bluuebunny/crossref_metadata_2025 huggingface dataset:
Longitudinal Analysis of the Evolution and Dissemination of Neisseria gonorrhoeae Strains (Saskatchewan, Canada, 2005 to 2008) Reveals Three Major Circulating Strains and Convergent Evolution of Ciprofloxacin and Azithromycin Resistance
Authors: Vidovic Sinisa , Thakur Sidharath D. , Horsman Greg B. , Levett Paul N. , Anvari Vahid , Dillon Jo-Anne R.
Published: September 2012

A longitudinal study combining multilocus sequence typing with molecular evolutionary analysis determined the distribution, population structure, and evolution of antibiotic resistance inNeisseria gonorrhoeaeisolates in Saskatchewan that were collected between 2005 and 2008. Of 195 gonococcal isolates examined, 29 sequence types (STs) were identified with 3 major circulating strains (ST-1 through ST-3) comprising 52% of all gonococcal isolates studied. The prevalences, persistence, distribution patterns, and clonalities of these isolates strongly suggest that gonorrhea endemicity within this broad geographic region was driven by these 3 circulating strains. ST-1 exhibited a significantly (P= 0.001) higher prevalence throughout the study than did the others, accounting for ∼25% of the tested isolates each year. The spatial distributions of the gonococcal strains indicated that ST-1 in 2007 entered a linear component of the sexual network, reaching the remote north and resulting in the further spread and maintenance of infection. Ciprofloxacin and azithromycin resistances were observed in distantly related gonococcal lineages, clearly indicating the convergent acquisition of these antibiotic-resistant phenotypes. In addition, all ciprofloxacin- and azithromycin-resistant lineages were found at the edges of the minimum spanning tree, far from the major lineages, suggesting that these antibiotic phenotypes were most likely introduced into the province. In contrast, resistance to penicillin was found mostly in the endemic gonococcal lineages, suggesting that penicillin resistance was probably acquired in Saskatchewan as a result of spontaneous mutations in already-established lineages. Tetracycline resistance was present in all STs except one, indicating its ubiquitous nature in the gonococcal population studied.
Molecular Epidemiology of Outbreak-Associated and Wild-Waterfowl-Derived Newcastle Disease Virus Strains in Finland, Including a Novel Class I Genotype
Authors: Lindh Erika , Ek-Kommonen Christine , Väänänen Veli-Matti , Alasaari Jukka , Vaheri Antti , Vapalahti Olli , Huovilainen Anita
Published: September 2012

Newcastle disease (ND) is a highly contagious, severe disease of poultry caused by pathogenic strains of Newcastle disease virus (NDV; or avian paramyxovirus-1). NDV is endemic in wild birds worldwide and one of the economically most important poultry pathogens. Most of the published strains are outbreak-associated strains, while the apathogenic NDV strains that occur in wild birds, posing a constant threat to poultry with their capability to convert into more virulent forms, have remained less studied. We screened for NDV RNA in cloacal and oropharyngeal samples from wild waterfowl in Finland during the years 2006 to 2010: 39 of 715 birds were positive (prevalence, 5.5%). The partial or full-length F genes of 37 strains were sequenced for phylogenetic purposes. We also characterized viruses derived from three NDV outbreaks in Finland and discuss the relationships between these outbreak-associated and the wild-bird-associated strains. We found that all waterfowl NDV isolates were lentogenic strains of class I or class II genotype I. We also isolated a genetically distinct class I strain (teal/Finland/13111/2008) grouping phylogenetically together with only strain HIECK87191, isolated in Northern Ireland in 1987. Together they seem to form a novel class I genotype genetically differing from other known NDVs by at least 12%.
Rapid Detection of Carbapenemase-Producing Pseudomonas spp
Authors: Dortet Laurent , Poirel Laurent , Nordmann Patrice
Published: September 2012

A novel biochemical technique, the Carba NP test, has been evaluated to detect carbapenemase production in
Pseudomonas
spp. This test was specific (100%), sensitive (94.4%), and rapid (<2 h). This cost-effective test, which could be implemented in any microbiology laboratory, offers a reliable technique for identification of carbapenemase-producing
Pseudomonas
spp.
Antimicrobial Treatment and Containment Measures for an Extremely Drug-Resistant Klebsiella pneumoniae ST101 Isolate Carrying pKPN101-IT, a Novel Fully Sequenced
bla
KPC-2
Plasmid
Authors: Frasson Ilaria , Lavezzo Enrico , Franchin Elisa , Toppo Stefano , Barzon Luisa , Cavallaro Antonietta , Richter Sara N. , Palù Giorgio
Published: September 2012

An extremely drug-resistant
Klebsiella pneumoniae
isolate, sequence type ST101, was isolated from a patient in Italy. We describe antibiotic treatment, measures to clear and contain the infection, and a complete sequence analysis of a novel large plasmid, pKPN101-IT, harboring the
bla
KPC-2
gene and arising from the threatening recombination of different worldwide-distributed backbones.
Next-Generation Ion Torrent Sequencing of Drug Resistance Mutations in Mycobacterium tuberculosis Strains
Authors: Daum Luke T. , Rodriguez John D. , Worthy Sue A. , Ismail Nazir A. , Omar Shaheed V. , Dreyer Andries W. , Fourie P. Bernard , Hoosen Anwar A. , Chambers James P. , Fischer Gerald W.
Published: September 2012

A novel protocol for full-length
Mycobacterium tuberculosis
gene analysis of first- and second-line drug resistance was developed using the Ion Torrent Personal Genome Machine (PGM). Five genes—
rpoB
(rifampin),
katG
(isoniazid),
pncA
(pyrazinamide),
gyrA
(ofloxacin/fluoroquinolone), and
rrs
(aminoglycosides)—were amplified and sequenced, and results were compared to those obtained by genotypic Hain line probe assay (LPA) and phenotypic Bactec MGIT 960 analysis using 26 geographically diverse South African clinical isolates collected between July and November 2011. Ion Torrent sequencing exhibited 100% (26/26) concordance to phenotypic resistance obtained by MGIT 960 culture and genotypic
rpoB
and
katG
results by LPA. In several rifampin-resistant isolates, Ion Torrent sequencing revealed uncommon substitutions (H526R and D516G) that did not have a defined mutation by LPA. Importantly, previously uncharacterized mutations in
rpoB
(V194I),
rrs
(G878A), and
pncA
(Q122Stop) genes were observed. Ion Torrent sequencing may facilitate tracking and monitoring geographically diverse multidrug-resistant and extensively drug-resistant strains and could potentially be integrated into selected regional and reference settings throughout Africa, India, and China.
Characterization of Methicillin-Resistant Staphylococcus aureus Strains Recovered from a Phase IV Clinical Trial for Linezolid versus Vancomycin for Treatment of Nosocomial Pneumonia
Authors: Mendes Rodrigo E. , Deshpande Lalitagauri M. , Smyth Davida S. , Shopsin Bo , Farrell David J. , Jones Ronald N.
Published: September 2012

A total of 434 methicillin-resistant
Staphylococcus aureus
(MRSA) baseline isolates were collected from subjects enrolled in a prospective, double-blind randomized trial comparing linezolid versus vancomycin for the treatment of nosocomial pneumonia. Isolates were susceptibility tested by broth microdilution, examined for inducible clindamycin resistance by D-test, and screened for heterogeneous resistance to vancomycin (hVISA) by the Etest macromethod. All strains were subjected to Panton-Valentine leukocidin (PVL) screening, and SCC
mec
, pulsed-field gel electrophoresis (PFGE), and
spa
typing. Selected strains were evaluated by multilocus sequence typing (MLST). Clonal complexes (CCs) were assigned based on the
spa
and/or MLST results. Most strains were CC5 (56.0%), which originated from North America (United States) (CC5-MRSA-SCC
mec
II/IV; 70.0%), Asia (CC5-MRSA-II; 14.0%) and Latin America (CC5-MRSA-I/II; 12.3%). The second- and third-most-prevalent clones were CC8-MRSA-IV (23.3%) and CC239-MRSA-III (11.3%), respectively. Furthermore, the CC5-MRSA-I/II clone predominated in Asia (50.7% within this region) and Latin America (66.7%), followed by CC239-MRSA-III (32.8% and 28.9%, respectively). The European strains were CC8-MRSA-IV (34.5%), CC22-MRSA-IV (18.2%), or CC5-MRSA-I/II/IV (16.4%), while the U.S. MRSA isolates were CC5-MRSA-II/IV (64.4%) or CC8-MRSA-IV (28.8%). Among the U.S. CC8-MRSA-II/IV strains, 73.7% (56/76 [21.2% of all U.S. MRSA strains]) clustered within USA300. One strain from the United States (USA800) was intermediate to vancomycin (MIC, 4 μg/ml). All remaining strains were susceptible to linezolid, daptomycin, vancomycin, and teicoplanin. hVISA strains (14.5%) were predominantly CC5-MRSA-II, from South Korea, and belonged to a single PFGE type. Overall, each region had two predominant clones. The USA300 rate corroborates previous reports describing increased prevalence of USA300 strains causing invasive infections. The prevalence of hVISA was elevated in Asia, and these strains were associated with CC5.
Isolation of Kerstersia gyiorum from a Patient with Cholesteatomatous Chronic Otitis Media
Authors: Almuzara Marisa N. , Barberis Claudia M. , Traglia German M. , Ordoñez Andrea Martinez , Famiglietti Angela M. R. , Ramirez Maria S. , Vay Carlos A.
Published: September 2012

We describe the first case of aKerstersia gyiorumstrain isolated from a patient with cholesteatomatous chronic otitis media. We emphasize the isolation of members of the familyAlcaligenaceaein serious infections and unusual sites and the importance of polyphasic identification addressing the definitive identification.
Novel Primer-Probe Sets for Detection and Identification of Mycobacteria by PCR-Microarray Assay
Authors: Lazzeri Elisa , Santoro Francesco , Oggioni Marco R. , Iannelli Francesco , Pozzi Gianni
Published: September 2012

A PCR-microarray assay was developed in which PCR primers and hybridization probes were designed for the 16S rRNA genes of 16 clinically relevant mycobacteria and for IS
6110
of
Mycobacterium tuberculosis
. The assay, based on a multiplex PCR followed by hybridization with oligonucleotide probes, was tested against 16
Mycobacterium
species and 70 clinical samples.
Dynamics of Memory B-Cell Populations in Blood, Lymph Nodes, and Bone Marrow during Antiretroviral Therapy and Envelope Boosting in Simian Immunodeficiency Virus SIVmac251-Infected Rhesus Macaques
Authors: Demberg Thorsten , Brocca-Cofano Egidio , Xiao Peng , Venzon David , Vargas-Inchaustegui Diego , Lee Eun Mi , Kalisz Irene , Kalyanaraman V. S. , DiPasquale Janet , McKinnon Katherine , Robert-Guroff Marjorie
Published: September 2012

Human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection causes B-cell dysregulation and the loss of memory B cells in peripheral blood mononuclear cells (PBMC). These effects are not completely reversed by antiretroviral treatment (ART). To further elucidate B-cell changes during chronic SIV infection and treatment, we investigated memory B-cell subpopulations and plasma cells/plasmablasts (PC/PB) in blood, bone marrow, and lymph nodes of rhesus macaques during ART and upon release from ART. Macaques previously immunized with SIV recombinants and the gp120 protein were included to assess the effects of prior vaccination. ART was administered for 11 weeks, with or without gp120 boosting at week 9. Naïve and resting, activated, and tissue-like memory B cells and PC/PB were evaluated by flow cytometry. Antibody-secreting cells (ASC) and serum antibody titers were assessed. No lasting changes in B-cell memory subpopulations occurred in bone marrow and lymph nodes, but significant decreases in numbers of activated memory B cells and increases in numbers of tissue-like memory B cells persisted in PBMC. Macaque PC/PB were found to be either CD27+or CD27−and therefore were defined as CD19+CD38hiCD138+. The numbers of these PC/PB were transiently increased in both PBMC and bone marrow following gp120 boosting of the unvaccinated and vaccinated macaque groups. Similarly, ASC numbers in PBMC and bone marrow of the two macaque groups also transiently increased following envelope boosting. Nevertheless, serum binding titers against SIVgp120 remained unchanged. Thus, even during chronic SIV infection, B cells respond to antigen, but long-term memory does not develop, perhaps due to germinal center destruction. Earlier and/or prolonged treatment to allow the generation of virus-specific long-term memory B cells should benefit ART/therapeutic vaccination regimens.
Authentic
In Vitro
Replication of Two Tombusviruses in Isolated Mitochondrial and Endoplasmic Reticulum Membranes
Authors: Xu Kai , Huang Tyng-Shyan , Nagy Peter D.
Published: September 2012

Replication of plus-stranded RNA viruses takes place on membranous structures derived from various organelles in infected cells. Previous works with
Tomato bushy stunt tombusvirus
(TBSV) revealed the recruitment of either peroxisomal or endoplasmic reticulum (ER) membranes for replication. In case of
Carnation Italian ringspot tombusvirus
(CIRV), the mitochondrial membranes supported CIRV replication. In this study, we developed ER and mitochondrion-based
in vitro
tombusvirus replication assays. Using purified recombinant TBSV and CIRV replication proteins, we showed that TBSV could use the purified yeast ER and mitochondrial preparations for complete viral RNA replication, while CIRV preferentially replicated in the mitochondrial membranes. The viral RNA became partly RNase resistant after ∼40 to 60 min of incubation in the purified ER and mitochondrial preparations, suggesting that assembly of TBSV and CIRV replicases could take place in the purified ER and mitochondrial membranes
in vitro
. Using chimeric and heterologous combinations of replication proteins, we showed that multiple domains within the replication proteins are involved in determining the efficiency of tombusvirus replication in the two subcellular membranes. Altogether, we demonstrated that TBSV is less limited while CIRV is more restricted in utilizing various intracellular membranes for replication. Overall, the current work provides evidence that tombusvirus replication could occur
in vitro
in isolated subcellular membranes, suggesting that tombusviruses have the ability to utilize alternative organellar membranes during infection that could increase the chance of mixed virus replication and rapid evolution during coinfection.
Amino Acid Residue-Specific Neutralization and Nonneutralization of Hepatitis C Virus by Monoclonal Antibodies to the E2 Protein
Authors: Duan Hongying , Kachko Alla , Zhong Lilin , Struble Evi , Pandey Shivani , Yan Hailing , Harman Christine , Virata-Theimer Maria Luisa , Deng Lu , Zhao Zhong , Major Marian , Feinstone Stephen , Zhang Pei
Published: September 2012

Antibodies to epitopes in the E2 protein of hepatitis C virus (HCV) reduce the viral infectivity
in vivo
and
in vitro
. However, the virus can persist in patients in the presence of neutralizing antibodies. In this study, we generated a panel of monoclonal antibodies that bound specifically to the region between residues 427 and 446 of the E2 protein of HCV genotype 1a, and we examined their capacity to neutralize HCV in a cell culture system. Of the four monoclonal antibodies described here, two were able to neutralize the virus in a genotype 1a-specific manner. The other two failed to neutralize the virus. Moreover, one of the nonneutralizing antibodies could interfere with the neutralizing activity of a chimpanzee polyclonal antibody at E2 residues 412 to 426, as it did with an HCV-specific immune globulin preparation, which was derived from the pooled plasma of chronic hepatitis C patients. Mapping the epitope-paratope contact interfaces revealed that these functionally distinct antibodies shared binding specificity for key amino acid residues, including W
437
, L
438
, L
441
, and F
442
, within the same epitope of the E2 protein. These data suggest that the effectiveness of antibody-mediated neutralization of HCV could be deduced from the interplay between an antibody and a specific set of amino acid residues. Further understanding of the molecular mechanisms of antibody-mediated neutralization and nonneutralization should provide insights for designing a vaccine to control HCV infection
in vivo
.
An Interaction between Human Papillomavirus 16 E2 and TopBP1 Is Required for Optimum Viral DNA Replication and Episomal Genome Establishment
Authors: Donaldson Mary M. , Mackintosh Lorna J. , Bodily Jason M. , Dornan Edward S. , Laimins Laimonis A. , Morgan Iain M.
Published: September 2012

In human papillomavirus DNA replication, the viral protein E2 forms homodimers and binds to 12-bp palindromic DNA sequences surrounding the origin of DNA replication. Via a protein-protein interaction, it then recruits the viral helicase E1 to an A/T-rich origin of replication, whereupon a dihexamer forms, resulting in DNA replication initiation. In order to carry out DNA replication, the viral proteins must interact with host factors that are currently not all known. An attractive cellular candidate for regulating viral replication is TopBP1, a known interactor of the E2 protein. In mammalian DNA replication, TopBP1 loads DNA polymerases onto the replicative helicase after the G
1
-to-S transition, and this process is tightly cell cycle controlled. The direct interaction between E2 and TopBP1 would allow E2 to bypass this cell cycle control, resulting in DNA replication more than once per cell cycle, which is a requirement for the viral life cycle. We report here the generation of an HPV16 E2 mutant compromised in TopBP1 interaction
in vivo
and demonstrate that this mutant retains transcriptional activation and repression functions but has suboptimal DNA replication potential. Introduction of this mutant into a viral life cycle model results in the failure to establish viral episomes. The results present a potential new antiviral target, the E2-TopBP1 interaction, and increase our understanding of the viral life cycle, suggesting that the E2-TopBP1 interaction is essential.
Impaired Surfactant Production by Alveolar Epithelial Cells in a SCID-hu Lung Mouse Model of Congenital Human Cytomegalovirus Infection
Authors: Maidji Ekaterina , Kosikova Galina , Joshi Pheroze , Stoddart Cheryl A.
Published: September 2012

Human cytomegalovirus (HCMV) is the leading viral cause of birth defects and life-threatening lung-associated diseases in premature infants and immunocompromised children. Although the fetal lung is a major target organ of the virus, HCMV lung pathogenesis has remained unexplored, possibly as a result of extreme host range restriction. To overcome this hurdle, we generated a SCID-hu lung mouse model that closely recapitulates the discrete stages of human lung developmentin utero. Human fetal lung tissue was implanted into severe combined immunodeficient (CB17-scid) mice and inoculated by direct injection with the VR1814 clinical isolate of HCMV. Virus replication in the fetal lung was assessed by the quantification of infectious virus titers and HCMV genome copies and the detection of HCMV proteins by immunohistochemistry and Western blotting. We show that HCMV efficiently replicated in the lung implants during a 2-week period, forming large viral lesions. The virus productively infected alveolar epithelial and mesenchymal cells, imitating congenital infection of the fetal lung. HCMV replication triggered apoptosis near and within the viral lesions and impaired the production of surfactant proteins in the alveolar epithelium. Our findings highlight that congenital and neonatal HCMV infection can adversely impact lung development, leading to pneumonia and acute lung injury. We have successfully developed a small-animal model that closely recapitulates fetal and neonatal lung development and provides a valuable, biologically relevant tool for an understanding of the lung pathogenesis of HCMV as well as other human respiratory viruses. Additionally, this model would greatly facilitate the development and testing of new antiviral therapies for HCMV along with select human pulmonary pathogens.
Herpes B Virus,Macacine Herpesvirus 1, Breaks Simplex Virus Tradition via Major Histocompatibility Complex Class I Expression in Cells from Human and Macaque Hosts
Authors: Vasireddi Mugdha , Hilliard Julia
Published: September 2012

B virus of the familyHerpesviridaeis endemic to rhesus macaques but results in 80% fatality in untreated humans who are zoonotically infected. Downregulation of major histocompatibility complex (MHC) class I in order to evade CD8+T-cell activation is characteristic of most herpesviruses. Here we examined the cell surface presence and total protein expression of MHC class I molecules in B virus-infected human foreskin fibroblast cells and macaque kidney epithelial cells in culture, which are representative of foreign and natural host initial target cells of B virus. Our results show <20% downregulation of surface MHC class I molecules in either type of host cells infected with B virus, which is statistically insignificantly different from that observed in uninfected cells. We also examined the surface expression of MHC class Ib molecules, HLA-E and HLA-G, involved in NK cell inhibition. Our results showed significant upregulation of HLA-E and HLA-G in host cells infected with B virus relative to the amounts observed in other herpesvirus-infected cells. These results suggest that B virus-infected cell surfaces maintain normal levels of MHC class Ia molecules, a finding unique among simplex viruses. This is a unique divergence in immune evasion for B virus, which, unlike human simplex viruses, does not inhibit the transport of peptides for loading onto MHC class Ia molecules because B virus ICP47 lacks a transporter-associated protein binding domain. The fact that MHC class Ib molecules were significantly upregulated has additional implications for host-pathogen interactions.
Suppression of Viral RNA Binding and the Assembly of Infectious Hepatitis C Virus Particles
In Vitro
by Cyclophilin Inhibitors
Authors: Nag Anita , Robotham Jason M. , Tang Hengli
Published: September 2012

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) is an indispensable component of the HCV replication and assembly machineries. Although its precise mechanism of action is not yet clear, current evidence indicates that its structure and function are regulated by the cellular peptidylprolyl isomerase cyclophilin A (CyPA). CyPA binds to proline residues in the C-terminal half of NS5A, in a distributed fashion, and modulates the structure of the disordered domains II and III. Cyclophilin inhibitors (CPIs), including cyclosporine (CsA) and its nonimmunosuppressive derivatives, inhibit HCV infection of diverse genotypes, both
in vitro
and
in vivo
. Here we report a mechanism by which CPIs inhibit HCV infection and demonstrate that CPIs can suppress HCV assembly in addition to their well-documented inhibitory effect on RNA replication. Although the interaction between NS5A and other viral proteins is not affected by CPIs, RNA binding by NS5A in cell culture-based HCV (HCVcc)-infected cells is significantly inhibited by CPI treatment, and sensitivity of RNA binding is correlated with previously characterized CyPA dependence or CsA sensitivity of HCV mutants. Furthermore, the difference in CyPA dependence between a subgenomic and a full-length replicon of JFH-1 was due, at least in part, to an additional role that CyPA plays in HCV assembly, a conclusion that is supported by experiments with the clinical CPI alisporivir. The host-directed nature and the ability to interfere with more than one step in the HCV life cycle may result in a higher genetic barrier to resistance for this class of HCV inhibitors.
Constraints on Viral Evolution during Chronic Hepatitis C Virus Infection Arising from a Common-Source Exposure
Authors: Bailey Justin R. , Laskey Sarah , Wasilewski Lisa N. , Munshaw Supriya , Fanning Liam J. , Kenny-Walsh Elizabeth , Ray Stuart C.
Published: September 2012

Extraordinary viral sequence diversity and rapid viral genetic evolution are hallmarks of hepatitis C virus (HCV) infection. Viral sequence evolution has previously been shown to mediate escape from cytotoxic T-lymphocyte (CTL) and neutralizing antibody responses in acute HCV infection. HCV evolution continues during chronic infection, but the pressures driving these changes are poorly defined. We analyzed plasma virus sequence evolution in 5.2-kb hemigenomes from multiple longitudinal time points isolated from individuals in the Irish anti-D cohort, who were infected with HCV from a common source in 1977 to 1978. We found phylogenetically distinct quasispecies populations at different plasma time points isolated late in chronic infection, suggesting ongoing viral evolution and quasispecies replacement over time. We saw evidence of early pressure driving net evolution away from a computationally reconstructed common ancestor, known as Bole1b, in predicted CTL epitopes and E1E2, with balanced evolution toward and away from the Bole1b amino acid sequence in the remainder of the genome. Late in chronic infection, the rate of evolution toward the Bole1b sequence increased, resulting in net neutral evolution relative to Bole1b across the entire 5.2-kb hemigenome. Surprisingly, even late in chronic infection, net amino acid evolution away from the infecting inoculum sequence still could be observed. These data suggest that, late in chronic infection, ongoing HCV evolution is not random genetic drift but rather the product of strong pressure toward a common ancestor and concurrent net ongoing evolution away from the inoculum virus sequence, likely balancing replicative fitness and ongoing immune escape.
Global Secretome Characterization of Herpes Simplex Virus 1-Infected Human Primary Macrophages
Authors: Miettinen Juho J. , Matikainen Sampsa , Nyman Tuula A.
Published: September 2012

Herpes simplex virus 1 (HSV-1) is a common pathogen infecting the majority of people worldwide at some stage in their lives. The early host response to viral infection is initiated by the cells of the innate immune response, including macrophages. Here, we have characterized the secretome of HSV-1-infected human primary macrophages using high-throughput quantitative proteomics. We identified and quantified 516 distinct human proteins with high confidence from the macrophage secretome upon HSV-1 infection, and the secretion of 411 proteins was >2-fold increased upon beta interferon (IFN-β) priming and/or HSV-1 infection. Bioinformatics analysis of the secretome data revealed that most of the secreted proteins were intracellular, and almost 80% of the proteins whose secretion increased more than 2-fold were known exosomal proteins. This strongly suggests that nonclassical, vesicle-mediated protein secretion is activated in IFN-β-primed and HSV-1-infected macrophages. Proteins related to immune and inflammatory responses, interferon-induced proteins, and endogenous danger signal proteins were efficiently secreted upon IFN-β priming and HSV-1 infection. The secreted IFN-induced proteins include interferon-induced tetratricopeptide protein 2 (IFIT2), IFIT3, signal transducer and activator of transcription 1 (STAT1), and myxovirus resistance protein A (MxA), implicating that these proteins also have important extracellular antiviral functions. Proinflammatory cytokine interleukin-1β was not released by HSV-1-infected macrophages, demonstrating that HSV-1 can antagonize inflammasome function. In conclusion, our results provide a global view of the secretome of HSV-1-infected macrophages, revealing host factors possibly having a role in antiviral defense.
The Crystal Structure of a Coxsackievirus B3-RD Variant and a Refined 9-Angstrom Cryo-Electron Microscopy Reconstruction of the Virus Complexed with Decay-Accelerating Factor (DAF) Provide a New Footprint of DAF on the Virus Surface
Authors: Yoder Joshua D. , Cifuente Javier O. , Pan Jieyan , Bergelson Jeffrey M. , Hafenstein Susan
Published: September 2012

The coxsackievirus-adenovirus receptor (CAR) and decay-accelerating factor (DAF) have been identified as cellular receptors for coxsackievirus B3 (CVB3). The first described DAF-binding isolate was obtained during passage of the prototype strain, Nancy, on rhabdomyosarcoma (RD) cells, which express DAF but very little CAR. Here, the structure of the resulting variant, CVB3-RD, has been solved by X-ray crystallography to 2.74 Å, and a cryo-electron microscopy reconstruction of CVB3-RD complexed with DAF has been refined to 9.0 Å. This new high-resolution structure permits us to correct an error in our previous view of DAF-virus interactions, providing a new footprint of DAF that bridges two adjacent protomers. The contact sites between the virus and DAF clearly encompass CVB3-RD residues recently shown to be required for binding to DAF; these residues interact with DAF short consensus repeat 2 (SCR2), which is known to be essential for virus binding. Based on the new structure, the mode of the DAF interaction with CVB3 differs significantly from the mode reported previously for DAF binding to echoviruses.
CTCF Occupation of the Herpes Simplex Virus 1 Genome Is Disrupted at Early Times Postreactivation in a Transcription-Dependent Manner
Authors: Ertel Monica K. , Cammarata Amy L. , Hron Rebecca J. , Neumann Donna M.
Published: September 2012

In herpes simplex virus 1 (HSV-1), binding clusters enriched in CTCF during latency have been previously identified. We hypothesized that CTCF binding to CTCF clusters in HSV-1 would be disrupted in a reactivation event. To investigate, CTCF occupation of three CTCF binding clusters in HSV-1 was analyzed following sodium butyrate (NaB)- and explant-induced reactivation in the mouse. Our data show that the CTCF domains positioned within the HSV-1 genome, specifically around the latency-associated transcript (LAT) and ICP0 and ICP4 regions of the genome, lose CTCF occupancy following the application of reactivation stimuli in wild-type virus. We also found that CTCF binding clusters upstream of the ICP0 and ICP4 promoters both function as classical insulators capable of acting as enhancer blockers of the LAT enhancer. Finally, our results suggest that CTCF occupation of domains in HSV-1 may be differentially regulated both during latency and at early times following reactivation by the presence of lytic transcripts and further implicate epigenetic regulation of HSV-1 as a critical component of the latency-reactivation transition.
The Vpx Lentiviral Accessory Protein Targets SAMHD1 for Degradation in the Nucleus
Authors: Hofmann Henning , Logue Eric C. , Bloch Nicolin , Daddacha Waaqo , Polsky Sylvie B. , Schultz Megan L. , Kim Baek , Landau Nathaniel R.
Published: September 2012

Sterile alpha motif domain- and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase that restricts the replication of lentiviruses in myeloid cells by hydrolyzing the cellular deoxynucleotide triphosphates to a level below that which is required for reverse transcription. Human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency viruses (SIVs) encode the accessory protein viral protein X (Vpx) that counteracts SAMHD1. Vpx recruits SAMHD1 to a cullin4A-RING E3 ubiquitin ligase (CRL4), which targets the enzyme for proteasomal degradation. Vpx and SAMHD1 both localize to the nucleus of the cell. We identified the nuclear localization sequence (NLS) of SAMHD1 as a conserved KRPR sequence at amino acid residues 11 to 14. SAMHD1 lacking a functional NLS localized to the cytoplasm but retained its triphosphohydrolase and antiviral activities. However, cytoplasmic SAMHD1 was resistant to Vpx-induced degradation, and its antiviral activity was not counteracted by Vpx. Cytoplasmic SAMHD1 interacted with Vpx and retained it in the cytoplasm. The inhibition of nuclear export with leptomycin B did not impair the ability of Vpx to degrade SAMHD1. These findings suggest that SAMHD1 is targeted by Vpx for ubiquitination and degradation in the nucleus.
Characterization of the Genome, Proteome, and Structure of Yersiniophage ϕR1-37
Authors: Skurnik Mikael , Hyytiäinen Heidi J. , Happonen Lotta J. , Kiljunen Saija , Datta Neeta , Mattinen Laura , Williamson Kirsty , Kristo Paula , Szeliga Magdalena , Kalin-Mänttäri Laura , Ahola-Iivarinen Elina , Kalkkinen Nisse , Butcher Sarah J.
Published: September 2012

The bacteriophage vB_YecM-ϕR1-37 (ϕR1-37) is a lytic yersiniophage that can propagate naturally in different
Yersinia
species carrying the correct lipopolysaccharide receptor. This large-tailed phage has deoxyuridine (dU) instead of thymidine in its DNA. In this study, we determined the genomic sequence of phage ϕR1-37, mapped parts of the phage transcriptome, characterized the phage particle proteome, and characterized the virion structure by cryo-electron microscopy and image reconstruction. The 262,391-bp genome of ϕR1-37 is one of the largest sequenced phage genomes, and it contains 367 putative open reading frames (ORFs) and 5 tRNA genes. Mass-spectrometric analysis identified 69 phage particle structural proteins with the genes scattered throughout the genome. A total of 269 of the ORFs (73%) lack homologues in sequence databases. Based on terminator and promoter sequences identified from the intergenic regions, the phage genome was predicted to consist of 40 to 60 transcriptional units. Image reconstruction revealed that the ϕR1-37 capsid consists of hexameric capsomers arranged on a T=27 lattice similar to the bacteriophage ϕKZ. The tail of ϕR1-37 has a contractile sheath. We conclude that phage ϕR1-37 is a representative of a novel phage type that carries the dU-containing genome in a ϕKZ-like head.
Reovirus Uses Multiple Endocytic Pathways for Cell Entry
Authors: Schulz Wade L. , Haj Amelia K. , Schiff Leslie A.
Published: September 2012

Entry of reovirus virions has been well studied in several tissue culture systems. After attachment to junctional adhesion molecule A (JAM-A), virions undergo clathrin-mediated endocytosis followed by proteolytic disassembly of the capsid and penetration to the cytoplasm. However, during
in vivo
infection of the intestinal tract, and likely in the tumor microenvironment, capsid proteolysis (uncoating) is initiated extracellularly. We used multiple approaches to determine if uncoated reovirus particles, called intermediate subviral particles (ISVPs), enter cells by directly penetrating the limiting membrane or if they take advantage of endocytic pathways to establish productive infection. We found that entry and infection by reovirus ISVPs was inhibited by dynasore, an inhibitor of dynamin-dependent endocytosis, as well as by genistein and dominant-negative caveolin-1, which block caveolar endocytosis. Inhibition of caveolar endocytosis also reduced infection by reovirus virions. Extraction of membrane cholesterol with methyl-β-cyclodextrin inhibited infection by virions but had no effect when infection was initiated with ISVPs. We found this pathway to be independent of both clathrin and caveolin. Together, these data suggest that reovirus virions can use both dynamin-dependent and dynamin-independent endocytic pathways during cell entry, and they reveal that reovirus ISVPs can take advantage of caveolar endocytosis to establish productive infection.
Broadly Neutralizing Antibodies Developed by an HIV-Positive Elite Neutralizer Exact a Replication Fitness Cost on the Contemporaneous Virus
Authors: Sather D. Noah , Carbonetti Sara , Kehayia Jenny , Kraft Zane , Mikell Iliyana , Scheid Johannes F. , Klein Florian , Stamatatos Leonidas
Published: September 2012

Approximately 1% of those infected with HIV-1 develop broad and potent serum cross-neutralizing antibody activities. It is unknown whether or not the development of such immune responses affects the replication of the contemporaneous autologous virus. Here, we defined a pathway of autologous viral escape from contemporaneous potent and broad serum neutralizing antibodies developed by an elite HIV-1-positive (HIV-1
+
) neutralizer. These antibodies potently neutralize diverse isolates from different clades and target primarily the CD4-binding site (CD4-BS) of the viral envelope glycoprotein. Viral escape required mutations in the viral envelope glycoprotein which limited the accessibility of the CD4-binding site to the autologous broadly neutralizing anti-CD4-BS antibodies but which allowed the virus to infect cells by utilizing CD4 receptors on their surface. The acquisition of neutralization resistance, however, resulted in reduced cell entry potential and slower viral replication kinetics. Our results indicate that
in vivo
escape from autologous broadly neutralizing antibodies exacts fitness costs to HIV-1.
Gamma Interferon (IFN-γ) Receptor Restricts Systemic Dengue Virus Replication and Prevents Paralysis in IFN-α/β Receptor-Deficient Mice
Authors: Prestwood Tyler R. , Morar Malika M. , Zellweger Raphaël M. , Miller Robyn , May Monica M. , Yauch Lauren E. , Lada Steven M. , Shresta Sujan
Published: September 2012

We previously reported that mice lacking alpha/beta and gamma interferon receptors (IFN-α/βR and -γR) uniformly exhibit paralysis following infection with the dengue virus (DENV) clinical isolate PL046, while only a subset of mice lacking the IFN-γR alone and virtually no mice lacking the IFN-α/βR alone develop paralysis. Here, using a mouse-passaged variant of PL046, strain S221, we show that in the absence of the IFN-α/βR, signaling through the IFN-γR confers approximately 140-fold greater resistance against systemic vascular leakage-associated dengue disease and virtually complete protection from dengue-induced paralysis. Viral replication in the spleen was assessed by immunohistochemistry and flow cytometry, which revealed a reduction in the number of infected cells due to IFN-γR signaling by 2 days after infection, coincident with elevated levels of IFN-γ in the spleen and serum. By 4 days after infection, IFN-γR signaling was found to restrict DENV replication systemically. Clearance of DENV, on the other hand, occurred in the absence of IFN-γR, except in the central nervous system (CNS) (brain and spinal cord), where clearance relied on IFN-γ from CD8
+
T cells. These results demonstrate the roles of IFN-γR signaling in protection from initial systemic and subsequent CNS disease following DENV infection and demonstrate the importance of CD8
+
T cells in preventing DENV-induced CNS disease.
Genetic Control of Translesion Synthesis on Leading and Lagging DNA Strands in Plasmids Derived from Epstein-Barr Virus in Human Cells
Authors: Yoon Jung-Hoon , Prakash Satya , Prakash Louise
Published: September 2012

DNA lesions in the template strand block synthesis by replicative DNA polymerases (Pols). Eukaryotic cells possess a number of specialized translesion synthesis (TLS) Pols with the ability to replicate through DNA lesions. The Epstein-Barr virus (EBV), a member of the herpesvirus family, infects human B cells and is maintained there as an extrachromosomal replicon, replicating once per cycle during S phase. Except for the requirement of the virus-encoded origin-binding protein EBNA1, replication of plasmids containing the EBV origin of replication (
oriP
) is controlled by the same cellular processes that govern chromosomal replication. Since replication of EBV plasmid closely mimics that of human chromosomal DNA, in this study we examined the genetic control of TLS in a duplex plasmid in which bidirectional replication initiates from an EBV
oriP
origin and a UV-induced
cis-
syn
TT dimer is placed on the leading- or the lagging-strand DNA template. Here we show that TLS occurs equally frequently on both the DNA strands of EBV plasmid and that the requirements of TLS Pols are the same regardless of which DNA strand carries the lesion. We discuss the implications of these observations for TLS mechanisms that operate on the two DNA strands during chromosomal replication and conclude that the same genetic mechanisms govern TLS during the replication of the leading and the lagging DNA strands in human cells.

IMPORTANCE
Since replication of EBV (Epstein-Barr virus) origin-based plasmids appropriates the cellular machinery for all the steps of replication, our observations that the same genetic mechanisms govern translesion synthesis (TLS) on the two DNA strands of EBV plasmids imply that the requirements of TLS Pols are not affected by any of the differences in the replicative Pols or in other proteins that may be used for the replication of the two DNA strands in human cells. These findings also have important implications for evaluating the significance of results of TLS studies with the SV40 origin-based plasmids that we have reported previously, in which we showed that TLS occurs similarly on the two DNA strands. Since the genetic control of TLS in SV40 plasmids resembles that in EBV plasmids, we conclude that TLS studies with the SV40 plasmids are as informative of TLS mechanisms that operate during cellular replication as those with the EBV plasmids.
Repression by Cyclic AMP Receptor Protein at a Distance
Authors: Lee David J. , Busby Stephen J. W.
Published: September 2012

In a previous study of promoters dependent on the
Escherichia coli
cyclic AMP receptor protein (CRP), carrying tandem DNA sites for CRP, we found that the upstream-bound CRP could either enhance or repress transcription, depending on its location. Here, we have analyzed the interactions between CRP and the C-terminal domains of the RNA polymerase α subunits at some of these promoters. We report that the upstream-bound CRP interacts with these domains irrespective of whether it up- or downregulates promoter activity. Hence, disruption of this interaction can lead to either down- or upregulation, depending on its location.

IMPORTANCE
Many bacterial promoters carry multiple DNA sites for transcription factors. While most factors that downregulate promoter activity bind to targets that overlap or are downstream of the transcription start and −10 element, very few cases of repression from upstream locations have been reported. Since more
Escherichia coli
promoters are regulated by cyclic AMP receptor protein (CRP) than by any other transcription factor, and since multiple DNA sites for CRP are commonplace at promoters, our results suggest that promoter downregulation by transcription factors may be more prevalent than hitherto thought, and this will have implications for the annotation of promoters from new bacterial genome sequences.
Disseminated tuberculosis in pregnancy unknown to doctors in Western Europe case presentation: ‘part of the routine study in infertility’
Authors: Jacquemyn Yves , Van Casteren Christelle , Luijks Marloes , Colpaert Cecile
Published: September 2012

Tuberculosis in pregnancy is possibly increasing in Western Europe due to a higher incidence in immigrant women from endemic regions. Common in recent immigrants. Diagnosis during pregnancy is delayed because the disease is more frequently extra-pulmonary with few symptoms and western doctors are no longer familiar with signs and symptoms of tuberculosis. We report the case of a 28-year-old woman presenting in the 13th week of pregnancy with vaginal bleeding, respiratory symptoms including persistent coughing and fever since 1 month. Diagnosis was delayed because of doubts on taking x-rays during pregnancy, and only 6 days after admission chest x-ray and blood-cultures led to diagnose miliary tuberculosis. Despite tuberculostatic medication expulsion of an infected fetus and placenta occurred. Histological examination of the placenta confirmed tuberculosis in the placental tissue.
Bilateral deep brain stimulation of the nucleus accumbens for comorbid obsessive compulsive disorder and Tourette's syndrome
Authors: Sachdev Perminder Singh , Cannon Elisabeth , Coyne Terry J , Silburn Peter
Published: September 2012

We present the case of a 32-year-old Caucasian woman with severe treatment-refractory obsessive compulsive disorder (OCD) and Tourette's syndrome. Both conditions were present prior to age 5 and impacted significantly on the patient's functioning. Multiple trials of evidence-based pharmacological and behavioural therapies had not achieved remission of symptoms. Bilateral deep brain stimulation of the nucleus accumbens was undertaken to treat both illnesses but with a particular focus on OCD, as the patient identified this as the more debilitating of the two disorders. Following surgery there was an immediate improvement in OCD and tic severity. At follow-up 8 months later, there was a 90% improvement in OCD symptoms and a 57% improvement in tic severity. No intraoperative or postoperative complications or adverse events occurred and there were no undesired effects of stimulation.
Gastrobronchial fistula following laparoscopic sleeve gastrectomy
Authors: Abraham Albin , Virdi Ravi Paul Singh , Rajan Dhyan , Singh Jaspreet , Mustacchia Paul , Iqbal Javed , Rizvon Kaleem
Published: September 2012

Acquired fistulas between the tracheobronchial tree and the gastrointestinal tract are rare but serious complications of laparoscopic sleeve gastrectomies with significant morbidity and mortality. With the rising popularity and widespread acceptance of bariatric surgery techniques, the occurrence of gastrobronchial fistulas is being increasingly recognised. We present the case of a 26-year-old woman who underwent laparoscopic sleeve gastrectomy for morbid obesity and presented later with a history of chronic productive cough. Upper gastrointestinal series showed the presence of a communicating fistula between the stomach and the lung, with extravasation of contrast into the lung. The aim of this paper is to highlight the importance of considering the diagnosis of a gastrobronchial fistula in cases of persistent respiratory infections in the postoperative period following bariatric surgery and to review its incidence, clinical manifestations and treatment.
FIFA Women's World Cup 2011: Pre-Competition Medical Assessment of female referees and assistant referees
Authors: Keller Dagmar I , Bizzini Mario , Feddermann Nina , Junge Astrid , Dvorak Jiri
Published: September 2012

Background

Precompetition screening was implemented for male referees during the 2010 Fédération Internationale de Football Association (FIFA) Word Cup. In contrast, female football referees have been neglected in this respect although they experience similar physical work loads compared to male referees.

Methods

The standardised football-specific Pre-Competition Medical Assessment (PCMA) was performed in 51 referees and assistant referees selected for the 2011 FIFA Women's World Cup.

Results

Family history for sudden cardiac death (SCD) was positive in four referees (7.8%), but cardiac examinations did not reveal any pathological findings. Training-unrelated ECG changes were identified in three referees (5.9%), all without correlates in echocardiography or clinical examination. Most common echocardiography findings (66.6%, n=34) were asymptomatic tricuspid and mitral regurgitations.

Conclusions

During the present screening, no elite female referee was identified being at risk for SCD, and no referee had to be excluded from participating in the 2011 FIFA Women's World Cup.