bluuebunny/crossref_metadata_2025 huggingface dataset:
High-Throughput Assay Optimization and Statistical Interpolation of Rubella-Specific Neutralizing Antibody Titers
Authors: Lambert Nathaniel D. , Pankratz V. Shane , Larrabee Beth R. , Ogee-Nwankwo Adaeze , Chen Min-hsin , Icenogle Joseph P. , Poland Gregory A.
Published: January 2014

Rubella remains a social and economic burden due to the high incidence of congenital rubella syndrome (CRS) in some countries. For this reason, an accurate and efficient high-throughput measure of antibody response to vaccination is an important tool. In order to measure rubella-specific neutralizing antibodies in a large cohort of vaccinated individuals, a high-throughput immunocolorimetric system was developed. Statistical interpolation models were applied to the resulting titers to refine quantitative estimates of neutralizing antibody titers relative to the assayed neutralizing antibody dilutions. This assay, including the statistical methods developed, can be used to assess the neutralizing humoral immune response to rubella virus and may be adaptable for assessing the response to other viral vaccines and infectious agents.
Topical CpG Adjuvantation of a Protein-Based Vaccine Induces Protective Immunity to Listeria monocytogenes
Authors: Cheng Wing Ki , Wee Kathleen , Kollmann Tobias R. , Dutz Jan P.
Published: January 2014

Robust CD8+T cell responses are essential for immune protection against intracellular pathogens. Using parenteral administration of ovalbumin (OVA) protein as a model antigen, the effect of the Toll-like receptor 9 (TLR9) agonist, CpG oligodeoxynucleotide (ODN) 1826, as an adjuvant delivered either topically, subcutaneously, or intramuscularly on antigen-specific CD8+T cell responses in a mouse model was evaluated. Topical CpG adjuvant increased the frequency of OVA-specific CD8+T cells in the peripheral blood and in the spleen. The more effective strategy to administer topical CpG adjuvant to enhance CD8+T cell responses was single-dose administration at the time of antigen injection with a prime-boost regimen. Topical CpG adjuvant conferred both rapid and long-lasting protection against systemic challenge with recombinantListeria monocytogenesexpressing the cytotoxic T lymphocyte (CTL) epitope of OVA257–264(strainLm-OVA) in a TLR9-dependent manner. Topical CpG adjuvant induced a higher proportion of CD8+effector memory T cells than parenteral administration of the adjuvant. Although traditional vaccination strategies involve coformulation of antigen and adjuvant, split administration using topical adjuvant is effective and has advantages of safety and flexibility. Split administration of topical CpG ODN 1826 with parenteral protein antigen is superior to other administration strategies in enhancing both acute and memory protective CD8+T cell immune responses to subcutaneous protein vaccines. This vaccination strategy induces rapid and persistent protective immune responses against the intracellular organismL. monocytogenes.
A Retinoblastoma Orthologue Is Required for the Sensing of a Chalone in Dictyostelium discoideum
Authors: Bakthavatsalam Deenadayalan , White Michael J. V. , Herlihy Sarah E. , Phillips Jonathan E. , Gomer Richard H.
Published: January 2014

Retinoblastoma-like proteins regulate cell differentiation and inhibit cell proliferation. The
Dictyostelium discoideum
retinoblastoma orthologue RblA affects the differentiation of cells during multicellular development, but it is unclear whether RblA has a significant effect on
Dictyostelium
cell proliferation, which is inhibited by the secreted proteins AprA and CfaD. We found that
rblA
−
cells in shaking culture proliferate to a higher density, die faster after reaching stationary density, and, after starvation, have a lower spore viability than wild-type cells, possibly because in shaking culture,
rblA
−
cells have both increased cytokinesis and lower extracellular accumulation of CfaD. However,
rblA
−
cells have abnormally slow proliferation on bacterial lawns. Recombinant AprA inhibits the proliferation of wild-type cells but not that of
rblA
−
cells, whereas CfaD inhibits the proliferation of both wild-type cells and
rblA
−
cells. Similar to
aprA
−
cells,
rblA
−
cells have a normal mass and protein accumulation rate on a per-nucleus basis, indicating that RblA affects cell proliferation but not cell growth. AprA also functions as a chemorepellent, and RblA is required for proper AprA chemorepellent activity despite the fact that RblA does not affect cell speed. Together, our data indicate that an autocrine proliferation-inhibiting factor acts through RblA to regulate cell density in
Dictyostelium
, suggesting that such factors may signal through retinoblastoma-like proteins to control the sizes of structures such as developing organs or tumors.
A Visual Screen of Protein Localization during Sporulation Identifies New Components of Prospore Membrane-Associated Complexes in Budding Yeast
Authors: Lam Chien , Santore Ethan , Lavoie Elizabeth , Needleman Leor , Fiacco Nicholas , Kim Carey , Neiman Aaron M.
Published: January 2014

During ascospore formation in
Saccharomyces cerevisiae
, the secretory pathway is reorganized to create new intracellular compartments, termed prospore membranes. Prospore membranes engulf the nuclei produced by the meiotic divisions, giving rise to individual spores. The shape and growth of prospore membranes are constrained by cytoskeletal structures, such as septin proteins, that associate with the membranes. Green fluorescent protein (GFP) fusions to various proteins that associate with septins at the bud neck during vegetative growth as well as to proteins encoded by genes that are transcriptionally induced during sporulation were examined for their cellular localization during prospore membrane growth. We report localizations for over 100 different GFP fusions, including over 30 proteins localized to the prospore membrane compartment. In particular, the screen identified
IRC10
as a new component of the leading-edge protein complex (LEP), a ring structure localized to the lip of the prospore membrane. Localization of Irc10 to the leading edge is dependent on
SSP1
, but not
ADY3
. Loss of
IRC10
caused no obvious phenotype, but an
ady3 irc10
mutant was completely defective in sporulation and displayed prospore membrane morphologies similar to those of an
ssp1
strain. These results reveal the architecture of the LEP and provide insight into the evolution of this membrane-organizing complex.
CodY-Mediated Regulation of the Staphylococcus aureus Agr System Integrates Nutritional and Population Density Signals
Authors: Roux Agnès , Todd Daniel A. , Velázquez Jose V. , Cech Nadja B. , Sonenshein Abraham L.
Published: January 2014

The
Staphylococcus aureus
Agr system regulates virulence gene expression by responding to cell population density (quorum sensing). When an extracellular peptide signal (AIP-III in strain UAMS-1, used for these experiments) reaches a concentration threshold, the AgrC-AgrA two-component regulatory system is activated through a cascade of phosphorylation events, leading to induction of the divergently transcribed
agrBDCA
operon and the RNAIII gene. RNAIII is a posttranscriptional regulator of numerous metabolic and pathogenesis genes. CodY, a global regulatory protein, is known to repress
agrBDCA
and RNAIII transcription during exponential growth in rich medium, but the mechanism of this regulation has remained elusive. Here we report that phosphorylation of AgrA by the AgrC protein kinase is required for the overexpression of the
agrBDCA
operon and the RNAIII gene in a
codY
mutant during the exponential-growth phase, suggesting that the quorum-sensing system, which normally controls AgrC activation, is active even in exponential-phase cells in the absence of CodY. In part, such premature expression of RNAIII was attributable to higher-than-normal accumulation of AIP-III in a
codY
mutant strain, as determined using ultrahigh-performance liquid chromatography coupled to mass spectrometry. Although CodY is a strong repressor of the
agr
locus, CodY bound only weakly to the
agrBDCA
-RNAIII promoter region, suggesting that direct regulation by CodY is unlikely to be the principal mechanism by which CodY regulates
agr
and RNAIII expression. Taken together, these results strongly suggest that cell population density signals inducing virulence gene expression can be overridden by nutrient availability, a condition monitored by CodY.
Myxobacteria Produce Outer Membrane-Enclosed Tubes in Unstructured Environments
Authors: Wei Xueming , Vassallo Christopher N. , Pathak Darshankumar T. , Wall Daniel
Published: January 2014

Myxobacteria are social microbes that exhibit complex multicellular behaviors. By use of fluorescent reporters, we show that
Myxococcus xanthus
isolates produce long narrow filaments that are enclosed by the outer membrane (OM) and contain proteins. We show that these OM tube (OMT) structures are produced at surprisingly high levels when cells are placed in liquid medium or buffer without agitation. OMTs can be long and easily exceed multiple cell lengths. When viewed by transmission electron microscopy, their morphology varies between tubes and chain-like structures. Intermediate-like structures are also found, suggesting that OMTs may transition between these two morphotypes. In support of this, video epifluorescence microscopy found that OMTs in solution dynamically twist and jiggle. On hard surfaces, myxobacteria glide, and upon cell-cell contact, they can efficiently exchange their OM proteins and lipids by a TraAB-dependent mechanism. Although the structure of OMTs hints at a possible role as conduits for exchange, evidence is presented to the contrary. For example, abundant OMT production occurs in
traA
or
traB
mutants and when cells are grown in liquid medium, yet transfer cannot occur under these conditions. Thus, genetic and environmental conditions that promote OMT production are incongruent with OM exchange.
YfbA, a Yersinia pestis Regulator Required for Colonization and Biofilm Formation in the Gut of Cat Fleas
Authors: Tam Christina , Demke Owen , Hermanas Timothy , Mitchell Anthony , Hendrickx Antoni P. A. , Schneewind Olaf
Published: January 2014

For transmission to new hosts,Yersinia pestis, the causative agent of plague, replicates as biofilm in the foregut of fleas that feed on plague-infected animals or humans.Y. pestisbiofilm formation has been studied in the rat flea; however, little is known about the cat flea, a species that may bridge zoonotic and anthroponotic plague cycles. Here, we show thatY. pestisinfects and replicates as a biofilm in the foregut of cat fleas in a manner requiringhmsFR, two determinants for extracellular biofilm matrix. Examining a library of transposon insertion mutants, we identified the LysR-type transcriptional regulator YfbA, which is essential forY. pestiscolonization and biofilm formation in cat fleas.
Genetic Redundancy, Proximity, and Functionality of
lspA
, the Target of Antibiotic TA, in the Myxococcus xanthus Producer Strain
Authors: Xiao Yao , Wall Daniel
Published: January 2014

We recently showed that type II signal peptidase (SPaseII) encoded by
lspA
is the target of an antibiotic called TA (myxovirescin), which is made by
Myxococcus xanthus
. SPaseII cleaves the signal peptide during bacterial lipoprotein processing. Bacteria typically contain one
lspA
gene; however, strikingly, the
M. xanthus
DK1622 genome contains four (
lspA1
to
lspA4
). Since two of these genes,
lspA3
and
lspA4
, are located in the giant TA biosynthetic gene cluster, we hypothesized they may play a role in TA resistance. To investigate the functions of the four
M. xanthus
lspA
(
lspA

Mx

) genes, we conducted sequence comparisons and found that they contained nearly all the conserved residues characteristic of SPaseII family members. Genetic studies found that an
Escherichia coli
Δ
lspA
mutation could be complemented by any of the
lspA

Mx

genes in an
lpp
mutant background, but not in an
E. coli lpp

+

background. Because Lpp is the most abundant
E. coli
lipoprotein, these results suggest the
M. xanthus
proteins do not function as efficiently as the host enzyme. In
E. coli
, overexpression of each of the LspA

Mx

proteins conferred TA and globomycin resistance, although LspA3 conferred the highest degree of resistance. In
M. xanthus
, each
lspA

Mx

gene could be deleted and was therefore dispensable for growth. However,
lspA3
or
lspA4
deletion mutants each exhibited a tan phase variation bias, which likely accounts for their reduced-swarming and delayed-development phenotypes. In summary, we propose that all four LspA

Mx

proteins function as SPaseIIs and that LspA3 and LspA4 might also have roles in TA resistance and regulation, respectively.
Comparison of the BD Veritor System for Flu A+B with the Alere BinaxNOW Influenza A&B Card for Detection of Influenza A and B Viruses in Respiratory Specimens from Pediatric Patients
Authors: Hassan Ferdaus , Nguyen Ashley , Formanek Ashley , Bell James J. , Selvarangan Rangaraj
Published: January 2014

The performance characteristics of two commercially available rapid tests for influenza, the BD Veritor System for Flu A+B (BD) and the Alere BinaxNOW influenza A&B card (BN), were evaluated using 200 frozen clinical specimens collected from January 2011 to June 2012 from pediatric patients. Real-time reverse transcriptase PCR (RT-PCR) was used as the gold standard to evaluate the results obtained by the two different assays. Of the 200 specimens tested, real-time RT-PCR assay detected influenza A or B virus in 116 samples, while BD detected 104 samples and BN detected 84 samples as positive. The overall sensitivity and specificity for detection of both influenza A and B virus in comparison to those of real-time RT-PCR were 89.6% (95% confidence interval [CI], 82.2 to 94.3) and 98.8% (95% CI, 92.6 to 99.9) for BD Veritor and 72.4% (95% CI, 63.2 to 80.0) and 100% (95% CI, 94.5 to 100.0) for BinaxNOW. Workflow analysis indicated that overall processing times for a batch size of 10 specimens were virtually identical between both systems. Overall, these results indicate that the BD Veritor assay was more sensitive than the BinaxNOW assay in detection of influenza A and B viruses in respiratory specimens from pediatric patients.
Three Cases of Postoperative Septic Arthritis Caused by Mycobacterium conceptionense in the Shoulder Joints of Immunocompetent Patients
Authors: Lee Keun Hwa , Heo Sang Taek , Choi Sung-Wook , Park Da Hee , Kim Young Ree , Yoo Seung Jin
Published: January 2014

Mycobacterium conceptionense
is a species member of
Mycobacterium fortuitum
complex, a potential pathogen of increasing clinical importance among opportunistic infections. This species causes a wide spectrum of cutaneous and extracutaneous diseases. In this report, we describe three patients who underwent shoulder surgery with postoperative joint infection by
M. conceptionense
.
Fluoroquinolone Susceptibility Testing of Salmonella enterica: Detection of Acquired Resistance and Selection of Zone Diameter Breakpoints for Levofloxacin and Ofloxacin
Authors: Sjölund-Karlsson Maria , Howie Rebecca L. , Crump John A. , Whichard Jean M.
Published: January 2014

Fluoroquinolones (e.g., ciprofloxacin) have become a mainstay for treating severe
Salmonella
infections in adults. Fluoroquinolone resistance in
Salmonella
is mostly due to mutations in the topoisomerase genes, but plasmid-mediated quinolone resistance (PMQR) mechanisms have also been described. In 2012, the Clinical and Laboratory Standards Institute (CLSI) revised the ciprofloxacin interpretive criteria (breakpoints) for disk diffusion and MIC test methods for
Salmonella
. In 2013, the CLSI published MIC breakpoints for
Salmonella
to levofloxacin and ofloxacin, but breakpoints for assigning disk diffusion results to susceptible (S), intermediate (I), and resistant (R) categories are still needed. In this study, the MICs and inhibition zone diameters for nalidixic acid, ciprofloxacin, levofloxacin, and ofloxacin were determined for 100 clinical isolates of nontyphi
Salmonella
with or without resistance mechanisms. We confirmed that the new levofloxacin MIC breakpoints resulted in the highest category agreement (94%) when plotted against the ciprofloxacin MICs and that the new ofloxacin MIC breakpoints resulted in 92% category agreement between ofloxacin and ciprofloxacin. By applying the new MIC breakpoints in the MIC zone scattergrams for levofloxacin and ofloxacin, the following disk diffusion breakpoints generated the least number of errors: ≥28 mm (S), 19 to 27 mm (I), and ≤18 mm (R) for levofloxacin and ≥25 mm (S), 16 to 24 mm (I), and ≤15 mm (R) for ofloxacin. Neither the levofloxacin nor the ofloxacin disk yielded good separation of isolates with and without resistance mechanisms. Further studies will be needed to develop a disk diffusion assay that efficiently detects all isolates with acquired resistance to fluoroquinolones.
Effect of the Initial Specimen Diversion Technique on Blood Culture Contamination Rates
Authors: Binkhamis Khalifa , Forward Kevin
Published: January 2014

The initial specimen diversion technique (ISDT) was first described by Patton and Schmitt (J. Clin. Microbiol.
48:
4501–4503, 2010, doi:10.1128/JCM.00910-10). This study looked at the effect of implementation of the ISDT on blood culture contamination rates at our center. We found a reduction of 30.34% in potential blood culture contaminants.
Detection of Trichomonas vaginalis DNA by Use of Self-Obtained Vaginal Swabs with the BD ProbeTec Q
x
Assay on the BD Viper System
Authors: Van Der Pol Barbara , Williams James A. , Taylor Stephanie N. , Cammarata Catherine L. , Rivers Charles A. , Body Barbara A. , Nye Melinda , Fuller Deanna , Schwebke Jane R. , Barnes Mathilda , Gaydos Charlotte A.
Published: January 2014

Trichomonas vaginalis
is the most prevalent nonviral sexually transmitted infection worldwide, and improved diagnostic methods are critical for controlling this pathogen. Diagnostic assays that can be used in conjunction with routine chlamydia/gonorrhea nucleic acid-based screening are likely to have the most impact on disease control. Here we describe the performance of the new BD
T. vaginalis
Q
x
(TVQ) amplified DNA assay, which can be performed on the automated BD Viper system. We focus on data from vaginal swab samples, since this is the specimen type routinely used for traditional trichomonas testing and the recommended specimen type for chlamydia/gonorrhea screening. Vaginal swabs were obtained from women attending sexually transmitted disease or family planning clinics at 7 sites. Patient-collected vaginal swabs were tested by the TVQ assay, and the Aptima
T. vaginalis
(ATV) assay was performed using clinician-collected vaginal swabs. Additional clinician-collected vaginal swabs were used for the wet mount and culture methods. Analyses included comparisons versus the patient infection status (PIS) defined by positive results with the wet mount method or culture, direct comparisons assessed with κ scores, and latent class analysis (LCA) as an unbiased estimator of test accuracy. Data from 838 women, 116 of whom were infected with
T. vaginalis
, were analyzed. The TVQ assay sensitivity and specificity estimates based on the PIS were 98.3% and 99.0%, respectively. The TVQ assay was similar to the ATV assay (κ = 0.938) in direct analysis. LCA estimated the TVQ sensitivity and specificity as 98.3 and 99.6%, respectively. The TVQ assay performed well using self-collected vaginal swabs, the optimal sample type, as recommended by the CDC for chlamydia/gonorrhea screening among women.
Clinical Validation of the HPV-Risk Assay, a Novel Real-Time PCR Assay for Detection of High-Risk Human Papillomavirus DNA by Targeting the E7 Region
Authors: Hesselink A. T. , Berkhof J. , van der Salm M. L. , van Splunter A. P. , Geelen T. H. , van Kemenade F. J. , Bleeker M. G. B. , Heideman D. A. M.
Published: January 2014

The HPV-Risk assay is a novel real-time PCR assay targeting the E7 region of 15 high-risk human papillomavirus (HPV) types (i.e., HPV16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -67, and −68), and provides additional genotype information for HPV16 and HPV18. This study evaluated the clinical performance and reproducibility of the HPV-Risk assay with cervical scraping specimens and its utility with self-collected (cervico)vaginal specimens. The clinical performance of the HPV-Risk assay for cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) with cervical scraping specimens was evaluated by a noninferiority analysis, relative to high-risk HPV GP5+/6+ PCR, following international guidelines for HPV test requirements for cervical cancer screening. The HPV-Risk assay showed clinical sensitivity for CIN2+ of 97.1% (95% confidence interval [CI], 89.1 to 99.3%; 67/69 samples) and a clinical specificity for CIN2+ of 94.3% (95% CI, 92.5 to 95.7%; 777/824 samples). The clinical sensitivity and specificity were noninferior to those of GP5+/6+ PCR (noninferiority score test,
P
= 0.006 and 0.0003, respectively). Intralaboratory reproducibility over time (99.5% [95% CI, 98.6 to 99.8%]; 544/547 samples, kappa = 0.99) and interlaboratory agreement (99.2% [95% CI, 98.6 to 99.8%]; 527/531 samples, kappa = 0.98) for the HPV-Risk assay with cervical scraping specimens were high. The agreement of the HPV-Risk assay results for self-collected (cervico)vaginal specimens and clinician-obtained cervical scraping specimens was also high, i.e., 95.9% (95% CI, 85.1 to 99.0%; 47/49 samples, kappa = 0.90) for self-collected lavage samples and 91.6% (95% CI, 84.6 to 95.6%; 98/107 samples, kappa = 0.82) for self-collected brush samples. In conclusion, the HPV-Risk assay meets the cross-sectional clinical and reproducibility criteria of the international guidelines for HPV test requirements and can be considered clinically validated for cervical screening purposes. The compatibility of the HPV-Risk assay with self-collected specimens supports its utility for HPV self-sampling.
Novel Real-Time PCR Assays Using TaqMan Minor Groove Binder Probes for Identification of Fecal Carriage of Streptococcus bovis/Streptococcus equinus Complex from Rectal Swab Specimens
Authors: Lopes Paulo Guilherme Markus , Cantarelli Vlademir Vicente , Agnes Grasiela , Costabeber Ane Micheli , d'Azevedo Pedro Alves
Published: January 2014

Real-time PCR based on the
recN
and
gyrB
genes was developed to detect four
Streptococcus bovis
/
Streptococcus equinus
complex (SBEC) subspecies from rectal swab specimens. The overall prevalence was 35.2%:
Streptococcus gallolyticus
subsp.
gallolyticus
(11.1%),
S. gallolyticus
subsp.
pasteurianus
(13%),
Streptococcus infantarius
subsp.
coli
(20.4%), and
S. infantarius
subsp.
infantarius
(11.1%). To conclude, these real-time PCR assays provide a reliable molecular method to detect SBEC pathogenic subspecies from rectal swab specimens.
Rapid Ertapenem Susceptibility Testing and Klebsiella pneumoniae Carbapenemase Phenotype Detection in Klebsiella pneumoniae Isolates by Use of Automated Microscopy of Immobilized Live Bacterial Cells
Authors: Burnham Carey-Ann D. , Frobel Rachel A. , Herrera Monica L. , Wickes Brian L.
Published: January 2014

We evaluated detection of ertapenem (ETP) resistance and
Klebsiella pneumoniae
carbapenemase (KPC) in 47
Klebsiella pneumoniae
isolates using a novel automated microscopy system. Automated microscopy correctly classified 22/23 isolates as ETP resistant and 24/24 as ETP susceptible and correctly classified 21/21 isolates as KPC positive and 26/26 as KPC negative.
Human Herpesvirus 7 U21 Tetramerizes To Associate with Class I Major Histocompatibility Complex Molecules
Authors: May Nathan A. , Wang Qiuhong , Balbo Andrea , Konrad Sheryl L. , Buchli Rico , Hildebrand William H. , Schuck Peter , Hudson Amy W.
Published: January 2014

The U21 gene product from human herpesvirus 7 binds to and redirects class I major histocompatibility complex (MHC) molecules to a lysosomal compartment. The molecular mechanism by which U21 reroutes class I MHC molecules to lysosomes is not known. Here, we have reconstituted the interaction between purified soluble U21 and class I MHC molecules, suggesting that U21 does not require additional cellular proteins to interact with class I MHC molecules. Our results demonstrate that U21, itself predicted to contain an MHC class I-like protein fold, interacts tightly with class I MHC molecules as a tetramer, in a 4:2 stoichiometry. These observations have helped to elucidate a refined model describing the mechanism by which U21 escorts class I MHC molecules to the lysosomal compartment.

IMPORTANCEIn this report, we show that the human herpesvirus 7 (HHV-7) immunoevasin U21, itself a class I MHC-like protein, binds with high affinity to class I MHC molecules as a tetramer and escorts them to lysosomes, where they are degraded. While many class I MHC-like molecules have been described in detail, this unusual viral class I-like protein functions as a tetramer, associating with class I MHC molecules in a 4:2 ratio, illuminating a functional significance of homooligomerization of a class I MHC-like protein.
Hantavirus GnT Elements Mediate TRAF3 Binding and Inhibit RIG-I/TBK1-Directed Beta Interferon Transcription by Blocking IRF3 Phosphorylation
Authors: Matthys Valery S. , Cimica Velasco , Dalrymple Nadine A. , Glennon Nicole B. , Bianco Chris , Mackow Erich R.
Published: January 2014

Hantaviruses successfully replicate in primary human endothelial cells by restricting the early induction of beta interferon (IFN-β) and interferon-stimulated genes (ISGs). Gn proteins from NY-1V, ANDV, and TULV, but not PHV, harbor elements in their 142-residue cytoplasmic tails (GnTs) that inhibit RIG-I/MAVS/TBK1-TRAF3-directed IFN-β induction. Here, we define GnT interactions and residues required to inhibit TRAF3-TBK1-directed IFN-β induction and IRF3 phosphorylation. We observed that GnTs bind TRAF3 via residues within the TRAF-N domain (residues 392 to 415) and that binding is independent of the MAVS-interactive TRAF-C domain (residues 415 to 568). We determined that GnT binding to TRAF3 is mediated by C-terminal degrons within NY-1V or ANDV GnTs and that mutations that add degrons to TULV or PHV GnTs confer TRAF3 binding. Further analysis of GnT domains revealed that TRAF3 binding is a discrete GnT function, independent of IFN regulation, and that residues 15 to 42 from the NY-1V GnT C terminus are required for inhibiting TBK1-directed IFN-β transcription. Mutagenesis of the NY-1V GnT revealed that altering tyrosine 627 (Y627A/S/F) abolished GnT regulation of RIG-I/TBK1-directed IRF3 phosphorylation and transcriptional responses of ISRE, κB, and IFN-β promoters. Moreover, GnTs from NY-1V, ANDV, and TULV, but not PHV, inhibited RIG-I-directed IRF3 phosphorylation. Collectively, these findings suggest a novel role for GnTs in regulating RIG-I/TBK1 pathway-directed IRF3 phosphorylation and IFN-β induction and define virulence determinants within GnTs that may permit the attenuation of pathogenic hantaviruses.

IMPORTANCE
These findings provide a mechanism for selected hantavirus GnT interactions to regulate RIG-I/TBK1 signaling responses required for IFN-β induction by inhibiting TBK1 phosphorylation of IRF3. These studies culminate in showing that a single GnT residue, Y627, is required for the NY-1V GnT to inhibit RIG-I/TBK1-directed IRF3 phosphorylation and IFN-β induction. These findings define a potential virulence determinant within the NY-1V GnT that may permit hantavirus attenuation.
Deletion of the Vaccinia Virus N2L Gene Encoding an Inhibitor of IRF3 Improves the Immunogenicity of Modified Vaccinia Virus Ankara Expressing HIV-1 Antigens
Authors: García-Arriaza Juan , Gómez Carmen E. , Sorzano Carlos Óscar S. , Esteban Mariano
Published: January 2014

A modified vaccinia virus Ankara poxvirus vector expressing the HIV-1 Env, Gag, Pol, and Nef antigens from clade B (MVA-B) is currently being tested in clinical trials. To improve its immunogenicity, we have generated and characterized the immune profile of MVA-B containing a deletion of the vaccinia viral gene
N2L
, which codes for an inhibitor of IRF3 (MVA-B ΔN2L). Deletion of
N2L
had no effect on virus growth kinetics or on the expression of HIV-1 antigens; hence, the N2 protein is not essential for MVA replication. The innate immune responses triggered by MVA-B ΔN2L revealed an increase in beta interferon, proinflammatory cytokines, and chemokines. Mouse prime-boost protocols showed that MVA-B ΔN2L improves the magnitude and polyfunctionality of HIV-1-specific CD4
+
and CD8
+
T cell adaptive and memory immune responses, with most of the HIV-1 responses mediated by CD8
+
T cells. In the memory phase, HIV-1-specific CD8
+
T cells with an effector phenotype were predominant and in a higher percentage with MVA-B ΔN2L than with MVA-B. In both immunization groups, CD4
+
and CD8
+
T cell responses were directed mainly against Env. Furthermore, MVA-B ΔN2L in the memory phase enhanced levels of antibody against Env. For the vector immune responses, MVA-B ΔN2L induced a greater magnitude and polyfunctionality of VACV-specific CD8
+
T memory cells than MVA-B, with an effector phenotype. These results revealed the immunomodulatory role of
N2L
, whose deletion enhanced the innate immunity and improved the magnitude and quality of HIV-1-specific T cell adaptive and memory immune responses. These findings are relevant for the optimization of poxvirus vectors as vaccines.

IMPORTANCE
On the basis of the limited efficacy of the RV144 phase III clinical trial, new optimized poxvirus vectors as vaccines against HIV/AIDS are needed. Here we have generated and characterized a new HIV/AIDS vaccine candidate on the basis of the poxvirus MVA vector expressing HIV-1 Env, Gag, Pol, and Nef antigens (MVA-B) and containing a deletion in the vaccinia virus
N2L
gene. Our findings revealed the immunomodulatory role of
N2L
and proved that its deletion from the MVA-B vector triggered an enhanced innate immune response in human macrophages and monocyte-derived dendritic cells. Furthermore, in immunized mice, MVA-B ΔN2L induced improvements in the magnitude and quality of adaptive and memory HIV-1-specific CD4
+
and CD8
+
T cell immune responses, together with an increase in the memory phase of levels of antibody against Env. Thus, the selective deletion of the
N2L
viral immunomodulatory gene is important for the optimization of MVA vectors as HIV-1 vaccines.
Dimerization of Flavivirus NS4B Protein
Authors: Zou Jing , Xie Xuping , Lee Le Tian , Chandrasekaran Ramya , Reynaud Aline , Yap Lijian , Wang Qing-Yin , Dong Hongping , Kang Congbao , Yuan Zhiming , Lescar Julien , Shi Pei-Yong
Published: January 2014

Flavivirus replication is mediated by a complex machinery that consists of viral enzymes, nonenzymatic viral proteins, and host factors. Many of the nonenzymatic viral proteins, such as NS4B, are associated with the endoplasmic reticulum membrane. How these membrane proteins function in viral replication is poorly understood. Here we report a robust method to express and purify dengue virus (DENV) and West Nile virus NS4B proteins. The NS4B proteins were expressed in
Escherichia coli
, reconstituted in dodecyl maltoside (DDM) detergent micelles, and purified to >95% homogeneity. The recombinant NS4B proteins dimerized
in vitro
, as evidenced by gel filtration, chemical cross-linking, and multiangle light scattering experiments. The dimeric form of NS4B was also detected when the protein was expressed alone in cells as well as in cells infected with DENV type 2 (DENV-2). Mutagenesis analysis showed that the cytosolic loop (amino acids 129 to 165) and the C-terminal region (amino acids 166 to 248) are responsible for NS4B dimerization.
trans
-Complementation experiments showed that (i) two genome-length RNAs containing distinct NS4B lethal mutations could not
trans
-complement each other, (ii) the replication defect of NS4B mutant RNA could be restored in cells containing DENV-2 replicons, and (iii) expression of wild-type NS4B protein alone was not sufficient to restore the replication of the NS4B mutant RNA. Collectively, the results indicate that
trans
-complementation of a lethal NS4B mutant RNA requires wild-type NS4B presented from a replication complex.

IMPORTANCE
The reported expression and purification system has made it possible to study the biochemistry and structure of flavivirus NS4B proteins. The finding of flavivirus NS4B dimerization and the mapping of regions important for NS4B dimerization provide the possibility to inhibit viral replication through blocking NS4B dimerization. The requirement of NS4B in the context of the replication complex for successful
trans
-complementation enhances our understanding of NS4B in flavivirus replication.
Activity of Hexokinase Is Increased by Its Interaction with Hepatitis C Virus Protein NS5A
Authors: Ramière Christophe , Rodriguez Jonathan , Enache Liviu S. , Lotteau Vincent , André Patrice , Diaz Olivier
Published: January 2014

The study of cellular central carbon metabolism modulations induced by viruses is an emerging field. Human cytomegalovirus (HCMV), herpes simplex virus (HSV), Kaposi's sarcoma-associated herpesvirus (KSHV), and hepatitis C virus (HCV) have been shown recently to reprogram cell metabolism to support their replication. During HCV infection the global glucidolipidic metabolism of hepatocytes is highly impacted. It was suggested that HCV might modify glucose uptake and glycolysis to increase fatty acids synthesis, but underlying mechanisms have not been completely elucidated. We thus investigated how HCV may modulate glycolysis. We observed that in infected Huh7.5 cells and in subgenomic replicon-positive Huh9.13 cells, glucose consumption as well as lactate secretion was increased. Using protein complementation assays and coimmunoprecipitation, we identified a direct interaction between the HCV NS5A protein and cellular hexokinase 2 (HK2), the first rate-limiting enzyme of glycolysis. NS5A expression was sufficient to enhance glucose consumption and lactate secretion in Huh7.5 cells. Moreover, determination of HK activity in cell homogenates revealed that addition of exogenous NS5A protein, either the full-length protein or its D2 or D3, but not D1, domain, was sufficient to increase enzyme activity. Finally, determination of recombinant HK2 catalytic parameters (
V
max
and

K
m

) in the presence of NS5A identified this viral protein as an activator of the enzyme. In summary, this study describes a direct interaction between HCV NS5A protein and cellular HK2 which is accompanied by an increase in HK2 activity that might contribute to an increased glycolysis rate during HCV infection.

IMPORTANCE
Substantial evidence indicates that viruses reprogram the central carbon metabolism of the cell to support their replication. Nevertheless, precise underlying mechanisms are poorly described. Metabolic pathways are structured as connected enzymatic cascades providing elemental biomolecular blocks necessary for cell life and viral replication. In this study, we observed an increase in glucose consumption and lactate secretion in HCV-infected cells, revealing higher glycolytic activity. We also identified an interaction between the HCV NS5A nonstructural protein and cellular hexokinase 2, the first rate-limiting enzyme of glycolysis. This interaction results in an enhancement of catalytic parameters of the enzyme, which might explain, at least in part, the aerobic glycolysis shift observed in HCV-infected cells.
T Cells Modulate Epstein-Barr Virus Latency Phenotypes during Infection of Humanized Mice
Authors: Heuts Frank , Rottenberg Martin E. , Salamon Daniel , Rasul Eahsan , Adori Monika , Klein George , Klein Eva , Nagy Noemi
Published: January 2014

Human B cells, the main target of Epstein-Barr virus (EBV), can display several types of latent viral protein expression, denoted 0, I, IIa, IIb, or III. Of these, only type III expression induces proliferation of cellsin vitro. These latency types are present at specific stages of infection and are also characteristic of different tumor types, but their generation is not fully understood. In this study, we analyzed the role of T cells in the regulation of EBV viral latency by using humanized NOD/SCID/IL2Rγ−/−mice. Several spleens presented macroscopic tumors 4 weeks after infection. Explanted spleen B cells from some of the EBV-infected mice proliferatedin vitro, but this was usually lowered when cyclosporine was added to the cultures. This suggested that thein vitrogrowth of EBV-infected B cells required T cell help; thus, cells other than type III cells were also present in the spleens. Quantitative PCR analysis of promoter activities specific for the different EBV latency types confirmed that in addition to type III cells, type IIa and type I cells were present in the spleen. The relative usage of the viral promoter specific for I and IIa latency types (Q promoter) was higher in CD8+cell-depleted mice, and it was absent from CD4+cell-depleted mice. These results indicate that CD4+T cells are necessary for the generation/maintenance of cells with latency I/IIa in the humanized mice. CD4+T cells contributed to this process through their CD40L expression.

IMPORTANCEAt primary infection with EBV, the infected B cells are proliferating and express viral proteins that have transforming potential. However, when the acute infection is resolved, in healthy individuals EBV is carried by a small fraction of B cells that express a restricted number of viral proteins unable to induce proliferation. Understanding the details of this transition is of fundamental importance. We studied this question in humanized mice by manipulating their different T cell compartments before and during infection with EBV. Our results indicate that CD4+T cells are responsible for the switch to a nonproliferating EBV program during primary infection with EBV.
Ecological and Genetic Determinants of Pepino Mosaic Virus Emergence
Authors: Moreno-Pérez Manuel G. , Pagán Israel , Aragón-Caballero Liliana , Cáceres Fátima , Fraile Aurora , García-Arenal Fernando
Published: January 2014

Virus emergence is a complex phenomenon, which generally involves spread to a new host from a wild host, followed by adaptation to the new host. Although viruses account for the largest fraction of emerging crop pathogens, knowledge about their emergence is incomplete. We address here the question of whether
Pepino Mosaic Virus
(PepMV) emergence as a major tomato pathogen worldwide could have involved spread from wild to cultivated plant species and host adaptation. For this, we surveyed natural populations of wild tomatoes in southern Peru for PepMV infection. PepMV incidence, genetic variation, population structure, and accumulation in various hosts were analyzed. PepMV incidence in wild tomatoes was high, and a strain not yet reported in domestic tomato was characterized. This strain had a wide host range within the
Solanaceae
, multiplying efficiently in most assayed
Solanum
species and being adapted to wild tomato hosts. Conversely, PepMV isolates from tomato crops showed evidence of adaptation to domestic tomato, possibly traded against adaptation to wild tomatoes. Phylogenetic reconstructions indicated that the most probable ancestral sequence came from a wild
Solanum
species. A high incidence of PepMV in wild tomato relatives would favor virus spread to crops and its efficient multiplication in different
Solanum
species, including tomato, allowing its establishment as an epidemic pathogen. Later, adaptation to tomato, traded off against adaptation to other
Solanum
species, would isolate tomato populations from those in other hosts.

IMPORTANCE
Virus emergence is a complex phenomenon involving multiple ecological and genetic factors and is considered to involve three phases: virus encounter with the new host, virus adaptation to the new host, and changes in the epidemiological dynamics. We analyze here if this was the case in the recent emergence of
Pepino Mosaic Virus
(PepMV) in tomato crops worldwide. We characterized a new strain of PepMV infecting wild tomato populations in Peru. Comparison of this strain with PepMV isolates from tomato crops, plus phylogenetic reconstructions, supports a scenario in which PepMV would have spread to crops from wild tomato relatives, followed by adaptation to the new host and eventually leading to population isolation. Our data, which derive from the analysis of field isolates rather than from experimental evolution approaches, significantly contribute to understanding of plant virus emergence, which is necessary for its anticipation and prevention.
Rice Stripe Tenuivirus NSvc2 Glycoproteins Targeted to the Golgi Body by the N-Terminal Transmembrane Domain and Adjacent Cytosolic 24 Amino Acids via the COP I- and COP II-Dependent Secretion Pathway
Authors: Yao Min , Liu Xiaofan , Li Shuo , Xu Yi , Zhou Yijun , Zhou Xueping , Tao Xiaorong
Published: January 2014

The NSvc2 glycoproteins encoded by
Rice stripe tenuivirus
(RSV) share many characteristics common to the glycoproteins found among
Bunyaviridae
. Within this viral family, glycoproteins targeting to the Golgi apparatus play a pivotal role in the maturation of the enveloped spherical particles. RSV particles, however, adopt a long filamentous morphology. Recently, RSV NSvc2 glycoproteins were shown to localize exclusively to the ER in Sf9 insect cells. Here, we demonstrate that the amino-terminal NSvc2 (NSvc2-N) targets to the Golgi apparatus in
Nicotiana benthamiana
cells, whereas the carboxyl-terminal NSvc2 (NSvc2-C) accumulates in the endoplasmic reticulum (ER). Upon coexpression, NSvc2-N redirects NSvc2-C from the ER to the Golgi bodies. The NSvc2 glycoproteins move together with the Golgi stacks along the ER/actin network. The targeting of the NSvc2 glycoproteins to the Golgi bodies was strictly dependent on functional anterograde traffic out of the ER to the Golgi bodies or on a retrograde transport route from the Golgi apparatus. The analysis of truncated and chimeric NSvc2 proteins demonstrates that the Golgi targeting signal comprises amino acids 269 to 315 of NSvc2-N, encompassing the transmembrane domain and 24 adjacent amino acids in the cytosolic tail. Our findings demonstrate for the first time that the glycoproteins from an unenveloped
Tenuivirus
could target Golgi bodies in plant cells.

IMPORTANCE
NSvc2 glycoprotein encoded by unenveloped
Rice stripe tenuivirus
(RSV) share many characteristics in common with glycoprotein found among
Bunyaviridae
in which all members have membrane-enveloped sphere particle. Recently, RSV NSvc2 glycoproteins were shown to localize exclusively to the ER in Sf9 insect cells. In this study, we demonstrated that the RSV glycoproteins could target Golgi bodies in plant cells. The targeting of NSvc2 glycoproteins to the Golgi bodies was dependent on active COP II or COP I. The Golgi targeting signal was mapped to the 23-amino-acid transmembrane domain and the adjacent 24 amino acids of the cytosolic tail of the NSvc2-N. In light of the evidence from viruses in
Bunyaviridae
that targeting Golgi bodies is important for the viral particle assembly and vector transmission, we propose that targeting of RSV glycoproteins into Golgi bodies in plant cells represents a physiologically relevant mechanism in the maturation of RSV particle complex for insect vector transmission.
The Majority of CD4
+
T-Cell Depletion during Acute Simian-Human Immunodeficiency Virus SHIV89.6P Infection Occurs in Uninfected Cells
Authors: Matrajt Laura , Younan Patrick M. , Kiem Hans-Peter , Schiffer Joshua T.
Published: January 2014

Untreated human immunodeficiency virus (HIV) infection is characterized by depletion of CD4
+
T cells, ultimately leading to the impairment of host immune defenses and death. HIV-infected CD4
+
T cells die from direct virus-induced apoptosis and CD8 T-cell-mediated elimination, but a broader and more profound depletion occurs in uninfected CD4
+
T cells via multiple indirect effects of infection. We fit mathematical models to data from experiments that tested an HIV eradication strategy in which five macaques with a proportion of CD4
+
T cells resistant to simian-human immunodeficiency virus (SHIV) entry were challenged with SHIV89.6P, a highly pathogenic dual-tropic chimeric SIV-HIV viral strain that results in rapid loss of both SHIV-susceptible and SHIV-resistant CD4
+
T cells. Our results suggest that uninfected (bystander) cell death accounts for the majority of CD4
+
T-lymphocyte loss, with at least 60% and 99% of CD4
+
T cell death occurring in uninfected cells during acute and established infection, respectively. Mechanisms to limit the profound indirect killing effects associated with HIV infection may be associated with immune preservation and improved long-term survival.

IMPORTANCE
HIV infection induces a massive depletion of CD4
+
T cells, leading to profound immunodeficiency, opportunistic infections, and eventually death. While HIV induces apoptosis (programmed cell death) by directly entering and replicating in CD4
+
T cells, uninfected CD4
+
T cells also undergo apoptosis due to ongoing toxic inflammation in the region of infection. In this paper, we use mathematical models in conjunction with data from simian-human immunodeficiency virus SHIV89.6P infection in macaques (a model of HIV infection in humans) to estimate the percentage of cell death that occurs in uninfected cells during the initial period of infection. We reveal that the vast majority of cell death occurs in these cells, which are not infected. The “bystander effects” that lead to enormous reductions in the number of uninfected CD4
+
T cells may be a target for future interventions that aim to limit the extent of damage caused by HIV.
Viral Entry of Hepatitis B and D Viruses and Bile Salts Transportation Share Common Molecular Determinants on Sodium Taurocholate Cotransporting Polypeptide
Authors: Yan Huan , Peng Bo , Liu Yang , Xu Guangwei , He Wenhui , Ren Bijie , Jing Zhiyi , Sui Jianhua , Li Wenhui
Published: January 2014

The liver bile acids transporter sodium taurocholate cotransporting polypeptide (NTCP) is responsible for the majority of sodium-dependent bile salts uptake by hepatocytes. NTCP also functions as a cellular receptor for viral entry of hepatitis B virus (HBV) and hepatitis D virus (HDV) through a specific interaction between NTCP and the pre-S1 domain of HBV large envelope protein. However, it remains unknown if these two functions of NTCP are independent or if they interfere with each other. Here we show that binding of the pre-S1 domain to human NTCP blocks taurocholate uptake by the receptor; conversely, some bile acid substrates of NTCP inhibit HBV and HDV entry. Mutations of NTCP residues critical for bile salts binding severely impair viral infection by HDV and HBV; to a lesser extent, the residues important for sodium binding also inhibit viral infection. The mutation S267F, corresponding to a single nucleotide polymorphism (SNP) found in about 9% of the East Asian population, renders NTCP without either taurocholate transporting activity or the ability to support HBV or HDV infection in cell culture. These results demonstrate that molecular determinants critical for HBV and HDV entry overlap with that for bile salts uptake by NTCP, indicating that viral infection may interfere with the normal function of NTCP, and bile acids and their derivatives hold the potential for further development into antiviral drugs.

IMPORTANCE
Human hepatitis B virus (HBV) and its satellite virus, hepatitis D virus (HDV), are important human pathogens. Available therapeutics against HBV are limited, and there is no drug that is clinically available for HDV infection. A liver bile acids transporter (sodium taurocholate cotransporting polypeptide [NTCP]) critical for maintaining homeostasis of bile acids serves as a functional receptor for HBV and HDV. We report here that the NTCP-binding lipopeptide that originates from the first 47 amino acids of the pre-S1 domain of the HBV L protein blocks taurocholate transport. Some bile salts dose dependently inhibit HBV and HDV infection mediated by NTCP; molecular determinants of NTCP critical for HBV and HDV entry overlap with that for bile acids transport. This work advances our understanding of NTCP-mediated HBV and HDV infection in relation to NTCP's physiological function. Our results also suggest that bile acids or their derivatives hold potential for development into novel drugs against HBV and HDV infection.