bluuebunny/crossref_metadata_2025 huggingface dataset:
Prediction of Gas Turbine Oil Scoop Capture Efficiency
Authors: Prasad Santosh Kumar , Sangli Pradeep , Buyukisik Osman , Pugh Dave
Published: February 2015

The lubrication system in a gas turbine engine is akin to the human blood circulatory system. Providing right quantities of oil to the right components for cooling and lubrication is the primary function of the lubrication system. In the current analysis, at the downstream end of the lube oil supply line, a stationary oil nozzle sprays a jet of oil to a high speed rotating component called an oil scoop. The function of the oil scoop which rotates at speeds usually greater than 10000 RPM is to ‘Scoop’ or capture the oil and provide an under race oil transfer mechanism to the bearings rotating especially at such high speeds. If the oil capture is less than required by the downstream bearing components, it could lead to diminished bearing lives in the gas turbine.

The oil scoop consists of two or more blades that are angled with respect to the radius of the Scoop to provide an entry to the oil jet. The ‘window’ of open space between the blades is important to capture the oil. The ratio of quantity of oil captured to the total oil sprayed on to oil scoop is termed as the oil capture efficiency. Several parameters like oil nozzle distance from the blade tip, spray characteristics, jet velocity, number of blades, blade angle, window width, rotational speeds, oil temperature etc. are important factors that determine the capture efficiency of the oil scoop. Prior to the availability of efficient CFD methodologies, it was extremely difficult to develop an oil scoop capture efficiency predictive tool that involves a complex 3D fluid flow from a stationary to a rotating component. The typical Reynolds number of the jet is around 13000 and the oil scoop tip speeds of the order of Mach 0.2 to 0.4. To evaluate various scoop design configurations and enhancements, a transient CFD methodology was developed using multiphase Volume of Fluid approach available in FLUENT® software. In this paper a technique or process is described and demonstrated to simulate the right ‘periodic’ nature of the oil capture and transfer mechanism. It is shown that the CFD methodology described compares well with experimental data.

This robust CFD methodology predicts the complex 3D flow with sufficient accuracy and has the potential to be used to optimize the geometry for maximum oil capture efficiency of oil scoops in gas turbine lubrication system. Significant reduction of costly experiments is also an important benefit of developing this predictive methodology.
Investigation of Flame Dynamics in a V - Flame Combustor During Combustion Instability
Authors: Vishnu R. , Sujith R. I. , Aghalayam Preeti
Published: February 2015

Propulsion systems such as gas turbines are susceptible to combustion instability, when operated at lean equivalence ratio [1]. During combustion instability, there is a nonlinear interaction between combustion and acoustics leading to large amplitude acoustic oscillations. These large amplitude oscillations are detrimental to the stability of the combustor and can cause damages to the structural integrity of the combustor, flame flash back or blow off. The main source of nonlinearity is in the heat release rate caused due to the velocity perturbations at the flame holder [2]. The heat release rate fluctuations are due to the variation in the flame surface area. Hence there is a need to understand the flame dynamics that contributes to the heat release rate fluctuations. The present study aims in understanding the stability of a V - flame combustor by varying the flame location inside an acoustic resonator. By varying the flame location the instability regimes of the combustor are identified. At the flame locations where the system exhibits combustion instability, acoustic pressure oscillations are acquired simultaneously with high speed images of the flame front fluctuations so that a correlation can be made between them. Tools from dynamical systems theory are applied to the pressure signal to quantify different dynamical states of the system during combustion instability. Moreover the flame dynamics at each dynamical state are investigated. It is observed that combustion instability is characterized by interesting dynamical states such as frequency locked state, quasi-periodic oscillations, period 3 oscillations and chaotic oscillations. High speed imaging of the flame reveals different interesting patterns of flame behavior during combustion instability. Flame wrinkling, roll up of flame elements, separation as islands of the flame elements and mutual annihilation of flame elements were some of the interesting flame behavior observed. This study helps in understanding the role of nonlinear heat release rate mechanism in establishing different dynamical states during combustion instability.
Structural Assessment of Torque Converter at Operating Conditions: A Numerical Study
Authors: Shivaramaiah Subbaramu , Banthia Vinod K. , Kumar S. M. Vijay
Published: February 2015

A single stage torque converter consists of three elements — pump, stator and turbine. Pump and turbine are coupled by transmission fluid. Unlike a fluid coupling, however, a torque converter is able to multiply torque when there is a substantial difference between input and output rotational speed, thus providing the equivalent of a reduction gear. During its operation all these elements are subjected to centrifugal load, fluid pressure load and heat generated in transmission fluid. Overloading a converter can result in several failure modes, some of them potentially dangerous in nature: ballooning, blade deformation and defragmentation, overheating.

In the current work a single stage torque converter, was modelled and analysed numerically for evaluating stress distribution and deformation. The engine operating speed at 2000 rpm was considered for analysis. For static analysis of torque converter components centrifugal load and fluid pressure load were considered. Analysis was carried out for six different speed ratios varying from zero to one. Variation of principal stresses (hoop stress and radial stress) and von-Mises stress has been discussed. Maximum stresses are found to be in pump at speed ratio of one and in turbine at speed ratio of zero. Maximum stresses are at shell core that is near to hub. Blade deformation in pump is maximum at coupling phase and in turbine it is maximum at stall condition. From these results it helps to predict the failure of torque converter components under different operating conditions.
NCAA Division I Female Head Coaches’ Experiences of Exemplary Care Within Coaching
Authors: Knust Susannah K. , Fisher Leslee Anne
Published: February 2015

In this paper, the experiences of 12 NCAA Division I female head coaches exemplifying care in their coaching are described. After a brief review of literature and terms, coaches’ own words from interview transcripts are used to illustrate four major themes: (a) team as “family”; (b) holistic care of student-athletes; (c) development of the “self-as-coach”; and (d) institutional care. We conclude by addressing why we believe that care is a coach education issue and why coaches should engage with the ongoing development of exemplary care.
Development and Implementation of an Autonomy Supportive Training Program among Youth Sport Coaches
Authors: Langdon Jody , Harris Brandonn S. , Burdette Glenn P. , Rothberger Sara
Published: February 2015

Studying perceived autonomy support, a basic tenet of self-determination theory (Deci & Ryan, 2000), provides some understanding as to how coaches can more positively influence youth athletes to enjoy and persist in youth sport. Borrowing insights from success in physical education and coaching-oriented interventions, the purpose of this paper was to highlight positive aspects and challenges of an innovative youth sport autonomy supportive training program for coaches. Positives included the initial training session and the use of an online training component. Challenges were the structure of the season, other coaches, and possibly the age of the athletes. Future training programs in youth sport coaching should increase in duration, provide specific examples of how to implement autonomy supportive coaching behaviors, as well as address solutions to the time constraints of the youth sport setting.
Examining High School Teacher-Coaches’ Perspective on Relationship Building With Student-Athletes
Authors: Camiré Martin
Published: February 2015

Adult leaders in sport can exert considerable influence on young athletes’ development but this influence is mediated by the quality of the relationship that is formed between both parties. The purpose of the current study was to examine high school teacher-coaches’ perspective on relationship building with student-athletes. Teacher-coaches (20 men, 5 women, Mage = 37.0 years, age range: 25–56 years) from Canada took part in semistructured interviews. Results indicated how the participants believed being both a teacher and a coach was advantageous because it allowed them to interact regularly with student-athletes. The teacher-coaches devised a number of strategies (e.g., early-season tournaments, regular team meetings) to nurture relationships and believed their recurrent interactions allowed them to exert a more positive influence on student-athletes than adult leaders in a single role. In terms of outcomes, the teacher-coaches believed their dual role helped increase their job satisfaction, positively influenced their identity, and allowed them to help student-athletes through critical family (e.g., alcoholism, divorce) and personal issues (e.g., suicide). The current study suggests that the dual role of teacher-coach is beneficial to both teacher-coaches and student-athletes. However, future work is needed, paying attention to how teacher-coaches can further nurture quality relationships with student-athletes.
Glycoprotein-Based Enzyme-Linked Immunosorbent Assays for Serodiagnosis of Infectious Laryngotracheitis
Authors: Kanabagatte Basavarajappa Mallikarjuna , Song Haichen , Lamichhane Chinta , Samal Siba K.
Published: February 2015

For detection of infectious laryngotracheitis virus (ILTV) antibody, glycoprotein B-, C-, and D-based enzyme-linked immunosorbent assays (B-, C-, and D-ELISAs, respectively) were developed. The B- and D-ELISAs showed enhanced detection of anti-ILTV antibodies in infected chickens compared to that of the commercial ELISA. Furthermore, the D-ELISA was efficient in detecting seroconversion with vectored vaccine, using recombinant Newcastle disease virus (rNDV) expressing glycoprotein D (gD) as the vaccine vector.
Presence of Virulent Newcastle Disease Virus in Vaccinated Chickens in Farms in Pakistan
Authors: Rehmani Shafqat Fatima , Wajid Abdul , Bibi Tasra , Nazir Bushra , Mukhtar Nadia , Hussain Abid , Lone Nazir Ahmad , Yaqub Tahir , Afonso Claudio L.
Published: February 2015

One year after a virulent Newcastle disease virus (vNDV) outbreak in Pakistan, the causative strain was present in vaccinated chickens of multiple farms despite the existence of high-average NDV-specific antibody titers (>4.75 log
2
). The data suggest a possible role of vaccinated birds as reservoirs of vNDV.
Efficient Isolation of Swine Influenza Viruses by Age-Targeted Specimen Collection
Authors: Ozawa Makoto , Matsuu Aya , Yonezawa Kouki , Igarashi Manabu , Okuya Kosuke , Kawabata Toshiko , Ito Kimihito , Tsukiyama-Kohara Kyoko , Taneno Akira , Deguchi Eisaburo
Published: February 2015

The control of swine influenza virus (SIV) infection is paramount for increasing the productivity of pig farming and minimizing the threat of pandemic outbreaks. Thus, SIV surveillance should be conducted by region and on a regular basis. Here, we established a microneutralization assay specific for SIV seroprevalence surveillance by using reporter gene-expressing recombinant influenza viruses. Growth-based SIV seroprevalence revealed that most sows and piglets were positive for neutralizing antibodies against influenza viruses. In contrast, the 90-day-old growing pigs exhibited limited neutralizing activity in their sera, suggesting that this particular age of population is most susceptible to SIV infection and thus is an ideal age group for SIV isolation. From nasal swab specimens of healthy pigs in this age population, we were able to isolate SIVs at a higher incidence (5.3%) than those of previous reports. Nucleotide sequencing and phylogenetic analysis of the hemagglutinin (HA) genes revealed that the isolated SIVs have circulated and evolved in pigs but not have been recently introduced from humans, implying that a large number of SIV lineages may remain “undiscovered” in the global porcine populations. We propose that the 90-day-old growing pig-targeted nasal swab collection presented in this study facilitates global SIV surveillance and contributes to the detection and control of SIV infection.
Single-Molecule Arrays for Ultrasensitive Detection of Host Immune Response to Dengue Virus Infection
Authors: Gaylord Shonda T. , Abdul-Aziz Sama , Walt David R.
Published: February 2015

Dengue virus serotype 2-positive plasma was applied to two indirect single-molecule arrays (Simoas) for the detection of anti-dengue virus IgG and IgM. The Simoas were 1,000 and 10,000 times more sensitive than enzyme-linked immunosorbent assays (ELISAs) for the detection of IgG and IgM, respectively. Using Simoas, serology may be used for the detection of dengue virus infection in the acute phase.
Gene Expression Profiling of Burkholderia cenocepacia at the Time of Cepacia Syndrome: Loss of Motility as a Marker of Poor Prognosis?
Authors: Kalferstova Lucie , Kolar Michal , Fila Libor , Vavrova Jolana , Drevinek Pavel
Published: February 2015

Cepacia syndrome (CS) is a fatal septic condition that develops in approximately 20% of cystic fibrosis (CF) patients chronically infected with the
Burkholderia cepacia
complex (Bcc). The most common causative agent is
Burkholderia cenocepacia
, a clinically dominant Bcc species that contains the globally distributed epidemic strain sequence type 32 (ST32). Using microarrays, we compared the transcriptomes of ST32 isolates from the bloodstream at the time of CS with their sputum counterparts recovered 1 to 2 months prior to the development of CS. Global gene expression profiles of blood isolates revealed greater activities of the virulence genes involved in the type III secretion system, the bacterial exopolysaccharide cepacian, and quorum sensing, while reduced expression was demonstrated for flagellar genes. Furthermore, a nonmotile phenotype (as evaluated by a swimming motility assay) was identified in blood isolates from 6 out of 8 patients with CS; this phenotype was traceable to 24 months prior to the onset of CS. Loss of motility was not observed in any of the 89 ST32 isolates recovered over the course of chronic infection from 17 patients without CS. In conclusion, the gene expression of Bcc bacteria disseminated during CS has been elucidated for the first time. This study demonstrated marked differences at the transcriptome level between isogenic ST32 isolates that are attributable to the stage and site of infection. The finding of a nonmotile
B. cenocepacia
isolate may serve as a warning sign for the development of CS in the near future.
Systematic Serological Testing for Hepatitis E Virus in Kidney Transplant Recipients
Authors: Moal Valérie , Legris Tristan , Motte Anne , Vacher-Coponat Henri , Fages Lucie , Jourde-Chiche Noémie , Borentain Patrick , Jaubert Dominique , Gerolami René , Colson Philippe
Published: February 2015

Hepatitis E virus (HEV) genotype 3 is endemic in Europe and hyperendemic in southern France. Recent reports of a high prevalence of HEV RNA in blood donations and in culinary specialties from this geographical area confirmed the endemicity of HEV and sources of viral transmission in this geographical area. HEV causes acute and chronic hepatitis in solid organ transplant recipients. Since March 2012, we have implemented systematic HEV serological testing in our cohort of kidney transplant recipients (KTRs) in Marseille in southeastern France. The aim of our study was to assess HEV exposure in this cohort between March 2012 and May 2014. During these 27 months, we found that 39% of the patients who underwent kidney transplantation had an anti-HEV IgG response using a sensitive microplate enzyme immunoassay. This seroprevalence was approximately 43% at both 1 and 8 years after, using the same assay. In addition, systematic HEV serological testing detected 6 cases of HEV infection among 578 KTRs (1%) during the 27 months of the study, with 5 at an acute stage and 1 at a chronic stage. In conclusion, continuous HEV monitoring in this population is useful for better understanding the epidemiology of HEV in France, because these patients are a well-monitored population. Moreover, HEV monitoring in KTRs is clinically relevant because HEV represents a clinical threat in these patients. Nevertheless, HEV serological testing may be more fruitful for identifying HEV infections when performed in cases of biological liver abnormalities than when performed systematically.
Impact of Revised Cefepime CLSI Breakpoints on Escherichia coli and Klebsiella pneumoniae Susceptibility and Potential Impact If Applied to Pseudomonas aeruginosa
Authors: Hamada Yukihiro , Sutherland Christina A. , Nicolau David P.
Published: February 2015

The CLSI reduced the cefepime
Enterobacteriaceae
susceptibility breakpoint and introduced the susceptible-dose-dependent (S-DD) category. In this study, MICs were determined for a Gram-negative collection to assess the impact of this change. For
Enterobacteriaceae
, this resulted in <2% reduction in susceptibility, with 1% being S-DD. If applied to
Pseudomonas aeruginosa
, the % susceptibility (%S) dropped from 77% to 43%, with 34% being S-DD. The new breakpoints did little to the
Enterobacteriaceae
%S, but for
P. aeruginosa
, a profound reduction was seen in %S. The recognition of a S-DD response to cefepime should alert clinicians to the possible need for higher doses.
A New Real-Time Reverse Transcription-PCR Assay for Detection of Human Enterovirus 68 in Respiratory Samples
Authors: Piralla Antonio , Girello Alessia , Premoli Marta , Baldanti Fausto
Published: February 2015

A global reemergence of human enterovirus 68 (EV-D68) associated with severe respiratory illness occurred in 2014. We developed and validated an EV-D68-specific real-time reverse transcription-PCR (RT-PCR) for the detection of EV-D68 in respiratory samples. The rapid diagnosis of EV-D68 may contribute to better management of EV-D68 infections.
The Laminin Receptor Is a Cellular Attachment Receptor for Classical Swine Fever Virus
Authors: Chen Jianing , He Wen-Rui , Shen Liang , Dong Hong , Yu Jiahui , Wang Xiao , Yu Shaoxiong , Li Yongfeng , Li Su , Luo Yuzi , Sun Yuan , Qiu Hua-Ji
Published: February 2015

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious, economically important viral disease in many countries. The E
rns
and E2 envelope glycoproteins are responsible for the binding to and entry into the host cell by CSFV. To date, only one cellular receptor, heparan sulfate (HS), has been identified as being involved in CSFV attachment. HS is also present on the surface of various cells that are nonpermissive to CSFV. Hence, there must be another receptor(s) that has been unidentified to date. In this study, we used a set of small interfering RNAs (siRNAs) against a number of porcine cell membrane protein genes to screen cellular proteins involved in CSFV infection. This approach resulted in the identification of several proteins, and of these, the laminin receptor (LamR) has been demonstrated to be a cellular receptor for several viruses. Confocal analysis showed that LamR is colocalized with CSFV virions on the membrane, and a coimmunoprecipitation assay indicated that LamR interacts with the CSFV E
rns
protein. In inhibition assays, anti-LamR antibodies, soluble laminin, or LamR protein significantly inhibited CSFV infection in a dose-dependent manner. Transduction of PK-15 cells with a recombinant lentivirus expressing LamR yielded higher viral titers. Moreover, an attachment assay demonstrated that LamR functions during virus attachment. We also demonstrate that LamR acts as an alternative attachment receptor, especially in SK6 cells. These results indicate that LamR is a cellular attachment receptor for CSFV.

IMPORTANCE
Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only heparan sulfate (HS) has been identified to be an attachment receptor for CSFV. Here, using RNA interference screening with small interfering RNAs (siRNAs) against a number of porcine membrane protein genes, we identified the laminin receptor (LamR) to be another attachment receptor. We demonstrate the involvement of LamR together with HS in virus attachment, and we elucidate the relationship between LamR and HS. LamR also serves as an attachment receptor for many viral pathogens, including dengue virus, a fatal human flavivirus. The study will help to enhance our understanding of the life cycle of flaviviruses and the development of antiviral strategies for flaviviruses.
Adenovirus Replaces Mitotic Checkpoint Controls
Authors: Turner Roberta L. , Groitl Peter , Dobner Thomas , Ornelles David A.
Published: February 2015

Infection with adenovirus triggers the cellular DNA damage response, elements of which include cell death and cell cycle arrest. Early adenoviral proteins, including the E1B-55K and E4orf3 proteins, inhibit signaling in response to DNA damage. A fraction of cells infected with an adenovirus mutant unable to express the E1B-55K and E4orf3 genes appeared to arrest in a mitotic-like state. Cells infected early in G
1
of the cell cycle were predisposed to arrest in this state at late times of infection. This arrested state, which displays hallmarks of mitotic catastrophe, was prevented by expression of either the E1B-55K or the E4orf3 genes. However, E1B-55K mutant virus-infected cells became trapped in a mitotic-like state in the presence of the microtubule poison colcemid, suggesting that the two viral proteins restrict entry into mitosis or facilitate exit from mitosis in order to prevent infected cells from arresting in mitosis. The E1B-55K protein appeared to prevent inappropriate entry into mitosis through its interaction with the cellular tumor suppressor protein p53. The E4orf3 protein facilitated exit from mitosis by possibly mislocalizing and functionally inactivating cyclin B1. When expressed in noninfected cells, E4orf3 overcame the mitotic arrest caused by the degradation-resistant R42A cyclin B1 variant.

IMPORTANCE
Cells that are infected with adenovirus type 5 early in G
1
of the cell cycle are predisposed to arrest in a mitotic-like state in a p53-dependent manner. The adenoviral E1B-55K protein prevents entry into mitosis. This newly described activity for the E1B-55K protein appears to depend on the interaction between the E1B-55K protein and the tumor suppressor p53. The adenoviral E4orf3 protein facilitates exit from mitosis, possibly by altering the intracellular distribution of cyclin B1. By preventing entry into mitosis and by promoting exit from mitosis, these adenoviral proteins act to prevent the infected cell from arresting in a mitotic-like state.
Expression of the Novel Hepatitis C Virus Core+1/ARF Protein in the Context of JFH1-Based Replicons
Authors: Kotta-Loizou Ioly , Karakasiliotis Ioannis , Vassilaki Niki , Sakellariou Panagiotis , Bartenschlager Ralf , Mavromara Penelope
Published: February 2015

Hepatitis C virus contains a second open reading frame within the core gene, designated core+1/ARF. Here we demonstrate for the first time expression of core+1/ARF protein in the context of a bicistronic JFH1-based replicon and report the production of two isoforms, core+1/L (long) and core+1/S (short), with different kinetics.
A Survey of the Interactome of Kaposi's Sarcoma-Associated Herpesvirus ORF45 Revealed Its Binding to Viral ORF33 and Cellular USP7, Resulting in Stabilization of ORF33 That Is Required for Production of Progeny Viruses
Authors: Gillen Joseph , Li Wenwei , Liang Qiming , Avey Denis , Wu Jianjun , Wu Fayi , Myoung JinJong , Zhu Fanxiu
Published: February 2015

The ORF45 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus-specific immediate-early tegument protein. Our previous studies have revealed its crucial roles in both early and late stages of KSHV infection. In this study, we surveyed the interactome of ORF45 using a panel of monoclonal antibodies. In addition to the previously identified extracellular regulated kinase (ERK) and p90 ribosomal S6 kinase (RSK) proteins, we found several other copurified proteins, including prominent ones of ∼38 kDa and ∼130 kDa. Mass spectrometry revealed that the 38-kDa protein is viral ORF33 and the 130-kDa protein is cellular USP7 (ubiquitin-specific protease 7). We mapped the ORF33-binding domain to the highly conserved carboxyl-terminal 19 amino acids (aa) of ORF45 and the USP7-binding domain to the reported consensus motif in the central region of ORF45. Using immunofluorescence staining, we observed colocalization of ORF45 with ORF33 or USP7 both under transfected conditions and in KSHV-infected cells. Moreover, we noticed ORF45-dependent relocalization of a portion of ORF33/USP7 from the nucleus to the cytoplasm. We found that ORF45 caused an increase in ORF33 protein accumulation that was abolished if either the ORF33- or USP7-binding domain in ORF45 was deleted. Furthermore, deletion of the conserved carboxyl terminus of ORF45 in the KSHV genome drastically reduced the level of ORF33 protein in KSHV-infected cells and abolished production of progeny virions. Collectively, our results not only reveal new components of the ORF45 interactome, but also demonstrate that the interactions among these proteins are crucial for KSHV lytic replication.

IMPORTANCE
Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several human cancers. KSHV ORF45 is a multifunctional protein that is required for KSHV lytic replication, but the exact mechanisms by which ORF45 performs its critical functions are unclear. Our previous studies revealed that all ORF45 protein in cells exists in high-molecular-weight complexes. We therefore sought to characterize the interactome of ORF45 to provide insights into its roles during lytic replication. Using a panel of monoclonal antibodies, we surveyed the ORF45 interactome in KSHV-infected cells. We identified two new binding partners of ORF45: the viral protein ORF33 and cellular ubiquitin-specific protease 7 (USP7). We further demonstrate that the interaction between ORF45 and ORF33 is crucial for the efficient production of KSHV viral particles, suggesting that the targeted interference with this interaction may represent a novel strategy to inhibit KSHV lytic replication.
Transfection of Sclerotinia sclerotiorum with
In Vitro
Transcripts of a Naturally Occurring Interspecific Recombinant of Sclerotinia sclerotiorum Hypovirus 2 Significantly Reduces Virulence of the Fungus
Authors: Marzano Shin-Yi Lee , Hobbs Houston A. , Nelson Berlin D. , Hartman Glen L. , Eastburn Darin M. , McCoppin Nancy K. , Domier Leslie L.
Published: February 2015

A recombinant strain of
Sclerotinia sclerotiorum
hypovirus 2 (SsHV2) was identified from a North American
Sclerotinia sclerotiorum
isolate (328) from lettuce (
Lactuca sativa
L.) by high-throughput sequencing of total RNA. The 5′- and 3′-terminal regions of the genome were determined by rapid amplification of cDNA ends. The assembled nucleotide sequence was up to 92% identical to two recently reported SsHV2 strains but contained a deletion near its 5′ terminus of more than 1.2 kb relative to the other SsHV2 strains and an insertion of 524 nucleotides (nt) that was distantly related to
Valsa ceratosperma
hypovirus 1. This suggests that the new isolate is a heterologous recombinant of SsHV2 with a yet-uncharacterized hypovirus. We named the new strain
Sclerotinia sclerotiorum
hypovirus 2 Lactuca (SsHV2L) and deposited the sequence in GenBank with accession number
KF898354
.
Sclerotinia sclerotiorum
isolate 328 was coinfected with a strain of
Sclerotinia sclerotiorum
endornavirus 1 and was debilitated compared to cultures of the same isolate that had been cured of virus infection by cycloheximide treatment and hyphal tipping. To determine whether SsHV2L alone could induce hypovirulence in
S. sclerotiorum
, a full-length cDNA of the 14,538-nt viral genome was cloned. Transcripts corresponding to the viral RNA were synthesized
in vitro
and transfected into a virus-free isolate of
S. sclerotiorum
, DK3. Isolate DK3 transfected with SsHV2L was hypovirulent on soybean and lettuce and exhibited delayed maturation of sclerotia relative to virus-free DK3, completing Koch's postulates for the association of hypovirulence with SsHV2L.

IMPORTANCE
A cosmopolitan fungus,
Sclerotinia sclerotiorum
infects more than 400 plant species and causes a plant disease known as white mold that produces significant yield losses in major crops annually. Mycoviruses have been used successfully to reduce losses caused by fungal plant pathogens, but definitive relationships between hypovirus infections and hypovirulence in
S. sclerotiorum
were lacking. By establishing a cause-and-effect relationship between
Sclerotinia sclerotiorum
hypovirus Lactuca (SsHV2L) infection and the reduction in host virulence, we showed direct evidence that hypoviruses have the potential to reduce the severity of white mold disease. In addition to intraspecific recombination, this study showed that recent interspecific recombination is an important factor shaping viral genomes. The construction of an infectious clone of SsHV2L allows future exploration of the interactions between SsHV2L and
S. sclerotiorum
, a widespread fungal pathogen of plants.
ORF57 Overcomes the Detrimental Sequence Bias of Kaposi's Sarcoma-Associated Herpesvirus Lytic Genes
Authors: Vogt Carolin , Hackmann Christian , Rabner Alona , Koste Lars , Santag Susann , Kati Semra , Mandel-Gutfreund Yael , Schulz Thomas F. , Bohne Jens
Published: February 2015

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which enhances the expression of intronless KSHV genes on multiple posttranscriptional levels. However, it remains elusive how ORF57 recognizes viral RNAs. Here, we demonstrate that ORF57 also increases the expression of the multiple intron-containing K15 gene. The nucleotide bias of the K15 cDNA revealed an unusual high AT content. Thus, we optimized the K15 cDNA by raising the frequency of GC nucleotides, yielding an ORF57-independent version. To further prove the importance of the sequence bias of ORF57-dependent RNAs, we grouped KSHV mRNAs according to their AT content and found a correlation between AT-richness and ORF57 dependency. More importantly, latent genes, which have to be expressed in the absence of ORF57, have a low AT content and are indeed ORF57 independent. The nucleotide composition of K15 resembles that of HIV
gag
, which cannot be expressed unless RNA export is facilitated by the HIV Rev protein. Interestingly, ORF57 can partially rescue HIV Gag expression. Thus, the KSHV target RNAs of ORF57 and HIV
gag
RNA may share certain motifs based on the nucleotide bias. A bioinformatic comparison between wild-type and sequence-optimized K15 revealed a higher density for hnRNP-binding motifs in the former. We speculate that binding of particular hnRNPs to KSHV lytic transcripts is the prerequisite for ORF57 to enhance their expression.

IMPORTANCE
The mostly intronless genes of KSHV are only expressed in the presence of the viral regulator protein ORF57, but how ORF57 recognizes viral RNAs remains elusive. We focused on the multiple intron-containing KSHV gene K15 and revealed that its expression is also increased by ORF57. Moreover, sequences in the K15 cDNA mediate this enhancement. The quest for a target sequence or a response element for ORF57 in the lytic genes was not successful. Instead, we found the nucleotide bias to be the critical determinant of ORF57 dependency. Based on the fact that ORF57 has only a weak affinity for nucleic acids, we speculate that a cellular RNA-binding protein provides the sequence preference for ORF57. This study provides evidence that herpesviral RNA regulator proteins use the sequence bias of lytic genes and the resulting composition of the viral mRNP to distinguish between viral and cellular mRNAs.
Peptides Designed To Spatially Depict the Epstein-Barr Virus Major Virion Glycoprotein gp350 Neutralization Epitope Elicit Antibodies That Block Virus-Neutralizing Antibody 72A1 Interaction with the Native gp350 Molecule
Authors: Tanner Jerome E. , Coinçon Mathieu , Leblond Valérie , Hu Jing , Fang Janey M. , Sygusch Jurgen , Alfieri Caroline
Published: February 2015

Epstein-Barr virus (EBV) is the etiologic agent of infectious mononucleosis and the root cause of B-cell lymphoproliferative disease in individuals with a weakened immune system, as well as a principal cofactor in nasopharyngeal carcinoma, various lymphomas, and other cancers. The EBV major virion surface glycoprotein gp350 is viewed as the best vaccine candidate to prevent infectious mononucleosis in healthy EBV-naive persons and EBV-related cancers in at-risk individuals. Previous epitope mapping of gp350 revealed only one dominant neutralizing epitope, which has been shown to be the target of the monoclonal antibody 72A1. Computer modeling of the 72A1 antibody interaction with the gp350 amino terminus was used to identify gp350 amino acids that could form strong ionic, electrostatic, or hydrogen bonds with the 72A1 antibody. Peptide DDRTTLQLAQNPVYIPETYPYIKWDN (designated peptide 2) and peptide GSAKPGNGSYFASVKTEMLGNEID (designated peptide 3) were designed to spatially represent the gp350 amino acids predicted to interact with the 72A1 antibody paratope. Peptide 2 bound to the 72A1 antibody and blocked 72A1 antibody recognition of the native gp350 molecule. Peptide 2 and peptide 3 were recognized by human IgG and shown to elicit murine antibodies that could target gp350 and block its recognition by the 72A1 antibody. This work provides a structural mapping of the interaction between the EBV-neutralizing antibody 72A1 and the major virion surface protein gp350. gp350 mimetic peptides that spatially depict the EBV-neutralizing epitope would be useful as a vaccine to focus the immune system exclusively to this important virus epitope.

IMPORTANCEThe production of virus-neutralizing antibodies targeting the Epstein-Barr virus (EBV) major surface glycoprotein gp350 is important for the prevention of infectious mononucleosis and EBV-related cancers. The data presented here provide the firstin silicomap of the gp350 interaction with a virus-blocking monoclonal antibody. Immunization with gp350 peptides identified byin silicomapping generated antibodies that cross-react with the EBV gp350 molecule and block recognition of the gp350 molecule by a virus-neutralizing antibody. Through its ability to focus the immune system exclusively on the gp350 sequence important for viral entry, these peptides may form the basis of an EBV vaccine candidate. This strategy would sidestep the production of other irrelevant gp350 antibodies that divert the immune system from generating a protective antiviral response or that impede access to the virus-blocking epitope by protective antibodies.
Host Proteome Correlates of Vaccine-Mediated Enhanced Disease in a Mouse Model of Respiratory Syncytial Virus Infection
Authors: van Diepen Angela , Brand H. Kim , de Waal Leon , Bijl Maarten , Jong Victor L. , Kuiken Thijs , van Amerongen Geert , van den Ham Henk-Jan , Eijkemans Marinus J. , Osterhaus Albert D. M. E. , Hermans Peter W. M. , Andeweg Arno C.
Published: February 2015

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants. Despite over 50 years of research, to date no safe and efficacious RSV vaccine has been licensed. Many experimental vaccination strategies failed to induce balanced T-helper (Th) responses and were associated with adverse effects such as hypersensitivity and immunopathology upon challenge. In this study, we explored the well-established recombinant vaccinia virus (rVV) RSV-F/RSV-G vaccination-challenge mouse model to study phenotypically distinct vaccine-mediated host immune responses at the proteome level. In this model, rVV-G priming and not rVV-F priming results in the induction of Th2 skewed host responses upon RSV challenge. Mass spectrometry-based spectral count comparisons enabled us to identify seven host proteins for which expression in lung tissue is associated with an aberrant Th2 skewed response characterized by the influx of eosinophils and neutrophils. These proteins are involved in processes related to the direct influx of eosinophils (eosinophil peroxidase [Epx]) and to chemotaxis and extravasation processes (Chil3 [chitinase-like-protein 3]) as well as to eosinophil and neutrophil homing signals to the lung (Itgam). In addition, the increased levels of Arg1 and Chil3 proteins point to a functional and regulatory role for alternatively activated macrophages and type 2 innate lymphoid cells in Th2 cytokine-driven RSV vaccine-mediated enhanced disease.

IMPORTANCERSV alone is responsible for 80% of acute bronchiolitis cases in infants worldwide and causes substantial mortality in developing countries. Clinical trials performed with formalin-inactivated RSV vaccine preparations in the 1960s failed to induce protection upon natural RSV infection and even predisposed patients for enhanced disease. Despite the clinical need, to date no safe and efficacious RSV vaccine has been licensed. Since RSV vaccines have a tendency to prime for unbalanced responses associated with an exuberant influx of inflammatory cells and enhanced disease, detailed characterization of primed host responses has become a crucial element in RSV vaccine research. We investigated the lung proteome of mice challenged with RSV upon priming with vaccine preparations known to induce phenotypically distinct host responses. Seven host proteins whose expression levels are associated with vaccine-mediated enhanced disease have been identified. The identified protein biomarkers support the development as well as detailed evaluation of next-generation RSV vaccines.
Functional Analysis of the Short Isoform of Orf Virus Protein OV20.0
Authors: Tseng Yeu-Yang , Lin Fong-Yuan , Cheng Sun-Fang , Tscharke David , Chulakasian Songkhla , Chou Chia-Chi , Liu Ya-Fen , Chang Wei-Shan , Wong Min-Liang , Hsu Wei-Li
Published: February 2015

Orf virus (ORFV)
OV20.0L
is an ortholog of vaccinia virus (VACV) gene
E3L
. The function of VACV E3 protein as a virulence factor is well studied, but OV20.0 has received less attention. Here we show that like VACV
E3L
,
OV20.0L
encodes two proteins, a full-length protein and a shorter form (sh20). The shorter sh20 is an N-terminally truncated OV20.0 isoform generated when a downstream AUG codon is used for initiating translation. These isoforms differed in cellular localization, with full-length OV20.0 and sh20 found throughout the cell and predominantly in the cytoplasm, respectively. Nonetheless, both OV20.0 isoforms were able to bind double-stranded RNA (dsRNA)-activated protein kinase (PKR) and dsRNA. Moreover, both isoforms strongly inhibited PKR activation as shown by decreased phosphorylation of the translation initiation factor eIF2α subunit and protection of Sindbis virus infection against the activity of interferon (IFN). In spite of this apparent conservation of function
in vitro
, a recombinant ORFV that was able to express only the sh20 isoform was attenuated in a mouse model.

IMPORTANCE
The OV20.0 protein of orf virus (ORFV) has two isoforms and contributes to virulence, but the roles of the two forms are not known. This study shows that the shorter isoform (sh20) arises due to use of a downstream initiation codon and is amino-terminally truncated. The sh20 form also differs in expression kinetics and cellular localization from full-length OV20.0. Similar to the full-length isoform, sh20 is able to bind dsRNA and PKR, inactivate PKR, and thus act as an antagonist of the interferon response
in vitro
.
In vivo
, however, wild-type OV20.0 could not be replaced with sh20 alone without a loss of virulence, suggesting that the functions of the isoforms are not simply redundant.
Archaeal Extrachromosomal Genetic Elements
Authors: Wang Haina , Peng Nan , Shah Shiraz A. , Huang Li , She Qunxin
Published: February 2015

Research on archaeal extrachromosomal genetic elements (ECEs) has progressed rapidly in the past decade. To date, over 60 archaeal viruses and 60 plasmids have been isolated. These archaeal viruses exhibit an exceptional diversity in morphology, with a wide array of shapes, such as spindles, rods, filaments, spheres, head-tails, bottles, and droplets, and some of these new viruses have been classified into one order, 10 families, and 16 genera. Investigation of model archaeal viruses has yielded important insights into mechanisms underlining various steps in the viral life cycle, including infection, DNA replication and transcription, and virion egression. Many of these mechanisms are unprecedented for any known bacterial or eukaryal viruses. Studies of plasmids isolated from different archaeal hosts have also revealed a striking diversity in gene content and innovation in replication strategies. Highly divergent replication proteins are identified in both viral and plasmid genomes. Genomic studies of archaeal ECEs have revealed a modular sequence structure in which modules of DNA sequence are exchangeable within, as well as among, plasmid families and probably also between viruses and plasmids. In particular, it has been suggested that ECE-host interactions have shaped the coevolution of ECEs and their archaeal hosts. Furthermore, archaeal hosts have developed defense systems, including the innate restriction-modification (R-M) system and the adaptive CRISPR (clustered regularly interspaced short palindromic repeats) system, to restrict invasive plasmids and viruses. Together, these interactions permit a delicate balance between ECEs and their hosts, which is vitally important for maintaining an innovative gene reservoir carried by ECEs. In conclusion, while research on archaeal ECEs has just started to unravel the molecular biology of these genetic entities and their interactions with archaeal hosts, it is expected to accelerate in the next decade.
Comparable mRNA expression of inflammatory markers but lower claudin-1 mRNA levels in foreskin tissue of HSV-2 seropositive versus seronegative asymptomatic Kenyan young men
Authors: Röhl Maria , Tjernlund Annelie , Mehta Supriya D , Pettersson Pernilla , Bailey Robert C , Broliden Kristina
Published: February 2015

Objectives

Skin biopsies from local sites of herpes simplex virus 2 (HSV-2)-induced ulcers can show infiltrates of inflammatory cells several months after macroscopic healing. We hypothesise that foreskin tissue samples of asymptomatic HSV-2 seropositive men had remaining signs of inflammation at the molecular level. Even in the absence of clinical lesions, genital inflammation may contribute to increased HIV susceptibility on sexual exposure to the virus.

Setting

Foreskin tissue samples were collected from men undergoing elective circumcision in Kisumu, Kenya.

Participants

The foreskin tissue samples (n=86) were stratified into study groups based on HSV-2 serology and assessed for mRNA expression of inflammatory markers. Markers of interest were further assessed by immunohistochemical staining within the tissue samples.

Results

The two study groups had comparable levels of all molecular markers (CD3, CD4, CD8, CD69, CCR5, HLA-DR, Langerin, DC-SIGN, Mannose Receptor 1, IL-1, IL-6, TNF-α, β7, IgA, IFN-α, CCL5, E-cadherin, ZO-1 and occludin), except for lower mRNA levels of the epithelial junction protein claudin-1 in the HSV-2 seropositive group (p=0.008). Although mRNA levels of claudin-1 were lower in HSV-2 seropositive individuals, the corresponding protein could be visualised in the foreskin epithelium of all samples tested.

Conclusions

Whereas no general inflammation was demonstrated in the foreskin of asymptomatic HSV-2 seropositive individuals, a decreased expression of claudin-1 indicates a less robust genital epithelial barrier. An intact epithelial barrier is essential for blocking mucosal entry of genital infections, including HIV.
A tool for prediction of risk of rehospitalisation and mortality in the hospitalised elderly: secondary analysis of clinical trial data
Authors: Alassaad Anna , Melhus Håkan , Hammarlund-Udenaes Margareta , Bertilsson Maria , Gillespie Ulrika , Sundström Johan
Published: February 2015

Objectives

To construct and internally validate a risk score, the ‘80+ score’, for revisits to hospital and mortality for older patients, incorporating aspects of pharmacotherapy. Our secondary aim was to compare the discriminatory ability of the score with that of three validated tools for measuring inappropriate prescribing: Screening Tool of Older Person's Prescriptions (STOPP), Screening Tool to Alert doctors to Right Treatment (START) and Medication Appropriateness Index (MAI).

Setting

Two acute internal medicine wards at Uppsala University hospital. Patient data were used from a randomised controlled trial investigating the effects of a comprehensive clinical pharmacist intervention.

Participants

Data from 368 patients, aged 80 years and older, admitted to one of the study wards.

Primary outcome measure

Time to rehospitalisation or death during the year after discharge from hospital. Candidate variables were selected among a large number of clinical and drug-specific variables. After a selection process, a score for risk estimation was constructed. The 80+ score was internally validated, and the discriminatory ability of the score and of STOPP, START and MAI was assessed using C-statistics.

Results

Seven variables were selected. Impaired renal function, pulmonary disease, malignant disease, living in a nursing home, being prescribed an opioid or being prescribed a drug for peptic ulcer or gastroesophageal reflux disease were associated with an increased risk, while being prescribed an antidepressant drug (tricyclic antidepressants not included) was linked to a lower risk of the outcome. These variables made up the components of the 80+ score. The C-statistics were 0.71 (80+), 0.57 (STOPP), 0.54 (START) and 0.63 (MAI).

Conclusions

We developed and internally validated a score for prediction of risk of rehospitalisation and mortality in hospitalised older people. The score discriminated risk better than available tools for inappropriate prescribing. Pending external validation, this score can aid in clinical identification of high-risk patients and targeting of interventions.