bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple Perspectives on Functional Coherence: Heterogeneity and Multiplexity in the Randstad
Authors: Burger Martijn J. , Meijers Evert J. , van Oort Frank G.
Published: December 2013

Measuring functional coherence in metropolitan regions, in particular polycentric ones, requires taking the issues of multiplexity and individual level heterogeneity more explicitly into account, as the spatial organisation of functional linkages is not necessarily identical. Based on the analysis of one type of functional linkage a region can appear to be integrated, but loosely connected based on another. We demonstrate the occurrence of multiplexity for the polycentric Dutch Randstad region, and focus particularly on the presence of individual level heterogeneity. Commuting patterns in the Randstad are strongly related to socio‐demographic variables and firm characteristics largely determine the scale of buyer‐supplier relationships. We present studies into functional coherence in the Randstad, and assess whether methods, including the interlocking network model, are sufficiently capable of accounting for multiplexity and individual level heterogeneity.
Market Reforms and Commodity Price Volatility: The Case of East African Coffee Market
Authors: Lukanima Benedicto , Swaray Raymond
Published: December 2013

The goal of this paper is to examine the impact of commodity market reforms on producer price volatility using evidence from the East African coffee market. The results, based on time‐varying volatility models and key summary statistics, show that coffee market reforms in the East African Community (EAC) are associated with changes in producer price volatility and volatility persistence at both country and regional levels. However, reforms were not the only cause of changes in price volatility. The study further shows that reforms had different effects on prices volatilities of Arabica and Robusta varieties of coffee grown in individual EAC countries. These findings have wider implication for commodity market reforms and producer price stabilisation policies in the EAC and coffee producing countries in sub‐Sahara Africa.
International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology: Cancun report (2012)
Authors: Storry J. R. , Castilho L. , Daniels G. , Flegel W. A. , Garratty G. , de Haas M. , Hyland C. , Lomas‐Francis C. , Moulds J. M. , Nogues N. , Olsson M. L. , Poole J. , Reid M. E. , Rouger P. , van der Schoot E. , Scott M. , Tani Y. , Yu L.‐C. , Wendel S. , Westhoff C. , Yahalom V. , Zelinski T.
Published: December 2013

The International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology convened during the International congress in Cancun, July 2012. This report details the newly identified antigens in existing blood group systems and presents three new blood group systems.
Effects of operational parameters on the treatment of nitrate‐rich wastewater by autohydrogenotrophic denitrifying bacteria
Authors: Mousavi Seyyedalireza , Ibrahim Shaliza , Aroua Mohamed Kheireddine
Published: December 2013

The effects of the C/N ratio, as well as hydrogen gas and substrate concentrations, on the treatment of nitrate‐rich wastewater by autohydrogenotrophic denitrifying bacteria in sequencing batch reactors were investigated. Among the various C/N ratios applied in this study, a C/N ratio of 4 was found to be the most suitable ratio that resulted in faster growth and adaptation. The denitrification rate improved with less accumulation of nitrite with increasing H2 sparging time. Moreover, an increase in nitrate concentration resulted in high nitrite accumulation. The nitrite accumulated to 601 mg/L NO2‐N when the nitrate concentration was 1000 mg/L NO3–‐N. The specific degradation rates for nitrate and nitrite were found to be 27.8 mg NO3−‐N/g MLVSS/d and 50 mg NO2−‐N/g MLVSS/d, respectively, when the C/N ratio was 2, and H2 as electron donor was sparged for 1 min with 2 h interval.
Stiffness of Parallel Manipulators With Serially Connected Legs
Authors: Sung Kim Han , Lipkin Harvey
Published: December 2013

A simple yet general method determines the stiffness matrix for parallel manipulators with serially connected legs. Link and actuator flexibilities for each leg are modeled by flexibility matrices that are additive. The effect of passive joints is implicitly included using reciprocal screws to yield a leg stiffness matrix that is generally singular. Since the legs act in parallel the leg stiffnesses are additive and yield the manipulator stiffness. The method is applicable to overconstrained, exactly constrained, and underconstrained robots in generic or singular configurations. It is illustrated using the Tricept robot containing a passive constraint leg and an overconstrained translating manipulator. Numerical results are confirmed using commercial structural analysis software.
Atmospheric pressure spatial atomic layer deposition web coating within situmonitoring of film thickness
Authors: Yersak Alexander S. , Lee Yung C. , Spencer Joseph A. , Groner Markus D.
Published: December 2013

Spectral reflectometry was implemented as a method for in situ thickness monitoring in a spatial atomic layer deposition (ALD) system. Al2O3 films were grown on a moving polymer web substrate at 100 °C using an atmospheric pressure ALD web coating system, with film growth of 0.11–0.13 nm/cycle. The modular coating head design and the in situ monitoring allowed for the characterization and optimization of the trimethylaluminum and water precursor exposures, purge flows, and web speed. A thickness uniformity of ±2% was achieved across the web. ALD cycle times as low as 76 ms were demonstrated with a web speed of 1 m/s and a vertical gap height of 0.5 mm. This atmospheric pressure ALD system with in situ process control demonstrates the feasibility of low-cost, high throughput roll-to-roll ALD.
Reactor concepts for atomic layer deposition on agitated particles: A review
Authors: Longrie Delphine , Deduytsche Davy , Detavernier Christophe
Published: December 2013

The number of possible applications for nanoparticles has strongly increased in the last decade. For many applications, nanoparticles with different surface and bulk properties are necessary. A popular surface modification technique is coating the particle surface with a nanometer thick layer. Atomic layer deposition (ALD) is known as a reliable method for depositing ultrathin and conformal coatings. In this article, agitation or fluidization of the particles is necessary for performing ALD on (nano)particles. The principles of gas fluidization of particles will be outlined, and a classification of the gas fluidization behavior of particles based on their size and density will be given. Following different reactor concepts that have been designed to conformally coat (nano)particles with ALD will be described, and a concise overview will be presented of the work that has been performed with each of them ending with a concept reactor for performing spatial ALD on fluidized particles.
Immunoproteomic Analysis of Antibody in Lymphocyte Supernatant in Patients with Typhoid Fever in Bangladesh
Authors: Charles Richelle C. , Liang Li , Khanam Farhana , Sayeed M. Abu , Hung Chris , Leung Daniel T. , Baker Stephen , Ludwig Albrecht , Harris Jason B. , LaRocque Regina C. , Calderwood Stephen B. , Qadri Firdausi , Felgner Philip L. , Ryan Edward T.
Published: December 2013

We have previously shown that an assay based on detection of anti-Salmonella entericaserotype Typhi antibodies in supernatant of lymphocytes harvested from patients presenting with typhoid fever (antibody in lymphocyte supernatant [ALS] assay) can identify 100% of patients with blood culture-confirmed typhoid fever in Bangladesh. In order to define immunodominant proteins within theS. Typhi membrane preparation used as antigen in these prior studies and to identify potential biomarkers unique toS. Typhi bacteremic patients, we probed microarrays containing 2,724S. Typhi proteins with ALS collected at the time of clinical presentation from 10 Bangladeshis with acute typhoid fever. We identified 62 immunoreactive antigens when evaluating both the IgG and IgA responses. Immune responses to 10 of these antigens discriminated between individuals with acute typhoid infection and healthy control individuals from areas where typhoid infection is endemic, as well as Bangladeshi patients presenting with fever who were subsequently confirmed to have a nontyphoid illness. Using an ALS enzyme-linked immunosorbent assay (ELISA) format and purified antigen, we then confirmed that immune responses against the antigen with the highest immunoreactivity (hemolysin E [HlyE]) correctly identified individuals with acute typhoid or paratyphoid fever in Dhaka, Bangladesh. These observations suggest that purified antigens could be used with ALS and corresponding acute-phase activated B lymphocytes in diagnostic platforms to identify acutely infected patients, even in areas where enteric fever is endemic.
Mutations in Toll-Like Receptor 3 Are Associated with Elevated Levels of Rotavirus-Specific IgG Antibodies in IgA-Deficient but Not IgA-Sufficient Individuals
Authors: Günaydın Gökçe , Nordgren Johan , Svensson Lennart , Hammarström Lennart
Published: December 2013

Double-stranded RNA (dsRNA) triggers immune-mediated responses through toll-like receptor 3 (TLR3), which is involved in innate antiviral defense. Low expression of TLR3 was recently suggested to contribute to susceptibility to rotavirus infection. Thus, we investigated the role of two TLR3 polymorphisms (rs3775291 and rs5743305), both of which resulted in reduced protein function or expression, in healthy blood donors and IgA-deficient (IgAD) individuals. These polymorphisms were associated with elevated rotavirus-specific IgG titers in IgAD individuals but not in healthy individuals. Thus, we propose that TLR3 signaling does not contribute to the rotavirus-specific antibody response in IgA-sufficient individuals, whereas it is associated with elevated antibody titers in IgAD individuals.
Time to Detection of Mycobacterium tuberculosis Using the MGIT 320 System Correlates with Colony Counting in Preclinical Testing of New Vaccines
Authors: Kolibab K. , Yang A. , Parra M. , Derrick S. C. , Morris S. L.
Published: December 2013

Clinical studies have suggested that the enumeration of mycobacteria by using automated liquid systems is a faster and simpler alternative to quantitative cultures. Here, we show that the time to detection ofM. tuberculosisgrowth as measured with the MGIT 320 liquid culture system inversely correlates with CFU determinations from culture on solid media and that mycobacterial quantification using the MGIT system is faster and easier to perform than CFU plating.
Draft Genome Sequence of Pseudomonas putida Strain S610, a Seed-Borne Bacterium of Wheat
Authors: Wang Dongping , Han Cliff S. , Dichosa Armand E. K. , Gleasner Cheryl D. , Johnson Shannon L. , Daligault Hajnalka E. , Davenport Karen W. , Li Po-E , Pierson Elizabeth A. , Pierson Leland S.
Published: December 2013

We report the genome sequence of a seed-borne bacterium,
Pseudomonas putida
strain S610. The size of the draft genome sequence is approximately 4.6 Mb, which is the smallest among all
P. putida
strains sequenced to date.
Genome Sequence of Sporolactobacillus laevolacticus DSM442, an Efficient Polymer-Grade
d
-Lactate Producer from Agricultural Waste Cottonseed as a Nitrogen Source
Authors: Wang Hongyan , Wang Limin , Ju Jiansong , Yu Bo , Ma Yanhe
Published: December 2013

Sporolactobacillus laevolacticus
DSM442 is an efficient polymer-grade
d
-lactic acid producer from low-cost agricultural waste cottonseed powder as the sole nitrogen source. Here we present a 3.59-Mb assembly of its genome sequence, which might provide useful information to further improve the strain for higher production titers.
Draft Genome Sequence of the Live Vaccine Strain Brucella abortus 82
Authors: Shevtsov Alexander , Tarlykov Pavel , Zholdybayeva Elena , Shevtsova Elena , Momynkulov Dauren , Sytnik Igor , Karibaev Talgat , Chsherbakov Andrey , Momynaliev Kuvat
Published: December 2013

Vaccination is a crucial part of the brucellosis eradication programs worldwide. A live vaccine strain of
Brucella abortus
82 has been successfully used for the vaccination of cattle against brucellosis in the former Soviet republics for the last 39 years. Here, we report the genome sequence of
Brucella abortus
82.
Draft Genome Sequence of an Obligate and Moderately Halophilic Bacterium, Thalassobacillus devorans Strain MSP14, the First Draft Genome of the Genus
Thalassobacillus
Authors: Pal Kamal Krishna , Dey Rinku , Sherathia Dharmesh , Sukhadiya Bhoomika , Dalsania Trupti , Patel Ilaxi , Savsani Kinjal , Thomas Manesh , Vanpariya Sejal , Mandaliya Mona , Rupapara Rupal , Rawal Priya , Ghorai Sucheta , Bhayani Sharmila , Shah Abhi , Saxena Anil Kumar
Published: December 2013

We report the 3.93-Mbp first draft genome sequence of a species of the genus
Thalassobacillus
,
Thalassobacillus devorans
strain MSP14, a moderate but obligate halophile, isolated from a salt crystallizer of the Little Rann of Kutch, India. Exploring the genome of this organism will facilitate understanding the mechanism(s) of its obligate halophilism.
Complete Genome Sequence of Pediococcus pentosaceus Strain SL4
Authors: Dantoft Shruti Harnal , Bielak Eliza Maria , Seo Jae-Gu , Chung Myung-Jun , Jensen Peter Ruhdal
Published: December 2013

Pediococcus pentosaceus
SL4 was isolated from a Korean fermented vegetable product, kimchi. We report here the whole-genome sequence (WGS) of
P. pentosaceus
SL4. The genome consists of a 1.79-Mb circular chromosome (G+C content of 37.3%) and seven distinct plasmids ranging in size from 4 kb to 50 kb.
Complete Genome Sequence of a Human Enterovirus 71 Strain Isolated in Wuhan, China, in 2010
Authors: Yang Zhu , Lu Songya , Xian Jianchun , Ye Jun , Xiao Li , Luo Jun , Zen Ke , Liu Fenyong
Published: December 2013

The complete genome sequence of a human enterovirus 71 strain (EV71/wuhan/3018/2010), which was isolated in Wuhan in 2010, was amplified by a reverse transcription-PCR method and sequenced. Phylogenetic analysis based on the complete genome sequence classified this strain into subgenogroup A.
Genome Sequence of the Pigment-Producing Bacterium
Pseudogulbenkiania ferrooxidans
, Isolated from Loktak Lake
Authors: Puranik Sampada , Talkal Reshma , Qureshi Asifa , Khardenavis Anshuman , Kapley Atya , Purohit Hemant J.
Published: December 2013

The whole genome of a pigment-producing isolate from a lake in northern India,
Pseudogulbenkiania ferrooxidans
strain EGD-HP2, has been sequenced to study the spectrum of biosynthesis of secondary metabolites. The genome annotation data revealed an operon for violacein, which showed homology with the reported operon of a
Chromobacterium
sp., and also a quinone cofactor.
Biofilm Formation Enhances Fomite Survival of Streptococcus pneumoniae and Streptococcus pyogenes
Authors: Marks Laura R. , Reddinger Ryan M. , Hakansson Anders P.
Published: December 2013

Both
Streptococcus pyogenes
and
Streptococcus pneumoniae
are widely thought to rapidly die outside the human host, losing infectivity following desiccation in the environment. However, to date, all literature investigating the infectivity of desiccated streptococci has used broth-grown, planktonic populations. In this study, we examined the impact of biofilm formation on environmental survival of clinical and laboratory isolates of
S. pyogenes
and
S. pneumoniae
as both organisms are thought to colonize the human host as biofilms. Results clearly demonstrate that while planktonic cells that are desiccated rapidly lose viability both on hands and abiotic surfaces, such as plastic, biofilm bacteria remain viable over extended periods of time outside the host and remain infectious in a murine colonization model. To explore the level and extent of streptococcal fomite contamination that children might be exposed to naturally, direct bacteriologic cultures of items in a day care center were conducted, which demonstrated high levels of viable streptococci of both species. These findings raise the possibility that streptococci may survive in the environment and be transferred from person to person via fomites contaminated with oropharyngeal secretions containing biofilm streptococci.
Microbial Biofilms on Needleless Connectors for Central Venous Catheters: Comparison of Standard and Silver-Coated Devices Collected from Patients in an Acute Care Hospital
Authors: Perez Elizabeth , Williams Margaret , Jacob Jesse T. , Reyes Mary Dent , Chernetsky Tejedor Sheri , Steinberg James P. , Rowe Lori , Ganakammal Satishkumar Ranganathan , Changayil Shankar , Weil M. Ryan , Donlan Rodney M.
Published: December 2013

Microorganisms may colonize needleless connectors (NCs) on intravascular catheters, forming biofilms and predisposing patients to catheter-associated infection (CAI). Standard and silver-coated NCs were collected from catheterized intensive care unit patients to characterize biofilm formation using culture-dependent and culture-independent methods and to investigate the associations between NC usage and biofilm characteristics. Viable microorganisms were detected by plate counts from 46% of standard NCs and 59% of silver-coated NCs (
P
= 0.11). There were no significant associations (
P
> 0.05, chi-square test) between catheter type, side of catheter placement, number of catheter lumens, site of catheter placement, or NC placement duration and positive NC findings. There was an association (
P
= 0.04, chi-square test) between infusion type and positive findings for standard NCs. Viable microorganisms exhibiting intracellular esterase activity were detected on >90% of both NC types (
P
= 0.751), suggesting that a large percentage of organisms were not culturable using the conditions provided in this study. Amplification of the 16S rRNA gene from selected NCs provided a substantially larger number of operational taxonomic units per NC than did plate counts (26 to 43 versus 1 to 4 operational taxonomic units/NC, respectively), suggesting that culture-dependent methods may substantially underestimate microbial diversity on NCs. NC bacterial communities were clustered by patient and venous access type and may reflect the composition of the patient's local microbiome but also may contain organisms from the health care environment. NCs provide a portal of entry for a wide diversity of opportunistic pathogens to colonize the catheter lumen, forming a biofilm and increasing the potential for CAI, highlighting the importance of catheter maintenance practices to reduce microbial contamination.
Evaluation of Two Commercial Real-Time PCR Assays for Detection of Mycoplasma genitalium in Urogenital Specimens
Authors: Le Roy Chloé , Pereyre Sabine , Bébéar Cécile
Published: December 2013

The performance of two commercial real-time PCR kits for the detection of
Mycoplasma genitalium
was evaluated in comparison to an in-house real-time PCR assay. Concordances of 96% and 93% were found for the TIB MOLBIOL and the Diagenode assays, respectively, compared to the results of the in-house assay.
Detection and Characterization of Mycoplasma pneumoniae during an Outbreak of Respiratory Illness at a University
Authors: Waller Jessica L. , Diaz Maureen H. , Petrone Brianna L. , Benitez Alvaro J. , Wolff Bernard J. , Edison Laura , Tobin-D'Angelo Melissa , Moore Ashley , Martyn Audrey , Dishman Hope , Drenzek Cherie L. , Turner Kim , Hicks Lauri A. , Winchell Jonas M.
Published: December 2013

An outbreak at a university in Georgia was identified after 83 cases of probable pneumonia were reported among students. Respiratory specimens were obtained from 21 students for the outbreak investigation. The TaqMan array card (TAC), a quantitative PCR (qPCR)-based multipathogen detection technology, was used to initially identify
Mycoplasma pneumoniae
as the causative agent in this outbreak. TAC demonstrated 100% diagnostic specificity and sensitivity compared to those of the multiplex qPCR assay for this agent. All
M. pneumoniae
specimens (
n
= 12) and isolates (
n
= 10) were found through genetic analysis to be susceptible to macrolide antibiotics. The strain diversity of
M. pneumoniae
associated with this outbreak setting was identified using a variety of molecular typing procedures, resulting in two P1 genotypes (types 1 [60%] and 2 [40%]) and seven different multilocus variable-number tandem-repeat analysis (MLVA) profiles. Continued molecular typing of this organism, particularly during outbreaks, may enhance the current understanding of the epidemiology of
M. pneumoniae
and may ultimately lead to a more effective public health response.
Hemolytic Uremic Syndrome following Infection with O111 Shiga Toxin-Producing Escherichia coli Revealed through Molecular Diagnostics
Authors: Operario Darwin J. , Moonah Shannon , Houpt Eric
Published: December 2013

We report a case of hemolytic uremic syndrome in a 69-year-old woman due to Shiga toxin-producing
Escherichia coli
, possibly serotype O111, to illustrate the potentially deleterious implications of a
Campylobacter
enzyme immunoassay (EIA) result and the increasing importance of molecular testing when conventional methods are limited.
Urine Is Not Sterile: Use of Enhanced Urine Culture Techniques To Detect Resident Bacterial Flora in the Adult Female Bladder
Authors: Hilt Evann E. , McKinley Kathleen , Pearce Meghan M. , Rosenfeld Amy B. , Zilliox Michael J. , Mueller Elizabeth R. , Brubaker Linda , Gai Xiaowu , Wolfe Alan J. , Schreckenberger Paul C.
Published: December 2013

Our previous study showed that bacterial genomes can be identified using 16S rRNA sequencing in urine specimens of both symptomatic and asymptomatic patients who are culture negative according to standard urine culture protocols. In the present study, we used a modified culture protocol that included plating larger volumes of urine, incubation under varied atmospheric conditions, and prolonged incubation times to demonstrate that many of the organisms identified in urine by 16S rRNA gene sequencing are, in fact, cultivable using an expanded quantitative urine culture (EQUC) protocol. Sixty-five urine specimens (from 41 patients with overactive bladder and 24 controls) were examined using both the standard and EQUC culture techniques. Fifty-two of the 65 urine samples (80%) grew bacterial species using EQUC, while the majority of these (48/52 [92%]) were reported as no growth at 10
3
CFU/ml by the clinical microbiology laboratory using the standard urine culture protocol. Thirty-five different genera and 85 different species were identified by EQUC. The most prevalent genera isolated were
Lactobacillus
(15%), followed by
Corynebacterium
(14.2%),
Streptococcus
(11.9%),
Actinomyces
(6.9%), and
Staphylococcus
(6.9%). Other genera commonly isolated include
Aerococcus
,
Gardnerella
,
Bifidobacterium
, and
Actinobaculum
. Our current study demonstrates that urine contains communities of living bacteria that comprise a resident female urine microbiota.
Fatal Subacute Myocarditis Associated with Human Bocavirus 2 in a 13-Month-Old Child
Authors: Brebion Amélie , Vanlieferinghen Philippe , Déchelotte Pierre , Boutry Morgane , Peigue-Lafeuille Hélène , Henquell Cécile
Published: December 2013

Human bocavirus has rarely been incriminated in fatal or life-threatening respiratory infections. We report a case of fatal disseminated infection with subacute lymphocytic myocarditis in a 13-month-old child. The human bocavirus 2 genome was detected by PCR analysis in nasal swab, plasma, urine, ascitic fluid, and mesenteric node, skeletal muscle, and lung tissue specimens.
Controlled Clinical Comparison of BacT/Alert FA Plus and FN Plus Blood Culture Media with BacT/Alert FA and FN Blood Culture Media
Authors: Kirn T. J. , Mirrett S. , Reller L. B. , Weinstein M. P.
Published: December 2013

New blood culture media containing antibiotic-binding polymeric beads have been developed for the BacT/Alert (bioMérieux, Inc., Durham, NC) blood culture system. To assess the performance of these new media, we compared the new BacT/Alert aerobic medium (FA Plus) with resins to BacT/Alert FA medium with activated charcoal and the new BacT/Alert anaerobic medium (FN Plus) to BacT/Alert FN medium at 3 tertiary care medical centers. Study bottle pairs were inoculated with a target volume of 6 to 10 ml of blood from adults and incubated in the BacT/Alert 3D blood culture instrument. In the FA Plus versus FA comparison, there were 1,507 study pairs. Among 170 isolates causing true bloodstream infections (BSIs), significantly more
Staphylococcus aureus
(
P
< 0.001) and total microorganisms (
P
< 0.01) grew in the FA Plus bottle than in the FA bottle. Fewer coagulase-negative staphylococcal (CoNS) contaminants grew in the FA Plus bottle than in the FA bottle (10 versus 22;
P
= 0.05). In addition, growth was detected earlier in the FA Plus bottle than in the FA bottle (
P
< 0.001). In the FN Plus versus FN comparison, there were 2,386 study pairs. Among 201 isolates causing true BSIs, significantly more
S. aureus
(
P
< 0.001), CoNS (
P
< 0.005), and total microorganisms (
P
< 0.001) grew in the FN Plus bottle than in the FN bottle. Also, significantly more CoNS contaminants grew in the FN Plus bottle than in the FN bottle (
P
< 0.001). Overall, microorganisms were detected earlier in the FN Plus than in the FN bottle (
P
< 0.001). Medical technologists at all 3 study sites preferred the new media for Gram stain interpretation. We conclude that the FA Plus and FN Plus media provide improved and earlier detection of microorganisms compared with the FA and FN media and are preferable for Gram stain interpretation as well.
Limited HIV-1 Superinfection in Seroconverters from the CAPRISA 004 Microbicide Trial
Authors: Redd Andrew D. , Mullis Caroline E. , Wendel Sarah K. , Sheward Daniel , Martens Craig , Bruno Daniel , Werner Lise , Garrett Nigel J. , Abdool Karim Quarraisha , Williamson Carolyn , Porcella Stephen F. , Quinn Thomas C. , Abdool Karim Salim S.
Published: December 2013

HIV-1 superinfection (SI) occurs when an infected individual acquires a distinct new viral strain. The rate of superinfection may be reflective of the underlying HIV risk in a population. The Centre for the AIDS Programme of Research in South Africa (CAPRISA) 004 clinical trial demonstrated that women who used a tenofovir-containing microbicide gel had lower rates of HIV infection than women using a placebo gel. Women who contracted HIV-1 during the trial were screened for the occurrence of superinfection by next-generation sequencing of the viral
gag
and
env
genes. There were two cases (one in each trial arm) of subtype C superinfection identified from the 76 women with primary infection screened at two time points (rate of superinfection, 1.5/100 person-years). Both women experienced a >0.5-log increase in viral load during the window when superinfection occurred. The rate of superinfection was significantly lower than the overall primary HIV incidence in the microbicide trial (incidence rate ratio [IRR], 0.20;
P
= 0.003). The women who seroconverted during the trial reported a significant increase in sexual contact with their stable partner 4 months after seroconversion (
P
< 0.001), which may have lowered the risk of superinfection in this population. The lower frequency of SI compared to the primary incidence is in contrast to a report from a general heterosexual African population but agrees with a study of high-risk women in Kenya. A better understanding of the rate of HIV superinfection could have important implications for ongoing HIV vaccine research.
H1N1, but Not H3N2, Influenza A Virus Infection Protects Ferrets from H5N1 Encephalitis
Authors: Bissel Stephanie J. , Wang Guoji , Carter Donald M. , Crevar Corey J. , Ross Ted M. , Wiley Clayton A.
Published: December 2013

Seasonal influenza causes substantial morbidity and mortality because of efficient human-to-human spread. Rarely, zoonotic strains of influenza virus spread to humans, where they have the potential to mediate new pandemics with high mortality. We studied systemic viral spread after intranasal infection with highly pathogenic avian influenza virus (H5N1 [A/Viet Nam/1203/2004]) in ferrets with or without prior pandemic H1N1pdm09 (A/Mexico/4108/2009) or H3N2 (A/Victoria/361/2011) infection. After intranasal challenge with H5N1 influenza virus, naive ferrets rapidly succumbed to systemic infection. Animals challenged with H5N1 influenza virus greater than 3 months after recovering from an initial H1N1pdm09 infection survived H5N1 virus challenge and cleared virus from the respiratory tract 4 days after infection. However, a prolonged low-level infection of hematopoietic elements in the small bowel lamina propria, liver, and spleen was present for greater than 2 weeks postinfection, raising the potential for reassortment of influenza genes in a host infected with multiple strains of influenza. Animals previously infected with an H3N2 influenza virus succumbed to systemic disease and encephalitis after H5N1 virus challenge. These results indicate prior infection with different seasonal influenza strains leads to radically different protection from H5N1 challenge and fatal encephalitis.

IMPORTANCE
Seasonal influenza is efficiently transmitted from human to human, causing substantial morbidity and mortality. Rarely, zoonotic strains of influenza virus spread to humans, where they have the potential to mediate new pandemics with high mortality. Infection of naive ferrets with H5N1 avian influenza virus causes a rapid and lethal systemic disease. We studied systemic H5N1 viral spread after infection of ferrets with or without prior exposure to either of two seasonal influenza virus strains, H1N1 and H3N2. Ferrets previously infected with H1N1 survive H5N1 challenge while those previously infected with H3N2 die of encephalitis. However ferrets protected from lethal H5N1 infection develop persistent low-level infection of the small intestine, liver, or spleen, providing a nidus for future viral strain recombination. The mechanism by which prior infection with specific strains of seasonal influenza virus protect from lethal H5N1 challenge needs to be elucidated in order to design effective immunization and treatments.
Determining the Breadth of the Respiratory Syncytial Virus-Specific T Cell Response
Authors: McDermott Daniel S. , Knudson Cory J. , Varga Steven M.
Published: December 2013

Respiratory syncytial virus (RSV) is the most common cause of viral lower respiratory tract infections in infants and children under the age of 5. Studies examining RSV infection in susceptible BALB/c mice indicate that both CD4 and CD8 T cells not only contribute to viral clearance but also facilitate RSV-induced disease. However, efforts to understand the mechanisms by which RSV-specific T cells mediate disease following acute RSV infection have been hampered by the lack of defined RSV-specific T cell epitopes. Using an overlapping peptide library spanning each of the RSV-derived proteins, intracellular cytokine staining for gamma interferon was utilized to identify novel RSV-specific CD4 and CD8 T cell epitopes. Five novel CD8 T cell epitopes were revealed within the RSV fusion (F) protein and glycoprotein (G). In addition, five previously unidentified CD4 T cell epitopes were discovered, including epitopes in the phosphoprotein (P), polymerase protein (L), M2-1 protein, and nucleoprotein (N). Though the initial CD4 T cell epitopes were 15 amino acids in length, synthesis of longer peptides increased the frequency of responding CD4 T cells. Our results indicate that CD4 T cell epitopes that are 17 amino acids in length result in more optimal CD4 T cell stimulation than the commonly used 15-mer peptides.

IMPORTANCERespiratory syncytial virus (RSV) is the leading cause of hospitalization for lower respiratory tract infection in children. T cells play a critical role in clearing an acute RSV infection, as well as contributing to RSV-induced disease. Here we examined the breadth of the RSV-specific T cell response, using for the first time an overlapping peptide library spanning the entire viral genome. We identified 5 new CD4 and 5 new CD8 T cell epitopes, including a CD8 T cell epitope within the G protein that was previously believed not to elicit a CD8 T cell response. Importantly, we also demonstrated that the use of longer, 17-mer peptides elicits a higher frequency of responding CD4 T cells than the more commonly used 15-mer peptides. Our results demonstrate the breadth of the CD4 and CD8 T cell response to RSV and demonstrate the importance of using longer peptides when stimulating CD4 T cell responses.
Genomewide Analysis of Reassortment and Evolution of Human Influenza A(H3N2) Viruses Circulating between 1968 and 2011
Authors: Westgeest Kim B. , Russell Colin A. , Lin Xudong , Spronken Monique I. J. , Bestebroer Theo M. , Bahl Justin , van Beek Ruud , Skepner Eugene , Halpin Rebecca A. , de Jong Jan C. , Rimmelzwaan Guus F. , Osterhaus Albert D. M. E. , Smith Derek J. , Wentworth David E. , Fouchier Ron A. M. , de Graaf Miranda
Published: December 2013

Influenza A(H3N2) viruses became widespread in humans during the 1968 H3N2 virus pandemic and have been a major cause of influenza epidemics ever since. These viruses evolve continuously by reassortment and genomic evolution. Antigenic drift is the cause for the need to update influenza vaccines frequently. Using two data sets that span the entire period of circulation of human influenza A(H3N2) viruses, it was shown that influenza A(H3N2) virus evolution can be mapped to 13 antigenic clusters. Here we analyzed the full genomes of 286 influenza A(H3N2) viruses from these two data sets to investigate the genomic evolution and reassortment patterns. Numerous reassortment events were found, scattered over the entire period of virus circulation, but most prominently in viruses circulating between 1991 and 1998. Some of these reassortment events persisted over time, and one of these coincided with an antigenic cluster transition. Furthermore, selection pressures and nucleotide and amino acid substitution rates of all proteins were studied, including those of the recently discovered PB1-N40, PA-X, PA-N155, and PA-N182 proteins. Rates of nucleotide and amino acid substitutions were most pronounced for the hemagglutinin, neuraminidase, and PB1-F2 proteins. Selection pressures were highest in hemagglutinin, neuraminidase, matrix 1, and nonstructural protein 1. This study of genotype in relation to antigenic phenotype throughout the period of circulation of human influenza A(H3N2) viruses leads to a better understanding of the evolution of these viruses.

IMPORTANCE
Each winter, influenza virus infects approximately 5 to 15% of the world's population, resulting in significant morbidity and mortality. Influenza A(H3N2) viruses evolve continuously by reassortment and genomic evolution. This leads to changes in antigenic recognition (antigenic drift) which make it necessary to update vaccines against influenza A(H3N2) viruses frequently. In this study, the relationship of genetic evolution to antigenic change spanning the entire period of A(H3N2) virus circulation was studied for the first time. The results presented in this study contribute to a better understanding of genetic evolution in correlation with antigenic evolution of influenza A(H3N2) viruses.
Enhanced Virulence of Sheep-Passaged Bovine Spongiform Encephalopathy Agent Is Revealed by Decreased Polymorphism Barriers in Prion Protein Conversion Studies
Authors: Priem Jan , Langeveld Jan P. M. , van Keulen Lucien J. M. , van Zijderveld Fred G. , Andreoletti Olivier , Bossers Alex
Published: December 2013

Bovine spongiform encephalopathy (BSE) can be efficiently transmitted to small ruminants (sheep and goats) with certain prion protein (PrP) genotypes. Polymorphisms in PrP of both the host and donor influence the transmission efficiency of transmissible spongiform encephalopathies (TSEs) in general. These polymorphisms in PrP also modulate the PrP conversion underlying TSE agent replication. Here we demonstrate that single-round protein misfolding cyclic amplification (PMCA) can be used to assess species and polymorphism barriers at the molecular level. We assessed those within and between the ovine and bovine species
in vitro
using a variety of natural scrapie and experimentally generated cross-species BSE agents. These BSE agents include ovBSE-ARQ isolates (BSE derived from sheep having the ARQ/ARQ PrP genotype), and two unique BSE-derived variants: BSE passaged in VRQ/VRQ sheep and a cow BSE agent isolate generated by back-transmission of ovBSE-ARQ into its original host. PMCA allowed us to quantitatively determine PrP conversion profiles that correlated with known
in vivo
transmissibility and susceptibility in the two ruminant species in which strain-specific molecular signatures, like its molecular weight after protease digestion, were maintained. Furthermore, both BSE agent isolates from ARQ and VRQ sheep demonstrated a surprising transmission profile in which efficient transmissions to both sheep and bovine variants was combined. Finally, all data support the notion that ARQ-derived sheep BSE points to a significant increase in virulence compared to all other tested scrapie- and BSE-derived variants reflected by the increased conversion efficiencies of previously inefficient convertible PrP variants (including the so-called “resistant” sheep ARR variant).

IMPORTANCE
Prion diseases such as scrapie in sheep and goats, BSE in cattle, and Creutzfeldt-Jakob disease (CJD) in humans are fatal neurodegenerative diseases caused by prions. BSE is known to be transmissible to a variety of hosts, including sheep and humans. Based on the typical BSE agent strain signatures and epidemiological data, the occurrence of a novel variant of CJD in humans was linked to BSE occurrence in the United Kingdom. Measures, including genetic selection of sheep toward less susceptible PrP genotypes, have been implemented to lower the risk of BSE transmission into sheep, since the disease could potentially spread into a natural reservoir. In this study, we demonstrated using molecular PrP conversion studies that when BSE is first transmitted through sheep, the host range is modified significantly and the PrP converting potency increased, allowing the ovine BSE to transmit more efficiently than cow BSE into supposedly less susceptible hosts.
Inhibition of Poliovirus-Induced Cleavage of Cellular Protein PCBP2 Reduces the Levels of Viral RNA Replication
Authors: Chase Amanda J. , Daijogo Sarah , Semler Bert L.
Published: December 2013

Due to their small genome size, picornaviruses must utilize host proteins to mediate cap-independent translation and viral RNA replication. The host RNA-binding protein poly(rC) binding protein 2 (PCBP2) is involved in both processes in poliovirus infected cells. It has been shown that the viral proteinase 3CD cleaves PCBP2 and contributes to viral translation inhibition. However, cleaved PCBP2 remains active in viral RNA replication. This would suggest that both cleaved and intact forms of PCBP2 have a role in the viral RNA replication cycle. The picornavirus genome must act as a template for both translation and RNA replication. However, a template that is actively being translated cannot function as a template for RNA replication, suggesting that there is a switch in template usage from translation to RNA replication. We demonstrate that the cleavage of PCBP2 by the poliovirus 3CD proteinase is a necessary step for efficient viral RNA replication and, as such, may be important for mediating a switch in template usage from translation to RNA replication.

IMPORTANCE
Poliovirus, like all positive-strand RNA viruses that replicate in the cytoplasm of eukaryotic cells, uses its genomic RNA as a template for both viral protein synthesis and RNA replication. Given that these processes cannot occur simultaneously on the same template, poliovirus has evolved a mechanism(s) to facilitate the switch from using templates for translation to using them for RNA synthesis. This study explores one possible scenario for how the virus alters the functions of a host cell RNA binding protein to mediate, in part, this important transition.
GS-8374, a Prototype Phosphonate-Containing Inhibitor of HIV-1 Protease, Effectively Inhibits Protease Mutants with Amino Acid Insertions
Authors: Grantz Šašková Klára , Kožíšek Milan , Stray Kirsten , de Jong Dorien , Řezáčová Pavlína , Brynda Jiří , van Maarseveen Noortje M. , Nijhuis Monique , Cihlář Tomáš , Konvalinka Jan
Published: December 2013

Insertions in the protease (PR) region of human immunodeficiency virus (HIV) represent an interesting mechanism of antiviral resistance against HIV PR inhibitors (PIs). Here, we demonstrate the improved ability of a phosphonate-containing experimental HIV PI, GS-8374, relative to that of other PIs, to effectively inhibit patient-derived recombinant HIV strains bearing PR insertions and numerous other mutations. We correlate enzyme inhibition with the catalytic activities of corresponding recombinant PRs
in vitro
and provide a biochemical and structural analysis of the PR-inhibitor complex.
Adenoviral Delivery of Recombinant Hepatitis B Virus Expressing Foreign Antigenic Epitopes for Immunotherapy of Persistent Viral Infection
Authors: Wang Zhuo , Zhu Kai , Bai Weiya , Jia Baosen , Hu Hao , Zhou Dongming , Zhang Xiaoming , Zhang Xinxin , Xie Youhua , Bourgine Maryline Mancini , Michel Marie-Louise , Lan Ke , Deng Qiang
Published: December 2013

We previously reported a proof-of-concept study for curing chronic hepatitis B virus (HBV) infection using a foreign-antigen recombinant HBV (rHBV) as a gene therapy vector. Targeted elimination of wild-type HBV (wtHBV)-infected cells could be achieved by functionally activating an
in situ
T-cell response against the foreign antigen. However, as chronic HBV infection spreads to all hepatocytes, specific targeting of virus-infected cells is thought to be less critical. It is also feared that rHBV may not induce active immunization in a setting resembling natural infection. For this immunotherapeutic approach to be practically viable, in the present study, we used a recombinant adenovirus (rAd) vector for rHBV delivery. The rAd vector allowed efficient transduction of wtHBV-producing HepG2 cells, with transferred rHBV undergoing dominant viral replication. Progeny rHBV virions proved to be infectious, as demonstrated in primary tupaia hepatocytes. These results greatly expanded the antiviral capacity of the replication-defective rAd/rHBV in wtHBV-infected liver tissue. With prior priming in the periphery, transduction with rAd/rHBV attracted a substantial influx of the foreign-antigen-specific T-effector cells into the liver. Despite the fully activated T-cell response, active expression of rHBV was observed for a prolonged time, which is essential for rHBV to achieve sustained expansion. In a mouse model of HBV persistence established by infection with a recombinant adeno-associated virus carrying the wtHBV genome, rAd/rHBV-based immunotherapy elicited a foreign-antigen-specific T-cell response that triggered effective viral clearance and subsequent seroconversion to HBV. It therefore represents an efficient strategy to overcome immune tolerance, thereby eliminating chronic HBV infection.

IMPORTANCE
Adenovirus-delivered rHBV activated a foreign-antigen-specific T-cell response that abrogated HBV persistence in a mouse model. Our study provides further evidence of the potential of foreign-antigen-based immunotherapy for the treatment of chronic HBV infection.
Antiviral Activity of Lambda Interferon in Chickens
Authors: Reuter Antje , Soubies Sebastien , Härtle Sonja , Schusser Benjamin , Kaspers Bernd , Staeheli Peter , Rubbenstroth Dennis
Published: December 2013

Interferons (IFNs) are essential components of the antiviral defense system of vertebrates. In mammals, functional receptors for type III IFN (lambda interferon [IFN-λ]) are found mainly on epithelial cells, and IFN-λ was demonstrated to play a crucial role in limiting viral infections of mucosal surfaces. To determine whether IFN-λ plays a similar role in birds, we produced recombinant chicken IFN-λ (chIFN-λ) and we used the replication-competent retroviral RCAS vector system to generate mosaic-transgenic chicken embryos that constitutively express chIFN-λ. We could demonstrate that chIFN-λ markedly inhibited replication of various virus strains, including highly pathogenic influenza A viruses,
in ovo
and
in vivo
, as well as in epithelium-rich tissue and cell culture systems. In contrast, chicken fibroblasts responded poorly to chIFN-λ. When applied
in vivo
to 3-week-old chickens, recombinant chIFN-λ strongly induced the IFN-responsive Mx gene in epithelium-rich organs, such as lungs, tracheas, and intestinal tracts. Correspondingly, these organs were found to express high transcript levels of the putative chIFN-λ receptor alpha chain (chIL28RA) gene. Transfection of chicken fibroblasts with a chIL28RA expression construct rendered these cells responsive to chIFN-λ treatment, indicating that receptor expression determines cell type specificity of IFN-λ action in chickens. Surprisingly, mosaic-transgenic chickens perished soon after hatching, demonstrating a detrimental effect of constitutive chIFN-λ expression. Our data highlight fundamental similarities between the IFN-λ systems of mammals and birds and suggest that type III IFN might play a role in defending mucosal surfaces against viral intruders in most if not all vertebrates.