bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutualism between armoured scale insects and ants: new species and observations on a unique trophobiosis (Hemiptera: Diaspididae; Hymenoptera: Formicidae: MelissotarsusEmery)
Authors: SCHNEIDER SCOTT A. , GILIOMEE JAN H. , DOOLEY JOHN W. , NORMARK BENJAMIN B.
Published: August 2013

The association between African armoured scale insects (Hemiptera: Coccoidea: Diaspididae) and ants belonging to Melissotarsus Emery (Hymenoptera: Formicidae: Myrmicinae) is the only trophobiosis known in which ants do not receive honeydew or nectar in exchange for protection and other services. Food reward for the ants in this mutualism remains unknown, despite repeated suggestions that diaspidids are consumed by the associated ants, thus serving as ‘domestic cattle’. We describe new observations on interactions between Melissotarsus emeryi Santschi and the diaspidid Morganella conspicua (Brain) from South Africa. Worker ants exhibited previously undescribed tending behaviours, most notably a ‘squeezing and licking’ performed on an adult female diaspidid and ‘culling’, in which a worker removed an adult female armoured scale from the host plant. These could represent the gathering of secretory products and the cultivation of an individual for consumption, respectively. An ant exclusion study over 12 days of isolation showed that adult female diaspidids and second‐instar nymphs secreted no wax or exudates that attending ants would ordinarily collect. Workers of M. emeryi did not defend their nest against invading colonies of Crematogaster and other unidentified ants: we hypothesize that the primary mode of defence is maintenance of isolation within galleries. We describe three new ant‐associated diaspidid species: Affirmaspis cederbergensis Schneider sp.n. from South Africa, Diaspis doumtsopi Schneider sp.n. from Cameroon, and Melissoaspis incola Schneider sp.n. from Madagascar. Melissoaspis formicaria (Ben‐Dov) comb.n. is transferred from Morganella (Brain). Diagnostic characteristics for Melissoaspis Ben‐Dov are revised, and additional taxonomic information defining this genus allows ease of identification. An updated identification key to the species of ant‐associated diaspidids is provided.

This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:632C1122-954A-4DE6-B946-8E6AB6A4D27F.
Antenatal and delivery services in Kinshasa, Democratic Republic of Congo: care‐seeking and experiences reported by women in a household‐based survey
Authors: Feinstein Lydia , Dimomfu Bruno Lapika , Mupenda Bavon , Duvall Sandra , Chalachala Jean Lambert , Edmonds Andrew , Behets Frieda
Published: August 2013

Objectives

Increasing coverage of quality reproductive health services, including prevention of mother‐to‐child transmission services, requires understanding where and how these services are provided. To inform scale‐up, we conducted a population‐based survey in Kinshasa, Democratic Republic of Congo.

Methods

Stratified two‐stage cluster sampling was used to select women ≥18 years old who had been pregnant within the prior three years. Participants were interviewed about their reproductive healthcare utilization and impressions of services received.

Results

We interviewed 1221 women, 98% of whom sought antenatal care (ANC). 78% of women began ANC after the first trimester and 22% reported <4 visits. Reasons for choosing an ANC facility included reputation (51%), friendly/accessible staff (39%), availability of comprehensive services (29%), medication access (26%), location (26%), and cost (21%). Most women reported satisfactory treatment by staff, but 47% reported that the ANC provider ignored their complaints, 23% had difficulty understanding responses to their questions, 22% wanted more time with the provider, 21% wanted more privacy, and 12% felt uncomfortable asking questions. Only 56% reported someone talked to them about HIV/AIDS. Strongest predictors of seeking inadequate ANC included low participant and partner education and lack of certain assets. Only 32% of women sought postnatal care. Some results varied by health zone.

Conclusions

Scaling‐up interventions to improve reproductive health services should include broad‐based health systems strengthening and promote equitable access to quality ANC, delivery, and postnatal services. Personal and structural‐level barriers to seeking ANC need to be addressed, with consideration given to local contexts.
Retroelement insertions at the Medicago FTa1 locus in spring mutants eliminate vernalisation but not long‐day requirements for early flowering
Authors: Jaudal Mauren , Yeoh Chin C. , Zhang Lulu , Stockum Christine , Mysore Kirankumar S. , Ratet Pascal , Putterill Joanna
Published: August 2013

Molecular‐genetic control of the flowering time of temperate‐climate plants is best understood in Arabidopsis and the cereals wheat and barley. However, key regulators such as FLC and cereal VRN2 are not found in legumes. Therefore, we used forward genetics to identify flowering time genes in the model legume Medicago truncatula (Medicago) which is induced to flower by vernalisation and long‐day photoperiods. A screen of a Tnt1 retroelement tagging population yielded two mutants, spring2 and spring3, with a dominant early flowering phenotype. These mutants overexpress the floral activator FTa1 and two candidate downstream flowering genes SOC1a and FULb, similar to the spring1 somaclonal variant that we identified previously. We demonstrate here that an increase in the expression of FTa1, SOC1a and FULb and early flowering does not occur in all conditions in the spring mutants. It depends on long‐day photoperiods but not on vernalisation. Isolation of flanking sequence tags and linkage analysis identified retroelement insertions at FTa1 that co‐segregated with the early flowering phenotype in all three spring mutants. These were Tnt1 insertions in the FTa1 third intron (spring3) or the 3′ intergenic region (spring2) and an endogenous MERE1‐4 retroelement in the 3′ intergenic region in spring1. Thus the spring mutants form an allelic series of gain‐of‐function mutations in FTa1 which confer a spring growth habit. The spring retroelement insertions at FTa1 separate long‐day input from vernalisation input into FTa1 regulation, but this is not due to large‐scale changes in FTa1 DNA methylation or transcript processing in the mutants.
Tissue Interactions in Neural Crest Cell Development and Disease
Authors: Takahashi Yoshiko , Sipp Douglas , Enomoto Hideki
Published: August 2013

Neural Crest Development

The vertebrate neural crest is characterized by a migratory population of multipotent cells that spread out from the dorsal side of the neural tube. Many different cell types and tissues originate here, including cells of the peripheral nervous system, the adrenal medulla, melanocytes, and some skeletal cells. Dysregulation of neural crest cells can lead to defects in cell differentiation and the cell cycle, as well as to the formation of ectopic tissue, which can result in various human diseases.

Takahashi
et al.

(p.
860
) review the normal development of neural crest cells, highlighting important associations of this cell population with local environments to influence tissue interactions and function, and describe pathogenesis that results when developmental events go awry.
Microscopic Evidence for Liquid-Liquid Separation in Supersaturated CaCO
3
Solutions
Authors: Wallace Adam F. , Hedges Lester O. , Fernandez-Martinez Alejandro , Raiteri Paolo , Gale Julian D. , Waychunas Glenn A. , Whitelam Stephen , Banfield Jillian F. , De Yoreo James J.
Published: August 2013

Making Crystals

The initial transition from a disordered solution to the formation of nuclei that grow into crystals continues to be a puzzle. Recent experiments suggested the formation of stable ordered clusters that appear prior to the formation of the first nuclei.

Wallace
et al.

(p.
885
; see the Perspective by

Myerson and Trout

) used molecular dynamics to look at the potential structure and dynamics of these clusters and lattice gas simulations to explore the population dynamics of the cluster populations prior to nucleation. A liquid-liquid phase separation process was observed whereby one phase becomes more concentrated in ions and becomes the precursor for nuclei to form.
A Critical Crossroad for BLM's Wild Horse Program
Authors: Garrott Robert A. , Oli Madan K.
Published: August 2013

Captive wild horses will cost the United States over $1 billion by 2030 unless management approaches change.
The Health Risk of Obesity—Better Metrics Imperative
Authors: Ahima Rexford S. , Lazar Mitchell A.
Published: August 2013

The impact of a high BMI on mortality is in question, calling for a rethinking of how metabolic health is assessed.
Abundant Porewater Mn(III) Is a Major Component of the Sedimentary Redox System
Authors: Madison Andrew S. , Tebo Bradley M. , Mucci Alfonso , Sundby Bjørn , Luther George W.
Published: August 2013

Mn at Work

Biogeochemical cycling involves oxidation-reduction reactions with common metals like iron and manganese. Depending on the oxygen concentration and pH of the surrounding water, these metals—which exist in multiple oxidation states—can either accept or donate electrons; however, detecting which chemical species is dominant in these reactions often proves difficult. Using a spectrophotometric method sensitive to Mn oxidation state,

Madison
et al.

(p.
875
) show that up to 90% of the total Mn in porewater collected from sediment cores in the St. Lawrence Estuary is soluble Mn(III)—a phase traditionally assumed to be unimportant in aqueous redox reactions except in a few niche environments.
Diplomatic Flap Threatens E.U.-Israeli Research Ties
Authors: Enserink Martin
Published: August 2013

Israel may forgo participation in Horizon 2020 over dispute on occupied territories.
How NASA Tried to Save Its Prime Planet-Spotter
Authors: Bhattacharjee Yudhijit
Published: August 2013

With 130 extrasolar planets to its credit, NASA's Kepler space telescope was going strong—until mechanical breakdowns crippled it and left astronomers scrambling to find ways to keep the mission doing science.
Can China Age Gracefully? A Massive Survey Aims to Find Out
Authors: Hvistendahl Mara
Published: August 2013

A massive survey on aging will chart China's shift from a nation of young workers to one of elderly pensioners.
Haplotypes of theIL10Gene as Potential Protection Factors in Leprosy Patients
Authors: Garcia Patricia , Alencar Dayse , Pinto Pablo , Santos Ney , Salgado Claudio , Sortica Vinicius A. , Hutz Mara H. , Ribeiro-dos-Santos Ândrea , Santos Sidney
Published: August 2013

Leprosy is an infectious disease caused byMycobacterium lepraecharacterized by dermatoneurological signs and symptoms that has a large number of new cases worldwide. Several studies have associated interleukin 10 with susceptibility/resistance to several diseases. We investigated haplotypes formed by three single nucleotide polymorphisms (SNPs) located in theIL10gene (A-1082G, C-819T, and C-592A) in order to better understand the susceptibility to and severity of leprosy in an admixed northern Brazil population, taking into account estimates of interethnic admixture. We observed the genotypes ACC/ACC (P= 0.021, odds ratio [OR] [95% confidence interval (CI)] = 0.290 [0.085 to 0823]) and ACC/GCC (P= 0.003, OR [95% CI] = 0.220 [0.504 to 0.040]) presenting significant results for protection against leprosy development, framed in the profiles of low and medium interleukin production, respectively. Therefore, we suggest that genotypes A-1082G, C-819T, and C-592A formed by interleukin-10 polymorphisms are closely related to protection of the leprosy development in an admixed northern Brazil population, in particular ACC/ACC and ACC/GCC genotypes.
Randomized, Controlled, Multicenter Study of the Immunogenicity and Safety of a Fully Liquid Combination Diphtheria–Tetanus Toxoid–Five-Component Acellular Pertussis (DTaP5), Inactivated Poliovirus (IPV), and Haemophilus influenzae Type b (Hib) Vaccine Compared with a DTaP3-IPV/Hib Vaccine Administered at 3, 5, and 12 Months of Age
Authors: Vesikari Timo , Silfverdal Sven Arne , Boisnard Florence , Thomas Stéphane , Mwawasi Grace , Reynolds Donna
Published: August 2013

This study compared the levels of immunogenicity and safety of diphtheria–tetanus toxoid–five-component acellular pertussis (DTaP5), inactivated poliovirus (IPV), andHaemophilus influenzaetype b (Hib) (DTaP5-IPV-Hib) and DTaP3-IPV/Hib vaccines for study participants 3, 5, and 12 months of age. Post-dose 3 noninferiority criteria comparing DTaP5-IPV-Hib to DTaP3-IPV/Hib using rates of seroprotection were demonstrated against diphtheria, tetanus, and polio types 1 to 3, but not for polyribosylribitol phosphate (PRP). While PRP did not meet noninferiority criteria, the seroprotection rate and geometric mean concentration (GMC) were high, indicating a clinically robust immune response. GMCs or titers for other antigens (including pertussis) and the safety profiles were generally similar between groups. Fully liquid DTaP5-IPV-Hib can be administered using the 3-, 5-, and 12-month vaccination schedule. (This study has been registered at ClinicalTrials.gov under registration no. NCT00287092.)
Assessment of the Phenotype and Functionality of Porcine CD8 T Cell Responses following Vaccination with Live Attenuated Classical Swine Fever Virus (CSFV) and Virulent CSFV Challenge
Authors: Franzoni Giulia , Kurkure Nitin V. , Edgar Daniel S. , Everett Helen E. , Gerner Wilhelm , Bodman-Smith Kikki B. , Crooke Helen R. , Graham Simon P.
Published: August 2013

Vaccination with live attenuated classical swine fever virus (CSFV) induces solid protection after only 5 days, which has been associated with virus-specific T cell gamma interferon (IFN-γ) responses. In this study, we employed flow cytometry to characterize T cell responses following vaccination and subsequent challenge infections with virulent CSFV. The CD3+CD4−CD8hiT cell population was the first and major source of CSFV-specific IFN-γ. A proportion of these cells showed evidence for cytotoxicity, as evidenced by CD107a mobilization, and coexpressed tumor necrosis factor alpha (TNF-α). To assess the durability and recall of these responses, a second experiment was conducted where vaccinated animals were challenged with virulent CSFV after 5 days and again after a further 28 days. While virus-specific CD4 T cell (CD3+CD4+CD8α+) responses were detected, the dominant response was again from the CD8 T cell population, with the highest numbers of these cells being detected 14 and 7 days after the primary and secondary challenges, respectively. These CD8 T cells were further characterized as CD44hiCD62L−and expressed variable levels of CD25 and CD27, indicative of a mixed effector and effector memory phenotype. The majority of virus-specific IFN-γ+CD8 T cells isolated at the peaks of the response after each challenge displayed CD107a on their surface, and subpopulations that coexpressed TNF-α and interleukin 2 (IL-2) were identified. While it is hoped that these data will aid the rational design and/or evaluation of next-generation marker CSFV vaccines, the novel flow cytometric panels developed should also be of value in the study of porcine T cell responses to other pathogens/vaccines.
All-trans-Retinoic Acid Imprints Expression of the Gut-Homing Marker α4β7 while Suppressing Lymph Node Homing of Dendritic Cells
Authors: Evans Tristan I. , Reeves R. Keith
Published: August 2013

Tissue-directed trafficking of dendritic cells (DCs) as natural adjuvants and/or direct vaccine carriers is highly attractive for the next generation of vaccines and immunotherapeutics. Since these types of studies would undoubtedly be first conducted using nonhuman primate models, we evaluated the ability of all-trans-retinoic acid (ATRA) to induce gut-homing α4β7 expression on rhesus macaque plasmacytoid and myeloid DCs (pDCs and mDCs, respectively). Induction of α4β7 occurred in both a time-dependent and a dose-dependent manner with up to 8-fold increases for mDCs and 2-fold increases for pDCs compared to medium controls. ATRA treatment was also specific in inducing α4β7 expression, but not expression of another mucosal trafficking receptor, CCR9. Unexpectedly, upregulation of α4β7 was associated with a concomitant downregulation of CD62L, a marker of lymph node homing, indicating an overall shift in the trafficking repertoire. These same phenomena occurred with ATRA treatment of human and chimpanzee DCs, suggesting a conserved mechanism among primates. Collectively, these data serve as a first evaluation forex vivomodification of primate DC homing patterns that could later be used in reinfusion studies for the purposes of immunotherapeutics or mucosa-directed vaccines.
Comparison of 14 Molecular Assays for Detection of Mycobacterium tuberculosis Complex in Bronchoalveolar Lavage Fluid
Authors: Akkerman Onno W. , van der Werf Tjip S. , de Boer Maria , de Beer Jessica L. , Rahim Zeaur , Rossen John W. A. , van Soolingen Dick , Kerstjens Huib A. M. , van der Zanden Adri G. M.
Published: August 2013

We compared 14 molecular assays for their ability to detect the
Mycobacterium tuberculosis
complex in bronchoalveolar lavage fluid samples. Three approaches were followed. First, by using DNA from
Mycobacterium bovis
BCG, we determined the detection limits of the assays using routine molecular methods. Second, in order to determine the analytical sensitivities of the assays, we added one of four
M. tuberculosis
isolates with various numbers of the insertion sequence IS
6110
to
N
-acetyl-
l
-cysteine (NALC)-NaOH-treated bronchoalveolar lavage fluid samples in dilutions of 1:10 to 1:10,000,000. Third, intertest variabilities were measured and defined by the standard deviations for the quantitation cycle (Cq) values of three positive test results per dilution per assay. The 14 assays tested had similar analytical sensitivities, except for GeneXpert, which had an analytical sensitivity that was 10- to 100-fold lower than that of the other assays. The MP MTB/NTM test and the in-house TaqMan-10 revealed the best performances for the detection limit and had the highest analytical sensitivities. Most of the tests performed well regarding detection limit and analytical sensitivity for the detection of the
M. tuberculosis
complex in serial dilutions, and the differences were small. The MP MTB/NTM and the in-house TaqMan-10 assays revealed the best, and GeneXpert the worst, overall performances.
Rapid Detection of Gram-Positive Organisms by Use of the Verigene Gram-Positive Blood Culture Nucleic Acid Test and the BacT/Alert Pediatric FAN System in a Multicenter Pediatric Evaluation
Authors: Sullivan K. V. , Turner N. N. , Roundtree S. S. , Young S. , Brock-Haag C. A. , Lacey D. , Abuzaid S. , Blecker-Shelly D. L. , Doern C. D.
Published: August 2013

Assays that expedite the reporting of organism identification and antibiotic susceptibility status in positive blood cultures can fast track interventions that improve clinical outcomes. We evaluated the Verigene Gram-positive blood culture nucleic acid test (BC-GP) in two pediatric hospitals. Positive BacT/Alert Pediatric FAN blood cultures with Gram-positive organisms were tested using the BC-GP in tandem with routine laboratory procedures. To test organisms underrepresented in the clinical blood culture evaluation, blood culture bottles were spiked with diluted organism suspensions at concentrations of 10 to 100 CFU per milliliter. A total of 249 Gram-positive bacterial isolates were recovered from 242 blood cultures. The BC-GP detected
Staphylococcus aureus
, methicillin-susceptible
S. aureus
, and methicillin-resistant
S. aureus
with sensitivities of 100%, 99%, and 100% and specificities of 100%, 100%, and 99.5%, respectively. The BC-GP detected
Staphylococcus epidermidis
, methicillin-susceptible
S. epidermidis
, and methicillin-resistant
S. epidermidis
with sensitivities of 95%, 80%, and 96%, respectively, and 100% specificity. The BC-GP correctly identified 14/15 cases of
Enterococcus faecalis
and
Enterococcus faecium
bacteremia and 9 cases of
Streptococcus pneumoniae
. It misidentified 5/15 clinical blood cultures with
Streptococcus mitis
/
Streptococcus oralis
and 1/3 blood cultures spiked with
Streptococcus anginosus
group as
S. pneumoniae
. The BC-GP detected a case of
Streptococcus pyogenes
bacteremia but failed to detect 2/3 clinical blood cultures with
Streptococcus agalactiae
. BC-GP's rapid accurate detection of
Staphylococcus
spp.,
E. faecium
, and
E. faecalis
and its ability to ascertain
mecA
,
vanA
, and
vanB
status may expedite clinical decisions pertaining to optimal antibiotic use. False-positive
S. pneumoniae
results may warrant reporting of only “
Streptococcus
spp.” when this organism is reported by the BC-GP.
Emergence, Circulation, and Spatiotemporal Phylogenetic Analysis of Coxsackievirus A6- and Coxsackievirus A10-Associated Hand, Foot, and Mouth Disease Infections from 2008 to 2012 in Shenzhen, China
Authors: He Ya-Qing , Chen Long , Xu Wen-Bo , Yang Hong , Wang Han-Zhong , Zong Wen-Ping , Xian Hui-Xia , Chen Hui-Ling , Yao Xiang-Jie , Hu Zhang-Li , Luo Min , Zhang Hai-Long , Ma Han-Wu , Cheng Jin-Quan , Feng Qian-Jin , Zhao De-Jian
Published: August 2013

Sporadic hand, foot, and mouth disease (HFMD) outbreaks and other infectious diseases in recent years have frequently been associated with certain human enterovirus (HEV) serotypes. This study explored the prevalences and genetic characteristics of non-HEV71 and non-coxsackievirus A16 (CV-A16) human enterovirus-associated HFMD infections in Shenzhen, China. A total of 2,411 clinical stool specimens were collected from hospital-based surveillance for HFMD from 2008 to 2012. The detection of HEV was performed by real-time reverse transcription-PCR (RT-PCR) and RT-seminested PCR, and spatiotemporal phylogenetic analysis was performed based on the VP1 genes. A total of 1,803 (74.8%) strains comprising 28 different serotypes were detected. In the past 5 years, the predominant serotypes were HEV71 (60.0%), followed by CV-A16 (21.2%) and two uncommon serotypes, CV-A6 (13.0%) and CV-A10 (3.3%). However, CV-A6 replaced CV-A16 as the second most common serotype between 2010 and 2012. As an emerging pathogen, CV-A6 became as common a causative agent of HFMD as HEV71 in Shenzhen in 2012. Phylogenetic analysis revealed that little variation occurred in the Chinese HEV71 and CV-A16 strains. The genetic characteristics of the Chinese CV-A6 and CV-A10 strains displayed geographic differences. The CV-A6 and CV-A10 strains circulating in Shenzhen likely originated in Europe. It was found that human enteroviruses have a high mutation rate due to evolutionary pressure and frequent recombination (3.2 × 10−3to 6.4 ×10−3substitutions per site per year for HEV71, CV-A6, CV-A16, and CV-A10). Since certain serotypes are potential threats to the public health, this study provides further insights into the significance of the epidemiological surveillance of HFMD.
Development of a Loop-Mediated Isothermal Amplification Assay Targeting the
mpb64
Gene for Diagnosis of Intraocular Tuberculosis
Authors: Balne Praveen Kumar , Barik Manas Ranjan , Sharma Savitri , Basu Soumyava
Published: August 2013

A loop-mediated isothermal amplification (LAMP) assay targeting the
mpb64
gene for the diagnosis of intraocular tuberculosis was highly specific (100%), sensitive (85.7%), rapid, and easy to perform. The LAMP assay can be an alternative to conventional PCR for the diagnosis of ocular tuberculosis in resource-limited settings.
Pharmacodynamics of Doxycycline and Tetracycline against Staphylococcus pseudintermedius: Proposal of Canine-Specific Breakpoints for Doxycycline
Authors: Maaland Marit Gaastra , Papich Mark G. , Turnidge John , Guardabassi Luca
Published: August 2013

Doxycycline is a tetracycline that has been licensed for veterinary use in some countries, but no clinical breakpoints are available for veterinary pathogens. The objectives of this study were (i) to establish breakpoints for doxycycline and (ii) to evaluate the use of tetracycline as a surrogate to predict the doxycycline susceptibility of
Staphylococcus pseudintermedius
isolates. MICs and inhibition zone diameters were determined for 168 canine
S. pseudintermedius
isolates according to Clinical and Laboratory Standards Institute (CLSI) standards. Tetracycline resistance genes were detected by PCR, and time-kill curves were determined for representative strains.
In vitro
pharmacodynamic and target animal pharmacokinetic data were analyzed by Monte Carlo simulation (MCS) for the development of MIC interpretive criteria. Optimal zone diameter breakpoints were defined using the standard error rate-bounded method. The two drugs displayed bacteriostatic activity and bimodal MIC distributions. Doxycycline was more active than tetracycline in non-wild-type strains. MCS and target attainment analysis indicated a certainty of ≥90% for attaining an area under the curve (AUC)/MIC ratio of >25 with a standard dosage of doxycycline (5 mg/kg of body weight every 12 h) for strains with MICs of ≤0.125 μg/ml. Tetracycline predicted doxycycline susceptibility, but current tetracycline breakpoints were inappropriate for the interpretation of doxycycline susceptibility results. Accordingly, canine-specific doxycycline MIC breakpoints (susceptible, ≤0.125 μg/ml; intermediate, 0.25 μg/ml; resistant, ≥0.5 μg/ml) and zone diameter breakpoints (susceptible, ≥25 mm; intermediate, 21 to 24 mm; resistant, ≤20 mm) and surrogate tetracycline MIC breakpoints (susceptible, ≤0.25 μg/ml; intermediate, 0.5 μg/ml; resistant, ≥1 μg/ml) and zone diameter breakpoints (susceptible, ≥23 mm; intermediate, 18 to 22 mm; resistant, ≤17 mm) were proposed based on the data generated in this study.
Tuberculosis-Spoligo-Rifampin-Isoniazid Typing: an All-in-One Assay Technique for Surveillance and Control of Multidrug-Resistant Tuberculosis on Luminex Devices
Authors: Gomgnimbou Michel Kiréopori , Hernández-Neuta Iván , Panaiotov Stefan , Bachiyska Elizabeta , Palomino Juan Carlos , Martin Anandi , del Portillo Patricia , Refregier Guislaine , Sola Christophe
Published: August 2013

As a follow-up of the “spoligoriftyping” development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (
n
= 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (
n
= 162 for
rpoB
gene sequencing and
n
= 76 for
katG
and
inhA
sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding
Mycobacterium tuberculosis
complex (
n
= 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere.
Impact of Blood Volume, Tube Shaking, and Incubation Time on Reproducibility of QuantiFERON-TB Gold In-Tube Assay
Authors: Gaur Rajiv L. , Pai Madhukar , Banaei Niaz
Published: August 2013

Gamma interferon (IFN-γ) release assays (IGRAs) are functional assays used serially to measure the efficacy of novel tuberculosis (TB) vaccines and to screen health care workers for latent tuberculosis infection (LTBI). However, studies have shown nonreproducible IGRA results. In this study, we investigated the effects of blood volume (0.8, 1.0, and 1.2 ml), tube shaking (gentle versus vigorous), and incubation duration (16, 20, and 24 h) on the reproducibility of QuantiFERON-TB Gold In-Tube (QFT-GIT) results for 50 subjects (33 uninfected and 17 infected). The median IFN-γ TB response (TB antigen [Ag] minus nil value) was significantly higher with 0.8 ml blood (1.04 IU/ml) than with 1.0 ml (0.85 IU/ml;
P
= 0.002) or 1.2 ml (0.49 IU/ml;
P
< 0.001) for subjects with LTBI. Compared with 0.8 ml (11.8%), there were larger proportions of false-negative results with 1.0 ml (29.4%;
P
= 0.2) and 1.2 ml (41.2%;
P
= 0.05) of blood for infected subjects. Blood volume did not significantly change the proportions of positive results in uninfected controls. Compared with gentle shaking, vigorous shaking increased the median IFN-γ response in nil (0.04 versus 0.06 IU/ml;
P
< 0.001) and TB Ag (0.12 versus 0.24 IU/ml;
P
= 0.004) tubes and increased TB responses (TB Ag
vigorous
minus nil
gentle
) (0.02 versus 0.08 IU/ml;
P
= 0.004). The duration of incubation did not have a significant impact on the proportion of positive results in uninfected or infected subjects. This study identified blood volume and tube shaking as novel preanalytical sources of variability which require further standardization in order to improve the quality and reproducibility of QFT-GIT results.
Gallibacterium anatis Bacteremia in a Human
Authors: Aubin Guillaume Ghislain , Haloun Alain , Treilhaud Michèle , Reynaud Alain , Corvec Stéphane
Published: August 2013

We describe the first case of bacteremia due to
Gallibacterium anatis
. The patient, a 26-year-old woman, developed bacteremia and diarrhea. The origin of infection was possibly due to a diet contaminated by
G. anatis
in this highly immunocompromised patient.
Molecular Characterization of Herpes Simplex Virus 2 Strains by Analysis of Microsatellite Polymorphism
Authors: Burrel Sonia , Ait-Arkoub Zaïna , Voujon Delphine , Deback Claire , Abrao Emiliana P. , Agut Henri , Boutolleau David
Published: August 2013

The complete 154-kbp linear double-stranded genomic DNA sequence of herpes simplex virus 2 (HSV-2), consisting of two extended regions of unique sequences bounded by a pair of inverted repeat elements, was published in 1998 and since then has been widely employed in a wide range of studies. Throughout the HSV-2 genome are scattered 150 microsatellites (also referred to as short tandem repeats) of 1- to 6-nucleotide motifs, mainly distributed in noncoding regions. Microsatellites are considered reliable markers for genetic mapping to differentiate herpesvirus strains, as shown for cytomegalovirus and HSV-1. The aim of this work was to characterize 12 polymorphic microsatellites within the HSV-2 genome by use of 3 multiplex PCR assays in combination with length polymorphism analysis for the rapid genetic differentiation of 56 HSV-2 clinical isolates and 2 HSV-2 laboratory strains (gHSV-2 and MS). This new system was applied to a specific new HSV-2 variant recently identified in HIV-1-infected patients originating from West Africa. Our results confirm that microsatellite polymorphism analysis is an accurate tool for studying the epidemiology of HSV-2 infections.
Detection of Influenza H7N9 Virus: All Molecular Tests Are Not Equal
Authors: Hatchette Todd F. , Drews Steven J. , Bastien Nathalie , Li Yan , German Gregory , Antonishyn Nick , Charest Hugues , Mazzulli Tony , Fonseca Kevin , Krajden Mel , Petric Martin , Dust Kerry , LeBlanc Jason J.
Published: August 2013

The recent emergence of influenza A virus (H7N9) emphasizes the need for its rapid detection. While commercial nucleic acid amplification tests (NAATs) are commonly used to detect seasonal influenza virus, this study demonstrated that the analytical sensitivity of commercial assays is highly variable compared to that of CDC-based in-house NAATs for the detection of H7N9.
Is Hydrogen Cyanide a Marker of Burkholderia cepacia Complex?
Authors: Gilchrist Francis J. , Sims Hayley , Alcock Alice , Jones Andrew M. , Bright-Thomas Rowland J. , Smith David , Španĕl Patrik , Webb A. Kevin , Lenney Warren
Published: August 2013

Biofilm cultures of
Burkholderia cepacia
complex (BCC) infection have been found to generate the nonvolatile cyanide ion. We investigated if gaseous hydrogen cyanide (HCN) was a marker of BCC infection. Selected ion flow tube mass spectrometry analysis showed HCN was not elevated in the headspace of planktonic or biofilm cultures or in the exhaled breath of adult cystic fibrosis patients with chronic BCC infection. HCN is therefore not an
in vitro
or
in vivo
marker of BCC.
Refrex-1, a Soluble Restriction Factor against Feline Endogenous and Exogenous Retroviruses
Authors: Ito Jumpei , Watanabe Shinya , Hiratsuka Takahiro , Kuse Kyohei , Odahara Yuka , Ochi Haruyo , Kawamura Maki , Nishigaki Kazuo
Published: August 2013

The host defense against viral infection is acquired during the coevolution or symbiosis of the host and pathogen. Several cellular factors that restrict retroviral infection have been identified in the hosts. Feline leukemia virus (FeLV) is a gammaretrovirus that is classified into several receptor interference groups, including a novel FeLV-subgroup D (FeLV-D) that we recently identified. FeLV-D is generated by transduction of the
env
gene of feline endogenous gammaretrovirus of the domestic cat (ERV-DCs) into FeLV. Some ERV-DCs are replication competent viruses which are present and hereditary in cats. We report here the determination of new viral receptor interference groups and the discovery of a soluble antiretroviral factor, termed Refrex-1. Detailed analysis of FeLV-D strains and ERV-DCs showed two receptor interference groups that are distinct from other FeLV subgroups, and Refrex-1 specifically inhibited one of them. Refrex-1 is characterized as a truncated envelope protein of ERV-DC and includes the N-terminal region of surface unit, which is a putative receptor-binding domain, but lacks the transmembrane region. Refrex-1 is efficiently secreted from the cells and appears to cause receptor interference extracellularly. Two variants of Refrex-1 encoded by provirus loci, ERV-DC7 and DC16, are expressed in a broad range of feline tissues. The host retains Refrex-1 as an antiretroviral factor, which may potentially prevent reemergence of the ERVs and the emergence of novel ERV-related viruses in cats. Refrex-1 may have been acquired during endogenization of ERV-DCs and may play an important role in retroviral restriction and antiviral defense in cats.
Neutralizing IgG at the Portal of Infection Mediates Protection against Vaginal Simian/Human Immunodeficiency Virus Challenge
Authors: Klein Katja , Veazey Ronald S. , Warrier Ranjit , Hraber Peter , Doyle-Meyers Lara A. , Buffa Viviana , Liao Hua-Xin , Haynes Barton F. , Shaw George M. , Shattock Robin J.
Published: August 2013

Neutralizing antibodies may have critical importance in immunity against human immunodeficiency virus type 1 (HIV-1) infection. However, the amount of protective antibody needed at mucosal surfaces has not been fully established. Here, we evaluated systemic and mucosal pharmacokinetics (PK) and pharmacodynamics (PD) of 2F5 IgG and 2F5 Fab fragments with respect to protection against vaginal challenge with simian-human immunodeficiency virus-BaL in macaques. Antibody assessment demonstrated that 2F5 IgG was more potent than polymeric forms (IgM and IgA) across a range of cellular and tissue models. Vaginal challenge studies demonstrated a dose-dependent protection for 2F5 IgG and no protection with 2F5 Fab despite higher vaginal Fab levels at the time of challenge. Animals receiving 50 or 25 mg/kg of body weight 2F5 IgG were completely protected, while 3/5 animals receiving 5 mg/kg were protected. In the control animals, infection was established by a minimum of 1 to 4 transmitted/founder (T/F) variants, similar to natural human infection by this mucosal route; in the two infected animals that had received 5 mg 2F5 IgG, infection was established by a single T/F variant. Serum levels of 2F5 IgG were more predictive of sterilizing protection than measured vaginal levels. Fc-mediated antiviral activity did not appear to influence infection of primary target cells in cervical explants. However, PK studies highlighted the importance of the Fc portion in tissue biodistribution. Data presented in this study may be important in modeling serum levels of neutralizing antibodies that need to be achieved by either vaccination or passive infusion to prevent mucosal acquisition of HIV-1 infection in humans.
KLRG1 Negatively Regulates Natural Killer Cell Functions through the Akt Pathway in Individuals with Chronic Hepatitis C Virus Infection
Authors: Wang Jia M. , Cheng Yong Q. , Shi Lei , Ying Ruo S. , Wu Xiao Y. , Li Guang Y. , Moorman Jonathan P. , Yao Zhi Q.
Published: August 2013

In this study, we demonstrate that killer cell lectin-like receptor subfamily G member 1 (KLRG1), a transmembrane protein preferentially expressed on T cells, is highly expressed on CD56
+
NK cells, which are significantly reduced in their numbers and functions in the peripheral blood of patients with chronic hepatitis C virus (HCV) infection compared to subjects without infection. KLRG1 expression is also upregulated on healthy NK cells exposed to Huh-7 hepatocytes infected with HCV
in vitro
. Importantly, the expression levels of KLRG1 are inversely associated with the capacity of NK cells to proliferate and to produce gamma interferon (IFN-γ) but positively associated with apoptosis of NK cells in response to inflammatory cytokine stimulation. KLRG1
+
NK cells, including CD56
bright
and CD56
dim
subsets, exhibit impaired cell activation and IFN-γ production but increased apoptosis compared to KLRG1
−
NK cells, particularly in HCV-infected individuals. Importantly, blockade of KLRG1 signaling significantly recovered the impaired IFN-γ production by NK cells from HCV-infected subjects. Blockade of KLRG1 also enhanced the impaired phosphorylation of Akt (Ser473) in NK cells from HCV-infected subjects. Taken together, these results indicate that KLRG1 negatively regulates NK cell numbers and functions via the Akt pathway, thus providing a novel marker and therapeutic target for HCV infection.
Global RNA Structure Analysis of Poliovirus Identifies a Conserved RNA Structure Involved in Viral Replication and Infectivity
Authors: Burrill Cecily P. , Westesson Oscar , Schulte Michael B. , Strings Vanessa R. , Segal Mark , Andino Raul
Published: August 2013

The genomes of RNA viruses often contain RNA structures that are crucial for translation and RNA replication and may play additional, uncharacterized roles during the viral replication cycle. For the picornavirus family member poliovirus, a number of functional RNA structures have been identified, but much of its genome, especially the open reading frame, has remained uncharacterized. We have now generated a global RNA structure map of the poliovirus genome using a chemical probing approach that interrogates RNA structure with single-nucleotide resolution. In combination with orthogonal evolutionary analyses, we uncover several conserved RNA structures in the open reading frame of the viral genome. To validate the ability of our global analyses to identify functionally important RNA structures, we further characterized one of the newly identified structures, located in the region encoding the RNA-dependent RNA polymerase, 3D
pol
, by site-directed mutagenesis. Our results reveal that the structure is required for viral replication and infectivity, since synonymous mutants are defective in these processes. Furthermore, these defects can be partially suppressed by mutations in the viral protein 3C
pro
, which suggests the existence of a novel functional interaction between an RNA structure in the 3D
pol
-coding region and the viral protein(s) 3C
pro
and/or its precursor 3CD
pro
.
Uptake of Rabies Virus into Epithelial Cells by Clathrin-Mediated Endocytosis Depends upon Actin
Authors: Piccinotti Silvia , Kirchhausen Tomas , Whelan Sean P. J.
Published: August 2013

Rabies virus (RABV) causes a fatal zoonotic encephalitis. Disease symptoms require replication and spread of the virus within neuronal cells; however, in infected animals as well as in cell culture the virus replicates in a broad range of cell types. Here we use a single-cycle RABV and a recombinant vesicular stomatitis virus (rVSV) in which the glycoprotein (G) was replaced with that of RABV (rVSV RABV G) to examine RABV uptake into the African green monkey kidney cell line BS-C-1. Combining biochemical studies and real-time spinning-disk confocal fluorescence microscopy, we show that the predominant entry pathway of RABV particles into BS-C-1 cells is clathrin dependent. Viral particles enter cells in pits with elongated structures and incomplete clathrin coats which depend upon actin to complete the internalization process. By measuring the time of internalization and the abundance of the clathrin adaptor protein AP2, we further show that the pits that internalize RABV particles are similar to those that internalize VSV particles. Pharmacological perturbations of dynamin or of actin polymerization inhibit productive infection, linking our observations on particle uptake with viral infectivity. This work extends to RABV particles the finding that clathrin-mediated endocytosis of rhabdoviruses proceeds through incompletely coated pits which depend upon actin.
Gag-Dependent Enrichment of HIV-1 RNA near the Uropod Membrane of Polarized T Cells
Authors: Hatch Steven C. , Sardo Luca , Chen Jianbo , Burdick Ryan , Gorelick Robert , Fivash Matthew J. , Pathak Vinay K. , Hu Wei-Shau
Published: August 2013

The enrichment of HIV-1 macromolecules at the uropod of polarized T cells can significantly promote virus assembly and cell-mediated infection. Using live-cell fluorescence microscopy, we demonstrate that full-length HIV-1 RNA is enriched at the uropod membrane; furthermore, the presence of HIV-1 Gag containing a functional nucleocapsid domain is necessary for this HIV-1 RNA enrichment. The results from these studies provide novel insights into the mechanism of HIV-1 replication in polarized T cells.
A Functional Interaction between gp41 and gp120 Is Observed for Monomeric but Not Oligomeric, Uncleaved HIV-1 Env gp140
Authors: Guttman Miklos , Lee Kelly K.
Published: August 2013

The envelope glycoprotein (Env) is the sole antigenic feature on the surface of HIV and the target for the humoral immune system. Soluble, uncleaved gp140 Env constructs truncated at the transmembrane domain are being investigated intensively as potential vaccine immunogens by many groups, and understanding their structural properties is essential. We used hydrogen/deuterium-exchange mass spectrometry and small-angle X-ray scattering to probe structural order in a panel of commonly used gp140 constructs and matched gp120 monomers. We observed that oligomeric forms of uncleaved gp140, generally presumed to be trimeric, contain a protease-resistant form of gp41 akin to the postfusion, helical bundle conformation and appear to lack specific interactions between gp120 and gp41. In contrast, the monomeric form of gp140 shows significant stabilization of the gp120 inner domain imparted by the gp41 region, demonstrating excellent agreement with past mutagenesis studies. Moreover, the gp140 monomers respond to CD4 binding in manner that is consistent with the initial stages of Env activation: CD4 binding induces structural ordering throughout gp120 while loosening its association with gp41. The results indicate that uncleaved gp140 oligomers do not represent an authentic prefusion form of Env, whereas gp140 monomers isolated from the same glycoprotein preparations in many ways exhibit function and internal structural order that are consistent with expectations for certain aspects of native Env. gp140 monomers may thus be a useful reagent for advancing structural and functional studies.
Multiple Heparan Sulfate Binding Site Engagements Are Required for the Infectious Entry of Human Papillomavirus Type 16
Authors: Richards Kathleen F. , Bienkowska-Haba Malgorzata , Dasgupta Jhimli , Chen Xiaojiang S. , Sapp Martin
Published: August 2013

Human papillomavirus (HPV) entry is accompanied by multiple receptor-induced conformational changes (CCs) affecting both the major and minor capsid proteins, L1 and L2. Interaction of heparan sulfate (HS) with L1 is essential for successful HPV16 entry. Recently, cocrystallization of HPV16 with heparin revealed four distinct binding sites. Here we characterize mutant HPV16 to delineate the role of engagement with HS binding sites during infectious internalization. Site 1 (Lys278, Lys361), which mediates primary binding, is sufficient to trigger an L2 CC, exposing the amino terminus. Site 2 (Lys54, Lys356) and site 3 (Asn57, Lys59, Lys442, Lys443) are engaged following primary attachment and are required for infectious entry. Site 2 mutant particles are efficiently internalized but fail to undergo an L1 CC on the cell surface and subsequent uncoating in the endocytic compartment. After initial attachment to the cell, site 3 mutants undergo L1 and L2 CCs and then accumulate on the extracellular matrix (ECM). We conclude that the induction of CCs following site 1 and site 2 interactions results in reduced affinity for the primary HS binding site(s) on the cell surface, which allows engagement with site 3. Taken together, our findings suggest that HS binding site engagement induces CCs that prepare the virus for downstream events, such as the exposure of secondary binding sites, CCs, transfer to the uptake receptor, and uncoating.
Processing of the VP1/2A Junction Is Not Necessary for Production of Foot-and-Mouth Disease Virus Empty Capsids and Infectious Viruses: Characterization of “Self-Tagged” Particles
Authors: Gullberg Maria , Polacek Charlotta , Bøtner Anette , Belsham Graham J.
Published: August 2013

The foot-and-mouth disease virus (FMDV) capsid protein precursor, P1-2A, is cleaved by 3C
pro
to generate VP0, VP3, VP1, and the peptide 2A. The capsid proteins self-assemble into empty capsid particles or viruses which do not contain 2A. In a cell culture-adapted strain of FMDV (O1 Manisa [Lindholm]), three different amino acid substitutions (E83K, S134C, and K210E) were identified within the VP1 region of the P1-2A precursor compared to the field strain (wild type [wt]). Expression of the O1 Manisa P1-2A (wt or with the S134C substitution in VP1) plus 3C
pro
, using a transient expression system, resulted in efficient capsid protein production and self-assembly of empty capsid particles. Removal of the 2A peptide from the capsid protein precursor had no effect on capsid protein processing or particle assembly. However, modification of E83K alone abrogated particle assembly with no apparent effect on protein processing. Interestingly, the K210E substitution, close to the VP1/2A junction, completely blocked processing by 3C
pro
at this cleavage site, but efficient assembly of “self-tagged” empty capsid particles, containing the uncleaved VP1-2A, was observed. These self-tagged particles behaved like the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction is not essential for virus viability. The production of such engineered self-tagged empty capsid particles may facilitate their purification for use as diagnostic reagents and vaccines.