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Geometry and Adhesion of Extracellular Domains of DC-SIGNR Neck Length Variants Analyzed by Force–Distance Measurements

[Image: see text] Force–distance measurements have been used to examine differences in the interaction of the dendritic cell glycan-binding receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR (L-SIGN) with membranes bearing glycan ligands. The results demonstrate that upon binding to membrane-anchored ligand, DC-SIGNR undergoes a conformational change similar to that previously observed for DC-SIGN. The results also validate a model for the extracellular domain of DC-SIGNR derived from crystallographic studies. Force measurements were performed with DC-SIGNR variants that differ in the length of the neck that result from genetic polymorphisms, which encode different numbers of the 23-amino acid repeat sequences that constitute the neck. The findings are consistent with an elongated, relatively rigid structure of the neck repeat observed in crystals. In addition, differences in the lengths of DC-SIGN and DC-SIGNR extracellular domains with equivalent numbers of neck repeats support a model in which the different dispositions of the carbohydrate-recognition domains in DC-SIGN and DC-SIGNR result from variations in the sequences of the necks.

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Global mRNA Degradation during Lytic Gammaherpesvirus Infection Contributes to Establishment of Viral Latency

During a lytic gammaherpesvirus infection, host gene expression is severely restricted by the global degradation and altered 3′ end processing of mRNA. This host shutoff phenotype is orchestrated by the viral SOX protein, yet its functional significance to the viral lifecycle has not been elucidated, in part due to the multifunctional nature of SOX. Using an unbiased mutagenesis screen of the murine gammaherpesvirus 68 (MHV68) SOX homolog, we isolated a single amino acid point mutant that is selectively defective in host shutoff activity. Incorporation of this mutation into MHV68 yielded a virus with significantly reduced capacity for mRNA turnover. Unexpectedly, the MHV68 mutant showed little defect during the acute replication phase in the mouse lung. Instead, the virus exhibited attenuation at later stages of in vivo infections suggestive of defects in both trafficking and latency establishment. Specifically, mice intranasally infected with the host shutoff mutant accumulated to lower levels at 10 days post infection in the lymph nodes, failed to develop splenomegaly, and exhibited reduced viral DNA levels and a lower frequency of latently infected splenocytes. Decreased latency establishment was also observed upon infection via the intraperitoneal route. These results highlight for the first time the importance of global mRNA degradation during a gammaherpesvirus infection and link an exclusively lytic phenomenon with downstream latency establishment.

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A modified MS2 bacteriophage plaque reduction assay for the rapid screening of antiviral plant extracts

INTRODUCTION: Traditional methods of screening plant extracts and purified components for antiviral activity require up to a week to perform, prompting the need to develop more rapid quantitative methods to measure the ability of plant based preparations to block viral replication. We describe an adaption of an MS2 plaque reduction assay for use in S. aureus. RESULTS: MS2 bacteriophage was capable of infecting and replicating in B. cereus, S. aureus and F + E. coli but not F- E. coli. Indeed, both B. cereus and S. aureus were more sensitive to MS2 induced lysis than F+ E. coli. When MS2 bacteriophage was mixed with Camellia sinensis extract (1 mg/ml), Scaevola spinescens extract (1 mg/ml) or Aloe barbadensis juice and the mixtures inoculated into S. aureus, the formation of plaques was reduced to 8.9 ± 3.8%, 5.4 ± 2.4% and 72.7 ± 20.9% of the untreated MS2 control values respectively. CONCLUSIONS: The ability of the MS2 plaque reduction assay to detect antiviral activity in these known antiviral plant preparations indicates its suitability as an antiviral screening tool. An advantage of this assay compared with traditionally used cytopathic effect reduction assays and replicon based assays is the more rapid acquisition of results. Antiviral activity was detected within 24 h of the start of testing. The MS2 assay is also inexpensive and non-pathogenic to humans making it ideal for initial screening studies or as a simulant for pathogenic viruses.

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Equal Graph Partitioning on Estimated Infection Network as an Effective Epidemic Mitigation Measure

Controlling severe outbreaks remains the most important problem in infectious disease area. With time, this problem will only become more severe as population density in urban centers grows. Social interactions play a very important role in determining how infectious diseases spread, and organization of people along social lines gives rise to non-spatial networks in which the infections spread. Infection networks are different for diseases with different transmission modes, but are likely to be identical or highly similar for diseases that spread the same way. Hence, infection networks estimated from common infections can be useful to contain epidemics of a more severe disease with the same transmission mode. Here we present a proof-of-concept study demonstrating the effectiveness of epidemic mitigation based on such estimated infection networks. We first generate artificial social networks of different sizes and average degrees, but with roughly the same clustering characteristic. We then start SIR epidemics on these networks, censor the simulated incidences, and use them to reconstruct the infection network. We then efficiently fragment the estimated network by removing the smallest number of nodes identified by a graph partitioning algorithm. Finally, we demonstrate the effectiveness of this targeted strategy, by comparing it against traditional untargeted strategies, in slowing down and reducing the size of advancing epidemics.

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Strengthening systems for communicable disease surveillance: creating a laboratory network in Rwanda

BACKGROUND: The recent emergence of a novel strain of influenza virus with pandemic potential underscores the need for quality surveillance and laboratory services to contribute to the timely detection and confirmation of public health threats. To provide a framework for strengthening disease surveillance and response capacities in African countries, the World Health Organization Regional Headquarters for Africa (AFRO) developed Integrated Disease Surveillance and Response (IDSR) aimed at improving national surveillance and laboratory systems. IDSR emphasizes the linkage of information provided by public health laboratories to the selection of relevant, appropriate and effective public health responses to disease outbreaks. METHODS: We reviewed the development of Rwanda's National Reference Laboratory (NRL) to understand essential structures involved in creating a national public health laboratory network. We reviewed documents describing the NRL's organization and record of test results, conducted site visits, and interviewed health staff in the Ministry of Health and in partner agencies. Findings were developed by organizing thematic categories and grouping examples within them. We purposefully sought to identify success factors as well as challenges inherent in developing a national public health laboratory system. RESULTS: Among the identified success factors were: a structured governing framework for public health surveillance; political commitment to promote leadership for stronger laboratory capacities in Rwanda; defined roles and responsibilities for each level; coordinated approaches between technical and funding partners; collaboration with external laboratories; and use of performance results in advocacy with national stakeholders. Major challenges involved general infrastructure, human resources, and budgetary constraints. CONCLUSIONS: Rwanda's experience with collaborative partnerships contributed to creation of a functional public health laboratory network.

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Prediction of outcome and prognosis of patients on mechanical ventilation using body mass index, SOFA score, C-Reactive protein, and serum albumin

CONTEXT: Body mass index (BMI), serum albumin, and C-reactive protein (CRP) appear to be major determinants of hospitalization. AIM: To determine the predictive ability of BMI, Sequential Organ Failure Assessment (SOFA score), serum albumin, and CRP to assess the duration and outcome of mechanical ventilation (MV). MATERIALS AND METHODS: Thirty patients aged >18 years who required mechanical ventilation (MV) were enrolled for the study. They were divided into two groups; patients who improved (Group 1), patients who expired (Group 2). Group 1 was further divided into two groups: patients on MV for <5 days (Group A), and patients on MV for >5days (Group B). BMI and SOFA score were calculated, and serum albumin and CRP were estimated. RESULTS AND DISCUSSION: Out of the 30 patients, 18 patients successfully improved after MV (Group 1) and 12 patients expired (Group 2). Among the 18 patients in group 1, ten patients improved within 5 days (Group A) and 8 patients after 5 days (Group B). SOFA score and CRP were significantly increased (P value 0.0003 and 0.0001, respectively) in group 2 when compared to group 1. CRP >24.2 mg/L or SOFA score >7 at the start of MV increases the probability of mortality by factor 13.08 or 3.92, respectively The above parameters did not show any statistical difference when group A was compared to group B. CONCLUSION: Simple, economic and easily accessible markers like CRP and assessment tools of critically ill patients with SOFA score are important determinants of possible outcomes of a patient from MV.

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Immunity Traits in Pigs: Substantial Genetic Variation and Limited Covariation

BACKGROUND: Increasing robustness via improvement of resistance to pathogens is a major selection objective in livestock breeding. As resistance traits are difficult or impossible to measure directly, potential indirect criteria are measures of immune traits (ITs). Our underlying hypothesis is that levels of ITs with no focus on specific pathogens define an individual's immunocompetence and thus predict response to pathogens in general. Since variation in ITs depends on genetic, environmental and probably epigenetic factors, our aim was to estimate the relative importance of genetics. In this report, we present a large genetic survey of innate and adaptive ITs in pig families bred in the same environment. METHODOLOGY/PRINCIPAL FINDINGS: Fifty four ITs were studied on 443 Large White pigs vaccinated against Mycoplasma hyopneumoniae and analyzed by combining a principal component analysis (PCA) and genetic parameter estimation. ITs include specific and non specific antibodies, seric inflammatory proteins, cell subsets by hemogram and flow cytometry, ex vivo production of cytokines (IFNα, TNFα, IL6, IL8, IL12, IFNγ, IL2, IL4, IL10), phagocytosis and lymphocyte proliferation. While six ITs had heritabilities that were weak or not significantly different from zero, 18 and 30 ITs had moderate (0.1<h2≤0.4) or high (h2>0.4) heritability values, respectively. Phenotypic and genetic correlations between ITs were weak except for a few traits that mostly include cell subsets. PCA revealed no cluster of innate or adaptive ITs. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that variation in many innate and adaptive ITs is genetically controlled in swine, as already reported for a smaller number of traits by other laboratories. A limited redundancy of the traits was also observed confirming the high degree of complementarity between innate and adaptive ITs. Our data provide a genetic framework for choosing ITs to be included as selection criteria in multitrait selection programmes that aim to improve both production and health traits.

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Demographic, etiological, and histological pulmonary analysis of patients with acute respiratory failure: a study of 19 years of autopsies

INTRODUCTION: Acute respiratory failure has been one of the most important causes of death in intensive care units, and certain aspects of its pulmonary pathology are currently unknown. OBJECTIVES: The objective was to describe the demographic data, etiology, and pulmonary histopathological findings of different diseases in the autopsies of patients with acute respiratory failure. METHOD: Autopsies of 4,710 patients with acute respiratory failure from 1990 to 2008 were reviewed, and the following data were obtained: age, sex, and major associated diseases. The pulmonary histopathology was categorized as diffuse alveolar damage, pulmonary edema, alveolar hemorrhage, and lymphoplasmacytic interstitial pneumonia. The odds ratio of the concordance between the major associated diseases and specific autopsy findings was calculated using logistic regression. RESULTS: Bacterial bronchopneumonia was present in 33.9% of the cases and cancer in 28.1%. The pulmonary histopathology showed diffuse alveolar damage in 40.7% (1,917) of the cases. A multivariate analysis showed a significant and powerful association between diffuse alveolar damage and bronchopneumonia, HIV/AIDS, sepsis, and septic shock, between liver cirrhosis and pulmonary embolism, between pulmonary edema and acute myocardial infarction, between dilated cardiomyopathy and cancer, between alveolar hemorrhage and bronchopneumonia and pulmonary embolism, and between lymphoplasmacytic interstitial pneumonia and HIV/AIDS and liver cirrhosis. CONCLUSIONS: Bronchopneumonia was the most common diagnosis in these cases. The most prevalent pulmonary histopathological pattern was diffuse alveolar damage, which was associated with different inflammatory conditions. Further studies are necessary to elucidate the complete pathophysiological mechanisms involved with each disease and the development of acute respiratory failure.

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Type 1 Interferon Induction of Natural Killer Cell Gamma Interferon Production for Defense during Lymphocytic Choriomeningitis Virus Infection

Natural killer (NK) cells are equipped to innately produce the cytokine gamma interferon (IFN-γ) in part because they basally express high levels of the signal transducer and activator of transcription 4 (STAT4). Type 1 interferons (IFNs) have the potential to activate STAT4 and promote IFN-γ expression, but concurrent induction of elevated STAT1 negatively regulates access to the pathway. As a consequence, it has been difficult to detect type 1 IFN stimulation of NK cell IFN-γ during viral infections in the presence of STAT1 and to understand the evolutionary advantage for maintaining the pathway. The studies reported here evaluated NK cell responses following infections with lymphocytic choriomeningitis virus (LCMV) in the compartment handling the earliest events after infection, the peritoneal cavity. The production of type 1 IFNs, both IFN-α and IFN-β, was shown to be early and of short duration, peaking at 30 h after challenge. NK cell IFN-γ expression was detected with overlapping kinetics and required activating signals delivered through type 1 IFN receptors and STAT4. It took place under conditions of high STAT4 levels but preceded elevated STAT1 expression in NK cells. The IFN-γ response reduced viral burdens. Interestingly, increases in STAT1 were delayed in NK cells compared to other peritoneal exudate cell (PEC) populations. Taken together, the studies demonstrate a novel mechanism for stimulating IFN-γ production and elucidate a biological role for type 1 IFN access to STAT4 in NK cells.

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The Interaction of LFA-1 on Mononuclear Cells and ICAM-1 on Tubular Epithelial Cells Accelerates TGF-β1-Induced Renal Epithelial-Mesenchymal Transition

The epithelial-mesenchymal transition (EMT) of renal epithelial cells (RTECs) has pivotal roles in the development of renal fibrosis. Although the interaction of lymphocyte function-associated antigen 1 (LFA-1) on leukocytes and its ligand, intracellular adhesion molecule 1 (ICAM-1), plays essential roles in most inflammatory reactions, its pathogenetic role in the EMT of RTECs remains to be clarified. In the present study, we investigated the effect of the interaction of LFA-1 on peripheral blood mononuclear cells (PBMCs) and ICAM-1 on HK-2 cells after stimulation with TGF-β(1) on the EMT of RTECs. ICAM-1 was highly expressed in HK-2 cells. After TGF-β(1) stimulation, the chemokines CCL3 and CXCL12 increased on HK-2 cells. After co-culture of PBMCs and HK-2 cells pre-stimulated with TGF-β(1) (0.1 ng/ml) (HK-2-TGF-β(1) (0.1)), the expression of the active form of LFA-1 increased on PBMCs; however, total LFA-1 expression did not change. The expression of the active form of LFA-1 on PBMCs did not increase after co-culture with not CCL3 but CXCL12 knockdown HK-2-TGF-β(1) (0.1). The expression of epithelial cell junction markers (E-cadherin and occludin) further decreased and that of mesenchymal markers (vimentin and fibronectin) further increased in HK-2-TGF-β(1) (0.1) after co-culture with PBMCs for 24 hrs (HK-2-TGF-β(1) (0.1)-PBMCs). The phosphorylation of ERK 1/2 but not smad2 and smad3 increased in HK-2-TGF-β(1) (0.1)-PBMCs. The snail and slug signaling did not increase HK-2-TGF-β(1) (0.1)-PBMCs. Although the migration and invasion of HK-2 cells induced full EMT by a high dose (10.0 ng/ml) and long-term (72–96 hrs) TGF-β(1) stimulation increased, that of HK-2-TGF-β(1) (0.1)-PBMCs did not increase. These results suggested that HK-2 cells stimulated with TGF-β(1) induced conformational activation of LFA-1 on PBMCs by increased CXCL12. Then, the direct interaction of LFA-1 on PBMCs and ICAM-1 on HK-2 cells activated ERK1/2 signaling to accelerate the part of EMT of HK-2 cells induced by TGF-β(1.)

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Histological study of the biodynamics of iron oxide nanoparticles with different diameters

The biodynamics of ultrasmall and small superparamagnetic iron oxide (USPIO and SPIO, respectively) particles that were injected intraperitoneally into 36 C57BL/6 mice were investigated chronologically. Their distribution was studied histologically at six time points by measuring iron-positive areas (μm(2)) in organ sections stained with Prussian blue. The uptake of the differently sized particles was also compared by cultured murine macrophages (J774.1). Iron-positive areas in the liver were significantly larger in the mice injected with USPIO than those injected with SPIO at the first three time points (P < 0.05). The amount of USPIO in the lung parenchyma around the airway was larger than that of SPIO at four time points (P < 0.05); distribution to the lymph nodes was not significantly different. The amount of iron was significantly larger in SPIO- than USPIO-treated cultured cells (P < 0.05). In conclusion, it is suggested that intra peritoneally injected USPIO particles could be used more quickly than SPIO to make Kupffer images of the liver and that both agents could help get lymph node images of similar quality.

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The Microbial Detection Array Combined with Random Phi29-Amplification Used as a Diagnostic Tool for Virus Detection in Clinical Samples

A common technique used for sensitive and specific diagnostic virus detection in clinical samples is PCR that can identify one or several viruses in one assay. However, a diagnostic microarray containing probes for all human pathogens could replace hundreds of individual PCR-reactions and remove the need for a clear clinical hypothesis regarding a suspected pathogen. We have established such a diagnostic platform for random amplification and subsequent microarray identification of viral pathogens in clinical samples. We show that Phi29 polymerase-amplification of a diverse set of clinical samples generates enough viral material for successful identification by the Microbial Detection Array, demonstrating the potential of the microarray technique for broad-spectrum pathogen detection. We conclude that this method detects both DNA and RNA virus, present in the same sample, as well as differentiates between different virus subtypes. We propose this assay for diagnostic analysis of viruses in clinical samples.

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B Cell Repertoire Analysis Identifies New Antigenic Domains on Glycoprotein B of Human Cytomegalovirus which Are Target of Neutralizing Antibodies

Human cytomegalovirus (HCMV), a herpesvirus, is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and infected newborns. Efforts are underway to prepare effective subunit vaccines and therapies including antiviral antibodies. However, current vaccine efforts are hampered by the lack of information on protective immune responses against HCMV. Characterizing the B-cell response in healthy infected individuals could aid in the design of optimal vaccines and therapeutic antibodies. To address this problem, we determined, for the first time, the B-cell repertoire against glycoprotein B (gB) of HCMV in different healthy HCMV seropositive individuals in an unbiased fashion. HCMV gB represents a dominant viral antigenic determinant for induction of neutralizing antibodies during infection and is also a component in several experimental HCMV vaccines currently being tested in humans. Our findings have revealed that the vast majority (>90%) of gB-specific antibodies secreted from B-cell clones do not have virus neutralizing activity. Most neutralizing antibodies were found to bind to epitopes not located within the previously characterized antigenic domains (AD) of gB. To map the target structures of these neutralizing antibodies, we generated a 3D model of HCMV gB and used it to identify surface exposed protein domains. Two protein domains were found to be targeted by the majority of neutralizing antibodies. Domain I, located between amino acids (aa) 133–343 of gB and domain II, a discontinuous domain, built from residues 121–132 and 344–438. Analysis of a larger panel of human sera from HCMV seropositive individuals revealed positivity rates of >50% against domain I and >90% against domain II, respectively. In accordance with previous nomenclature the domains were designated AD-4 (Dom II) and AD-5 (Dom I), respectively. Collectively, these data will contribute to optimal vaccine design and development of antibodies effective in passive immunization.

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Inflammatory Cytokine Expression Is Associated with Chikungunya Virus Resolution and Symptom Severity

The Chikungunya virus infection zones have now quickly spread from Africa to parts of Asia, North America and Europe. Originally thought to trigger a disease of only mild symptoms, recently Chikungunya virus caused large-scale fatalities and widespread economic loss that was linked to recent virus genetic mutation and evolution. Due to the paucity of information on Chikungunya immunological progression, we investigated the serum levels of 13 cytokines/chemokines during the acute phase of Chikungunya disease and 6- and 12-month post-infection follow-up from patients of the Italian outbreak. We found that CXCL9/MIG, CCL2/MCP-1, IL-6 and CXCL10/IP-10 were significantly raised in the acute phase compared to follow-up samples. Furthermore, IL-1β, TNF-α, Il-12, IL-10, IFN-γ and IL-5 had low initial acute phase levels that significantly increased at later time points. Analysis of symptom severity showed association with CXCL9/MIG, CXCL10/IP-10 and IgG levels. These data give insight into Chikungunya disease establishment and subsequent convalescence, which is imperative to the treatment and containment of this quickly evolving and frequently re-emerging disease.

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The Failure of R (0)

The basic reproductive ratio, R (0), is one of the fundamental concepts in mathematical biology. It is a threshold parameter, intended to quantify the spread of disease by estimating the average number of secondary infections in a wholly susceptible population, giving an indication of the invasion strength of an epidemic: if R (0) < 1, the disease dies out, whereas if R (0) > 1, the disease persists. R (0) has been widely used as a measure of disease strength to estimate the effectiveness of control measures and to form the backbone of disease-management policy. However, in almost every aspect that matters, R (0) is flawed. Diseases can persist with R (0) < 1, while diseases with R (0) > 1 can die out. We show that the same model of malaria gives many different values of R (0), depending on the method used, with the sole common property that they have a threshold at 1. We also survey estimated values of R (0) for a variety of diseases, and examine some of the alternatives that have been proposed. If R (0) is to be used, it must be accompanied by caveats about the method of calculation, underlying model assumptions and evidence that it is actually a threshold. Otherwise, the concept is meaningless.

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Identification of a Highly Conserved H1 Subtype-Specific Epitope with Diagnostic Potential in the Hemagglutinin Protein of Influenza A Virus

Subtype specificity of influenza A virus (IAV) is determined by its two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). For HA, 16 distinct subtypes (H1–H16) exist, while nine exist for NA. The epidemic strains of H1N1 IAV change frequently and cause annual seasonal epidemics as well as occasional pandemics, such as the notorious 1918 influenza pandemic. The recent introduction of pandemic A/H1N1 IAV (H1N1pdm virus) into humans re-emphasizes the public health concern about H1N1 IAV. Several studies have identified conserved epitopes within specific HA subtypes that can be used for diagnostics. However, immune specific epitopes in H1N1 IAV have not been completely assessed. In this study, linear epitopes on the H1N1pdm viral HA protein were identified by peptide scanning using libraries of overlapping peptides against convalescent sera from H1N1pdm patients. One epitope, P5 (aa 58–72) was found to be immunodominant in patients and to evoke high titer antibodies in mice. Multiple sequence alignments and in silico coverage analysis showed that this epitope is highly conserved in influenza H1 HA [with a coverage of 91.6% (9,860/10,767)] and almost completely absent in other subtypes [with a coverage of 3.3% (792/23,895)]. This previously unidentified linear epitope is located outside the five well-recognized antigenic sites in HA. A peptide ELISA method based on this epitope was developed and showed high correlation (χ(2) = 51.81, P<0.01, Pearson correlation coefficient R = 0.741) with a hemagglutination inhibition test. The highly conserved H1 subtype-specific immunodominant epitope may form the basis for developing novel assays for sero-diagnosis and active surveillance against H1N1 IAVs.

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Stimulation of stop codon readthrough: frequent presence of an extended 3′ RNA structural element

In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 3′-adjacent to the UGA. However, analysis of variability at synonymous sites revealed strikingly enhanced conservation within the ∼150 nt 3′-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem–loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem–loop increases readthrough by up to 10-fold. The same computational analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 3′ RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis.

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Antigen-expressing immunostimulatory liposomes as a genetically programmable synthetic vaccine

Liposomes are versatile (sub)micron-sized membrane vesicles that can be used for a variety of applications, including drug delivery and in vivo imaging but they also represent excellent models for artificial membranes or cells. Several studies have demonstrated that in vitro transcription and translation can take place inside liposomes to obtain compartmentalized production of functional proteins within the liposomes (Kita et al. in Chembiochem 9(15):2403–2410, 2008; Moritani et al.in FEBS J, 2010; Kuruma et al. in Methods Mol Biol 607:161–171, 2010; Murtas et al. in Biochem Biophys Res Commun 363(1):12–17, 2007; Sunami et al. in Anal Biochem 357(1):128–136, 2006; Ishikawa et al. in FEBS Lett 576(3):387–390, 2004; Oberholzer et al. in Biochem Biophys Res Commun 261(2):238–241, 1999). Such a minimal artificial cell-based model is ideal for synthetic biology based applications. In this study, we propose the use of liposomes as artificial microbes for vaccination. These artificial microbes can be genetically programmed to produce specific antigens at will. To show proof-of-concept for this artificial cell-based platform, a bacterial in vitro transcription and translation system together with a gene construct encoding the model antigen β-galactosidase were entrapped inside multilamellar liposomes. Vaccination studies in mice showed that such antigen-expressing immunostimulatory liposomes (AnExILs) elicited higher specific humoral immune responses against the produced antigen (β-galactosidase) than control vaccines (i.e. AnExILs without genetic input, liposomal β-galactosidase or pDNA encoding β-galactosidase). In conclusion, AnExILs present a new platform for DNA-based vaccines which combines antigen production, adjuvanticity and delivery in one system and which offer several advantages over existing vaccine formulations.

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Chinese Herbal Formula Xiao Yao San for Treatment of Depression: A Systematic Review of Randomized Controlled Trials

Objectives. To assess the beneficial and adverse effects of Xiaoyaosan for depression. Search Strategy. Electronic databases were searched until December 2009. Inclusion Criteria. We included randomized clinical trials testing Xiaoyaosan against placebo, antidepressants, or combined with antidepressants against antidepressants alone. Data Extraction and Analyses. Study selection, data extraction, quality assessment, and data analyses were conducted according to the Cochrane standards. Results. 26 randomized trials (involving 1837 patients) were included and the methodological quality was evaluated as generally low. The pooled results showed that Xiaoyaosan combined with antidepressants was more effective in comprehensive effect, the score of HAMD and the score of SDS compared with antidepressants alone. Xiaoyaosan was superior to antidepressants for the score of HAMD. However, Xiaoyaosan was not different from placebo for the score of SDS. There was no adverse effects reported in the trials from Xiaoyaosan. Conclusions. Xiaoyaosan appears to be effective on improving symptoms in patients with depression. However, due to poor methodological quality in the majority of included trials, the potential benefit from Xiaoyaosan need to be confirmed in rigorous trials and the design and reporting of trials should follow international standards.

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Preventing the next 'SARS' - European healthcare workers' attitudes towards monitoring their health for the surveillance of newly emerging infections: qualitative study

BACKGROUND: Hospitals are often the epicentres of newly circulating infections. Healthcare workers (HCWs) are at high risk of acquiring infectious diseases and may be among the first to contract emerging infections. This study aims to explore European HCWs' perceptions and attitudes towards monitoring their absence and symptom reports for surveillance of newly circulating infections. METHODS: A qualitative study with thematic analysis was conducted using focus group methodology. Forty-nine hospital-based HCWs from 12 hospitals were recruited to six focus groups; two each in England and Hungary and one each in Germany and Greece. RESULTS: HCWs perceived risk factors for occupationally acquired infectious diseases to be 1.) exposure to patients with undiagnosed infections 2.) break-down in infection control procedures 3.) immuno-naïvety and 4.) symptomatic colleagues. They were concerned that a lack of monitoring and guidelines for infectious HCWs posed a risk to staff and patients and felt employers failed to take a positive interest in their health. Staffing demands and loss of income were noted as pressures to attend work when unwell. In the UK, Hungary and Greece participants felt monitoring staff absence and the routine disclosure of symptoms could be appropriate provided the effectiveness and efficiency of such a system were demonstrable. In Germany, legislation, privacy and confidentiality were identified as barriers. All HCWs highlighted the need for knowledge and structural improvements for timelier recognition of emerging infections. These included increased suspicion and awareness among staff and standardised, homogenous absence reporting systems. CONCLUSIONS: Monitoring absence and infectious disease symptom reports among HCWs may be a feasible means of surveillance for emerging infections in some settings. A pre-requisite will be tackling the drivers for symptomatic HCWs to attend work.

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Informational needs assessment of non‐Hodgkin lymphoma survivors and their physicians

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Prediction of Peptide Reactivity with Human IVIg through a Knowledge-Based Approach

The prediction of antibody-protein (antigen) interactions is very difficult due to the huge variability that characterizes the structure of the antibodies. The region of the antigen bound to the antibodies is called epitope. Experimental data indicate that many antibodies react with a panel of distinct epitopes (positive reaction). The Challenge 1 of DREAM5 aims at understanding whether there exists rules for predicting the reactivity of a peptide/epitope, i.e., its capability to bind to human antibodies. DREAM 5 provided a training set of peptides with experimentally identified high and low reactivities to human antibodies. On the basis of this training set, the participants to the challenge were asked to develop a predictive model of reactivity. A test set was then provided to evaluate the performance of the model implemented so far. We developed a logistic regression model to predict the peptide reactivity, by facing the challenge as a machine learning problem. The initial features have been generated on the basis of the available knowledge and the information reported in the dataset. Our predictive model had the second best performance of the challenge. We also developed a method, based on a clustering approach, able to “in-silico” generate a list of positive and negative new peptide sequences, as requested by the DREAM5 “bonus round” additional challenge. The paper describes the developed model and its results in terms of reactivity prediction, and highlights some open issues concerning the propensity of a peptide to react with human antibodies.

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Description of two measles outbreaks in the Lazio Region, Italy (2006-2007). Importance of pockets of low vaccine coverage in sustaining the infection

BACKGROUND: Despite the launch of the national plan for measles elimination, in Italy, immunization coverage remains suboptimal and outbreaks continue to occur. Two measles outbreaks, occurred in Lazio region during 2006-2007, were investigated to identify sources of infection, transmission routes, and assess operational implications for elimination of the disease. METHODS: Data were obtained from several sources, the routine infectious diseases surveillance system, field epidemiological investigations, and molecular genotyping of virus by the national reference laboratory. RESULTS: Overall 449 cases were reported, sustained by two different stereotypes overlapping for few months. Serotype D4 was likely imported from Romania by a Roma/Sinti family and subsequently spread to the rest of the population. Serotype B3 was responsible for the second outbreak which started in a secondary school. Pockets of low vaccine coverage individuals (Roma/Sinti communities, high school students) facilitated the reintroduction of serotypes not endemic in Italy and facilitated the measles infection to spread. CONCLUSIONS: Communities with low vaccine coverage represent a more serious public health threat than do sporadic susceptible individuals. The successful elimination of measles will require additional efforts to immunize low vaccine coverage population groups, including hard-to-reach individuals, adolescents, and young adults. An enhanced surveillance systems, which includes viral genotyping to document chains of transmission, is an essential tool for evaluating strategy to control and eliminate measles

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Systematic Identification of Novel, Essential Host Genes Affecting Bromovirus RNA Replication

Positive-strand RNA virus replication involves viral proteins and cellular proteins at nearly every replication step. Brome mosaic virus (BMV) is a well-established model for dissecting virus-host interactions and is one of very few viruses whose RNA replication, gene expression and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. Previously, our laboratory identified ∼100 non-essential host genes whose loss inhibited or enhanced BMV replication at least 3-fold. However, our isolation of additional BMV-modulating host genes by classical genetics and other results underscore that genes essential for cell growth also contribute to BMV RNA replication at a frequency that may be greater than that of non-essential genes. To systematically identify novel, essential host genes affecting BMV RNA replication, we tested a collection of ∼900 yeast strains, each with a single essential gene promoter replaced by a doxycycline-repressible promoter, allowing repression of gene expression by adding doxycycline to the growth medium. Using this strain array of ∼81% of essential yeast genes, we identified 24 essential host genes whose depleted expression reproducibly inhibited or enhanced BMV RNA replication. Relevant host genes are involved in ribosome biosynthesis, cell cycle regulation and protein homeostasis, among other cellular processes. BMV 2a(Pol) levels were significantly increased in strains depleted for a heat shock protein (HSF1) or proteasome components (PRE1 and RPT6), suggesting these genes may affect BMV RNA replication by directly or indirectly modulating 2a(Pol) localization, post-translational modification or interacting partners. Investigating the diverse functions of these newly identified essential host genes should advance our understanding of BMV-host interactions and normal cellular pathways, and suggest new modes of virus control.

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Unusual association of ST-T abnormalities, myocarditis and cardiomyopathy with H(1)N(1 )influenza in pregnancy: two case reports and review of the literature

INTRODUCTION: Myocarditis is rarely reported as an extra-pulmonary manifestation of influenza while pregnancy is a rare cause of cardiomyopathy. Pregnancy was identified as a major risk factor for increased mortality and morbidity due to H(1)N(1 )influenza in the pandemic of 2009 to 2010. However, to the best of our knowledge there are no previous reports in the literature linking H(1)N(1 )with myocarditis in pregnancy. CASE PRESENTATION: We report the cases of two pregnant Caucasian women (aged 29 and 30), with no pre-existing illness, presenting with respiratory manifestations of H(1)N(1 )influenza virus infection in their third trimester. Both women developed evidence of myocarditis. One woman developed acute respiratory distress syndrome, almost reaching the point of requiring extra-corporeal membrane oxygenation, and subsequently developed persistent cardiomyopathy; the other recovered without any long-term consequence. CONCLUSIONS: While it is not possible to ascertain retrospectively if myocarditis was caused by either infection with H(1)N(1 )virus or as a result of pregnancy (in the absence of endomyocardial biopsies), the significant association with myocardial involvement in both women demonstrates the increased risk of exposure to H(1)N(1 )influenza virus in pregnant women. This highlights the need for health care providers to increase awareness amongst caregivers to target this 'at risk' group aggressively with vaccination and prompt treatment.

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Dominating Biological Networks

Proteins are essential macromolecules of life that carry out most cellular processes. Since proteins aggregate to perform function, and since protein-protein interaction (PPI) networks model these aggregations, one would expect to uncover new biology from PPI network topology. Hence, using PPI networks to predict protein function and role of protein pathways in disease has received attention. A debate remains open about whether network properties of “biologically central (BC)” genes (i.e., their protein products), such as those involved in aging, cancer, infectious diseases, or signaling and drug-targeted pathways, exhibit some topological centrality compared to the rest of the proteins in the human PPI network. To help resolve this debate, we design new network-based approaches and apply them to get new insight into biological function and disease. We hypothesize that BC genes have a topologically central (TC) role in the human PPI network. We propose two different concepts of topological centrality. We design a new centrality measure to capture complex wirings of proteins in the network that identifies as TC those proteins that reside in dense extended network neighborhoods. Also, we use the notion of domination and find dominating sets (DSs) in the PPI network, i.e., sets of proteins such that every protein is either in the DS or is a neighbor of the DS. Clearly, a DS has a TC role, as it enables efficient communication between different network parts. We find statistically significant enrichment in BC genes of TC nodes and outperform the existing methods indicating that genes involved in key biological processes occupy topologically complex and dense regions of the network and correspond to its “spine” that connects all other network parts and can thus pass cellular signals efficiently throughout the network. To our knowledge, this is the first study that explores domination in the context of PPI networks.

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Neglected Disease – African Sleeping Sickness: Recent Synthetic and Modeling Advances

Human African Trypanosomiasis (HAT) also called sleeping sickness is caused by subspecies of the parasitic hemoflagellate Trypanosoma brucei that mostly occurs in sub-Saharan Africa. The current chemotherapy of the human trypanosomiases relies on only six drugs, five of which have been developed more than 30 years ago, have undesirable toxic side effects and most of them show drug-resistance. Though development of new anti-trypanosomal drugs seems to be a priority area research in this area has lagged far behind. The given review mainly focus upon the recent synthetic and computer based approaches made by various research groups for the development of newer anti-trypanosomal analogues which may have improved efficacy and oral bioavailability than the present ones. The given paper also attempts to investigate the relationship between the various physiochemical parameters and anti-trypanosomal activity that may be helpful in development of potent anti-trypanosomal agents against sleeping sickness.

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NoD: a Nucleolar localization sequence detector for eukaryotic and viral proteins

BACKGROUND: Nucleolar localization sequences (NoLSs) are short targeting sequences responsible for the localization of proteins to the nucleolus. Given the large number of proteins experimentally detected in the nucleolus and the central role of this subnuclear compartment in the cell, NoLSs are likely to be important regulatory elements controlling cellular traffic. Although many proteins have been reported to contain NoLSs, the systematic characterization of this group of targeting motifs has only recently been carried out. RESULTS: Here, we describe NoD, a web server and a command line program that predicts the presence of NoLSs in proteins. Using the web server, users can submit protein sequences through the NoD input form and are provided with a graphical output of the NoLS score as a function of protein position. While the web server is most convenient for making prediction for just a few proteins, the command line version of NoD can return predictions for complete proteomes. NoD is based on our recently described human-trained artificial neural network predictor. Through stringent independent testing of the predictor using available experimentally validated NoLS-containing eukaryotic and viral proteins, the NoD sensitivity and positive predictive value were estimated to be 71% and 79% respectively. CONCLUSIONS: NoD is the first tool to provide predictions of nucleolar localization sequences in diverse eukaryotes and viruses. NoD can be run interactively online at http://www.compbio.dundee.ac.uk/nod or downloaded to use locally.

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Avian Influenza Risk Perception and Preventive Behavior among Traditional Market Workers and Shoppers in Taiwan: Practical Implications for Prevention

BACKGROUND: Avian influenza (AI) can be highly pathogenic and fatal. Preventive behavior such as handwashing and wearing face masks has been recommended. However, little is known about what psychosocial factors might influence people's decision to adopt such preventive behavior. This study aims to explore risk perception and other factors associated with handwashing and wearing face masks to prevent AI. METHODOLOGY/PRINCIPAL FINDINGS: An interviewer-administered survey was conducted among 352 traditional market workers and shoppers in Taiwan between December 2009 and January 2010. Factors associated with the recommended AI preventive behavior (i.e., when in a traditional market, wearing a face mask and also washing hands after any contact with poultry) included: having correct knowledge about the fatality rate of AI (adjusted odds ratio [AOR] = 4.18), knowing of severe cases of AI (AOR = 2.13), being informed of local AI outbreaks (AOR = 2.24), living in northeastern Taiwan (AOR = 6.01), having a senior high-school education (AOR = 3.33), and having a university or higher education (AOR = 6.86). Gender interactive effect was also found among participants with a senior high-school education, with males being less likely to engage in the recommended AI preventive behavior than their female counterparts (AOR = 0.34). CONCLUSIONS/SIGNIFICANCE: Specific information concerning AI risk perception was associated with the recommended AI preventive behavior. In particular, having correct knowledge about the fatality rate of AI and being informed of severe cases and local outbreaks of AI were linked to increased AI preventive behavior. These findings underscore the importance of transparency in dealing with epidemic information. These results also have practical implications for prevention and policy-making to more effectively promote the recommended AI preventive behavior in the public.

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In the name of the greater good?

In the event of a pandemic that poses widespread infection and high death rates, the utilitarian mandate to ‘reduce harm’ is the relevant moral value that trumps other ethical considerations. The primacy of a utilitarian approach dictates that those who are in a position to assist the cessation of the most serious outbreaks in whatever role they may have, must be present to provide their services, and those who administer health care must also be present to ensure that all responders are supported and protected to the highest degree.

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Emerging viral threats in Gabon: health capacities and response to the risk of emerging zoonotic diseases in Central Africa

Emerging infectious diseases (EID) are currently the major threat to public health worldwide and most EID events have involved zoonotic infectious agents. Central Africa in general and Gabon in particular are privileged areas for the emergence of zoonotic EIDs. Indeed, human incursions in Gabonese forests for exploitation purposes lead to intensified contacts between humans and wildlife thus generating an increased risk of emergence of zoonotic diseases. In Gabon, 51 endemic or potential endemic viral infectious diseases have been reported. Among them, 22 are of zoonotic origin and involve 12 families of viruses. The most notorious are dengue, yellow fever, ebola, marburg, Rift Valley fever and chikungunya viruses. Potential EID due to wildlife in Gabon are thereby plentiful and need to be inventoried. The Gabonese Public Health system covers geographically most of the country allowing a good access to sanitary information and efficient monitoring of emerging diseases. However, access to treatment and prevention is better in urban areas where medical structures are more developed and financial means are concentrated even though the population is equally distributed between urban and rural areas. In spite of this, Gabon could be a good field for investigating the emergence or re-emergence of zoonotic EID. Indeed Gabonese health research structures such as CIRMF, advantageously located, offer high quality researchers and facilities that study pathogens and wildlife ecology, aiming toward a better understanding of the contact and transmission mechanisms of new pathogens from wildlife to human, the emergence of zoonotic EID and the breaking of species barriers by pathogens.

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Poverty, Global Health, and Infectious Disease: Lessons from Haiti and Rwanda

Poverty and infectious diseases interact in complex ways. Casting destitution as intractable, or epidemics that afflict the poor as accidental, erroneously exonerates us from responsibility for caring for those most in need. Adequately addressing communicable diseases requires a biosocial appreciation of the structural forces that shape disease patterns. Most health interventions in resource-poor settings could garner support based on cost/benefit ratios with appropriately lengthy time horizons to capture the return on health investments and an adequate accounting of externalities; however, such a calculus masks the suffering of inaction and risks eroding the most powerful incentive to act: redressing inequality.

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Forecasting incidence of hemorrhagic fever with renal syndrome in China using ARIMA model

BACKGROUND: China is a country that is most seriously affected by hemorrhagic fever with renal syndrome (HFRS) with 90% of HFRS cases reported globally. At present, HFRS is getting worse with increasing cases and natural foci in China. Therefore, there is an urgent need for monitoring and predicting HFRS incidence to make the control of HFRS more effective. In this study, we applied a stochastic autoregressive integrated moving average (ARIMA) model with the objective of monitoring and short-term forecasting HFRS incidence in China. METHODS: Chinese HFRS data from 1975 to 2008 were used to fit ARIMA model. Akaike Information Criterion (AIC) and Ljung-Box test were used to evaluate the constructed models. Subsequently, the fitted ARIMA model was applied to obtain the fitted HFRS incidence from 1978 to 2008 and contrast with corresponding observed values. To assess the validity of the proposed model, the mean absolute percentage error (MAPE) between the observed and fitted HFRS incidence (1978-2008) was calculated. Finally, the fitted ARIMA model was used to forecast the incidence of HFRS of the years 2009 to 2011. All analyses were performed using SAS9.1 with a significant level of p < 0.05. RESULTS: The goodness-of-fit test of the optimum ARIMA (0,3,1) model showed non-significant autocorrelations in the residuals of the model (Ljung-Box Q statistic = 5.95,P = 0.3113). The fitted values made by ARIMA (0,3,1) model for years 1978-2008 closely followed the observed values for the same years, with a mean absolute percentage error (MAPE) of 12.20%. The forecast values from 2009 to 2011 were 0.69, 0.86, and 1.21per 100,000 population, respectively. CONCLUSION: ARIMA models applied to historical HFRS incidence data are an important tool for HFRS surveillance in China. This study shows that accurate forecasting of the HFRS incidence is possible using an ARIMA model. If predicted values from this study are accurate, China can expect a rise in HFRS incidence.

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Delivery Systems for Intradermal Vaccination

Intradermal (ID) vaccination can offer improved immunity and simpler logistics of delivery, but its use in medicine is limited by the need for simple, reliable methods of ID delivery. ID injection by the Mantoux technique requires special training and may not reliably target skin, but is nonetheless used currently for BCG and rabies vaccination. Scarification using a bifurcated needle was extensively used for smallpox eradication, but provides variable and inefficient delivery into the skin. Recently, ID vaccination has been simplified by introduction of a simple-to-use hollow microneedle that has been approved for ID injection of influenza vaccine in Europe. Various designs of hollow microneedles have been studied preclinically and in humans. Vaccines can also be injected into skin using needle-free devices, such as jet injection, which is receiving renewed clinical attention for ID vaccination. Projectile delivery using powder and gold particles (i.e., gene gun) have also been used clinically for ID vaccination. Building off the scarification approach, a number of preclinical studies have examined solid microneedle patches for use with vaccine coated onto metal microneedles, encapsulated within dissolving microneedles or added topically to skin after microneedle pretreatment, as well as adapting tattoo guns for ID vaccination. Finally, technologies designed to increase skin permeability in combination with a vaccine patch have been studied through the use of skin abrasion, ultrasound, electroporation, chemical enhancers, and thermal ablation. The prospects for bringing ID vaccination into more widespread clinical practice are encouraging, given the large number of technologies for ID delivery under development.

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Recurrent Recruitment Manoeuvres Improve Lung Mechanics and Minimize Lung Injury during Mechanical Ventilation of Healthy Mice

INTRODUCTION: Mechanical ventilation (MV) of mice is increasingly required in experimental studies, but the conditions that allow stable ventilation of mice over several hours have not yet been fully defined. In addition, most previous studies documented vital parameters and lung mechanics only incompletely. The aim of the present study was to establish experimental conditions that keep these parameters within their physiological range over a period of 6 h. For this purpose, we also examined the effects of frequent short recruitment manoeuvres (RM) in healthy mice. METHODS: Mice were ventilated at low tidal volume V(T) = 8 mL/kg or high tidal volume V(T) = 16 mL/kg and a positive end-expiratory pressure (PEEP) of 2 or 6 cmH(2)O. RM were performed every 5 min, 60 min or not at all. Lung mechanics were followed by the forced oscillation technique. Blood pressure (BP), electrocardiogram (ECG), heart frequency (HF), oxygen saturation and body temperature were monitored. Blood gases, neutrophil-recruitment, microvascular permeability and pro-inflammatory cytokines in bronchoalveolar lavage (BAL) and blood serum as well as histopathology of the lung were examined. RESULTS: MV with repetitive RM every 5 min resulted in stable respiratory mechanics. Ventilation without RM worsened lung mechanics due to alveolar collapse, leading to impaired gas exchange. HF and BP were affected by anaesthesia, but not by ventilation. Microvascular permeability was highest in atelectatic lungs, whereas neutrophil-recruitment and structural changes were strongest in lungs ventilated with high tidal volume. The cytokines IL-6 and KC, but neither TNF nor IP-10, were elevated in the BAL and serum of all ventilated mice and were reduced by recurrent RM. Lung mechanics, oxygenation and pulmonary inflammation were improved by increased PEEP. CONCLUSIONS: Recurrent RM maintain lung mechanics in their physiological range during low tidal volume ventilation of healthy mice by preventing atelectasis and reduce the development of pulmonary inflammation.

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Virtual High-Throughput Screening Identifies Mycophenolic Acid as a Novel RNA Capping Inhibitor

The RNA guanylyltransferase (GTase) is involved in the synthesis of the (m7)Gppp-RNA cap structure found at the 5′ end of eukaryotic mRNAs. GTases are members of the covalent nucleotidyl transferase superfamily, which also includes DNA and RNA ligases. GTases catalyze a two-step reaction in which they initially utilize GTP as a substrate to form a covalent enzyme-GMP intermediate. The GMP moiety is then transferred to the diphosphate end of the RNA transcript in the second step of the reaction to form the Gppp-RNA structure. In the current study, we used a combination of virtual database screening, homology modeling, and biochemical assays to search for novel GTase inhibitors. Using this approach, we demonstrate that mycophenolic acid (MPA) can inhibit the GTase reaction by preventing the catalytic transfer of the GMP moiety onto an acceptor RNA. As such, MPA represents a novel type of inhibitor against RNA guanylyltransferases that inhibits the second step of the catalytic reaction. Moreover, we show that the addition of MPA to S. cerevisiae cells leads to a reduction of capped mRNAs. Finally, biochemical assays also demonstrate that MPA can inhibit DNA ligases through inhibition of the second step of the reaction. The biological implications of these findings for the MPA-mediated inhibition of members of the covalent nucleotidyl superfamily are discussed.

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Long-term follow-up and treatment of congenital alveolar proteinosis

BACKGROUND: Clinical presentation, diagnosis, management and outcome of molecularly defined congenital pulmonary alveolar proteinosis (PAP) due to mutations in the GM-CSF receptor are not well known. CASE PRESENTATION: A 2 1/2 years old girl was diagnosed as having alveolar proteinosis. Whole lung lavages were performed with a new catheter balloon technique, feasible in small sized airways. Because of some interstitial inflammation in the lung biopsy and to further improve the condition, empirical therapy with systemic steroids and azathioprin, and inhaled and subcutaneous GMCSF, were used. Based on clinical measures, total protein and lipid recovered by whole lung lavages, all these treatments were without benefit. Conversely, severe respiratory viral infections and an invasive aspergillosis with aspergilloma formation occurred. Recently the novel homozygous stop mutation p.Ser25X of the GMCSF receptor alpha chain was identified in the patient. This mutation leads to a lack of functional GMCSF receptor and a reduced response to GMCSF stimulation of CD11b expression of mononuclear cells of the patient. Subsequently a very intense treatment with monthly lavages was initiated, resulting for the first time in complete resolution of partial respiratory insufficiency and a significant improvement of the overall somato-psychosocial condition of the child. CONCLUSIONS: The long term management from early childhood into young adolescence of severe alveolar proteinosis due to GMCSF receptor deficiency requires a dedicated specialized team to perform technically demanding whole lung lavages and cope with complications.

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Severe influenza cases in paediatric intensive care units in Germany during the pre-pandemic seasons 2005 to 2008

BACKGROUND: Data on complications in children with seasonal influenza virus infection are limited. We initiated a nation-wide three-year surveillance of children who were admitted to a paediatric intensive care unit (PICU) with severe seasonal influenza. METHODS: From October 2005 to July 2008, active surveillance was performed using an established reporting system for rare diseases (ESPED) including all paediatric hospitals in Germany. Cases to be reported were hospitalized children < 17 years of age with laboratory-confirmed influenza treated in a PICU or dying in hospital. RESULTS: Twenty severe influenza-associated cases were reported from 14 PICUs during three pre-pandemic influenza seasons (2005-2008). The median age of the patients (12 males/8 females) was 7.5 years (range 0.1-15 years). None had received vaccination against influenza. In 14 (70%) patients, the infection had been caused by influenza A and in five (25%) by influenza B; in one child (5%) the influenza type was not reported. Patients spent a median of 19 (IQR 12-38) days in the hospital and a median of 11 days (IQR 6-18 days) in the PICU; 10 (50%) needed mechanical ventilation. Most frequent diagnoses were influenza-associated pneumonia (60%), bronchitis/bronchiolitis (30%), encephalitis/encephalopathy (25%), secondary bacterial pneumonia (25%), and ARDS (25%). Eleven (55%) children had chronic underlying medical conditions, including 8 (40%) with chronic pulmonary diseases. Two influenza A- associated deaths were reported: i) an 8-year old boy with pneumococcal encephalopathy following influenza infection died from cerebral edema, ii) a 14-year-old boy with asthma bronchiale, cardiac malformation and Addison's disease died from cardiac and respiratory failure. For nine (45%) patients, possibly permanent sequelae were reported (3 neurological, 3 pulmonary, 3 other sequelae). CONCLUSIONS: Influenza-associated pneumonia and secondary bacterial infections are relevant complications of seasonal influenza in Germany. The incidence of severe influenza cases in PICUs was relatively low. This may be either due to the weak to moderate seasonal influenza activity during the years 2005 to 2008 or due to under-diagnosis of influenza by physicians. Fifty% of the observed severe cases might have been prevented by following the recommendations for vaccination of risk groups in Germany.

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Next-Generation Sequencing Reveals HIV-1-Mediated Suppression of T Cell Activation and RNA Processing and Regulation of Noncoding RNA Expression in a CD4(+) T Cell Line

Next-generation sequencing (NGS) enables the highly sensitive measurement of whole transcriptomes. We report the first application to our knowledge of this technology to the analysis of RNA from a CD4(+) T cell line infected with intact HIV. We sequenced the total mRNA from infected cells and detected differences in the expression of both host and viral mRNA. Viral reads represented a large portion of the total mapped sequencing reads: approximately 20% at 12 h postinfection (hpi) and 40% at 24 hpi. We also detected a small but significant suppression of T cell activation-related genes at 12 hpi. This suppression persisted and expanded by 24 hpi, providing new possible markers of virus-induced T cell cytopathology. By 24 hpi, the expression of over 50% of detectable host loci was also altered, indicating widespread alteration of host processes, including RNA processing, splicing, and transport to an extent not previously reported. In addition, next-generation sequencing provided insights into alternative viral RNA splice events and the expression of noncoding RNAs, including microRNA host genes.

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Epidemiological and clinical characteristics of childhood pandemic 2009 H1N1 virus infection: an observational cohort study

BACKGROUND: There was a pandemic influenza around the world in 2009 including South Korea since last pandemic occurred four decades ago. We aimed to evaluate the epidemiological and clinical characteristics of this infection in childhood. METHODS: We evaluated the epidemiologic characteristics of all the subjects infected with the 2009 H1N1 influenza A virus (2,971 patients, ≤ 15 years of age), and the clinical and laboratory findings of the inpatients (217 patients, 80 had pneumonia) between 1 September 2009 and 31 January 2010 in a single hospital throughout the epidemic. RESULTS: The age distribution of all the subjects was relatively even. Over 90% of cases occurred during a two-month period. Two hundred and five patients (94.5%) received oseltamivir within 48 h of fever onset, and 97% of inpatients defervesced within 48 h of medication. The group with pneumonia included more males than females, and had higher leukocytes counts with lower lymphocyte differentials than the group without pneumonia. The white blood cell count and lymphocyte differential were associated with the severity of pneumonia. Corticosteroid treatment for severe pneumonia patients was highly effective in preventing disease progression. CONCLUSION: Children of all ages affected with even rates of infection, but males were predominant in pneumonia patients. Pneumonia patients showed lymphopenia and its severity was associated with the severity of illness. Our results suggest that the mechanism of lung injury in 2009 H1N1 virus infection may be associated with the host immune response.

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Immunodetection of occult eosinophils in lung tissue biopsies may help predict survival in acute lung injury

BACKGROUND: Acute lung injury (ALI) is a serious respiratory disorder for which therapy is primarily supportive once infection is excluded. Surgical lung biopsy may rule out other diagnoses, but has not been generally useful for therapy decisions or prognosis in this setting. Importantly, tissue and peripheral blood eosinophilia, the hallmarks of steroid-responsive acute eosinophilic pneumonia, are not commonly linked with ALI. We hypothesized that occult eosinophilic pneumonia may explain better outcomes for some patients with ALI. METHODS: Immunohistochemistry using a novel monoclonal antibody recognizing eosinophil peroxidase (EPX-mAb) was used to assess intrapulmonary eosinophil accumulation/degranulation. Lung biopsies from ALI patients (n = 20) were identified following review of a pathology database; 45% of which (i.e., 9/20) displayed classical diffuse alveolar damage (ALI-DAD). Controls were obtained from uninvolved tissue in patients undergoing lobectomy for lung cancer (n = 10). Serial biopsy sections were stained with hematoxylin and eosin (H&E) and subjected to EPX-mAb immunohistochemistry. RESULTS: EPX-mAb immunohistochemistry provided a >40-fold increased sensitivity to detect eosinophils in the lung relative to H&E stained sections. This increased sensitivity led to the identification of higher numbers of eosinophils in ALI patients compared with controls; differences using H&E staining alone were not significant. Clinical assessments showed that lung infiltrating eosinophil numbers were higher in ALI patients that survived hospitalization compared with non-survivors. A similar conclusion was reached quantifying eosinophil degranulation in each biopsy. CONCLUSION: The enhanced sensitivity of EPX-mAb immunohistochemistry uniquely identified eosinophil accumulation/degranulation in patients with ALI relative to controls. More importantly, this method was a prognostic indicator of patient survival. These observations suggest that EPX-mAb immunohistochemistry may represent a diagnostic biomarker identifying a subset of ALI patients with improved clinical outcomes.

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Overlapping signals for translational regulation and packaging of influenza A virus segment 2

Influenza A virus segment 2 mRNA expresses three polypeptides: PB1, PB1-F2 and PB1-N40, from AUGs 1, 4 and 5 respectively. Two short open reading frames (sORFs) initiated by AUGs 2 and 3 are also present. To understand translational regulation in this system, we systematically mutated AUGs 1–4 and monitored polypeptide synthesis from plasmids and recombinant viruses. This identified sORF2 as a key regulatory element with opposing effects on PB1-F2 and PB1-N40 expression. We propose a model in which AUGs 1–4 are accessed by leaky ribosomal scanning, with sORF2 repressing synthesis of downstream PB1-F2. However, sORF2 also up-regulates PB1-N40 expression, most likely by a reinitiation mechanism that permits skipping of AUG4. Surprisingly, we also found that in contrast to plasmid-driven expression, viruses with improved AUG1 initiation contexts produced less PB1 in infected cells and replicated poorly, producing virions with elevated particle:PFU ratios. Analysis of the genome content of virus particles showed reduced packaging of the mutant segment 2 vRNAs. Overall, we conclude that segment 2 mRNA translation is regulated by a combination of leaky ribosomal scanning and reinitiation, and that the sequences surrounding the PB1 AUG codon are multifunctional, containing overlapping signals for translation initiation and for segment-specific packaging.

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The ferret as a model organism to study influenza A virus infection

Influenza is a human pathogen that continues to pose a public health threat. The use of small mammalian models has become indispensable for understanding the virulence of influenza viruses. Among numerous species used in the laboratory setting, only the ferret model is equally well suited for studying both the pathogenicity and transmissibility of human and avian influenza viruses. Here, we compare the advantages and limitations of the mouse, ferret and guinea pig models for research with influenza A viruses, emphasizing the multifarious uses of the ferret in the assessment of influenza viruses with pandemic potential. Research performed in the ferret model has provided information, support and guidance for the public health response to influenza viruses in humans. We highlight the recent and emerging uses of this species in influenza virus research that are advancing our understanding of virus-host interactions.

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Leukocyte- and Platelet-Derived Microvesicle Interactions following In Vitro and In Vivo Activation of Toll-Like Receptor 4 by Lipopolysaccharide

BACKGROUND: Pro-coagulant membrane microvesicles (MV) derived from platelets and leukocytes are shed into the circulation following receptor-mediated activation, cell-cell interaction, and apoptosis. Platelets are sentinel markers of toll-like receptor 4 (TLR4) activation. Experiments were designed to evaluate the time course and mechanism of direct interactions between platelets and leukocytes following acute activation of TLR4 by bacterial lipopolysaccharide (LPS). METHODOLOGY/PRINCIPAL FINDINGS: Blood from age-matched male and female wild type (WT) and TLR4 gene deleted (dTLR4) mice was incubated with ultra-pure E. coli LPS (500 ng/ml) for up to one hour. At designated periods, leukocyte antigen positive platelets, platelet antigen positive leukocytes and cell-derived MV were quantified by flow cytometry. Numbers of platelet- or leukocyte-derived MV did not increase within one hour following in vitro exposure of blood to LPS. However, with LPS stimulation numbers of platelets staining positive for both platelet- and leukocyte-specific antigens increased in blood derived from WT but not dTLR4 mice. This effect was blocked by inhibition of TLR4 signaling mediated by My88 and TRIF. Seven days after a single intravenous injection of LPS (500 ng/mouse or 20 ng/gm body wt) to WT mice, none of the platelets stained for leukocyte antigen. However, granulocytes, monocytes and apoptotic bodies stained positive for platelet antigens. CONCLUSIONS/SIGNIFICANCE: Within one hour of exposure to LPS, leukocytes exchange surface antigens with platelets through TLR4 activation. In vivo, leukocyte expression of platelet antigen is retained after a single exposure to LPS following turn over of the platelet pool. Acute expression of leukocyte antigen on platelets within one hour of exposure to LPS and the sustained expression of platelet antigen on leukocytes following a single acute exposure to LPS in vivo explains, in part, associations of platelets and leukocytes in response to bacterial infection and changes in thrombotic propensity of the blood.

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NKG2D contributes to efficient clearance of picornavirus from the acutely infected murine brain

Activated murine cytotoxic T cells express the NKG2D natural cytotoxicity receptor. This receptor recognizes MHC class I-like molecules expressed on the surface of infected cells and serves to augment T cell-mediated cytotoxicity. The role of NKG2D-mediated augmentation in the clearance of central nervous system viral infections has not been explored. Using the Theiler's murine encephalomyelitis virus model we found that NKG2D-positive CD8+ cytotoxic T cells enter the brain, that NKG2D ligands are expressed in the brain during acute infection, and that interruption of NKG2D ligand recognition via treatment with a function blocking antibody attenuates the efficacy of viral clearance from the central nervous system.

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Discovery and Genomic Characterization of a Novel Ovine Partetravirus and a New Genotype of Bovine Partetravirus

Partetravirus is a recently described group of animal parvoviruses which include the human partetravirus, bovine partetravirus and porcine partetravirus (previously known as human parvovirus 4, bovine hokovirus and porcine hokovirus respectively). In this report, we describe the discovery and genomic characterization of partetraviruses in bovine and ovine samples from China. These partetraviruses were detected by PCR in 1.8% of bovine liver samples, 66.7% of ovine liver samples and 71.4% of ovine spleen samples. One of the bovine partetraviruses detected in the present samples is phylogenetically distinct from previously reported bovine partetraviruses and likely represents a novel genotype. The ovine partetravirus is a novel partetravirus and phylogenetically most related to the bovine partetraviruses. The genome organization is conserved amongst these viruses, including the presence of a putative transmembrane protein encoded by an overlapping reading frame in ORF2. Results from the present study provide further support to the classification of partetraviruses as a separate genus in Parvovirinae.

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Reconstructing disease outbreaks from genetic data: a graph approach

Epidemiology and public health planning will increasingly rely on the analysis of genetic sequence data. In particular, genetic data coupled with dates and locations of sampled isolates can be used to reconstruct the spatiotemporal dynamics of pathogens during outbreaks. Thus far, phylogenetic methods have been used to tackle this issue. Although these approaches have proved useful for informing on the spread of pathogens, they do not aim at directly reconstructing the underlying transmission tree. Instead, phylogenetic models infer most recent common ancestors between pairs of isolates, which can be inadequate for densely sampled recent outbreaks, where the sample includes ancestral and descendent isolates. In this paper, we introduce a novel method based on a graph approach to reconstruct transmission trees directly from genetic data. Using simulated data, we show that our approach can efficiently reconstruct genealogies of isolates in situations where classical phylogenetic approaches fail to do so. We then illustrate our method by analyzing data from the early stages of the swine-origin A/H1N1 influenza pandemic. Using 433 isolates sequenced at both the hemagglutinin and neuraminidase genes, we reconstruct the likely history of the worldwide spread of this new influenza strain. The presented methodology opens new perspectives for the analysis of genetic data in the context of disease outbreaks.

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Human Bocavirus – Insights into a Newly Identified Respiratory Virus

Human Bocavirus (HBoV) was discovered in 2005 using a molecular virus screening technique. It is often found in respiratory samples and is a likely cause for respiratory diseases in children. HBoV is distributed worldwide and has been found not only in respiratory samples, but also in feces, urine and serum. HBoV infections are mostly found in young children and coinfections with other respiratory viruses are often found, exacerbating the efforts to link HBoV to specific symptoms. The purpose of this review is to give an overview of recent HBoV research, highlighting some recent findings.

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Plasmacytoid Dendritic Cells and the Control of Herpesvirus Infections

Type-I interferons (IFN-I) are cytokines essential for vertebrate antiviral defense, including against herpesviruses. IFN-I have potent direct antiviral activities and also mediate a multiplicity of immunoregulatory functions, which can either promote or dampen antiviral adaptive immune responses. Plasmacytoid dendritic cells (pDCs) are the professional producers of IFN-I in response to many viruses, including all of the herpesviruses tested. There is strong evidence that pDCs could play a major role in the initial orchestration of both innate and adaptive antiviral immune responses. Depending on their activation pattern, pDC responses may be either protective or detrimental to the host. Here, we summarize and discuss current knowledge regarding pDC implication in the physiopathology of mouse and human herpesvirus infections, and we discuss how pDC functions could be manipulated in immunotherapeutic settings to promote health over disease.

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Communicable Diseases Prioritized for Surveillance and Epidemiological Research: Results of a Standardized Prioritization Procedure in Germany, 2011

INTRODUCTION: To establish strategic priorities for the German national public health institute (RKI) and guide the institute's mid-term strategic decisions, we prioritized infectious pathogens in accordance with their importance for national surveillance and epidemiological research. METHODS: We used the Delphi process with internal (RKI) and external experts and a metric-consensus approach to score pathogens according to ten three-tiered criteria. Additional experts were invited to weight each criterion, leading to the calculation of a median weight by which each score was multiplied. We ranked the pathogens according to the total weighted score and divided them into four priority groups. RESULTS: 127 pathogens were scored. Eighty-six experts participated in the weighting; “Case fatality rate” was rated as the most important criterion. Twenty-six pathogens were ranked in the highest priority group; among those were pathogens with internationally recognised importance (e.g., Human Immunodeficiency Virus, Mycobacterium tuberculosis, Influenza virus, Hepatitis C virus, Neisseria meningitides), pathogens frequently causing large outbreaks (e.g., Campylobacter spp.), and nosocomial pathogens associated with antimicrobial resistance. Other pathogens in the highest priority group included Helicobacter pylori, Respiratory Syncytial Virus, Varicella zoster virus and Hantavirus. DISCUSSION: While several pathogens from the highest priority group already have a high profile in national and international health policy documents, high scores for other pathogens (e.g., Helicobacter pylori, Respiratory syncytial virus or Hantavirus) indicate a possible under-recognised importance within the current German public health framework. A process to strengthen respective surveillance systems and research has been started. The prioritization methodology has worked well; its modular structure makes it potentially useful for other settings.

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A Vesicular Stomatitis Virus Replicon-Based Bioassay for the Rapid and Sensitive Determination of Multi-Species Type I Interferon

Type I interferons (IFN) comprise a family of cytokines that signal through a common cellular receptor to induce a plethora of genes with antiviral and other activities. Recombinant IFNs are used for the treatment of hepatitis C virus infection, multiple sclerosis, and certain malignancies. The capability of type I IFN to suppress virus replication and resultant cytopathic effects is frequently used to measure their bioactivity. However, these assays are time-consuming and require appropriate biosafety containment. In this study, an improved IFN assay is presented which is based on a recombinant vesicular stomatitis virus (VSV) replicon encoding two reporter proteins, firefly luciferase and green fluorescent protein. The vector lacks the essential envelope glycoprotein (G) gene of VSV and is propagated on a G protein-expressing transgenic cell line. Several mammalian and avian cells turned out to be susceptible to infection with the complemented replicon particles. Infected cells readily expressed the reporter proteins at high levels five hours post infection. When human fibroblasts were treated with serial dilutions of human IFN-β prior to infection, reporter expression was accordingly suppressed. This method was more sensitive and faster than a classical IFN bioassay based on VSV cytopathic effects. In addition, the antiviral activity of human IFN-λ (interleukin-29), a type III IFN, was determined on Calu-3 cells. Both IFN-β and IFN-λ were acid-stable, but only IFN-β was resistant to alkaline treatment. The antiviral activities of canine, porcine, and avian type I IFN were analysed with cell lines derived from the corresponding species. This safe bioassay will be useful for the rapid and sensitive quantification of multi-species type I IFN and potentially other antiviral cytokines.

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Characterization of Neutralizing Profiles in HIV-1 Infected Patients from whom the HJ16, HGN194 and HK20 mAbs were Obtained

Several new human monoclonal antibodies (mAbs) with a neutralizing potential across different subtypes have recently been described. Three mAbs, HJ16, HGN194 and HK20, were obtained from patients within the HIV-1 cohort of the Institute of Tropical Medicine (ITM). Our aim was to generate immunization antibodies equivalent to those seen in plasma. Here, we describe the selection and characterization of patient plasma and their mAbs, using a range of neutralization assays, including several peripheral blood mononuclear cell (PBMC) based assays and replicating primary viruses as well as cell line based assays and pseudoviruses (PV). The principal criterion for selection of patient plasma was the activity in an ‘extended incubation phase’ PBMC assay. Neutralizing Abs, derived from their memory B cells, were then selected by ELISA with envelope proteins as solid phase. MAbs were subsequently tested in a high-throughput HOS-PV assay to assess functional neutralization. The present study indicates that the strong profiles in the patients' plasma were not solely due to antibodies represented by the newly isolated mAbs. Although results from the various assays were divergent, they by and large indicate that neutralizing Abs to other epitopes of the HIV-1 envelope are present in the plasma and synergy between Abs may be important. Thus, the spectrum of the obtained mAbs does not cover the range of cross-reactivity seen in plasma in these carefully selected patients irrespective of which neutralization assay is used. Nevertheless, these mAbs are relevant for immunogen discovery because they bind to the recombinant glycoproteins to which the immune response needs to be targeted in vivo. Our observations illustrate the remaining challenges required for successful immunogen design and development.

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EBV-gp350 Confers B-Cell Tropism to Tailored Exosomes and Is a Neo-Antigen in Normal and Malignant B Cells—A New Option for the Treatment of B-CLL

gp350, the major envelope protein of Epstein-Barr-Virus, confers B-cell tropism to the virus by interacting with the B lineage marker CD21. Here we utilize gp350 to generate tailored exosomes with an identical tropism. These exosomes can be used for the targeted co-transfer of functional proteins to normal and malignant human B cells. We demonstrate here the co-transfer of functional CD154 protein on tailored gp350+ exosomes to malignant B blasts from patients with B chronic lymphocytic leukemia (B-CLL), rendering B blasts immunogenic to tumor-reactive autologous T cells. Intriguingly, engulfment of gp350+ exosomes by B-CLL cells and presentation of gp350-derived peptides also re-stimulated EBV-specific T cells and redirected the strong antiviral cellular immune response in patients to leukemic B cells. In essence, we show that gp350 alone confers B-cell tropism to exosomes and that these exosomes can be further engineered to simultaneously trigger virus- and tumor-specific immune responses. The simultaneous exploitation of gp350 as a tropism molecule for tailored exosomes and as a neo-antigen in malignant B cells provides a novel attractive strategy for immunotherapy of B-CLL and other B-cell malignancies.

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The Ebola Virus Glycoprotein and HIV-1 Vpu Employ Different Strategies to Counteract the Antiviral Factor Tetherin

The antiviral protein tetherin/BST2/CD317/HM1.24 restricts cellular egress of human immunodeficiency virus (HIV) and of particles mimicking the Ebola virus (EBOV), a hemorrhagic fever virus. The HIV-1 viral protein U (Vpu) and the EBOV-glycoprotein (EBOV-GP) both inhibit tetherin. Here, we compared tetherin counteraction by EBOV-GP and Vpu. We found that EBOV-GP but not Vpu counteracted tetherin from different primate species, indicating that EBOV-GP and Vpu target tetherin differentially. Tetherin interacted with the GP2 subunit of EBOV-GP, which might encode the determinants for tetherin counteraction. Vpu reduced cell surface expression of tetherin while EBOV-GP did not, suggesting that both proteins employ different mechanisms to counteract tetherin. Finally, Marburg virus (MARV)–GP also inhibited tetherin and downregulated tetherin in a cell type–dependent fashion, indicating that tetherin antagonism depends on the cellular source of tetherin. Collectively, our results indicate that EBOV-GP counteracts tetherin by a novel mechanism and that tetherin inhibition is conserved between EBOV-GP and MARV-GP.

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Detection of a Fourth Orbivirus Non-Structural Protein

The genus Orbivirus includes both insect and tick-borne viruses. The orbivirus genome, composed of 10 segments of dsRNA, encodes 7 structural proteins (VP1–VP7) and 3 non-structural proteins (NS1–NS3). An open reading frame (ORF) that spans almost the entire length of genome segment-9 (Seg-9) encodes VP6 (the viral helicase). However, bioinformatic analysis recently identified an overlapping ORF (ORFX) in Seg-9. We show that ORFX encodes a new non-structural protein, identified here as NS4. Western blotting and confocal fluorescence microscopy, using antibodies raised against recombinant NS4 from Bluetongue virus (BTV, which is insect-borne), or Great Island virus (GIV, which is tick-borne), demonstrate that these proteins are synthesised in BTV or GIV infected mammalian cells, respectively. BTV NS4 is also expressed in Culicoides insect cells. NS4 forms aggregates throughout the cytoplasm as well as in the nucleus, consistent with identification of nuclear localisation signals within the NS4 sequence. Bioinformatic analyses indicate that NS4 contains coiled-coils, is related to proteins that bind nucleic acids, or are associated with membranes and shows similarities to nucleolar protein UTP20 (a processome subunit). Recombinant NS4 of GIV protects dsRNA from degradation by endoribonucleases of the RNAse III family, indicating that it interacts with dsRNA. However, BTV NS4, which is only half the putative size of the GIV NS4, did not protect dsRNA from RNAse III cleavage. NS4 of both GIV and BTV protect DNA from degradation by DNAse. NS4 was found to associate with lipid droplets in cells infected with BTV or GIV or transfected with a plasmid expressing NS4.

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Geographic Distribution and Risk Factors of the Initial Adult Hospitalized Cases of 2009 Pandemic Influenza A (H1N1) Virus Infection in Mainland China

BACKGROUND: As of 31(st) March 2010, more than 127,000 confirmed cases of 2009 pandemic influenza A (H1N1), including 800 deaths, were reported in mainland China. The distribution and characteristics of the confirmed cases in the initial phase of this pandemic in this country are largely unknown. The present study aimed to characterize the geographic distribution and patient characteristics of H1N1 infection in the 2009 pandemic as well as to identify potential risk factors associated with adverse patient outcome in China, through retrospective analyses of 885 hospitalized cases with confirmed H1N1 infection. METHODOLOGY/PRINCIPAL FINDINGS: The proportional hazards model was employed to detect risk factors for adverse outcome; the geo-statistical maps were used to characterize the distribution of all 2668 confirmed H1N1 patients throughout mainland China. The number of new cases increased slowly in May, 2009, but rapidly between June and August of the year. Confirmed cases were reported in 26 provinces; Beijing, Guangdong, Shanghai, Zhejiang and Fujian were the top five regions of the incidence of the virus infection. After being adjusted for gender, age, chronic pulmonary disease and other general symptoms, delay for more than two days before hospital admission (HR: 0.6; 95%CI: 0.5–0.7) and delayed onset of the H1N1-specific respiratory symptoms (HR: 0.3; 95%CI: 0.2–0.4) were associated with adverse patient outcome. CONCLUSIONS/SIGNIFICANCE: The 2009 pandemic influenza A affected east and southeast coastal provinces and most populous cities more severely than other regions in mainland China due to higher risk of high level traffic-, high population density-, and high population mobility-associated H1N1 transmission.The clinical symptoms were mild in the initial phase of infection. Delayed hospital admission and delayed appearance of respiratory symptoms were among the major risk factors for poor patient outcome. These findings may have significant implications in the future pandemic preparedness and response.

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Inferring viral quasispecies spectra from 454 pyrosequencing reads

BACKGROUND: RNA viruses infecting a host usually exist as a set of closely related sequences, referred to as quasispecies. The genomic diversity of viral quasispecies is a subject of great interest, particularly for chronic infections, since it can lead to resistance to existing therapies. High-throughput sequencing is a promising approach to characterizing viral diversity, but unfortunately standard assembly software was originally designed for single genome assembly and cannot be used to simultaneously assemble and estimate the abundance of multiple closely related quasispecies sequences. RESULTS: In this paper, we introduce a new Viral Spectrum Assembler (ViSpA) method for quasispecies spectrum reconstruction and compare it with the state-of-the-art ShoRAH tool on both simulated and real 454 pyrosequencing shotgun reads from HCV and HIV quasispecies. Experimental results show that ViSpA outperforms ShoRAH on simulated error-free reads, correctly assembling 10 out of 10 quasispecies and 29 sequences out of 40 quasispecies. While ShoRAH has a significant advantage over ViSpA on reads simulated with sequencing errors due to its advanced error correction algorithm, ViSpA is better at assembling the simulated reads after they have been corrected by ShoRAH. ViSpA also outperforms ShoRAH on real 454 reads. Indeed, 7 most frequent sequences reconstructed by ViSpA from a real HCV dataset are viable (do not contain internal stop codons), and the most frequent sequence was within 1% of the actual open reading frame obtained by cloning and Sanger sequencing. In contrast, only one of the sequences reconstructed by ShoRAH is viable. On a real HIV dataset, ShoRAH correctly inferred only 2 quasispecies sequences with at most 4 mismatches whereas ViSpA correctly reconstructed 5 quasispecies with at most 2 mismatches, and 2 out of 5 sequences were inferred without any mismatches. ViSpA source code is available at http://alla.cs.gsu.edu/~software/VISPA/vispa.html. CONCLUSIONS: ViSpA enables accurate viral quasispecies spectrum reconstruction from 454 pyrosequencing reads. We are currently exploring extensions applicable to the analysis of high-throughput sequencing data from bacterial metagenomic samples and ecological samples of eukaryote populations.

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Ebola Virion Attachment and Entry into Human Macrophages Profoundly Effects Early Cellular Gene Expression

Zaire ebolavirus (ZEBOV) infections are associated with high lethality in primates. ZEBOV primarily targets mononuclear phagocytes, which are activated upon infection and secrete mediators believed to trigger initial stages of pathogenesis. The characterization of the responses of target cells to ZEBOV infection may therefore not only further understanding of pathogenesis but also suggest possible points of therapeutic intervention. Gene expression profiles of primary human macrophages exposed to ZEBOV were determined using DNA microarrays and quantitative PCR to gain insight into the cellular response immediately after cell entry. Significant changes in mRNA concentrations encoding for 88 cellular proteins were observed. Most of these proteins have not yet been implicated in ZEBOV infection. Some, however, are inflammatory mediators known to be elevated during the acute phase of disease in the blood of ZEBOV-infected humans. Interestingly, the cellular response occurred within the first hour of Ebola virion exposure, i.e. prior to virus gene expression. This observation supports the hypothesis that virion binding or entry mediated by the spike glycoprotein (GP(1,2)) is the primary stimulus for an initial response. Indeed, ZEBOV virions, LPS, and virus-like particles consisting of only the ZEBOV matrix protein VP40 and GP(1,2) (VLP(VP40-GP)) triggered comparable responses in macrophages, including pro-inflammatory and pro-apoptotic signals. In contrast, VLP(VP40) (particles lacking GP(1,2)) caused an aberrant response. This suggests that GP(1,2) binding to macrophages plays an important role in the immediate cellular response.

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Case-based reported mortality associated with laboratory-confirmed influenza A(H1N1) 2009 virus infection in the Netherlands: the 2009-2010 pandemic season versus the 2010-2011 influenza season

BACKGROUND: In contrast to seasonal influenza epidemics, where the majority of deaths occur amongst elderly, a considerable part of the 2009 pandemic influenza related deaths concerned relatively young people. In the Netherlands, all deaths associated with laboratory-confirmed influenza A(H1N1) 2009 virus infection had to be notified, both during the 2009-2010 pandemic season and the 2010-2011 influenza season. To assess whether and to what extent pandemic mortality patterns were reverting back to seasonal patterns, a retrospective analyses of all notified fatal cases associated with laboratory-confirmed influenza A(H1N1) 2009 virus infection was performed. METHODS: The notification database, including detailed information about the clinical characteristics of all notified deaths, was used to perform a comprehensive analysis of all deceased patients with a laboratory-confirmed influenza A(H1N1) 2009 virus infection. Characteristics of the fatalities with respect to age and underlying medical conditions were analysed, comparing the 2009-2010 pandemic and the 2010-2011 influenza season. RESULTS: A total of 65 fatalities with a laboratory-confirmed influenza A(H1N1) 2009 virus infection were notified in 2009-2010 and 38 in 2010-2011. During the pandemic season, the population mortality rates peaked in persons aged 0-15 and 55-64 years. In the 2010-2011 influenza season, peaks in mortality were seen in persons aged 0-15 and 75-84 years. During the 2010-2011 influenza season, the height of first peak was lower compared to that during the pandemic season. Underlying immunological disorders were more common in the pandemic season compared to the 2010-2011 season (p = 0.02), and cardiovascular disorders were more common in the 2010-2011 season (p = 0.005). CONCLUSIONS: The mortality pattern in the 2010-2011 influenza season still resembled the 2009-2010 pandemic season with a peak in relatively young age groups, but concurrently a clear shift toward seasonal patterns was seen, with a peak in mortality in the elderly, i.e. ≥ 75 years of age.

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A Chinese Herbal Formula to Improve General Psychological Status in Posttraumatic Stress Disorder: A Randomized Placebo-Controlled Trial on Sichuan Earthquake Survivors

Introduction. Posttraumatic stress disorder (PTSD) is accompanied by poor general psychological status (GPS). In the present study, we investigated the effects of a Chinese herbal formula on GPS in earthquake survivors with PTSD. Methods. A randomized, double-blind, placebo-controlled trial compared a Chinese herbal formula, Xiao-Tan-Jie-Yu-Fang (XTJYF), to placebo in 2008 Sichuan earthquake survivors with PTSD. Patients were randomized into XTJYF (n = 123) and placebo (n = 122) groups. Baseline-to-end-point score changes in the three global indices of the Symptom Checklist-90-Revised (SCL-90-R) and rates of response in the SCL global severity index (GSI) were the primary endpoints. A subanalysis of the nine SCL factors and the sleep quality score were secondary endpoints. Results and Discussion. Compared to placebo, the XTJYF group was significantly improved in all three SCL global indices (P = 0.001~0.028). More patients in the XTJYF group reported “much improved” than the placebo group (P = 0.001). The XTJYF group performed significantly better than control in five out of nine SCL factors (somatization, obsessive-compulsive behavior, depression, anxiety, and hostility (P = 0.001~0.036)), and in sleep quality score (P < 0.001). XTJYF produced no serious adverse events. These findings suggest that XTJYF may be an effective and safe treatment option for improving GPS in patients with PTSD.

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Quercetin 7-rhamnoside reduces porcine epidemic diarrhea virus replication via independent pathway of viral induced reactive oxygen species

BACKGROUND: On the base of our previous study we were observed relevant studies on the hypothesis that the antiviral activity of quercetin 7-rhamnoside (Q7R), a flavonoid, won't relate ability of its antioxidant. METHODS: We were investigated the effects of Q7R on the cytopathic effects (CPE) by CPE reduction assay. Production of DNA fragment and reactive oxygen species (ROS) induced by PEDV infection were studied using DNA fragmentation assay and flow cytometry. RESULTS: In the course of this study it was discovered that Q7R is an extremely potent compound against PEDV. The addition of Q7R to PEDV-infected Vero cells directly reduced the formation of a visible cytopathic effect (CPE). Also, Q7R did not induce DNA fragmentation. Furthermore, ROS increased the infection of PEDV, which was strongly decreased by N-acetyl-L-cysteins (NAC). However, the increased ROS was not decreased by Q7R. Antiviral activity of antioxidants such as NAC, pyrrolidine dithiocarbamate (PDTC), and the vitamin E derivative, trolox, were hardly noticed. CONCLUSIONS: We concluded that the inhibition of PEDV production by Q7R is not simply due to a general action as an antioxidants and is highly specific, as several other antioxidants (NAC, PDTC, trolox) are inactive against PEDV infection.

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Use of Recombinant Adenovirus Vectored Consensus IFN-α to Avert Severe Arenavirus Infection

Several arenaviruses can cause viral hemorrhagic fever, a severe disease with case-fatality rates in hospitalized individuals ranging from 15-30%. Because of limited prophylaxis and treatment options, new medical countermeasures are needed for these viruses classified by the National Institutes of Allergy and Infectious Diseases (NIAID) as top priority biodefense Category A pathogens. Recombinant consensus interferon alpha (cIFN-α) is a licensed protein with broad clinical appeal. However, while cIFN-α has great therapeutic value, its utility for biodefense applications is hindered by its short in vivo half-life, mode and frequency of administration, and costly production. To address these limitations, we describe the use of DEF201, a replication-deficient adenovirus vector that drives the expression of cIFN-α, for pre- and post-exposure prophylaxis of acute arenaviral infection modeled in hamsters. Intranasal administration of DEF201 24 h prior to challenge with Pichindé virus (PICV) was highly effective at protecting animals from mortality and preventing viral replication and liver-associated disease. A significant protective effect was still observed with a single dosing of DEF201 given two weeks prior to PICV challenge. DEF201 was also efficacious when administered as a treatment 24 to 48 h post-virus exposure. The protective effect of DEF201 was largely attributed to the expression of cIFN-α, as dosing with a control empty vector adenovirus did not protect hamsters from lethal PICV challenge. Effective countermeasures that are highly stable, easily administered, and elicit long lasting protective immunity are much needed for arena and other viral infections. The DEF201 technology has the potential to address all of these issues and may serve as a broad-spectrum antiviral to enhance host defense against a number of viral pathogens.

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Protein Disulfide Isomerase and Host-Pathogen Interaction

Reactive oxygen species (ROS) production by immunological cells is known to cause damage to pathogens. Increasing evidence accumulated in the last decade has shown, however, that ROS (and redox signals) functionally regulate different cellular pathways in the host-pathogen interaction. These especially affect (i) pathogen entry through protein redox switches and redox modification (i.e., intra- and interdisulfide and cysteine oxidation) and (ii) phagocytic ROS production via Nox family NADPH oxidase enzyme and the control of phagolysosome function with key implications for antigen processing. The protein disulfide isomerase (PDI) family of redox chaperones is closely involved in both processes and is also implicated in protein unfolding and trafficking across the endoplasmic reticulum (ER) and towards the cytosol, a thiol-based redox locus for antigen processing. Here, we summarise examples of the cellular association of host PDI with different pathogens and explore the possible roles of pathogen PDIs in infection. A better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections.

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Improved Immunological Tolerance Following Combination Therapy with CTLA-4/Ig and AAV-Mediated PD-L1/2 Muscle Gene Transfer

Initially thought as being non-immunogenic, recombinant AAVs have emerged as efficient vector candidates for treating monogenic diseases. It is now clear however that they induce potent immune responses against transgene products which can lead to destruction of transduced cells. Therefore, developing strategies to circumvent these immune responses and facilitate long-term expression of transgenic therapeutic proteins is a main challenge in gene therapy. We evaluated herein a strategy to inhibit the undesirable immune activation that follows muscle gene transfer by administration of CTLA-4/Ig to block the costimulatory signals required early during immune priming and by using gene transfer of PD-1 ligands to inhibit T cell functions at the tissue sites. We provide the proof of principle that this combination immunoregulatory therapy targeting two non-redundant checkpoints of the immune response, i.e., priming and effector functions, can improve persistence of transduced cells in experimental settings where cytotoxic T cells escape initial blockade. Therefore, CTLA-4/Ig plus PD-L1/2 combination therapy represents a candidate approach to circumvent the bottleneck of immune responses directed toward transgene products.

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Comparison of Humoral Immune Responses to Epstein-Barr Virus and Kaposi’s Sarcoma–Associated Herpesvirus Using a Viral Proteome Microarray

Background. Epstein-Barr virus (EBV) is a ubiquitous herpesvirus, and Kaposi’s sarcoma–associated herpesvirus (KSHV) has a restricted seroprevalence. Both viruses are associated with malignancies that have an increased frequency in individuals who are coinfected with human immunodeficiency virus type 1 (HIV-1). Methods. To obtain an overview of humoral immune responses to these viruses, we generated a protein array that displayed 174 EBV and KSHV polypeptides purified from yeast. Antibody responses to EBV and KSHV were examined in plasma from healthy volunteers and patients with B cell lymphoma or with AIDS-related Kaposi’s sarcoma or lymphoma. Results. In addition to the commonly studied antigens, IgG responses were frequently detected to the tegument proteins KSHV ORF38 and EBV BBRF and BGLF2 and BNRF1 and to the EBV early lytic proteins BRRF1 and BORF2. The EBV vIL-10 protein was particularly well recognized by plasma IgA. The most intense IgG responses to EBV antigens occurred in HIV-1–positive patients. No clear correlation was observed between viral DNA load in plasma and antibody profile. Conclusions. The protein array provided a sensitive platform for global screening; identified new, frequently recognized viral antigens; and revealed a broader humoral response to EBV compared with KSHV in the same patients.

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Stem–loop structures can effectively substitute for an RNA pseudoknot in −1 ribosomal frameshifting

−1 Programmed ribosomal frameshifting (PRF) in synthesizing the gag-pro precursor polyprotein of Simian retrovirus type-1 (SRV-1) is stimulated by a classical H-type pseudoknot which forms an extended triple helix involving base–base and base–sugar interactions between loop and stem nucleotides. Recently, we showed that mutation of bases involved in triple helix formation affected frameshifting, again emphasizing the role of the triple helix in −1 PRF. Here, we investigated the efficiency of hairpins of similar base pair composition as the SRV-1 gag-pro pseudoknot. Although not capable of triple helix formation they proved worthy stimulators of frameshifting. Subsequent investigation of ∼30 different hairpin constructs revealed that next to thermodynamic stability, loop size and composition and stem irregularities can influence frameshifting. Interestingly, hairpins carrying the stable GAAA tetraloop were significantly less shifty than other hairpins, including those with a UUCG motif. The data are discussed in relation to natural shifty hairpins.

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Usefulness of Published PCR Primers in Detecting Human Rhinovirus Infection

We conducted a preliminary comparison of the relative sensitivity of a cross-section of published human rhinovirus (HRV)–specific PCR primer pairs, varying the oligonucleotides and annealing temperature. None of the pairs could detect all HRVs in 2 panels of genotyped clinical specimens; >1 PCR is required for accurate description of HRV epidemiology.

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NNAlign: A Web-Based Prediction Method Allowing Non-Expert End-User Discovery of Sequence Motifs in Quantitative Peptide Data

Recent advances in high-throughput technologies have made it possible to generate both gene and protein sequence data at an unprecedented rate and scale thereby enabling entirely new “omics”-based approaches towards the analysis of complex biological processes. However, the amount and complexity of data that even a single experiment can produce seriously challenges researchers with limited bioinformatics expertise, who need to handle, analyze and interpret the data before it can be understood in a biological context. Thus, there is an unmet need for tools allowing non-bioinformatics users to interpret large data sets. We have recently developed a method, NNAlign, which is generally applicable to any biological problem where quantitative peptide data is available. This method efficiently identifies underlying sequence patterns by simultaneously aligning peptide sequences and identifying motifs associated with quantitative readouts. Here, we provide a web-based implementation of NNAlign allowing non-expert end-users to submit their data (optionally adjusting method parameters), and in return receive a trained method (including a visual representation of the identified motif) that subsequently can be used as prediction method and applied to unknown proteins/peptides. We have successfully applied this method to several different data sets including peptide microarray-derived sets containing more than 100,000 data points. NNAlign is available online at http://www.cbs.dtu.dk/services/NNAlign.

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Red blood cell transfusion in the critically ill patient

Red blood cell (RBC) transfusion is a common intervention in intensive care unit (ICU) patients. Anemia is frequent in this population and is associated with poor outcomes, especially in patients with ischemic heart disease. Although blood transfusions are generally given to improve tissue oxygenation, they do not systematically increase oxygen consumption and effects on oxygen delivery are not always very impressive. Blood transfusion may be lifesaving in some circumstances, but many studies have reported increased morbidity and mortality in transfused patients. This review focuses on some important aspects of RBC transfusion in the ICU, including physiologic considerations, a brief description of serious infectious and noninfectious hazards of transfusion, and the effects of RBC storage lesions. Emphasis is placed on the importance of personalizing blood transfusion according to physiological endpoints rather than arbitrary thresholds.

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Respiratory support by neurally adjusted ventilatory assist (NAVA) in severe RSV-related bronchiolitis: a case series report

BACKGROUND: Neurally adjusted ventilatory assist (NAVA) is a new mode of mechanical ventilation controlled by diaphragmatic electrical signals. The electrical signals allow synchronization of ventilation to spontaneous breathing efforts of a child, as well as permitting pressure assistance proportional to the electrical signal. NAVA provides equally fine synchronization of respiratory support and pressure assistance varying with the needs of the child. NAVA has mainly been studied in children who underwent cardiac surgery during the period of weaning from a respirator. CASE PRESENTATION: We report here a series of 3 children (1 month, 3 years, and 28 days old) with severe respiratory distress due to RSV-related bronchiolitis requiring invasive mechanical ventilation with a high level of oxygen (FiO(2 )≥50%) for whom NAVA facilitated respiratory support. One of these children had diagnosis criteria for acute lung injury, another for acute respiratory distress syndrome. Establishment of NAVA provided synchronization of mechanical ventilatory support with the breathing efforts of the children. Respiratory rate and inspiratory pressure became extremely variable, varying at each cycle, while children were breathing easily and smoothly. All three children demonstrated less oxygen requirements after introducing NAVA (57 ± 6% to 42 ± 18%). This improvement was observed while peak airway pressure decreased (28 ± 3 to 15 ± 5 cm H(2)O). In one child, NAVA facilitated the management of acute respiratory distress syndrome with extensive subcutaneous emphysema. CONCLUSIONS: Our findings highlight the feasibility and benefit of NAVA in children with severe RSV-related bronchiolitis. NAVA provides a less aggressive ventilation requiring lower inspiratory pressures with good results for oxygenation and more comfort for the children.

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Evaluation of Approaches to Identify the Targets of Cellular Immunity on a Proteome-Wide Scale

BACKGROUND: Vaccine development against malaria and other complex diseases remains a challenge for the scientific community. The recent elucidation of the genome, proteome and transcriptome of many of these complex pathogens provides the basis for rational vaccine design by identifying, on a proteome-wide scale, novel target antigens that are recognized by T cells and antibodies from exposed individuals. However, there is currently no algorithm to effectively identify important target antigens from genome sequence data; this is especially challenging for T cell targets. Furthermore, for some of these pathogens, such as Plasmodium, protein expression using conventional platforms has been problematic but cell-free in vitro transcription translation (IVTT) strategies have recently proved successful. Herein, we report a novel approach for proteome-wide scale identification of the antigenic targets of T cell responses using IVTT products. PRINCIPAL FINDINGS: We conducted a series of in vitro and in vivo experiments using IVTT proteins either unpurified, absorbed to carboxylated polybeads, or affinity purified through nickel resin or magnetic beads. In vitro studies in humans using CMV, EBV, and Influenza A virus proteins showed antigen-specific cytokine production in ELIspot and Cytometric Bead Array assays with cells stimulated with purified or unpurified IVTT antigens. In vitro and in vivo studies in mice immunized with the Plasmodium yoelii circumsporozoite DNA vaccine with or without IVTT protein boost showed antigen-specific cytokine production using purified IVTT antigens only. Overall, the nickel resin method of IVTT antigen purification proved optimal in both human and murine systems. CONCLUSIONS: This work provides proof of concept for the potential of high-throughput approaches to identify T cell targets of complex parasitic, viral or bacterial pathogens from genomic sequence data, for rational vaccine development against emerging and re-emerging diseases that pose a threat to public health.

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Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities

In all vertebrate animals, CD8(+) cytotoxic T lymphocytes (CTLs) are controlled by major histocompatibility complex class I (MHC-I) molecules. These are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating CTLs. The polymorphism of the MHC effectively individualizes the immune response of each member of the species. We have recently developed efficient methods to generate recombinant human MHC-I (also known as human leukocyte antigen class I, HLA-I) molecules, accompanying peptide-binding assays and predictors, and HLA tetramers for specific CTL staining and manipulation. This has enabled a complete mapping of all HLA-I specificities (“the Human MHC Project”). Here, we demonstrate that these approaches can be applied to other species. We systematically transferred domains of the frequently expressed swine MHC-I molecule, SLA-1*0401, onto a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules. A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data indicate that it is possible to extend the biochemical and bioinformatics tools of the Human MHC Project to other vertebrate species. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00251-011-0555-3) contains supplementary material, which is available to authorized users.

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Immunogenetic Factors Associated with Severe Respiratory Illness Caused by Zoonotic H1N1 and H5N1 Influenza Viruses

Following the 2009 H1N1 pandemic and ongoing sporadic avian-to-human transmission of H5N1 viruses, an emphasis has been placed on better understanding the determinants and pathogenesis of severe influenza infections. Much of the current literature has focused on viral genetics and its impact on host immunity as well as novel risk factors for severe infection (particularly within the H1N1 pandemic). An understanding of the host genetic determinants of susceptibility and severe respiratory illness, however, is currently lacking. By better defining the role of genetic variability in influenza infection and identifying key polymorphisms that impair the host immune response or correlate with protection, we will be able to better identify at-risk populations and new targets for therapeutic interventions and vaccines. This paper will summarize known immunogenetic factors associated with susceptibility or severity of both pH1N1 and H5N1 infections and will also identify genetic pathways and polymorphisms of high relevance for future study.

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In China, Students in Crowded Dormitories with a Low Ventilation Rate Have More Common Colds: Evidence for Airborne Transmission

OBJECTIVE: To test whether the incidence of common colds among college students in China is associated with ventilation rates and crowdedness in dormitories. METHODS: In Phase I of the study, a cross-sectional study, 3712 students living in 1569 dorm rooms in 13 buildings responded to a questionnaire about incidence and duration of common colds in the previous 12 months. In Phase II, air temperature, relative humidity and CO(2) concentration were measured for 24 hours in 238 dorm rooms in 13 buildings, during both summer and winter. Out-to indoor air flow rates at night were calculated based on measured CO(2) concentrations. RESULTS: In Phase I, 10% of college students reported an incidence of more than 6 common colds in the previous 12 months, and 15% reported that each infection usually lasted for more than 2 weeks. Students in 6-person dorm rooms were about 2 times as likely to have an incidence of common colds ≥6 times per year and a duration ≥2 weeks, compared to students in 3-person rooms. In Phase II, 90% of the measured dorm rooms had an out-to indoor air flow rate less than the Chinese standard of 8.3 L/s per person during the heating season. There was a dose-response relationship between out-to indoor air flow rate per person in dorm rooms and the proportion of occupants with annual common cold infections ≥6 times. A mean ventilation rate of 5 L/(s•person) in dorm buildings was associated with 5% of self reported common cold ≥6 times, compared to 35% at 1 L/(s•person). CONCLUSION: Crowded dormitories with low out-to indoor airflow rates are associated with more respiratory infections among college students.

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Epidemiology and clinical characteristics of hospitalized patients with pandemic influenza A (H1N1) 2009 infections: the effects of bacterial coinfection

BACKGROUND: Numerous reports have described the epidemiological and clinical characteristics of influenza A (H1N1) 2009 infected patients. However, data on the effects of bacterial coinfection on these patients are very scarce. Therefore, this study explores the impact of bacterial coinfection on the clinical and laboratory parameters amongst H1N1 hospitalized patients. FINDINGS: This retrospective study involved hospitalized patients with laboratory-confirmed H1N1 infections (September 2009 to May 2010). Relevant clinical data and the detection of bacterial coinfection from respiratory or sterile site samples were obtained. Multiplex PCR was used to determine the co-existence of other respiratory viruses. Comparison was made between patients with and without bacterial coinfection. The occurrence of coinfection was 34%; 14 (28%) bacterial and only 3 (6%) viral. Mycoplasma pneumoniae (n = 5) was the commonest bacteria followed by Staphylococcus aureus (n = 3). In univariate analysis, clinical factors associated with bacterial coinfection were age > 50 years (p = 0.02), presence of comorbidity (p = 0.04), liver impairment (p = 0.02), development of complications (p = 0.004) and supplemental oxygen requirement (p = 0.02). Leukocytosis (p = 0.02) and neutrophilia (p = 0.004) were higher in bacterial coinfected patients. Multivariate logistic regression analysis revealed that age > 50 years and combined complications were predictive of bacterial coinfection. CONCLUSIONS: Bacterial coinfection is not uncommon in H1N1 infected patients and is more frequently noted in the older aged patients and is associated with higher rates of complications. Also, as adjunct to clinical findings, clinicians need to have a higher index of suspicion if neutrophilia was identified at admission as it may denote bacterial coinfection.

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Clinical Review: Gene-based therapies for ALI/ARDS: where are we now?

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) confer substantial morbidity and mortality, and have no specific therapy. The accessibility of the distal lung epithelium via the airway route, and the relatively transient nature of ALI/ARDS, suggest that the disease may be amenable to gene-based therapies. Ongoing advances in our understanding of the pathophysiology of ALI/ARDS have revealed multiple therapeutic targets for gene-based approaches. Strategies to enhance or restore lung epithelial and/or endothelial cell function, to strengthen lung defense mechanisms against injury, to speed clearance of infection and to enhance the repair process following ALI/ARDS have all demonstrated promise in preclinical models. Despite three decades of gene therapy research, however, the clinical potential for gene-based approaches to lung diseases including ALI/ARDS remains to be realized. Multiple barriers to effective pulmonary gene therapy exist, including the pulmonary architecture, pulmonary defense mechanisms against inhaled particles, the immunogenicity of viral vectors and the poor transfection efficiency of nonviral delivery methods. Deficits remain in our knowledge regarding the optimal molecular targets for gene-based approaches. Encouragingly, recent progress in overcoming these barriers offers hope for the successful translation of gene-based approaches for ALI/ARDS to the clinical setting.

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Critical care services and the H1N1 (2009) influenza epidemic in Australia and New Zealand in 2010: the impact of the second winter epidemic

INTRODUCTION: During the first winter of exposure, the H1N1 2009 influenza virus placed considerable strain on intensive care unit (ICU) services in Australia and New Zealand (ANZ). We assessed the impact of the H1N1 2009 influenza virus on ICU services during the second (2010) winter, following the implementation of vaccination. METHODS: A prospective, cohort study was conducted in all ANZ ICUs during the southern hemisphere winter of 2010. Data on demographic and clinical characteristics, including vaccination status and outcomes, were collected. The characteristics of patients admitted during the 2010 and 2009 seasons were compared. RESULTS: From 1 June to 15 October 2010, there were 315 patients with confirmed influenza A, of whom 283 patients (90%) had H1N1 2009 (10.6 cases per million inhabitants; 95% confidence interval (CI), 9.4 to 11.9) which was an observed incidence of 33% of that in 2009 (P < 0.001). The maximum daily ICU occupancy was 2.4 beds (95% CI, 1.8 to 3) per million inhabitants in 2010 compared with 7.5 (95% CI, 6.5 to 8.6) in 2009, (P < 0.001). The onset of the epidemic in 2010 was delayed by five weeks compared with 2009. The clinical characteristics were similar in 2010 and 2009 with no difference in the age distribution, proportion of patients treated with mechanical ventilation, duration of ICU admission, or hospital mortality. Unlike 2009 the incidence of critical illness was significantly greater in New Zealand (18.8 cases per million inhabitants compared with 9 in Australia, P < 0.001). Of 170 patients with known vaccination status, 26 (15.3%) had been vaccinated against H1N1 2009. CONCLUSIONS: During the 2010 ANZ winter, the impact of H1N1 2009 on ICU services was still appreciable in Australia and substantial in New Zealand. Vaccination failure occurred.

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Early corticosteroid treatment for severe pneumonia caused by 2009 H1N1 influenza virus

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Multinational, observational study of procalcitonin in ICU patients with pneumonia requiring mechanical ventilation: a multicenter observational study

INTRODUCTION: The intent of this study was to determine whether serum procalcitonin (PCT) levels are associated with prognosis, measured as organ dysfunctions and 28-day mortality, in patients with severe pneumonia. METHODS: This was a multicenter, observational study of critically ill adult patients with pneumonia requiring mechanical ventilation conducted in 10 academic hospitals in Canada, the United States, and Central Europe. PCT was measured daily for 14 days using an immuno-luminometric assay. RESULTS: We included 175 patients, 57 with community acquired pneumonia (CAP), 61 with ventilator associated pneumonia (VAP) and 57 with hospital acquired pneumonia (HAP). Initial PCT levels were higher in CAP than VAP patients (median (interquartile range: IQR); 2.4 (0.95 to 15.8) vs. 0.7 (0.3 to 2.15), ng/ml, P < 0.001) but not significantly different to HAP (2.2 (0.4 to 8.0) ng/ml). The 28-day ICU mortality rate for all patients was 18.3% with a median ICU length of stay of 16 days (range 1 to 142 days). PCT levels were higher in non-survivors than in survivors. Initial and maximum PCT levels correlated with maximum Sequential Organ Failure Assessment (SOFA) score r(2 )= 0.50 (95% CI: 0.38 to 0.61) and r(2 )= 0.57 (0.46 to 0.66), respectively. Receiver operating curve (ROC) analysis on discrimination of 28-day mortality showed areas under the curve (AUC) of 0.74, 0.70, and 0.69 for maximum PCT, initial PCT, and Acute Physiology and Chronic Health Evaluation (APACHE) II score, respectively. The optimal cut-off to predict mortality for initial PCT was 1.1 ng/ml (odds ratio: OD 7.0 (95% CI 2.6 to 25.2)) and that for maximum PCT was 7.8 ng/ml (odds ratio 5.7 (95% CI 2.5 to 13.1)). CONCLUSIONS: PCT is associated with the severity of illness in patients with severe pneumonia and appears to be a prognostic marker of morbidity and mortality comparable to the APACHE II score.

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The role of receptor for advanced glycation endproducts (RAGE) in infection

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Metagenomic Analysis of Fever, Thrombocytopenia and Leukopenia Syndrome (FTLS) in Henan Province, China: Discovery of a New Bunyavirus

Since 2007, many cases of fever, thrombocytopenia and leukopenia syndrome (FTLS) have emerged in Henan Province, China. Patient reports of tick bites suggested that infection could contribute to FTLS. Many tick-transmitted microbial pathogens were tested for by PCR/RT-PCR and/or indirect immunofluorescence assay (IFA). However, only 8% (24/285) of samples collected from 2007 to 2010 tested positive for human granulocytic anaplasmosis (HGA), suggesting that other pathogens could be involved. Here, we used an unbiased metagenomic approach to screen and survey for microbes possibly associated with FTLS. BLASTx analysis of deduced protein sequences revealed that a novel bunyavirus (36% identity to Tehran virus, accession: HQ412604) was present only in sera from FTLS patients. A phylogenetic analysis further showed that, although closely related to Uukuniemi virus of the Phlebovirus genus, this virus was distinct. The candidate virus was examined for association with FTLS among samples collected from Henan province during 2007–2010. RT-PCR, viral cultures, and a seroepidemiologic survey were undertaken. RT-PCR results showed that 223 of 285 (78.24%) acute-phase serum samples contained viral RNA. Of 95 patients for whom paired acute and convalescent sera were available, 73 had serologic evidence of infection, with 52 seroconversions and 21 exhibiting a 4-fold increase in antibody titer to the virus. The new virus was isolated from patient acute-phase serum samples and named Henan Fever Virus (HNF virus). Whole-genome sequencing confirmed that the virus was a novel bunyavirus with genetic similarity to known bunyaviruses, and was most closely related to the Uukuniemi virus (34%, 24%, and 29% of maximum identity, respectively, for segment L, M, S at maximum query coverage). After the release of the GenBank sequences of SFTSV, we found that they were nearly identical (>99% identity). These results show that the novel bunyavirus (HNF virus) is strongly correlated with FTLS.

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Clinical aspects and cytokine response in severe H1N1 influenza A virus infection

INTRODUCTION: The immune responses in patients with novel A(H1N1) virus infection (nvA(H1N1)) are incompletely characterized. We investigated the profile of Th1 and Th17 mediators and interferon-inducible protein-10 (IP-10) in groups with severe and mild nvA(H1N1) disease and correlated them with clinical aspects. METHODS: Thirty-two patients hospitalized with confirmed nvA(H1N1) infection were enrolled in the study: 21 patients with nvA(H1N1)-acute respiratory distress syndrome (ARDS) and 11 patients with mild disease. One group of 20 patients with bacterial sepsis-ARDS and another group of 15 healthy volunteers were added to compare their cytokine levels with pandemic influenza groups. In the nvA(H1N1)-ARDS group, the serum cytokine samples were obtained on admission and 3 days later. The clinical aspects were recorded prospectively. RESULTS: In the nvA(H1N1)-ARDS group, obesity and lymphocytopenia were more common and IP-10, interleukin (IL)-12, IL-15, tumor necrosis factor (TNF)α, IL-6, IL-8 and IL-9 were significantly increased versus control. When comparing mild with severe nvA(H1N1) groups, IL-6, IL-8, IL-15 and TNFα were significantly higher in the severe group. In nonsurvivors versus survivors, IL-6 and IL-15 were increased on admission and remained higher 3 days later. A positive correlation of IL-6, IL-8 and IL-15 levels with C-reactive protein and with > 5-day interval between symptom onset and admission, and a negative correlation with the PaO(2):FiO(2 )ratio, were found in nvA(H1N1) groups. In obese patients with influenza disease, a significant increased level of IL-8 was found. When comparing viral ARDS with bacterial ARDS, the level of IL-8, IL-17 and TNFα was significantly higher in bacterial ARDS and IL-12 was increased only in viral ARDS. CONCLUSIONS: In our critically ill patients with novel influenza A(H1N1) virus infection, the hallmarks of the severity of disease were IL-6, IL-15, IL-8 and TNFα. These cytokines, except TNFα, had a positive correlation with the admission delay and C-reactive protein, and a negative correlation with the PaO(2):FiO(2 )ratio. Obese patients with nvA(H1N1) disease have a significant level of IL-8. There are significant differences in the level of cytokines when comparing viral ARDS with bacterial ARDS.

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Clinical review: Idiopathic pulmonary fibrosis acute exacerbations - unravelling Ariadne's thread

Idiopathic pulmonary fibrosis (IPF) is a dreadful, chronic, and irreversibly progressive fibrosing disease leading to death in all patients affected, and IPF acute exacerbations constitute the most devastating complication during its clinical course. IPF exacerbations are subacute/acute, clinically significant deteriorations of unidentifiable cause that usually transform the slow and more or less steady disease decline to the unexpected appearance of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) ending in death. The histological picture is that of diffuse alveolar damage (DAD), which is the tissue counterpart of ARDS, upon usual interstitial pneumonia, which is the tissue equivalent of IPF. ALI/ARDS and acute interstitial pneumonia share with IPF exacerbations the tissue damage pattern of DAD. 'Treatment' with high-dose corticosteroids with or without an immunosuppressant proved ineffective and represents the coup de grace for these patients. Provision of excellent supportive care and the search for and treatment of the 'underlying cause' remain the only options. IPF exacerbations require rapid decisions about when and whether to initiate mechanical support. Admission to an intensive care unit (ICU) is a particular clinical and ethical challenge because of the extremely poor outcome. Transplantation in the ICU setting often presents insurmountable difficulties.

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Influence of genetic variability at the surfactant proteins A and D in community-acquired pneumonia: a prospective, observational, genetic study

INTRODUCTION: Genetic variability of the pulmonary surfactant proteins A and D may affect clearance of microorganisms and the extent of the inflammatory response. The genes of these collectins (SFTPA1, SFTPA2 and SFTPD) are located in a cluster at 10q21-24. The objective of this study was to evaluate the existence of linkage disequilibrium (LD) among these genes, and the association of variability at these genes with susceptibility and outcome of community-acquired pneumonia (CAP). We also studied the effect of genetic variability on SP-D serum levels. METHODS: Seven non-synonymous polymorphisms of SFTPA1, SFTPA2 and SFTPD were analyzed. For susceptibility, 682 CAP patients and 769 controls were studied in a case-control study. Severity and outcome were evaluated in a prospective study. Haplotypes were inferred and LD was characterized. SP-D serum levels were measured in healthy controls. RESULTS: The SFTPD aa11-C allele was significantly associated with lower SP-D serum levels, in a dose-dependent manner. We observed the existence of LD among the studied genes. Haplotypes SFTPA1 6A(2 )(P = 0.0009, odds ration (OR) = 0.78), SFTPA2 1A(0 )(P = 0.002, OR = 0.79), SFTPA1-SFTPA2 6A(2)-1A(0 )(P = 0.0005, OR = 0.77), and SFTPD-SFTPA1-SFTPA2 C-6A(2)-1A(0 )(P = 0.00001, OR = 0.62) were underrepresented in patients, whereas haplotypes SFTPA2 1A(10 )(P = 0.00007, OR = 6.58) and SFTPA1-SFTPA2 6A(3)-1A (P = 0.0007, OR = 3.92) were overrepresented. Similar results were observed in CAP due to pneumococcus, though no significant differences were now observed after Bonferroni corrections. 1A(10 )and 6A-1A were associated with higher 28-day and 90-day mortality, and with multi-organ dysfunction syndrome (MODS) and acute respiratory distress syndrome (ARDS) respectively. SFTPD aa11-C allele was associated with development of MODS and ARDS. CONCLUSIONS: Our study indicates that missense single nucleotide polymorphisms and haplotypes of SFTPA1, SFTPA2 and SFTPD are associated with susceptibility to CAP, and that several haplotypes also influence severity and outcome of CAP.

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Survival of Influenza A(H1N1) on Materials Found in Households: Implications for Infection Control

BACKGROUND: The majority of influenza transmission occurs in homes, schools and workplaces, where many frequently touched communal items are situated. However the importance of transmission via fomites is unclear since few data exist on the survival of virus on commonly touched surfaces. We therefore measured the viability over time of two H1N1 influenza strains applied to a variety of materials commonly found in households and workplaces. METHODOLOGY AND PRINCIPAL FINDINGS: Influenza A/PuertoRico/8/34 (PR8) or A/Cambridge/AHO4/2009 (pandemic H1N1) viruses were inoculated onto a wide range of surfaces used in home and work environments, then sampled at set times following incubation at stabilised temperature and humidity. Virus genome was measured by RT-PCR; plaque assay (for PR8) or fluorescent focus formation (for pandemic H1N1) was used to assess the survival of viable virus. CONCLUSIONS/SIGNIFICANCE: The genome of either virus could be detected on most surfaces 24 h after application with relatively little drop in copy number, with the exception of unsealed wood surfaces. In contrast, virus viability dropped much more rapidly. Live virus was recovered from most surfaces tested four hours after application and from some non-porous materials after nine hours, but had fallen below the level of detection from all surfaces at 24 h. We conclude that influenza A transmission via fomites is possible but unlikely to occur for long periods after surface contamination (unless re-inoculation occurs). In situations involving a high probability of influenza transmission, our data suggest a hierarchy of priorities for surface decontamination in the multi-surface environments of home and hospitals.

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Respiratory failure presenting in H1N1 influenza with Legionnaires disease: two case reports

INTRODUCTION: Media sensationalism on the H1N1 outbreak may have influenced decisional processes and clinical diagnosis. CASE PRESENTATION: We report two cases of patients who presented in 2009 with coexisting H1N1 virus and Legionella infections: a 69-year-old Caucasian man and a 71-year-old Caucasian woman. In our cases all the signs and symptoms, including vomiting, progressive respiratory disease leading to respiratory failure, refractory hypoxemia, leukopenia, lymphopenia, thrombocytopenia, and elevated levels of creatine kinase and hepatic aminotransferases, were consistent with critical illness due to 2009 H1N1 virus infection. Other infectious disorders may mimic H1N1 viral infection especially Legionnaires' disease. Because the swine flu H1N1 pandemic occurred in Autumn in Italy, Legionnaires disease was to be highly suspected since the peak incidence usually occurs in early fall. We do think that our immediate suspicion of Legionella infection based on clinical history and X-ray abnormalities was fundamental for a successful resolution. CONCLUSION: Our two case reports suggest that patients with H1N1 should be screened for Legionella, which is not currently common practice. This is particularly important since the signs and symptoms of both infections are similar.

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Agricultural intensification, priming for persistence and the emergence of Nipah virus: a lethal bat-borne zoonosis

Emerging zoonoses threaten global health, yet the processes by which they emerge are complex and poorly understood. Nipah virus (NiV) is an important threat owing to its broad host and geographical range, high case fatality, potential for human-to-human transmission and lack of effective prevention or therapies. Here, we investigate the origin of the first identified outbreak of NiV encephalitis in Malaysia and Singapore. We analyse data on livestock production from the index site (a commercial pig farm in Malaysia) prior to and during the outbreak, on Malaysian agricultural production, and from surveys of NiV's wildlife reservoir (flying foxes). Our analyses suggest that repeated introduction of NiV from wildlife changed infection dynamics in pigs. Initial viral introduction produced an explosive epizootic that drove itself to extinction but primed the population for enzootic persistence upon reintroduction of the virus. The resultant within-farm persistence permitted regional spread and increased the number of human infections. This study refutes an earlier hypothesis that anomalous El Niño Southern Oscillation-related climatic conditions drove emergence and suggests that priming for persistence drove the emergence of a novel zoonotic pathogen. Thus, we provide empirical evidence for a causative mechanism previously proposed as a precursor to widespread infection with H5N1 avian influenza and other emerging pathogens.

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Do expert assessments converge? An exploratory case study of evaluating and managing a blood supply risk

BACKGROUND: Examining professional assessments of a blood product recall/withdrawal and its implications for risk and public health, the paper introduces ideas about perceptions of minimal risk and its management. It also describes the context of publicly funded blood transfusion in Canada and the withdrawal event that is the basis of this study. METHODS: Interviews with 45 experts from administration, medicine, blood supply, laboratory services and risk assessment took place using a multi-level sampling framework in the aftermath of the recall. These experts either directly dealt with the withdrawal or were involved in the management of the blood supply at the national level. Data from these interviews were coded in NVivo for analysis and interpretation. Analytically, data were interpreted to derive typifications to relate interview responses to risk management heuristics. RESULTS: While all those interviewed agreed on the importance of patient safety, differences in the ways in which the risk was contextualized and explicated were discerned. Risk was seen in terms of patient safety, liability or precaution. These different risk logics are illustrated by selected quotations. CONCLUSIONS: Expert assessments did not fully converge and it is possible that these different risk logics and discourses may affect the risk management process more generally, although not necessarily in a negative way. Patient safety is not to be compromised but management of blood risk in publicly funded systems may vary. We suggest ways of managing blood risk using formal and safety case approaches.

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Noninvasive positive pressure ventilation for acute respiratory failure in children: a concise review

Noninvasive positive pressure ventilation (NPPV) refers to the delivery of mechanical respiratory support without the use of endotracheal intubation (ETI). The present review focused on the effectiveness of NPPV in children > 1 month of age with acute respiratory failure (ARF) due to different conditions. ARF is the most common cause of cardiac arrest in children. Therefore, prompt recognition and treatment of pediatric patients with pending respiratory failure can be lifesaving. Mechanical respiratory support is a critical intervention in many cases of ARF. In recent years, NPPV has been proposed as a valuable alternative to invasive mechanical ventilation (IMV) in this acute setting. Recent physiological studies have demonstrated beneficial effects of NPPV in children with ARF. Several pediatric clinical studies, the majority of which were noncontrolled or case series and of small size, have suggested the effectiveness of NPPV in the treatment of ARF due to acute airway (upper or lower) obstruction or certain primary parenchymal lung disease, and in specific circumstances, such as postoperative or postextubation ARF, immunocompromised patients with ARF, or as a means to facilitate extubation. NPPV was well tolerated with rare major complications and was associated with improved gas exchange, decreased work of breathing, and ETI avoidance in 22-100% of patients. High FiO(2 )needs or high PaCO(2 )level on admission or within the first hours after starting NPPV appeared to be the best independent predictive factors for the NPPV failure in children with ARF. However, many important issues, such as the identification of the patient, the right time for NPPV application, and the appropriate setting, are still lacking. Further randomized, controlled trials that address these issues in children with ARF are recommended.

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Pandemic A/H1N1v influenza 2009 in hospitalized children: a multicenter Belgian survey

BACKGROUND: During the 2009 influenza A/H1N1v pandemic, children were identified as a specific "at risk" group. We conducted a multicentric study to describe pattern of influenza A/H1N1v infection among hospitalized children in Brussels, Belgium. METHODS: From July 1, 2009, to January 31, 2010, we collected epidemiological and clinical data of all proven (positive H1N1v PCR) and probable (positive influenza A antigen or culture) pediatric cases of influenza A/H1N1v infections, hospitalized in four tertiary centers. RESULTS: During the epidemic period, an excess of 18% of pediatric outpatients and emergency department visits was registered. 215 children were hospitalized with proven/probable influenza A/H1N1v infection. Median age was 31 months. 47% had ≥ 1 comorbid conditions. Febrile respiratory illness was the most common presentation. 36% presented with initial gastrointestinal symptoms and 10% with neurological manifestations. 34% had pneumonia. Only 24% of the patients received oseltamivir but 57% received antibiotics. 10% of children were admitted to PICU, seven of whom with ARDS. Case fatality-rate was 5/215 (2%), concerning only children suffering from chronic neurological disorders. Children over 2 years of age showed a higher propensity to be admitted to PICU (16% vs 1%, p = 0.002) and a higher mortality rate (4% vs 0%, p = 0.06). Infants less than 3 months old showed a milder course of infection, with few respiratory and neurological complications. CONCLUSION: Although influenza A/H1N1v infections were generally self-limited, pediatric burden of disease was significant. Compared to other countries experiencing different health care systems, our Belgian cohort was younger and received less frequently antiviral therapy; disease course and mortality were however similar.

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A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture

Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses of neurodegenerative disorders such as Alzheimer's and Huntington disease. Commercially available assays to measure caspase-6 activity commonly use the VEID peptide as a substrate. However these methods are not well suited to specifically assess caspase-6 activity in the presence of other, confounding protease activities, as often encountered in cell and tissue samples. Here we report the development of a method that overcomes this limitation by using a protein substrate, lamin A, which is highly specific for caspase-6 cleavage at amino acid 230. Using a neo-epitope antibody against cleaved lamin A, we developed an electrochemiluminescence-based ELISA assay that is suitable to specifically detect and quantify caspase-6 activity in highly apoptotic cell extracts. The method is more sensitive than VEID-based assays and can be adapted to a high-content imaging platform for high-throughput screening. This method should be useful to screen for and characterize caspase-6 inhibitor compounds and other interventions to decrease intracellular caspase-6 activity for applications in neurodegenerative disorders.

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Quantifying social distancing arising from pandemic influenza

Local epidemic curves during the 1918–1919 influenza pandemic were often characterized by multiple epidemic waves. Identifying the underlying cause(s) of such waves may help manage future pandemics. We investigate the hypothesis that these waves were caused by people avoiding potentially infectious contacts—a behaviour termed ‘social distancing’. We estimate the effective disease reproduction number and from it infer the maximum degree of social distancing that occurred during the course of the multiple-wave epidemic in Sydney, Australia. We estimate that, on average across the city, people reduced their infectious contact rate by as much as 38%, and that this was sufficient to explain the multiple waves of this epidemic. The basic reproduction number, R(0), was estimated to be in the range of 1.6–2.0 with a preferred estimate of 1.8, in line with other recent estimates for the 1918–1919 influenza pandemic. The data are also consistent with a high proportion (more than 90%) of the population being initially susceptible to clinical infection, and the proportion of infections that were asymptomatic (if this occurs) being no higher than approximately 9%. The observed clinical attack rate of 36.6% was substantially lower than the 59% expected based on the estimated value of R(0), implying that approximately 22% of the population were spared from clinical infection. This reduction in the clinical attack rate translates to an estimated 260 per 100 000 lives having been saved, and suggests that social distancing interventions could play a major role in mitigating the public health impact of future influenza pandemics.

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Novel Inhibitor Design for Hemagglutinin against H1N1 Influenza Virus by Core Hopping Method

The worldwide spread of H1N1 avian influenza and the increasing reports about its resistance to the current drugs have made a high priority for developing new anti-influenza drugs. Owing to its unique function in assisting viruses to bind the cellular surface, a key step for them to subsequently penetrate into the infected cell, hemagglutinin (HA) has become one of the main targets for drug design against influenza virus. To develop potent HA inhibitors, the ZINC fragment database was searched for finding the optimal compound with the core hopping technique. As a result, the Neo6 compound was obtained. It has been shown through the subsequent molecular docking studies and molecular dynamic simulations that Neo6 not only assumes more favorable conformation at the binding pocket of HA but also has stronger binding interaction with its receptor. Accordingly, Neo6 may become a promising candidate for developing new and more powerful drugs for treating influenza. Or at the very least, the findings reported here may provide useful insights to stimulate new strategy in this area.

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Tamiflu-Resistant but HA-Mediated Cell-to-Cell Transmission through Apical Membranes of Cell-Associated Influenza Viruses

The infection of viruses to a neighboring cell is considered to be beneficial in terms of evasion from host anti-virus defense systems. There are two pathways for viral infection to “right next door”: one is the virus transmission through cell-cell fusion by forming syncytium without production of progeny virions, and the other is mediated by virions without virus diffusion, generally designated cell-to-cell transmission. Influenza viruses are believed to be transmitted as cell-free virus from infected cells to uninfected cells. Here, we demonstrated that influenza virus can utilize cell-to-cell transmission pathway through apical membranes, by handover of virions on the surface of an infected cell to adjacent host cells. Live cell imaging techniques showed that a recombinant influenza virus, in which the neuraminidase gene was replaced with the green fluorescence protein gene, spreads from an infected cell to adjacent cells forming infected cell clusters. This type of virus spreading requires HA activation by protease treatment. The cell-to-cell transmission was also blocked by amantadine, which inhibits the acidification of endosomes required for uncoating of influenza virus particles in endosomes, indicating that functional hemagglutinin and endosome acidification by M2 ion channel were essential for the cell-to-cell influenza virus transmission. Furthermore, in the cell-to-cell transmission of influenza virus, progeny virions could remain associated with the surface of infected cell even after budding, for the progeny virions to be passed on to adjacent uninfected cells. The evidence that cell-to-cell transmission occurs in influenza virus lead to the caution that local infection proceeds even when treated with neuraminidase inhibitors.

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Persistent Expression of Hepatitis C Virus Non-Structural Proteins Leads to Increased Autophagy and Mitochondrial Injury in Human Hepatoma Cells

HCV infection is a major cause of chronic liver disease and liver cancer in the United States. To address the pathogenesis caused by HCV infection, recent studies have focused on the direct cytopathic effects of individual HCV proteins, with the objective of identifying their specific roles in the overall pathogenesis. However, this approach precludes examination of the possible interactions between different HCV proteins and organelles. To obtain a better understanding of the various cytopathic effects of and cellular responses to HCV proteins, we used human hepatoma cells constitutively replicating HCV RNA encoding either the full-length polyprotein or the non-structural proteins, or cells constitutively expressing the structural protein core, to model the state of persistent HCV infection and examined the combination of various HCV proteins in cellular pathogenesis. Increased reactive oxygen species (ROS) generation in the mitochondria, mitochondrial injury and degeneration, and increased lipid accumulation were common among all HCV protein-expressing cells regardless of whether they expressed the structural or non-structural proteins. Expression of the non-structural proteins also led to increased oxidative stress in the cytosol, membrane blebbing in the endoplasmic reticulum, and accumulation of autophagocytic vacuoles. Alterations of cellular redox state, on the other hand, significantly changed the level of autophagy, suggesting a direct link between oxidative stress and HCV-mediated activation of autophagy. With the wide-spread cytopathic effects, cells with the full-length HCV polyprotein showed a modest antioxidant response and exhibited a significant increase in population doubling time and a concomitant decrease in cyclin D1. In contrast, cells expressing the non-structural proteins were able to launch a vigorous antioxidant response with up-regulation of antioxidant enzymes. The population doubling time and cyclin D1 level were also comparable to that of control cells. Finally, the cytopathic effects of core protein appeared to focus on the mitochondria without remarkable disturbances in the cytosol.

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Neighborhood Properties Are Important Determinants of Temperature Sensitive Mutations

Temperature-sensitive (TS) mutants are powerful tools to study gene function in vivo. These mutants exhibit wild-type activity at permissive temperatures and reduced activity at restrictive temperatures. Although random mutagenesis can be used to generate TS mutants, the procedure is laborious and unfeasible in multicellular organisms. Further, the underlying molecular mechanisms of the TS phenotype are poorly understood. To elucidate TS mechanisms, we used a machine learning method–logistic regression–to investigate a large number of sequence and structure features. We developed and tested 133 features, describing properties of either the mutation site or the mutation site neighborhood. We defined three types of neighborhood using sequence distance, Euclidean distance, and topological distance. We discovered that neighborhood features outperformed mutation site features in predicting TS mutations. The most predictive features suggest that TS mutations tend to occur at buried and rigid residues, and are located at conserved protein domains. The environment of a buried residue often determines the overall structural stability of a protein, thus may lead to reversible activity change upon temperature switch. We developed TS prediction models based on logistic regression and the Lasso regularized procedure. Through a ten-fold cross-validation, we obtained the area under the curve of 0.91 for the model using both sequence and structure features. Testing on independent datasets suggested that the model predicted TS mutations with a 50% precision. In summary, our study elucidated the molecular basis of TS mutants and suggested the importance of neighborhood properties in determining TS mutations. We further developed models to predict TS mutations derived from single amino acid substitutions. In this way, TS mutants can be efficiently obtained through experimentally introducing the predicted mutations.

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Missing and accounted for: gaps and areas of wealth in the public health review literature

BACKGROUND: High-quality review evidence is useful for informing and influencing public health policy and practice decisions. However, certain topic areas lack representation in terms of the quantity and quality of review literature available. The objectives of this paper are to identify the quantity, as well as quality, of review-level evidence available on the effectiveness of public health interventions for public health decision makers. METHODS: Searches conducted on http://www.health-evidence.ca produced an inventory of public health review literature in 21 topic areas. Gaps and areas of wealth in the review literature, as well as the proportion of reviews rated methodologically strong, moderate, or weak were identified. The top 10 topic areas of interest for registered users and visitors of http://www.health-evidence.ca were extracted from user profile data and Google Analytics. RESULTS: Registered users' top three interests included: 1) healthy communities, 2) chronic diseases, and 3) nutrition. The top three preferences for visitors included: 1) chronic diseases, 2) physical activity, and 3) addiction/substance use. All of the topic areas with many (301+) available reviews were of interest to registered users and/or visitors (mental health, physical activity, addiction/substance use, adolescent health, child health, nutrition, adult health, and chronic diseases). Conversely, the majority of registered users and/or visitors did not have preference for topic areas with few (≤ 150) available reviews (food safety and inspection, dental health, environmental health) with the exception of social determinants of health and healthy communities. Across registered users' and visitors' topic areas of preference, 80.2% of the reviews were of well-done methodological quality, with 43.5% of reviews having a strong quality rating and 36.7% a moderate review quality rating. CONCLUSIONS: In topic areas in which many reviews are available, higher level syntheses are needed to guide policy and practice. For other topic areas with few reviews, it is necessary to determine whether primary study evidence exists, or is needed, so that reviews can be conducted in the future. Considering that less than half of the reviews available on http://www.health-evidence.ca are of strong methodological quality, the quality of the review-level evidence needs to improve across the range of public health topic areas.

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Inhibition of Interferon Induction and Action by the Nairovirus Nairobi Sheep Disease Virus/Ganjam Virus

The Nairoviruses are an important group of tick-borne viruses that includes pathogens of man (Crimean Congo hemorrhagic fever virus) and livestock animals (Dugbe virus, Nairobi sheep disease virus (NSDV)). NSDV is found in large parts of East Africa and the Indian subcontinent (where it is known as Ganjam virus). We have investigated the ability of NSDV to antagonise the induction and actions of interferon. Both pathogenic and apathogenic isolates could actively inhibit the induction of type 1 interferon, and also blocked the signalling pathways of both type 1 and type 2 interferons. Using transient expression of viral proteins or sections of viral proteins, these activities all mapped to the ovarian tumour-like protease domain (OTU) found in the viral RNA polymerase. Virus infection, or expression of this OTU domain in transfected cells, led to a great reduction in the incorporation of ubiquitin or ISG15 protein into host cell proteins. Point mutations in the OTU that inhibited the protease activity also prevented it from antagonising interferon induction and action. Interestingly, a mutation at a peripheral site, which had little apparent effect on the ability of the OTU to inhibit ubiquitination and ISG15ylation, removed the ability of the OTU to block the induction of type 1 and the action of type 2 interferons, but had a lesser effect on the ability to block type 1 interferon action, suggesting that targets other than ubiquitin and ISG15 may be involved in the actions of the viral OTU.

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A Human Monoclonal Antibody with Neutralizing Activity against Highly Divergent Influenza Subtypes

The interest in broad-range anti-influenza A monoclonal antibodies (mAbs) has recently been strengthened by the identification of anti-hemagglutinin (HA) mAbs endowed with heterosubtypic neutralizing activity to be used in the design of “universal” prophylactic or therapeutic tools. However, the majority of the single mAbs described to date do not bind and neutralize viral isolates belonging to highly divergent subtypes clustering into the two different HA-based influenza phylogenetic groups: the group 1 including, among others, subtypes H1, H2, H5 and H9 and the group 2 including, among others, H3 subtype. Here, we describe a human mAb, named PN-SIA28, capable of binding and neutralizing all tested isolates belonging to phylogenetic group 1, including H1N1, H2N2, H5N1 and H9N2 subtypes and several isolates belonging to group 2, including H3N2 isolates from the first period of the 1968 pandemic. Therefore, PN-SIA28 is capable of neutralizing isolates belonging to subtypes responsible of all the reported pandemics, as well as other subtypes with pandemic potential. The region recognized by PN-SIA28 has been identified on the stem region of HA and includes residues highly conserved among the different influenza subtypes. A deep characterization of PN-SIA28 features may represent a useful help in the improvement of available anti-influenza therapeutic strategies and can provide new tools for the development of universal vaccinal strategies.

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Tissue Tropism and Target Cells of NSs-Deleted Rift Valley Fever Virus in Live Immunodeficient Mice

BACKGROUND: Rift Valley fever virus (RVFV) causes disease in livestock and humans. It can be transmitted by mosquitoes, inhalation or physical contact with the body fluids of infected animals. Severe clinical cases are characterized by acute hepatitis with hemorrhage, meningoencephalitis and/or retinitis. The dynamics of RVFV infection and the cell types infected in vivo are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: RVFV strains expressing humanized Renilla luciferase (hRLuc) or green fluorescent protein (GFP) were generated and inoculated to susceptible Ifnar1-deficient mice. We investigated the tissue tropism in these mice and the nature of the target cells in vivo using whole-organ imaging and flow cytometry. After intraperitoneal inoculation, hRLuc signal was observed primarily in the thymus, spleen and liver. Macrophages infiltrating various tissues, in particular the adipose tissue surrounding the pancreas also expressed the virus. The liver rapidly turned into the major luminescent organ and the mice succumbed to severe hepatitis. The brain remained weakly luminescent throughout infection. FACS analysis in RVFV-GFP-infected mice showed that the macrophages, dendritic cells and granulocytes were main target cells for RVFV. The crucial role of cells of the monocyte/macrophage/dendritic lineage during RVFV infection was confirmed by the slower viral dissemination, decrease in RVFV titers in blood, and prolonged survival of macrophage- and dendritic cell-depleted mice following treatment with clodronate liposomes. Upon dermal and nasal inoculations, the viral dissemination was primarily observed in the lymph node draining the injected ear and in the lungs respectively, with a significant increase in survival time. CONCLUSIONS/SIGNIFICANCE: These findings reveal the high levels of phagocytic cells harboring RVFV during viral infection in Ifnar1-deficient mice. They demonstrate that bioluminescent and fluorescent viruses can shed new light into the pathogenesis of RVFV infection.