P-M interaction is critical for packaging N and P into infectious viral particles.

IMPORTANCE
Human parainfluenza virus type 3 (HPIV3) is a nonsegmented, negative-sense, single-stranded RNA virus that belongs to the
Paramyxoviridae
family and can cause lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. However, no effective vaccine has been developed or licensed. We used virus-like particle (VLP) incorporation and coimmunoprecipitation assays to determine how the M protein assembles internal viral proteins. We demonstrate that both nucleoprotein (N) and phosphoprotein (P) can incorporate into M-VLPs and N inhibits the M-P interaction via the binding of N to the C terminus of P. We also provide additional evidence that the N-M interaction but not the P-M interaction is critical for the regulation of HPIV3 assembly. Our studies provide a more complete characterization of HPIV3 virion assembly and substantiation that N interaction with M regulates internal viral organization.
Transcriptional Regulation in Ebola Virus: Effects of Gene Border Structure and Regulatory Elements on Gene Expression and Polymerase Scanning Behavior
Authors: Brauburger Kristina , Boehmann Yannik , Krähling Verena , Mühlberger Elke 
Published: December 2015

ABSTRACT

The highly pathogenic Ebola virus (EBOV) has a nonsegmented negative-strand (NNS) RNA genome containing seven genes. The viral genes either are separated by intergenic regions (IRs) of variable length or overlap. The structure of the EBOV gene overlaps is conserved throughout all filovirus genomes and is distinct from that of the overlaps found in other NNS RNA viruses. Here, we analyzed how diverse gene borders and noncoding regions surrounding the gene borders influence transcript levels and govern polymerase behavior during viral transcription. Transcription of overlapping genes in EBOV bicistronic minigenomes followed the stop-start mechanism, similar to that followed by IR-containing gene borders. When the gene overlaps were extended, the EBOV polymerase was able to scan the template in an upstream direction. This polymerase feature seems to be generally conserved among NNS RNA virus polymerases. Analysis of IR-containing gene borders showed that the IR sequence plays only a minor role in transcription regulation. Changes in IR length were generally well tolerated, but specific IR lengths led to a strong decrease in downstream gene expression. Correlation analysis revealed that these effects were largely independent of the surrounding gene borders. Each EBOV gene contains exceptionally long untranslated regions (UTRs) flanking the open reading frame. Our data suggest that the UTRs adjacent to the gene borders are the main regulators of transcript levels. A highly complex interplay between the different
cis
-acting elements to modulate transcription was revealed for specific combinations of IRs and UTRs, emphasizing the importance of the noncoding regions in EBOV gene expression control.

IMPORTANCE
Our data extend those from previous analyses investigating the implication of noncoding regions at the EBOV gene borders for gene expression control. We show that EBOV transcription is regulated in a highly complex yet not easily predictable manner by a set of interacting
cis
-active elements. These findings are important not only for the design of recombinant filoviruses but also for the design of other replicon systems widely used as surrogate systems to study the filovirus replication cycle under low biosafety levels. Insights into the complex regulation of EBOV transcription conveyed by noncoding sequences will also help to interpret the importance of mutations that have been detected within these regions, including in isolates of the current outbreak.
Type I Interferon Response Limits Astrovirus Replication and Protects against Increased Barrier Permeability
In Vitro
and
In Vivo
Authors: Marvin Shauna A. , Huerta C. Theodore , Sharp Bridgett , Freiden Pamela , Cline Troy D. , Schultz-Cherry Stacey 
Published: December 2015

ABSTRACT

Little is known about intrinsic epithelial cell responses against astrovirus infection. Here we show that human astrovirus type 1 (HAstV-1) infection induces type I interferon (beta interferon [IFN-β]) production in differentiated Caco2 cells, which not only inhibits viral replication by blocking positive-strand viral RNA and capsid protein synthesis but also protects against HAstV-1-increased barrier permeability. Excitingly, we found similar results
in vivo
using a murine astrovirus (MuAstV) model, providing new evidence that virus-induced type I IFNs may protect against astrovirus replication and pathogenesis
in vivo
.

IMPORTANCE
Human astroviruses are a major cause of pediatric diarrhea, yet little is known about the immune response. Here we show that type I interferon limits astrovirus infection and preserves barrier permeability both
in vitro
and
in vivo
. Importantly, we characterized a new mouse model for studying astrovirus replication and pathogenesis.
Crystal Structure of Feline Infectious Peritonitis Virus Main Protease in Complex with Synergetic Dual Inhibitors
Authors: Wang Fenghua , Chen Cheng , Liu Xuemeng , Yang Kailin , Xu Xiaoling , Yang Haitao 
Published: December 2015

ABSTRACT

Coronaviruses (CoVs) can cause highly prevalent diseases in humans and animals. Feline infectious peritonitis virus (FIPV) belongs to the genus
Alphacoronavirus
, resulting in a lethal systemic granulomatous disease called feline infectious peritonitis (FIP), which is one of the most important fatal infectious diseases of cats worldwide. No specific vaccines or drugs have been approved to treat FIP. CoV main proteases (M
pro
s) play a pivotal role in viral transcription and replication, making them an ideal target for drug development. Here, we report the crystal structure of FIPV M
pro
in complex with dual inhibitors, a zinc ion and a Michael acceptor. The complex structure elaborates a unique mechanism of two distinct inhibitors synergizing to inactivate the protease, providing a structural basis to design novel antivirals and suggesting the potential to take advantage of zinc as an adjunct therapy against CoV-associated diseases.

IMPORTANCE
Coronaviruses (CoVs) have the largest genome size among all RNA viruses. CoV infection causes various diseases in humans and animals, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). No approved specific drugs or vaccinations are available to treat their infections. Here, we report a novel dual inhibition mechanism targeting CoV main protease (M
pro
) from feline infectious peritonitis virus (FIPV), which leads to lethal systemic granulomatous disease in cats. M
pro
, conserved across all CoV genomes, is essential for viral replication and transcription. We demonstrated that zinc ion and a Michael acceptor-based peptidomimetic inhibitor synergistically inactivate FIPV M
pro
. We also solved the structure of FIPV M
pro
complexed with two inhibitors, delineating the structural view of a dual inhibition mechanism. Our study provides new insight into the pharmaceutical strategy against CoV M
pro
through using zinc as an adjuvant therapy to enhance the efficacy of an irreversible peptidomimetic inhibitor.
Sensing of HIV-1 Infection in Tzm-bl Cells with Reconstituted Expression of STING
Authors: Trotard Maud , Tsopoulidis Nikolaos , Tibroni Nadine , Willemsen Joschka , Binder Marco , Ruggieri Alessia , Fackler Oliver T. 
Published: December 2015

ABSTRACT
Production of proinflammatory cytokines indicative of potent recognition by the host innate immune system has long been recognized as a hallmark of the acute phase of HIV-1 infection. The first components of the machinery by which primary HIV target cells sense infection have recently been described; however, the mechanistic dissection of innate immune recognition and viral evasion would be facilitated by an easily accessible cell line model. Here we describe that reconstituted expression of the innate signaling adaptor STING enhanced the ability of the well-established HIV reporter cell line Tzm-bl to sense HIV infection and to convert this information into nuclear translocation of IRF3 as well as expression of cytokine mRNA. STING-dependent immune sensing of HIV-1 required virus entry and reverse transcription but not genome integration. Particularly efficient recognition was observed for an HIV-1 variant lacking expression of the accessory protein Vpr, suggesting a role of the viral protein in circumventing STING-mediated immune signaling. Vpr as well as STING significantly impacted the magnitude and breadth of the cytokine mRNA expression profile induced upon HIV-1 infection. However, cytoplasmic DNA sensing did not result in detectable cytokine secretion in this cell system, and innate immune recognition did not affect infection rates. Despite these deficits in eliciting antiviral effector functions, these results establish Tzm-bl STING and Tzm-bl STING IRF3.GFP cells as useful tools for studies aimed at dissecting mechanisms and regulation of early innate immune recognition of HIV infection.

IMPORTANCE
Cell-autonomous immune recognition of HIV infection was recently established as an important aspect by which the host immune system attempts to fend off HIV-1 infection. Mechanistic studies on host cell recognition and viral evasion are hampered by the resistance of many primary HIV target cells to detailed experimental manipulation. We describe here that expression of the signaling adaptor STING renders the well-established HIV reporter cell line Tzm-bl competent for innate recognition of HIV infection. Key characteristics reflected in this cell model include nuclear translocation of IRF3, expression of a broad range of cytokine mRNAs, and an antagonistic activity of the HIV-1 protein Vpr. These results establish Tzm-bl STING and Tzm-bl STING IRF3.GFP cells as a useful tool for studies of innate recognition of HIV infection.