bluuebunny/crossref_metadata_2025_split huggingface dataset:
Standardization of Nucleic Acid Tests for Clinical Measurements of Bacteria and Viruses
Authors: Pavšič Jernej , Devonshire Alison S. , Parkes Helen , Schimmel Heinz , Foy Carole A. , Karczmarczyk Maria , Gutiérrez-Aguirre Ion , Honeyborne Isobella , Huggett Jim F. , McHugh Timothy D. , Milavec Mojca , Zeichhardt Heinz , Žel Jana 
Published: November 2014

ABSTRACT

Nucleic acid-based tests for infectious diseases currently used in the clinical laboratory and in point-of-care devices are diverse. Measurement challenges associated with standardization of quantitative viral load testing are discussed in relation to human cytomegalovirus, BK virus, and Epstein-Barr virus, while the importance of defining the performance of qualitative methods is illustrated with
Mycobacterium tuberculosis
and influenza virus. The development of certified reference materials whose values are traceable to higher-order standards and reference measurement procedures, using, for instance, digital PCR, will further contribute to the understanding of analytical performance characteristics and promote clinical data comparability.
Evaluation of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Vibrio cholerae
Authors: Rychert Jenna , Creely David , Mayo-Smith Leslie M. , Calderwood Stephen B. , Ivers Louise C. , Ryan Edward T. , Boncy Jacques , Qadri Firdausi , Ahmed Dilruba , Ferraro Mary Jane , Harris Jason B. 
Published: November 2014

ABSTRACTWe evaluated the use of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MS) for the identification ofVibrio cholerae. MS identified all 42 isolates ofV. choleraeO1 and O139 and 7 of 9 non-O1/O139 isolates. MS correctly discriminated between allAeromonasandV. choleraeisolates. Overall, MS performed as well as or better than biochemical methods.
Evaluation of an Online Program To Teach Microbiology to Internal Medicine Residents
Authors: Guarner Jeannette , Burd Eileen M. , Kraft Colleen S. , Armstrong Wendy S. , Lenorr Kenya , Spicer Jennifer O. , Martin Donna , del Rio Carlos 
Published: November 2014

ABSTRACT

Microbiology rounds are an integral part of infectious disease consultation service. During microbiology rounds, we highlight microbiology principles using vignettes. We created case-based, interactive, microbiology online modules similar to the vignettes presented during microbiology rounds. Since internal medicine residents rotating on our infectious disease elective have limited time to participate in rounds and learn microbiology, our objective was to evaluate the use of the microbiology online modules by internal medicine residents. We asked residents to complete 10 of 25 online modules during their infectious disease elective. We evaluated which modules they chose and the change in their knowledge level. Forty-six internal medicine residents completed assessments given before and after accessing the modules with an average of 11/20 (range, 6 to 19) and 16/20 (range, 9 to 20) correct questions, respectively (average improvement, 5 questions;
P
= 0.0001). The modules accessed by more than 30 residents included those related to
Clostridium difficile
, anaerobes,
Candida
spp.,
Streptococcus pneumoniae
, influenza,
Mycobacterium tuberculosis
, and
Neisseria meningitidis
. We demonstrated improved microbiology knowledge after completion of the online modules. This improvement may not be solely attributed to completing the online modules, as fellows and faculty may have provided additional microbiology education during the rotation.
The Nature of the N-Terminal Amino Acid Residue of HIV-1 RNase H Is Critical for the Stability of Reverse Transcriptase in Viral Particles
Authors: Boso Guney , Örvell Claes , Somia Nikunj V. 
Published: November 2014

ABSTRACT

Reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is synthesized and packaged into the virion as a part of the GagPol polyprotein. Mature RT is released by the action of viral protease. However, unlike other viral proteins, RT is subject to an internal cleavage event leading to the formation of two subunits in the virion: a p66 subunit and a p51 subunit that lacks the RNase H domain. We have previously identified RNase H to be an HIV-1 protein that has the potential to be a substrate for the N-end rule pathway, which is an ubiquitin-dependent proteolytic system in which the identity of the N-terminal amino acid determines the half-life of a protein. Here we examined the importance of the N-terminal amino acid residue of RNase H in the early life cycle of HIV-1. We show that changing this residue to an amino acid structurally different from the conserved residue leads to the degradation of RT and, in some cases, integrase in the virus particle and this abolishes infectivity. Using intravirion complementation and
in vitro
protease cleavage assays, we show that degradation of RT in RNase H N-terminal mutants occurs in the absence of active viral protease in the virion. Our results also indicate the importance of the RNase H N-terminal residue in the dimerization of RT subunits.

IMPORTANCE
HIV-1 proteins are initially made as part of a polyprotein that is cleaved by the viral protease into the proteins that form the virus particle. We were interested in one particular protein, RNase H, that is cleaved from reverse transcriptase. In particular, we found that the first amino acid of RNase H never varied in over 1,850 isolates of HIV-1 that we compared. When we changed the first amino acid, we found that the reverse transcriptase in the virus was degraded. While other studies have implied that the viral protease can degrade mutant RT proteins, we show here that this may not be the case for our mutants. Our results suggest that the presence of active viral protease is not required for the degradation of RT in RNase H N-terminal mutants, suggesting a role for a cellular protease in this process.
The Murine Norovirus Core Subgenomic RNA Promoter Consists of a Stable Stem-Loop That Can Direct Accurate Initiation of RNA Synthesis
Authors: Yunus Muhammad Amir , Lin Xiaoyan , Bailey Dalan , Karakasiliotis Ioannis , Chaudhry Yasmin , Vashist Surender , Zhang Guo , Thorne Lucy , Kao C. Cheng , Goodfellow Ian 
Published: November 2014

ABSTRACT

All members of the
Caliciviridae
family of viruses produce a subgenomic RNA during infection. The subgenomic RNA typically encodes only the major and minor capsid proteins, but in murine norovirus (MNV), the subgenomic RNA also encodes the VF1 protein, which functions to suppress host innate immune responses. To date, the mechanism of norovirus subgenomic RNA synthesis has not been characterized. We have previously described the presence of an evolutionarily conserved RNA stem-loop structure on the negative-sense RNA, the complementary sequence of which codes for the viral RNA-dependent RNA polymerase (NS7). The conserved stem-loop is positioned 6 nucleotides 3′ of the start site of the subgenomic RNA in all caliciviruses. We demonstrate that the conserved stem-loop is essential for MNV viability. Mutant MNV RNAs with substitutions in the stem-loop replicated poorly until they accumulated mutations that revert to restore the stem-loop sequence and/or structure. The stem-loop sequence functions in a noncoding context, as it was possible to restore the replication of an MNV mutant by introducing an additional copy of the stem-loop between the NS7- and VP1-coding regions. Finally,
in vitro
biochemical data suggest that the stem-loop sequence is sufficient for the initiation of viral RNA synthesis by the recombinant MNV RNA-dependent RNA polymerase, confirming that the stem-loop forms the core of the norovirus subgenomic promoter.

IMPORTANCE
Noroviruses are a significant cause of viral gastroenteritis, and it is important to understand the mechanism of norovirus RNA synthesis. Here we describe the identification of an RNA stem-loop structure that functions as the core of the norovirus subgenomic RNA promoter in cells and
in vitro
. This work provides new insights into the molecular mechanisms of norovirus RNA synthesis and the sequences that determine the recognition of viral RNA by the RNA-dependent RNA polymerase.
Tulane Virus Recognizes the A Type 3 and B Histo-Blood Group Antigens
Authors: Zhang Dongsheng , Huang Pengwei , Zou Lu , Lowary Todd L. , Tan Ming , Jiang Xi 
Published: November 2014

ABSTRACT

Tulane virus (TV), the prototype of the
Recovirus
genus in the calicivirus family, was isolated from the stools of rhesus monkeys and can be cultivated
in vitro
in monkey kidney cells. TV is genetically closely related to the genus
Norovirus
and recognizes the histo-blood group antigens (HBGAs), similarly to human noroviruses (NoVs), making it a valuable surrogate for human NoVs. However, the precise structures of HBGAs recognized by TV remain elusive. In this study, we performed binding and blocking experiments on TV with extended HBGA types and showed that, while TV binds all four types (types 1 to 4) of the B antigens, it recognizes only the A type 3 antigen among four types of A antigens tested. The requirements for HBGAs in TV replication were demonstrated by blocking of TV replication in cell culture using the A type 3/4 and B saliva samples. Similar results were also observed in oligosaccharide-based blocking assays. Importantly, the previously reported, unexplained increase in TV replication by oligosaccharide in cell-based blocking assays has been clarified, which will facilitate the application of TV as a surrogate for human NoVs.

IMPORTANCE
Our understanding of the role of HBGAs in NoV infection has been significantly advanced in the past decade, but direct evidence for HBGAs as receptors for human NoVs remains lacking due to a lack of a cell culture method. TV recognizes HBGAs and can replicate
in vitro
, providing a valuable surrogate for human NoVs. However, TV binds to some but not all saliva samples from A-positive individuals, and an unexplained observation of synthetic oligosaccharide blocking of TV binding has been reported. These issues have been resolved in this study.
Measles Virus Glycoprotein Complexes Preassemble Intracellularly and Relax during Transport to the Cell Surface in Preparation for Fusion
Authors: Brindley Melinda A. , Chaudhury Sukanya , Plemper Richard K. 
Published: November 2014

ABSTRACT
Measles virus (MeV), a morbillivirus within the paramyxovirus family, expresses two envelope glycoproteins. The attachment (H) protein mediates receptor binding, followed by triggering of the fusion (F) protein, which leads to merger of the viral envelope with target cell membranes. Receptor binding by members of related paramyxovirus genera rearranges the head domains of the attachment proteins, liberating an F-contact domain within the attachment protein helical stalk. However, morbillivirus glycoproteins first assemble intracellularly prior to receptor binding, raising the question of whether alternative protein-protein interfaces are involved or whether an entirely distinct triggering principle is employed. To test these possibilities, we generated headless H stem mutants of progressively shorter length. Conformationally restricted H stems remained capable of intracellular assembly with a standard F protein and a soluble MeV F mutant. Proteolytic maturation of F, but not the altered biochemical conditions at the cell surface, reduces the strength of glycoprotein interaction, readying the complexes for triggering. F mutants stabilized in the prefusion conformation interact with H intracellularly and at the cell surface, while destabilized F mutants interact only intracellularly, prior to F maturation. These results showcase an MeV entry machinery that functionally varies conserved motifs of the proposed paramyxovirus infection pathway. Intracellular and plasma membrane-resident MeV glycoprotein complexes employ the same protein-protein interface. F maturation prepares for complex separation after triggering, and the H head domains in prereceptor-bound conformation prevent premature stalk rearrangements and F activation. Intracellular preassembly affects MeV fusion profiles and may contribute to the high cell-to-cell fusion activity characteristic of the morbillivirus genus.

IMPORTANCE
Paramyxoviruses of the morbillivirus genus, such as measles, are highly contagious, major human and animal pathogens. MeV envelope glycoproteins preassemble intracellularly into tightly associated hetero-oligomers. To address whether preassembly reflects a unique measles virus entry strategy, we characterized the protein-protein interface of intracellular and surface-exposed fusion complexes and investigated the effect of the attachment protein head domains, glycoprotein maturation, and altered biochemical conditions at the cell surface on measles virus fusion complexes. Our results demonstrate that measles virus functionally varies conserved elements of the paramyxovirus entry pathway, providing a possible explanation for the high cell-to-cell fusion activity of morbilliviruses. Insight gained from these data affects the design of effective broad-spectrum paramyxovirus entry inhibitors.
Structure-Based Functional Analyses of Domains II and III of Pseudorabies Virus Glycoprotein H
Authors: Böhm Sebastian W. , Eckroth Elisa , Backovic Marija , Klupp Barbara G. , Rey Felix A. , Mettenleiter Thomas C. , Fuchs Walter 
Published: November 2014

ABSTRACT

Enveloped viruses utilize membrane fusion for entry into, and release from, host cells. For entry, members of the
Herpesviridae
require at least three envelope glycoproteins: the homotrimeric gB and a heterodimer of gH and gL. The crystal structures of three gH homologues, including pseudorabies virus (PrV) gH, revealed four conserved domains. Domain II contains a planar β-sheet (“fence”) and a syntaxin-like bundle of three α-helices (SLB), similar to those found in eukaryotic fusion proteins, potentially executing an important role in gH function. To test this hypothesis, we introduced targeted mutations into the PrV gH gene, which either disrupt the helices of the SLB by introduction of proline residues or covalently join them by artificial intramolecular disulfide bonds between themselves, to the adjacent fence region, or to domain III. Disruption of either of the three α-helices of the SLB (A250P, V275P, V298P) severely affected gH function in
in vitro
fusion assays and replication of corresponding PrV mutants. Considerable defects in fusion activity of gH, as well as in penetration kinetics and cell-to-cell spread of PrV mutants, were also observed after disulfide linkage of two α-helices within the SLB (A284C-S291C) or between SLB and domain III (H251C-L432C), as well as by insertions of additional cysteine pairs linking fence, SLB, and domain III.
In vitro
fusion activity of mutated gH could be partly restored by reduction of the artificial disulfide bonds. Our results indicate that the structure and flexibility of the SLB are relevant for the function of PrV gH in membrane fusion.

IMPORTANCE
Mutational analysis based on crystal structures of proteins is a powerful tool to understand protein function. Here, we continued our study of pseudorabies virus gH, a part of the core fusion machinery of herpesviruses. We previously showed that the “flap” region in domain IV of PrV gH is important for its function. We now demonstrate that mutations within domain II that interfere with integrity or flexibility of a syntaxin-like three-helix bundle also significantly impair gH function during fusion. These studies provide important insights into the structural requirements of gH for function in fusion.
Ebola Virus Transmission in Guinea Pigs
Authors: Wong Gary , Qiu Xiangguo , Richardson Jason S. , Cutts Todd , Collignon Brad , Gren Jason , Aviles Jenna , Embury-Hyatt Carissa , Kobinger Gary P. 
Published: November 2014

ABSTRACT

Ebola virus (EBOV) transmission is currently poorly characterized and is thought to occur primarily by direct contact with infectious material; however transmission from swine to nonhuman primates via the respiratory tract has been documented. To establish an EBOV transmission model for performing studies with statistical significance, groups of six guinea pigs (gps) were challenged intranasally (i.n.) or intraperitoneally (i.p.) with 10,000 times the 50% lethal dose (LD
50
) of gp-adapted EBOV, and naive gps were then introduced as cage mates for contact exposure at 1 day postinfection (p.i.). The animals were monitored for survival and clinical signs of disease and quantitated for virus shedding postexposure. Changes in the duration of contact of naive gps with infected animals were evaluated for their impact on transmission efficiency. Transmission was more efficient from i.n.- than from i.p.-challenged gps, with 17% versus 83% of naive gps surviving exposure, respectively. Virus shedding was detected beginning at 3 days p.i. from both i.n.- and i.p.-challenged animals. Contact duration positively correlated with transmission efficiency, and the abrogation of direct contact between infected and naive animals through the erection of a steel mesh was effective at stopping virus spread, provided that infectious animal bedding was absent from the cages. Histopathological and immunohistochemical findings show that i.n.-infected gps display enhanced lung pathology and EBOV antigen in the trachea, which supports increased virus transmission from these animals. The results suggest that i.n.-challenged gps are more infectious to naive animals than their systemically infected counterparts and that transmission occurs through direct contact with infectious materials, including those transported through air movement over short distances.

IMPORTANCE
Ebola is generally thought to be spread between humans though infectious bodily fluids. However, a study has shown that Ebola can be spread from pigs to monkeys without direct contact. Further studies have been hampered, because an economical animal model for Ebola transmission is not available. To address this, we established a transmission model in guinea pigs and determined the mechanisms behind virus spread. The survival data, in addition to microscopic examination of lung and trachea sections, show that mucosal infection of guinea pigs is an efficient model for Ebola transmission. Virus spread is increased with longer contact times with an infected animal and is possible without direct contact between an infected and a naive host but can be stopped if infectious materials are absent. These results warrant consideration for the development of future strategies against Ebola transmission and for a better understanding of the parameters involved in virus spread.
A Cryo-Electron Microscopy Study Identifies the Complete H16.V5 Epitope and Reveals Global Conformational Changes Initiated by Binding of the Neutralizing Antibody Fragment
Authors: Lee Hyunwook , Brendle Sarah A. , Bywaters Stephanie M. , Guan Jian , Ashley Robert E. , Yoder Joshua D. , Makhov Alexander M. , Conway James F. , Christensen Neil D. , Hafenstein Susan 
Published: November 2014

ABSTRACT
Human papillomavirus 16 (HPV16) is a worldwide health threat and an etiologic agent of cervical cancer. To understand the antigenic properties of HPV16, we pursued a structural study to elucidate HPV capsids and antibody interactions. The cryo-electron microscopy (cryo-EM) structures of a mature HPV16 particle and an altered capsid particle were solved individually and as complexes with fragment of antibody (Fab) from the neutralizing antibody H16.V5. Fitted crystal structures provided a pseudoatomic model of the virus-Fab complex, which identified a precise footprint of H16.V5, including previously unrecognized residues. The altered-capsid–Fab complex map showed that binding of the Fab induced significant conformational changes that were not seen in the altered-capsid structure alone. These changes included more ordered surface loops, consolidated so-called “invading-arm” structures, and tighter intercapsomeric connections at the capsid floor. The H16.V5 Fab preferentially bound hexavalent capsomers likely with a stabilizing effect that directly correlated with the number of bound Fabs. Additional cryo-EM reconstructions of the virus-Fab complex for different incubation times and structural analysis provide a model for a hyperstabilization of the capsomer by H16.V5 Fab and showed that the Fab distinguishes subtle differences between antigenic sites.

IMPORTANCE
Our analysis of the cryo-EM reconstructions of the HPV16 capsids and virus-Fab complexes has identified the entire HPV.V5 conformational epitope and demonstrated a detailed neutralization mechanism of this clinically important monoclonal antibody against HPV16. The Fab bound and ordered the apical loops of HPV16. This conformational change was transmitted to the lower region of the capsomer, resulting in enhanced intercapsomeric interactions evidenced by the more ordered capsid floor and “invading-arm” structures. This study advances the understanding of the neutralization mechanism used by H16.V5.
Three Novel Virophage Genomes Discovered from Yellowstone Lake Metagenomes
Authors: Zhou Jinglie , Sun Dawei , Childers Alyson , McDermott Timothy R. , Wang Yongjie , Liles Mark R. 
Published: November 2014

ABSTRACT
Virophages are a unique group of circular double-stranded DNA viruses that are considered parasites of giant DNA viruses, which in turn are known to infect eukaryotic hosts. In this study, the genomes of three novel Yellowstone Lake virophages (YSLVs)—YSLV5, YSLV6, and YSLV7—were identified from Yellowstone Lake through metagenomic analyses. The relative abundance of these three novel virophages and previously identified Yellowstone Lake virophages YSLV1 to -4 were determined in different locations of the lake, revealing that most of the sampled locations in the lake, including both mesophilic and thermophilic habitats, had multiple virophage genotypes. This likely reflects the diverse habitats or diversity of the eukaryotic hosts and their associated giant viruses that serve as putative hosts for these virophages. YSLV5 has a 29,767-bp genome with 32 predicted open reading frames (ORFs), YSLV6 has a 24,837-bp genome with 29 predicted ORFs, and YSLV7 has a 23,193-bp genome with 26 predicted ORFs. Based on multilocus phylogenetic analysis, YSLV6 shows a close evolutionary relationship with YSLV1 to -4, whereas YSLV5 and YSLV7 are distantly related to the others, and YSLV7 represents the fourth novel virophage lineage. In addition, the genome of YSLV5 has a G+C content of 51.1% that is much higher than all other known virophages, indicating a unique host range for YSLV5. These results suggest that virophages are abundant and have diverse genotypes that likely mirror diverse giant viral and eukaryotic hosts within the Yellowstone Lake ecosystem.

IMPORTANCE
This study discovered novel virophages present within the Yellowstone Lake ecosystem using a conserved major capsid protein as a phylogenetic anchor for assembly of sequence reads from Yellowstone Lake metagenomic samples. The three novel virophage genomes (YSLV5 to -7) were completed by identifying specific environmental samples containing these respective virophages, and closing gaps by targeted PCR and sequencing. Most of the YSLV genotypes were associated primarily with photic-zone and nonhydrothermal samples; however, YSLV5 had a unique distribution with an occurrence in vent samples similar to that in photic-zone samples and with a higher GC content that suggests a distinct host and habitat compared to other YSLVs. In addition, genome content and phylogenetic analyses indicate that YSLV5 and YSLV7 are distinct from known virophages and that additional as-yet-uncharacterized virophages are likely present within the Yellowstone Lake ecosystem.