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A cross-sectional survey to evaluate knowledge, attitudes and practices (KAP) regarding seasonal influenza vaccination among European travellers to resource-limited destinations

BACKGROUND: Influenza is one of the most common vaccine-preventable diseases in travellers. By performing two cross-sectional questionnaire surveys during winter 2009 and winter 2010 among European travellers to resource-limited destinations, we aimed to investigate knowledge, attitudes and practices (KAP) regarding seasonal influenza vaccination. METHODS: Questionnaires were distributed in the waiting room to the visitors of the University of Zurich Centre for Travel' Health (CTH) in January and February 2009 and January 2010 prior to travel health counselling (CTH09 and CTH10). Questions included demographic data, travel-related characteristics and KAP regarding influenza vaccination. Data were analysed by using SPSS(® )version 14.0 for Windows. Differences in proportions were compared using the Chi-square test and the significance level was set at p ≤ 0.05. Predictors for seasonal and pandemic influenza vaccination were determined by multiple logistic regression analyses. RESULTS: With a response rate of 96.6%, 906 individuals were enrolled and 868 (92.5%) provided complete data. Seasonal influenza vaccination coverage was 13.7% (n = 119). Only 43 (14.2%) participants were vaccinated against pandemic influenza A/H1N1, mostly having received both vaccines simultaneously, the seasonal and pandemic one. Job-related purposes (44, 37%), age > 64 yrs (25, 21%) and recommendations of the family physician (27, 22.7%) were the most often reported reasons for being vaccinated. In the multiple logistic regression analyses of the pooled data increasing age (OR = 1.03, 95% CI 1.01 - 1.04), a business trip (OR = 0.39, 95% CI 0.17 - 0.92) and seasonal influenza vaccination in the previous winter seasons (OR = 12.91, 95% CI 8.09 - 20.58) were independent predictors for seasonal influenza vaccination in 2009 or 2010. Influenza vaccination recommended by the family doctor (327, 37.7%), travel to regions with known high risk of influenza (305, 35.1%), and influenza vaccination required for job purposes (233, 26.8%) were most frequently mentioned to consider influenza vaccination. CONCLUSIONS: Risk perception and vaccination coverage concerning seasonal and pandemic influenza was very poor among travellers to resource-limited destinations when compared to traditional at-risk groups. Previous access to influenza vaccination substantially facilitated vaccinations in the subsequent year. Information strategies about influenza should be intensified and include health professionals, e.g. family physicians, travel medicine practitioners and business enterprises.

Serodiagnosis of Echinococcus spp. Infection: Explorative Selection of Diagnostic Antigens by Peptide Microarray

BACKGROUND: Production of native antigens for serodiagnosis of helminthic infections is laborious and hampered by batch-to-batch variation. For serodiagnosis of echinococcosis, especially cystic disease, most screening tests rely on crude or purified Echinococcus granulosus hydatid cyst fluid. To resolve limitations associated with native antigens in serological tests, the use of standardized and highly pure antigens produced by chemical synthesis offers considerable advantages, provided appropriate diagnostic sensitivity and specificity is achieved. METHODOLOGY/PRINCIPAL FINDINGS: Making use of the growing collection of genomic and proteomic data, we applied a set of bioinformatic selection criteria to a collection of protein sequences including conceptually translated nucleotide sequence data of two related tapeworms, Echinococcus multilocularis and Echinococcus granulosus. Our approach targeted alpha-helical coiled-coils and intrinsically unstructured regions of parasite proteins potentially exposed to the host immune system. From 6 proteins of E. multilocularis and 5 proteins of E. granulosus, 45 peptides between 24 and 30 amino acids in length were designed. These peptides were chemically synthesized, spotted on microarrays and screened for reactivity with sera from infected humans. Peptides reacting above the cut-off were validated in enzyme-linked immunosorbent assays (ELISA). Peptides identified failed to differentiate between E. multilocularis and E. granulosus infection. The peptide performing best reached 57% sensitivity and 94% specificity. This candidate derived from Echinococcus multilocularis antigen B8/1 and showed strong reactivity to sera from patients infected either with E. multilocularis or E. granulosus. CONCLUSIONS/SIGNIFICANCE: This study provides proof of principle for the discovery of diagnostically relevant peptides by bioinformatic selection complemented with screening on a high-throughput microarray platform. Our data showed that a single peptide cannot provide sufficient diagnostic sensitivity whereas pooling several peptide antigens improved sensitivity; thus combinations of several peptides may lead the way to new diagnostic tests that replace, or at least complement conventional immunodiagnosis of echinococcosis. Our strategy could prove useful for diagnostic developments in other pathogens.

Protective Efficacy of Cross-Reactive CD8(+) T Cells Recognising Mutant Viral Epitopes Depends on Peptide-MHC-I Structural Interactions and T Cell Activation Threshold

Emergence of a new influenza strain leads to a rapid global spread of the virus due to minimal antibody immunity. Pre-existing CD8(+) T-cell immunity directed towards conserved internal viral regions can greatly ameliorate the disease. However, mutational escape within the T cell epitopes is a substantial issue for virus control and vaccine design. Although mutations can result in a loss of T cell recognition, some variants generate cross-reactive T cell responses. In this study, we used reverse genetics to modify the influenza NP(336–374) peptide at a partially-solvent exposed residue (N->A, NPN3A mutation) to assess the availability, effectiveness and mechanism underlying influenza-specific cross-reactive T cell responses. The engineered virus induced a diminished CD8(+) T cell response and selected a narrowed T cell receptor (TCR) repertoire within two Vβ regions (Vβ8.3 and Vβ9). This can be partially explained by the H-2D(b)NPN3A structure that showed a loss of several contacts between the NPN3A peptide and H-2D(b), including a contact with His155, a position known to play an important role in mediating TCR-pMHC-I interactions. Despite these differences, common cross-reactive TCRs were detected in both the naïve and immune NPN3A-specific TCR repertoires. However, while the NPN3A epitope primes memory T-cells that give an equivalent recall response to the mutant or wild-type (wt) virus, both are markedly lower than wt->wt challenge. Such decreased CD8(+) responses elicited after heterologous challenge resulted in delayed viral clearance from the infected lung. Furthermore, mice first exposed to the wt virus give a poor, low avidity response following secondary infection with the mutant. Thus, the protective efficacy of cross-reactive CD8(+) T cells recognising mutant viral epitopes depend on peptide-MHC-I structural interactions and functional avidity. Our study does not support vaccine strategies that include immunization against commonly selected cross-reactive variants with mutations at partially-solvent exposed residues that have characteristics comparable to NPN3A.

Development of a Symptom Score for Clinical Studies to Identify Children With a Documented Viral Upper Respiratory Tract Infection

The objective of this study was to develop a symptom scoring system for use in clinical studies that differentiates children with cold symptoms who have an identifiable viral etiology for their upper respiratory tract infection (URI) from those in whom no virus is detected. Nasal swabs for PCR testing for identification of respiratory viruses were obtained on children aged 2–11 y at baseline and when parents thought their child was developing a cold. Parental-recorded severity of specific symptoms in children with and without a documented viral URI were compared. Nasal swabs were obtained on 108 children whose parents reported their child was developing a cold. A viral etiology was identified in 62 of 108 (57.4%) samples. Symptom measures that best differentiated children with a viral etiology from those without were significant runny nose and significant cough on days 1–4 of the illness. A URI symptom score was developed based on these symptoms, with a sensitivity of 81.4%, specificity of 61.9%, and accuracy of 73.3%. Parental impression is only a moderately accurate predictor of viral URI in children. Our URI symptom score provided a more accurate method for identifying children with viral URIs for clinical studies.

Cancer Biomarker Discovery: The Entropic Hallmark

BACKGROUND: It is a commonly accepted belief that cancer cells modify their transcriptional state during the progression of the disease. We propose that the progression of cancer cells towards malignant phenotypes can be efficiently tracked using high-throughput technologies that follow the gradual changes observed in the gene expression profiles by employing Shannon's mathematical theory of communication. Methods based on Information Theory can then quantify the divergence of cancer cells' transcriptional profiles from those of normally appearing cells of the originating tissues. The relevance of the proposed methods can be evaluated using microarray datasets available in the public domain but the method is in principle applicable to other high-throughput methods. METHODOLOGY/PRINCIPAL FINDINGS: Using melanoma and prostate cancer datasets we illustrate how it is possible to employ Shannon Entropy and the Jensen-Shannon divergence to trace the transcriptional changes progression of the disease. We establish how the variations of these two measures correlate with established biomarkers of cancer progression. The Information Theory measures allow us to identify novel biomarkers for both progressive and relatively more sudden transcriptional changes leading to malignant phenotypes. At the same time, the methodology was able to validate a large number of genes and processes that seem to be implicated in the progression of melanoma and prostate cancer. CONCLUSIONS/SIGNIFICANCE: We thus present a quantitative guiding rule, a new unifying hallmark of cancer: the cancer cell's transcriptome changes lead to measurable observed transitions of Normalized Shannon Entropy values (as measured by high-througput technologies). At the same time, tumor cells increment their divergence from the normal tissue profile increasing their disorder via creation of states that we might not directly measure. This unifying hallmark allows, via the the Jensen-Shannon divergence, to identify the arrow of time of the processes from the gene expression profiles, and helps to map the phenotypical and molecular hallmarks of specific cancer subtypes. The deep mathematical basis of the approach allows us to suggest that this principle is, hopefully, of general applicability for other diseases.

SYSGENET: a meeting report from a new European network for systems genetics

The first scientific meeting of the newly established European SYSGENET network took place at the Helmholtz Centre for Infection Research (HZI) in Braunschweig, April 7-9, 2010. About 50 researchers working in the field of systems genetics using mouse genetic reference populations (GRP) participated in the meeting and exchanged their results, phenotyping approaches, and data analysis tools for studying systems genetics. In addition, the future of GRP resources and phenotyping in Europe was discussed.

Adaptive Contact Networks Change Effective Disease Infectiousness and Dynamics

Human societies are organized in complex webs that are constantly reshaped by a social dynamic which is influenced by the information individuals have about others. Similarly, epidemic spreading may be affected by local information that makes individuals aware of the health status of their social contacts, allowing them to avoid contact with those infected and to remain in touch with the healthy. Here we study disease dynamics in finite populations in which infection occurs along the links of a dynamical contact network whose reshaping may be biased based on each individual's health status. We adopt some of the most widely used epidemiological models, investigating the impact of the reshaping of the contact network on the disease dynamics. We derive analytical results in the limit where network reshaping occurs much faster than disease spreading and demonstrate numerically that this limit extends to a much wider range of time scales than one might anticipate. Specifically, we show that from a population-level description, disease propagation in a quickly adapting network can be formulated equivalently as disease spreading on a well-mixed population but with a rescaled infectiousness. We find that for all models studied here – SI, SIS and SIR – the effective infectiousness of a disease depends on the population size, the number of infected in the population, and the capacity of healthy individuals to sever contacts with the infected. Importantly, we indicate how the use of available information hinders disease progression, either by reducing the average time required to eradicate a disease (in case recovery is possible), or by increasing the average time needed for a disease to spread to the entire population (in case recovery or immunity is impossible).

The role of infections in the pathogenesis and course of multiple sclerosis

Interplay between susceptibility genes and environmental factors is considered important player in the genesis of multiple sclerosis (MS). Among environmental factors, a role for an infectious pathogen has long been considered central to the disease process. This opinion has support both from epidemiological data and the findings of immunological abnormalities in spinal fluid that reflect an immune response to an as yet undetermined antigen, possibly a pathogen, in the cerebrospinal fluid. Our review will outline the current understanding of the role of infection in the causation and progression of MS. We will review the data that point to an infectious cause of MS and consider the specific agents Chlamydophila (Chlamydia) pneumoniae, Human Herpes Virus 6, and Epstein-Barr Virus, that are implicated in either the development or progression of MS.

Calciomics: prediction and analysis of EF-hand calcium binding proteins by protein engineering

Ca(2+) plays a pivotal role in the physiology and biochemistry of prokaryotic and mammalian organisms. Viruses also utilize the universal Ca(2+) signal to create a specific cellular environment to achieve coexistence with the host, and to propagate. In this paper we first describe our development of a grafting approach to understand site-specific Ca(2+) binding properties of EF-hand proteins with a helix-loop-helix Ca(2+) binding motif, then summarize our prediction and identification of EF-hand Ca(2+) binding sites on a genome-wide scale in bacteria and virus, and next report the application of the grafting approach to probe the metal binding capability of predicted EF-hand motifs within the streptococcal hemoprotein receptor (Shr) of Streptococcus pyrogenes and the nonstructural protein 1 (nsP1) of Sindbis virus. When methods such as the grafting approach are developed in conjunction with prediction algorithms we are better able to probe continuous Ca(2+)-binding sites that have been previously underrepresented due to the limitation of conventional methodology.

Proteomics Comparison of Cerebrospinal Fluid of Relapsing Remitting and Primary Progressive Multiple Sclerosis

BACKGROUND: Based on clinical representation of disease symptoms multiple sclerosis (MScl) patients can be divided into two major subtypes; relapsing remitting (RR) MScl (85–90%) and primary progressive (PP) MScl (10–15%). Proteomics analysis of cerebrospinal fluid (CSF) has detected a number of proteins that were elevated in MScl patients. Here we specifically aimed to differentiate between the PP and RR subtypes of MScl by comparing CSF proteins. METHODOLOGY/PRINCIPAL FINDINGS: CSF samples (n = 31) were handled according to the same protocol for quantitative mass spectrometry measurements we reported previously. In the comparison of PP MScl versus RR MScl we observed a number of differentially abundant proteins, such as protein jagged-1 and vitamin D-binding protein. Protein jagged-1 was over three times less abundant in PP MScl compared to RR MScl. Vitamin D-binding protein was only detected in the RR MScl samples. These two proteins were validated by independent techniques (western blot and ELISA) as differentially abundant in the comparison between both MScl types. CONCLUSIONS/SIGNIFICANCE: The main finding of this comparative study is the observation that the proteome profiles of CSF in PP and RR MScl patients overlap to a large extent. Still, a number of differences could be observed. Protein jagged-1 is a ligand for multiple Notch receptors and involved in the mediation of Notch signaling. It is suggested in literature that the Notch pathway is involved in the remyelination of MScl lesions. Aberration of normal homeostasis of Vitamin D, of which approximately 90% is bound to vitamin D-binding protein, has been widely implicated in MScl for some years now. Vitamin D directly and indirectly regulates the differentiation, activation of CD4+ T-lymphocytes and can prevent the development of autoimmune processes, and so it may be involved in neuroprotective elements in MScl.

Interstitial lung diseases in children

Interstitial lung disease (ILD) in infants and children comprises a large spectrum of rare respiratory disorders that are mostly chronic and associated with high morbidity and mortality. These disorders are characterized by inflammatory and fibrotic changes that affect alveolar walls. Typical features of ILD include dyspnea, diffuse infiltrates on chest radiographs, and abnormal pulmonary function tests with restrictive ventilatory defect and/or impaired gas exchange. Many pathological situations can impair gas exchange and, therefore, may contribute to progressive lung damage and ILD. Consequently, diagnosis approach needs to be structured with a clinical evaluation requiring a careful history paying attention to exposures and systemic diseases. Several classifications for ILD have been proposed but none is entirely satisfactory especially in children. The present article reviews current concepts of pathophysiological mechanisms, etiology and diagnostic approaches, as well as therapeutic strategies. The following diagnostic grouping is used to discuss the various causes of pediatric ILD: 1) exposure-related ILD; 2) systemic disease-associated ILD; 3) alveolar structure disorder-associated ILD; and 4) ILD specific to infancy. Therapeutic options include mainly anti-inflammatory, immunosuppressive, and/or anti-fibrotic drugs. The outcome is highly variable with a mortality rate around 15%. An overall favorable response to corticosteroid therapy is observed in around 50% of cases, often associated with sequelae such as limited exercise tolerance or the need for long-term oxygen therapy.

Predictive Power of Air Travel and Socio-Economic Data for Early Pandemic Spread

BACKGROUND: Controlling the pandemic spread of newly emerging diseases requires rapid, targeted allocation of limited resources among nations. Critical, early control steps would be greatly enhanced if the key risk factors can be identified that accurately predict early disease spread immediately after emergence. METHODOLOGY/PRINCIPAL FINDINGS: Here, we examine the role of travel, trade, and national healthcare resources in predicting the emergence and initial spread of 2009 A/H1N1 influenza. We find that incorporating national healthcare resource data into our analyses allowed a much greater capacity to predict the international spread of this virus. In countries with lower healthcare resources, the reporting of 2009 A/H1N1 cases was significantly delayed, likely reflecting a lower capacity for testing and reporting, as well as other socio-political issues. We also report substantial international trade in live swine and poultry in the decade preceding the pandemic which may have contributed to the emergence and mixed genotype of this pandemic strain. However, the lack of knowledge of recent evolution of each H1N1 viral gene segment precludes the use of this approach to determine viral origins. CONCLUSIONS/SIGNIFICANCE: We conclude that strategies to prevent pandemic influenza virus emergence and spread in the future should include: 1) enhanced surveillance for strains resulting from reassortment in traded livestock; 2) rapid deployment of control measures in the initial spreading phase to countries where travel data predict the pathogen will reach and to countries where lower healthcare resources will likely cause delays in reporting. Our results highlight the benefits, for all parties, when higher income countries provide additional healthcare resources for lower income countries, particularly those that have high air traffic volumes. In particular, international authorities should prioritize aid to those poorest countries where both the risk of emerging infectious diseases and air traffic volume is highest. This strategy will result in earlier detection of pathogens and a reduction in the impact of future pandemics.

Evolutionary Entropy Determines Invasion Success in Emergent Epidemics

BACKGROUND: Standard epidemiological theory claims that in structured populations competition between multiple pathogen strains is a deterministic process which is mediated by the basic reproduction number ([Image: see text]) of the individual strains. A new theory based on analysis, simulation and empirical study challenges this predictor of success. PRINCIPAL FINDINGS: We show that the quantity [Image: see text] is a valid predictor in structured populations only when size is infinite. In this article we show that when population size is finite the dynamics of infection by multi-strain pathogens is a stochastic process whose outcome can be predicted by evolutionary entropy, S, an information theoretic measure which describes the uncertainty in the infectious age of an infected parent of a randomly chosen new infective. Evolutionary entropy characterises the demographic stability or robustness of the population of infectives. This statistical parameter determines the duration of infection and thus provides a quantitative index of the pathogenicity of a strain. Standard epidemiological theory based on [Image: see text] as a measure of selective advantage is the limit as the population size tends to infinity of the entropic selection theory. The standard model is an approximation to the entropic selection theory whose validity increases with population size. CONCLUSION: An epidemiological analysis based on entropy is shown to explain empirical observations regarding the emergence of less pathogenic strains of human influenza during the antigenic drift phase. Furthermore, we exploit the entropy perspective to discuss certain epidemiological patterns of the current H1N1 swine 'flu outbreak.

The Nature of Protein Domain Evolution: Shaping the Interaction Network

The proteomes that make up the collection of proteins in contemporary organisms evolved through recombination and duplication of a limited set of domains. These protein domains are essentially the main components of globular proteins and are the most principal level at which protein function and protein interactions can be understood. An important aspect of domain evolution is their atomic structure and biochemical function, which are both specified by the information in the amino acid sequence. Changes in this information may bring about new folds, functions and protein architectures. With the present and still increasing wealth of sequences and annotation data brought about by genomics, new evolutionary relationships are constantly being revealed, unknown structures modeled and phylogenies inferred. Such investigations not only help predict the function of newly discovered proteins, but also assist in mapping unforeseen pathways of evolution and reveal crucial, co-evolving inter- and intra-molecular interactions. In turn this will help us describe how protein domains shaped cellular interaction networks and the dynamics with which they are regulated in the cell. Additionally, these studies can be used for the design of new and optimized protein domains for therapy. In this review, we aim to describe the basic concepts of protein domain evolution and illustrate recent developments in molecular evolution that have provided valuable new insights in the field of comparative genomics and protein interaction networks.

Quantitative Phosphoproteomics of Proteasome Inhibition in Multiple Myeloma Cells

BACKGROUND: The proteasome inhibitor bortezomib represents an important advance in the treatment of multiple myeloma (MM). Bortezomib inhibits the activity of the 26S proteasome and induces cell death in a variety of tumor cells; however, the mechanism of cytotoxicity is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the differential phosphoproteome upon proteasome inhibition by using stable isotope labeling by amino acids in cell culture (SILAC) in combination with phosphoprotein enrichment and LC-MS/MS analysis. In total 233 phosphoproteins were identified and 72 phosphoproteins showed a 1.5-fold or greater change upon bortezomib treatment. The phosphoproteins with expression alterations encompass all major protein classes, including a large number of nucleic acid binding proteins. Site-specific phosphopeptide quantitation revealed that Ser38 phosphorylation on stathmin increased upon bortezomib treatment, suggesting new mechanisms associated to bortezomib-induced apoptosis in MM cells. Further studies demonstrated that stathmin phosphorylation profile was modified in response to bortezomib treatment and the regulation of stathmin by phosphorylation at specific Ser/Thr residues participated in the cellular response induced by bortezomib. CONCLUSIONS/SIGNIFICANCE: Our systematic profiling of phosphorylation changes in response to bortezomib treatment not only advanced the global mechanistic understanding of the action of bortezomib on myeloma cells but also identified previously uncharacterized signaling proteins in myeloma cells.

Insights into the Evolution and Emergence of a Novel Infectious Disease

Many zoonotic, novel infectious diseases in humans appear as sporadic infections with spatially and temporally restricted outbreaks, as seen with influenza A(H5N1). Adaptation is often a key factor for successfully establishing sustained human-to-human transmission. Here we use simple mathematical models to describe different adaptation scenarios with particular reference to spatial heterogeneity within the human population. We present analytical expressions for the probability of emergence per introduction, as well as the waiting time to a successful emergence event. Furthermore, we derive general analytical results for the statistical properties of emergence events, including the probability distribution of outbreak sizes. We compare our analytical results with a stochastic model, which has previously been studied computationally. Our results suggest that, for typical connection strengths between communities, spatial heterogeneity has only a weak effect on outbreak size distributions, and on the risk of emergence per introduction. For example, if [Image: see text] or larger, any village connected to a large city by just ten commuters a day is, effectively, just a part of the city when considering the chances of emergence and the outbreak size distribution. We present empirical data on commuting patterns and show that the vast majority of communities for which such data are available are at least this well interconnected. For plausible parameter ranges, the effects of spatial heterogeneity are likely to be dominated by the evolutionary biology of host adaptation. We conclude by discussing implications for surveillance and control of emerging infections.

Role of Host Immune Response and Viral Load in the Differential Outcome of Pandemic H1N1 (2009) Influenza Virus Infection in Indian Patients

BACKGROUND: An unusually high number of severe pneumonia cases with considerable mortality is being observed with the pandemic H1N1 2009 virus infections globally. In India, all mild as well as critically ill cases were admitted and treated in the government hospitals during the initial phase of the pandemic. The present study was undertaken during this early phase of the pandemic. METHODOLOGY: The role of viral load and host factors in the pathogenesis were assessed by examining 26 mild (MP), 15 critically ill patients (CIP) and 20 healthy controls from Pune, India. Sequential blood and lung aspirate samples were collected from CIP. Viral load and cytokines/chemokine levels were determined from the plasma and lung aspirates of the patients. TLR levels were determined by staining and FACS analysis. Gene profiling was done for both cells in the lung aspirates and PBMCs using TaqMan Low Density arrays. Antibody titres and isotyping was done using HA protein based ELISAs. PRINCIPAL FINDINGS: 13/15 critically ill patients expired. All plasma samples were negative for the virus irrespective of the patient's category. Sequential lung samples from CIP showed lower viral loads questioning association of viral replication with the severity. Anti-rpH1N1-09-HA-IgG titres were significantly higher in critically ill patients and both categories circulated exclusively IgG1 isotype. Critically ill patients exhibited increase in TLR-3, 4, 7 and decrease in TLR-2 expressions. The disease severity correlated with increased plasma levels of IL1RA, IL2, IL6, CCL3, CCL4 and IL10. Majority of the immune-function genes were down-regulated in the PBMCs and up-regulated in the cells from lung aspirates of critically ill patients. No distinct pattern differentiating fatal and surviving patients was observed when sequential samples were examined for various parameters. CONCLUSIONS: Disease severity was associated with pronounced impairment of host immune response.

Generation of Human Antigen-Specific Monoclonal IgM Antibodies Using Vaccinated “Human Immune System” Mice

BACKGROUND: Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the ‘humanization’ of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique. METHODOLOGY/PRINCIPAL FINDINGS: After transplantation with CD34(+)CD38(−) human hematopoietic progenitor cells, BALB/c Rag2(−/−)IL-2Rγc(−/−) mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. “Human Immune System” mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19(+)CD27(+) B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively. CONCLUSION/SIGNIFICANCE: This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens.

Reflections on Pandemic (H1N1) 2009 and the International Response

Gabriel Leung and Angus Nicoll provide their reflections on the international response to the 2009 H1N1 influenza pandemic, including what went well and what changes need to be made in anticipation of future flu pandemics.

Development of a large-scale isolation chamber system for the safe and humane care of medium-sized laboratory animals harboring infectious diseases

The close phylogenetic relationship between humans and non-human primates makes non-human primates an irreplaceable model for the study of human infectious diseases. In this study, we describe the development of a large-scale automatic multi-functional isolation chamber for use with medium-sized laboratory animals carrying infectious diseases. The isolation chamber, including the transfer chain, disinfection chain, negative air pressure isolation system, animal welfare system, and the automated system, is designed to meet all biological safety standards. To create an internal chamber environment that is completely isolated from the exterior, variable frequency drive blowers are used in the air-intake and air-exhaust system, precisely controlling the filtered air flow and providing an air-barrier protection. A double door transfer port is used to transfer material between the interior of the isolation chamber and the outside. A peracetic acid sterilizer and its associated pipeline allow for complete disinfection of the isolation chamber. All of the isolation chamber parameters can be automatically controlled by a programmable computerized menu, allowing for work with different animals in different-sized cages depending on the research project. The large-scale multi-functional isolation chamber provides a useful and safe system for working with infectious medium-sized laboratory animals in high-level bio-safety laboratories.

Proteomics: Challenges, Techniques and Possibilities to Overcome Biological Sample Complexity

Proteomics is the large-scale study of the structure and function of proteins in complex biological sample. Such an approach has the potential value to understand the complex nature of the organism. Current proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. Advances in protein fractionation and labeling techniques have improved protein identification to include the least abundant proteins. In addition, proteomics has been complemented by the analysis of posttranslational modifications and techniques for the quantitative comparison of different proteomes. However, the major limitation of proteomic investigations remains the complexity of biological structures and physiological processes, rendering the path of exploration paved with various difficulties and pitfalls. The quantity of data that is acquired with new techniques places new challenges on data processing and analysis. This article provides a brief overview of currently available proteomic techniques and their applications, followed by detailed description of advantages and technical challenges. Some solutions to circumvent technical difficulties are proposed.

A Systematic Molecular Pathology Study of a Laboratory Confirmed H5N1 Human Case

Autopsy studies have shown that human highly pathogenic avian influenza virus (H5N1) can infect multiple human organs other than just the lungs, and that possible causes of organ damage are either viral replication and/or dysregulation of cytokines and chemokines. Uncertainty still exists, partly because of the limited number of cases analysed. In this study, a full autopsy including 5 organ systems was conducted on a confirmed H5N1 human fatal case (male, 42 years old) within 18 hours of death. In addition to the respiratory system (lungs, bronchus and trachea), virus was isolated from cerebral cortex, cerebral medullary substance, cerebellum, brain stem, hippocampus ileum, colon, rectum, ureter, aortopulmonary vessel and lymph-node. Real time RT-PCR evidence showed that matrix and hemagglutinin genes were positive in liver and spleen in addition to positive tissues with virus isolation. Immunohistochemistry and in-situ hybridization stains showed accordant evidence of viral infection with real time RT-PCR except bronchus. Quantitative RT-PCR suggested that a high viral load was associated with increased host responses, though the viral load was significantly different in various organs. Cells of the immunologic system could also be a target for virus infection. Overall, the pathogenesis of HPAI H5N1 virus was associated both with virus replication and with immunopathologic lesions. In addition, immune cells cannot be excluded from playing a role in dissemination of the virus in vivo.

Situational Awareness and Health Protective Responses to Pandemic Influenza A (H1N1) in Hong Kong: A Cross-Sectional Study

BACKGROUND: Whether information sources influence health protective behaviours during influenza pandemics or other emerging infectious disease epidemics is uncertain. METHODOLOGY: Data from cross-sectional telephone interviews of 1,001 Hong Kong adults in June, 2009 were tested against theory and data-derived hypothesized associations between trust in (formal/informal) information, understanding, self-efficacy, perceived susceptibility and worry, and hand hygiene and social distancing using Structural Equation Modelling with multigroup comparisons. PRINCIPAL FINDINGS: Trust in formal (government/media) information about influenza was associated with greater reported understanding of A/H1N1 cause (β = 0.36) and A/H1N1 prevention self-efficacy (β = 0.25), which in turn were associated with more hand hygiene (β = 0.19 and β = 0.23, respectively). Trust in informal (interpersonal) information was negatively associated with perceived personal A/H1N1 susceptibility (β = −0.21), which was negatively associated with perceived self-efficacy (β = −0.42) but positively associated with influenza worry (β = 0.44). Trust in informal information was positively associated with influenza worry (β = 0.16) which was in turn associated with greater social distancing (β = 0.36). Multigroup comparisons showed gender differences regarding paths from trust in formal information to understanding of A/H1N1 cause, trust in informal information to understanding of A/H1N1 cause, and understanding of A/H1N1 cause to perceived self-efficacy. CONCLUSIONS/SIGNIFICANCE: Trust in government/media information was more strongly associated with greater self-efficacy and handwashing, whereas trust in informal information was strongly associated with perceived health threat and avoidance behaviour. Risk communication should consider the effect of gender differences.

Human Anti-Plague Monoclonal Antibodies Protect Mice from Yersinia pestis in a Bubonic Plague Model

Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr) V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs) against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252) and two anti-V-specific human mAb (m253, m254) by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans.

Diagnostic value of triggering receptor expressed on myeloid cells-1 and C-reactive protein for patients with lung infiltrates: an observational study

BACKGROUND: Differential diagnosis of patients with lung infiltrates remains a challenge. Triggering receptor expressed on myeloid cells (TREM)-1 is a neutrophil and monocyte receptor up-regulated during infection. The aim of this study was to evaluate the diagnostic accuracy of TREM-1 and of C-reactive protein (CRP) from patients with lung infiltrates to discern community acquired lung infections. METHODS: 68 patients admitted to a medical ward with acute respiratory illness were enrolled in the study. Neutrophil and monocyte TREM-1 expression were measured by flow cytometry, sTREM-1 by an enzyme immunoassay and C-reactive protein by nephelometry. Clinical pulmonary infection score was recorded. RESULTS: 34 patients were diagnosed with bacterial community acquired pneumonia (group A) and 34 with non-bacterial pulmonary disease (group B). Median serum TREM-1 concentration was 102.09 pg/ml in group A and lower than 15.10 pg/ml (p < 0.0001) in group B. Mean±SE neutrophil TREM-1 expression was 4.67 ± 0.53 MFI in group A and 2.64 ± 0.25 MFI (p = 0.001) in group B. Monocyte TREM-1 expression was 4.2 ± 0.42 MFI in group A and 2.64 ± 0.35 MFI (p = 0.007) in group B and mean±SE CRP was 18.03 ± 2 mg/ml in group A and 7.1 ± 1.54 mg/ml (p < 0.001) in group B. A cut-off of 19.53 pg/ml of sTREM-1 with sensitivity 82.6% and specificity 63% to discriminate between infectious and non-infectious pulmonary infiltrates was found. sTREM-1 at admission greater than 180 pg/ml was accompanied with unfavourable outcome. CONCLUSION: TREM-1 myeloid expression and sTREM-1 are reliable markers of bacterial infection among patients with pulmonary infiltrates; sTREM-1 is a predictor of final outcome.

Nodeomics: Pathogen Detection in Vertebrate Lymph Nodes Using Meta-Transcriptomics

The ongoing emergence of human infections originating from wildlife highlights the need for better knowledge of the microbial community in wildlife species where traditional diagnostic approaches are limited. Here we evaluate the microbial biota in healthy mule deer (Odocoileus hemionus) by analyses of lymph node meta-transcriptomes. cDNA libraries from five individuals and two pools of samples were prepared from retropharyngeal lymph node RNA enriched for polyadenylated RNA and sequenced using Roche-454 Life Sciences technology. Protein-coding and 16S ribosomal RNA (rRNA) sequences were taxonomically profiled using protein and rRNA specific databases. Representatives of all bacterial phyla were detected in the seven libraries based on protein-coding transcripts indicating that viable microbiota were present in lymph nodes. Residents of skin and rumen, and those ubiquitous in mule deer habitat dominated classifiable bacterial species. Based on detection of both rRNA and protein-coding transcripts, we identified two new proteobacterial species; a Helicobacter closely related to Helicobacter cetorum in the Helicobacter pylori/Helicobacter acinonychis complex and an Acinetobacter related to Acinetobacter schindleri. Among viruses, a novel gamma retrovirus and other members of the Poxviridae and Retroviridae were identified. We additionally evaluated bacterial diversity by amplicon sequencing the hypervariable V6 region of 16S rRNA and demonstrate that overall taxonomic diversity is higher with the meta-transcriptomic approach. These data provide the most complete picture to date of the microbial diversity within a wildlife host. Our research advances the use of meta-transcriptomics to study microbiota in wildlife tissues, which will facilitate detection of novel organisms with pathogenic potential to human and animals.

In Vitro and In Vivo Studies Identify Important Features of Dengue Virus pr-E Protein Interactions

Flaviviruses bud into the endoplasmic reticulum and are transported through the secretory pathway, where the mildly acidic environment triggers particle rearrangement and allows furin processing of the prM protein to pr and M. The peripheral pr peptide remains bound to virus at low pH and inhibits virus-membrane interaction. Upon exocytosis, the release of pr at neutral pH completes virus maturation to an infectious particle. Together this evidence suggests that pr may shield the flavivirus fusion protein E from the low pH environment of the exocytic pathway. Here we developed an in vitro system to reconstitute the interaction of dengue virus (DENV) pr with soluble truncated E proteins. At low pH recombinant pr bound to both monomeric and dimeric forms of E and blocked their membrane insertion. Exogenous pr interacted with mature infectious DENV and specifically inhibited virus fusion and infection. Alanine substitution of E H244, a highly conserved histidine residue in the pr-E interface, blocked pr-E interaction and reduced release of DENV virus-like particles. Folding, membrane insertion and trimerization of the H244A mutant E protein were preserved, and particle release could be partially rescued by neutralization of the low pH of the secretory pathway. Thus, pr acts to silence flavivirus fusion activity during virus secretion, and this function can be separated from the chaperone activity of prM. The sequence conservation of key residues involved in the flavivirus pr-E interaction suggests that this protein-protein interface may be a useful target for broad-spectrum inhibitors.

Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF

CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200(tr)) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200.

Efficacy of Oseltamivir-Zanamivir Combination Compared to Each Monotherapy for Seasonal Influenza: A Randomized Placebo-Controlled Trial

BACKGROUND: Neuraminidase inhibitors are thought to be efficacious in reducing the time to alleviation of symptoms in outpatients with seasonal influenza. The objective of this study was to compare the short-term virological efficacy of oseltamivir-zanamivir combination versus each monotherapy plus placebo. METHODS AND FINDINGS: We conducted a randomized placebo-controlled trial with 145 general practitioners throughout France during the 2008–2009 seasonal influenza epidemic. Patients, general practitioners, and outcome assessors were all blinded to treatment assignment. Adult outpatients presenting influenza-like illness for less than 36 hours and a positive influenza A rapid test diagnosis were randomized to oseltamivir 75 mg orally twice daily plus zanamivir 10 mg by inhalation twice daily (OZ), oseltamivir plus inhaled placebo (O), or zanamivir plus oral placebo (Z). Treatment efficacy was assessed virologically according to the proportion of patients with nasal influenza reverse transcription (RT)-PCR below 200 copies genome equivalent (cgeq)/µl at day 2 (primary outcome), and clinically to the time to alleviation of symptoms until day 14. Overall 541 patients (of the 900 planned) were included (OZ, n = 192; O, n = 176; Z, n = 173), 49% male, mean age 39 years. In the intention-to-treat analysis conducted in the 447 patients with RT-PCR-confirmed influenza A, 46%, 59%, and 34% in OZ (n = 157), O (n = 141), and Z (n = 149) arms had RT-PCR<200 cgeq/µl (−13.0%, 95% confidence interval [CI] −23.1 to −2.9, p = 0.025; +12.3%, 95% CI 2.39–22.2, p = 0.028 for OZ/O and OZ/Z comparisons). Mean day 0 to day 2 viral load decrease was 2.14, 2.49, and 1.68 log(10) cgeq/µl (p = 0.060, p = 0.016 for OZ/O and OZ/Z). Median time to alleviation of symptoms was 4.0, 3.0, and 4.0 days (+1.0, 95% CI 0.0–4.0, p = 0.018; +0.0, 95% CI −3.0 to 3.0, p = 0.960 for OZ/O and OZ/Z). Four severe adverse events were observed. Nausea and/or vomiting tended to be more frequent in the combination arm (OZ, n = 13; O, n = 4; and Z, n = 5 patients, respectively). CONCLUSIONS: In adults with seasonal influenza A mainly H3N2 virus infection, the oseltamivir-zanamivir combination appeared less effective than oseltamivir monotherapy, and not significantly more effective than zanamivir monotherapy. Despite the theoretical potential for the reduction of the emergence of antiviral resistance, the lower effectiveness of this combination calls for caution in its use in clinical practice. TRIAL REGISTRATION: www.ClinicalTrials.gov NCT00799760 Please see later in the article for the Editors' Summary

A comparison of methods for purification and concentration of norovirus GII-4 capsid virus-like particles

Noroviruses (NoVs) are one of the leading causes of acute gastroenteritis worldwide. NoV GII-4 VP1 protein was expressed in a recombinant baculovirus system using Sf9 insect cells. Several methods for purification and concentration of virus-like particles (VLPs) were evaluated. Electron microscopy (EM) and histo-blood group antigen (HBGA) binding assays showed that repeated sucrose gradient purification followed by ultrafiltration resulted in intact VLPs with excellent binding to H type 3 antigens. VLPs were stable for at least 12 months at 4°C, and up to 7 days at ambient temperature. These findings indicate that this method yielded stable and high-quality VLPs.

Urine Peptidomic and Targeted Plasma Protein Analyses in the Diagnosis and Monitoring of Systemic Juvenile Idiopathic Arthritis

PURPOSE: Systemic juvenile idiopathic arthritis is a chronic pediatric disease. The initial clinical presentation can mimic other pediatric inflammatory conditions, which often leads to significant delays in diagnosis and appropriate therapy. SJIA biomarker development is an unmet diagnostic/prognostic need to prevent disease complications. EXPERIMENTAL DESIGN: We profiled the urine peptidome to analyze a set of 102 urine samples, from patients with SJIA, Kawasaki disease (KD), febrile illnesses (FI), and healthy controls. A set of 91 plasma samples, from SJIA flare and quiescent patients, were profiled using a customized antibody array against 43 proteins known to be involved in inflammatory and protein catabolic processes. RESULTS: We identified a 17-urine-peptide biomarker panel that could effectively discriminate SJIA patients at active, quiescent, and remission disease states, and patients with active SJIA from confounding conditions including KD and FI. Targeted sequencing of these peptides revealed that they fall into several tight clusters from seven different proteins, suggesting disease-specific proteolytic activities. The antibody array plasma profiling identified an SJIA plasma flare signature consisting of tissue inhibitor of metalloproteinase-1 (TIMP1), interleukin (IL)-18, regulated upon activation, normal T cell expressed and secreted (RANTES), P-Selectin, MMP9, and L-Selectin. CONCLUSIONS AND CLINICAL RELEVANCE: The urine peptidomic and plasma protein analyses have the potential to improve SJIA care and suggest that SJIA urine peptide biomarkers may be an outcome of inflammation-driven effects on catabolic pathways operating at multiple sites. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12014-010-9058-8) contains supplementary material, which is available to authorized users.

Segmented Helical Structure of the Neck Region of the Glycan-Binding Receptor DC-SIGNR

Carbohydrate-recognition domains (CRDs) in the glycan-binding receptors DC-SIGN (dendritic-cell-specific intercellular adhesion molecule 1-grabbing nonintegrin; CD209) and DC-SIGNR (DC-SIGN-related receptor, also known as L-SIGN and variously designated CD209L and CD299) are projected from the membrane surface by extended neck domains containing multiple repeats of a largely conserved 23-amino-acid sequence motif. Crystals of a fragment of the neck domain of DC-SIGNR containing multiple repeats in which each molecule extends through multiple unit cells, such that the observed crystallographic asymmetric unit represents one repeat averaged over six repeats of the protein, have been obtained. The repeats are largely α-helical. Based on the structure and arrangement of the repeats in the crystal, the neck region can be described as a series of four-helix bundles connected by short, non-helical linkers. Combining the structure of the isolated neck domain with a previously determined overlapping structure of the distal end of the neck region with the CRDs attached provides a model of the almost-complete extracellular portion of the receptor. The results are consistent with previous characterization of the extended structure for the isolated neck region and the extracellular domain. The organization of the neck suggests how CRDs may be disposed differently in DC-SIGN compared with DC-SIGNR and in variant forms of DC-SIGNR assembled from polypeptides with different numbers of repeats in the neck domain.

Translation Elongation Factor 1A Facilitates the Assembly of the Tombusvirus Replicase and Stimulates Minus-Strand Synthesis

Replication of plus-strand RNA viruses depends on host factors that are recruited into viral replicase complexes. Previous studies showed that eukaryotic translation elongation factor (eEF1A) is one of the resident host proteins in the highly purified tombusvirus replicase complex. Using a random library of eEF1A mutants, we identified one mutant that decreased and three mutants that increased Tomato bushy stunt virus (TBSV) replication in a yeast model host. Additional in vitro assays with whole cell extracts prepared from yeast strains expressing the eEF1A mutants demonstrated several functions for eEF1A in TBSV replication: facilitating the recruitment of the viral RNA template into the replicase complex; the assembly of the viral replicase complex; and enhancement of the minus-strand synthesis by promoting the initiation step. These roles for eEF1A are separate from its canonical role in host and viral protein translation, emphasizing critical functions for this abundant cellular protein during TBSV replication.

The calculation of information and organismal complexity

BACKGROUND: It is difficult to measure precisely the phenotypic complexity of living organisms. Here we propose a method to calculate the minimal amount of genomic information needed to construct organism (effective information) as a measure of organismal complexity, by using permutation and combination formulas and Shannon's information concept. RESULTS: The results demonstrate that the calculated information correlates quite well with the intuitive organismal phenotypic complexity defined by traditional taxonomy and evolutionary theory. From viruses to human beings, the effective information gradually increases, from thousands of bits to hundreds of millions of bits. The simpler the organism is, the less the information; the more complex the organism, the more the information. About 13% of human genome is estimated as effective information or functional sequence. CONCLUSIONS: The effective information can be used as a quantitative measure of phenotypic complexity of living organisms and also as an estimate of functional fraction of genome. REVIEWERS: This article was reviewed by Dr. Lavanya Kannan (nominated by Dr. Arcady Mushegian), Dr. Chao Chen, and Dr. ED Rietman (nominated by Dr. Marc Vidal).

Environmental factors preceding illness onset differ in phenotypes of the juvenile idiopathic inflammatory myopathies

Objective. To assess whether certain environmental factors temporally associated with the onset of juvenile idiopathic inflammatory myopathies (JIIMs) differ between phenotypes. Methods. Physicians completed questionnaires regarding documented infections, medications, immunizations and an open-ended question about other noted exposures within 6 months before illness onset for 285 patients with probable or definite JIIM. Medical records were reviewed for 81% of the patients. Phenotypes were defined by standard clinical and laboratory measures. Results. Sixty per cent of JIIM patients had a reported exposure within 6 months before illness onset. Most patients (62%) had one recorded exposure, 26% had two and 12% had three to five exposures. Patients older than the median age at diagnosis, those with a longer delay to diagnosis and those with anti-signal recognition particle autoantibodies had a higher frequency of documented exposures [odds ratios (ORs) 95% CI 3.4, 31]. Infections were the most common exposure and represented 44% of the total number of reported exposures. Non-infectious exposures included medications (18%), immunizations (11%), stressful life events (11%) and unusual sun exposure (7%). Exposures varied by age at diagnosis, race, disease course and the presence of certain myositis autoantibodies. Conclusion. The JIIMs may be related to multiple exposures and these appear to vary among phenotypes.

Network Analysis of Global Influenza Spread

Although vaccines pose the best means of preventing influenza infection, strain selection and optimal implementation remain difficult due to antigenic drift and a lack of understanding global spread. Detecting viral movement by sequence analysis is complicated by skewed geographic and seasonal distributions in viral isolates. We propose a probabilistic method that accounts for sampling bias through spatiotemporal clustering and modeling regional and seasonal transmission as a binomial process. Analysis of H3N2 not only confirmed East-Southeast Asia as a source of new seasonal variants, but also increased the resolution of observed transmission to a country level. H1N1 data revealed similar viral spread from the tropics. Network analysis suggested China and Hong Kong as the origins of new seasonal H3N2 strains and the United States as a region where increased vaccination would maximally disrupt global spread of the virus. These techniques provide a promising methodology for the analysis of any seasonal virus, as well as for the continued surveillance of influenza.

Willingness to accept H1N1 pandemic influenza vaccine: A cross-sectional study of Hong Kong community nurses

BACKGROUND: The 2009 pandemic of influenza A (H1N1) infection has alerted many governments to make preparedness plan to control the spread of influenza A (H1N1) infection. Vaccination for influenza is one of the most important primary preventative measures to reduce the disease burden. Our study aims to assess the willingness of nurses who work for the community nursing service (CNS) in Hong Kong on their acceptance of influenza A (H1N1) influenza vaccination. METHODS: 401 questionnaires were posted from June 24, 2009 to June 30, 2009 to community nurses with 67% response rate. Results of the 267 respondents on their willingness to accept influenza A (H1N1) vaccine were analyzed. RESULTS: Twenty-seven percent of respondents were willing to accept influenza vaccination if vaccines were available. Having been vaccinated for seasonable influenza in the previous 12 months were significantly independently associated with their willingness to accept influenza A (H1N1) vaccination (OR = 4.03; 95% CI: 2.03-7.98). CONCLUSIONS: Similar to previous findings conducted in hospital healthcare workers and nurses, we confirmed that the willingness of community nurses to accept influenza A (H1N1) vaccination is low. Future studies that evaluate interventions to address nurses' specific concerns or interventions that aim to raise the awareness among nurses on the importance of influenza A (H1N1) vaccination to protect vulnerable patient populations is needed.

Development of an optimized RNA-based murine norovirus reverse genetics system

Murine norovirus (MNV), identified in 2003, is the only norovirus which replicates efficiently in tissue culture and as a result has been used extensively as a model for human noroviruses, a major cause of acute gastroenteritis. The current report describes the generation of a new approach to reverse genetics recovery of genetically defined MNV that relies on the transfection of in vitro transcribed capped RNA directly into cells. The use of the recently developed ScriptCap post-transcriptional enzymatic capping system, followed by optimized Neon mediated electroporation of the highly permissive RAW 264.7 cells, resulted in the rapid and robust recovery of infectious MNV. Transfection of cells capable of supporting virus replication but not permissive to virus infection, namely human or hamster kidney cells, also resulted in robust recovery of infectious virus without subsequent amplification by multiple rounds of re-infection. This latter system may provide a reproducible method to measure the specific infectivity of mutant norovirus RNA allowing the accurate quantitation of the effect of mutations on norovirus replication.

On epidemic modeling in real time: An application to the 2009 Novel A (H1N1) influenza outbreak in Canada

BACKGROUND: Management of emerging infectious diseases such as the 2009 influenza pandemic A (H1N1) poses great challenges for real-time mathematical modeling of disease transmission due to limited information on disease natural history and epidemiology, stochastic variation in the course of epidemics, and changing case definitions and surveillance practices. FINDINGS: The Richards model and its variants are used to fit the cumulative epidemic curve for laboratory-confirmed pandemic H1N1 (pH1N1) infections in Canada, made available by the Public Health Agency of Canada (PHAC). The model is used to obtain estimates for turning points in the initial outbreak, the basic reproductive number (R(0)), and for expected final outbreak size in the absence of interventions. Confirmed case data were used to construct a best-fit 2-phase model with three turning points. R(0 )was estimated to be 1.30 (95% CI 1.12-1.47) for the first phase (April 1 to May 4) and 1.35 (95% CI 1.16-1.54) for the second phase (May 4 to June 19). Hospitalization data were also used to fit a 1-phase model with R(0 )= 1.35 (1.20-1.49) and a single turning point of June 11. CONCLUSIONS: Application of the Richards model to Canadian pH1N1 data shows that detection of turning points is affected by the quality of data available at the time of data usage. Using a Richards model, robust estimates of R(0 )were obtained approximately one month after the initial outbreak in the case of 2009 A (H1N1) in Canada.

Antiviral and Neuroprotective Role of Octaguanidinium Dendrimer-Conjugated Morpholino Oligomers in Japanese Encephalitis

BACKGROUND: Japanese encephalitis (JE), caused by a mosquito-borne flavivirus, is endemic to the entire south-east Asian and adjoining regions. Currently no therapeutic interventions are available for JE, thereby making it one of the most dreaded encephalitides in the world. An effective way to counter the virus would be to inhibit viral replication by using anti-sense molecules directed against the viral genome. Octaguanidinium dendrimer-conjugated Morpholino (or Vivo-Morpholino) are uncharged anti-sense oligomers that can enter cells of living organisms by endocytosis and subsequently escape from endosomes into the cytosol/nuclear compartment of cells. We hypothesize that Vivo-Morpholinos generated against specific regions of 3′ or 5′ untranslated regions of JEV genome, when administered in an experimental model of JE, will have significant antiviral and neuroprotective effect. METHODOLOGY/PRINCIPAL FINDINGS: Mice were infected with JEV (GP78 strain) followed by intraperitoneal administration of Morpholinos (5 mg/kg body weight) daily for up to five treatments. Survivability of the animals was monitored for 15 days (or until death) following which they were sacrificed and their brains were processed either for immunohistochemical staining or protein extraction. Plaque assay and immunoblot analysis performed from brain homogenates showed reduced viral load and viral protein expression, resulting in greater survival of infected animals. Neuroprotective effect was observed by thionin staining of brain sections. Cytokine bead array showed reduction in the levels of proinflammatory cytokines in brain following Morpholino treatment, which were elevated after infection. This corresponded to reduced microglial activation in brain. Oxidative stress was reduced and certain stress-related signaling molecules were found to be positively modulated following Morpholino treatment. In vitro studies also showed that there was decrease in infective viral particle production following Morpholino treatment. CONCLUSIONS/SIGNIFICANCE: Administration of Vivo-Morpholino effectively resulted in increased survival of animals and neuroprotection in a murine model of JE. Hence, these oligomers represent a potential antiviral agent that merits further evaluation.

Severe novel influenza A (H1N1) infection in cancer patients

Background: The natural history and consequences of severe H1N1 influenza infection among cancer patients are not yet fully characterized. We describe eight cases of H1N1 infection in cancer patients admitted to the intensive care unit of a referral cancer center. Patients and methods: Clinical data from all patients admitted with acute respiratory failure due to novel viral H1N1 infection were reviewed. Lung tissue was submitted for viral and bacteriological analyses by real-time RT-PCR, and autopsy was conducted on all patients who died. Results: Eight patients were admitted, with ages ranging from 55 to 65 years old. There were five patients with solid organ tumors (62.5%) and three with hematological malignancies (37.5%). Five patients required mechanical ventilation and all died. Four patients had bacterial bronchopneumonia. All deaths occurred due to multiple organ failure. A milder form of lung disease was present in the three cases who survived. Lung tissue analysis was performed in all patients and showed diffuse alveolar damage in most patients. Other lung findings were necrotizing bronchiolitis or extensive hemorrhage. Conclusions: H1N1 viral infection in patients with cancer can cause severe illness, resulting in acute respiratory distress syndrome and death. More data are needed to identify predictors of unfavorable evolution in these patients.

The Tennessee Children's Respiratory Initiative: Objectives, design and recruitment results of a prospective cohort study investigating infant viral respiratory illness and the development of asthma and allergic diseases

Background and objective: The ‘attack rate’ of asthma following viral lower respiratory tract infections (LRTI) is about 3–4 fold higher than that of the general population; however, the majority of children who develop viral LRTI during infancy do not develop asthma, and asthma incidence has been observed to continuously decrease with age. Thus, we do not understand how viral LRTI either predispose or serve as a marker of children to develop asthma. The Tennessee Children's Respiratory Initiative has been established as a longitudinal prospective investigation of infants and their biological mothers. The primary goals are to investigate both the acute and the long‐term health consequences of varying severity and aetiology of clinically significant viral respiratory tract infections on early childhood outcomes. Methods: Over four respiratory viral seasons, 2004–2008, term, non‐low birth weight previously healthy infants and their biological mothers were enrolled during an infant's acute viral respiratory illness. Longitudinal follow up to age 6 years is ongoing. Results: This report describes the study objectives, design and recruitment results of the over 650 families enrolled in this longitudinal investigation. The Tennessee Children's Respiratory Initiative is additionally unique because it is designed in parallel with a large retrospective birth cohort of over 95 000 mother–infant dyads with similar objectives to investigate the role of respiratory viral infection severity and aetiology in the development of asthma. Conclusions: Future reports from this cohort will help to clarify the complex relationship between infant respiratory viral infection severity, aetiology, atopic predisposition and the subsequent development of early childhood asthma and atopic diseases.

Eosinophilic infiltrate in a patient with severe Legionella pneumonia as a levofloxacin-related complication: a case report

INTRODUCTION: Legionella pneumonia can appear with different levels of severity and it can often present with complications such as acute respiratory distress syndrome. CASE PRESENTATION: We report the case of a 44-year-old Caucasian man with Legionella pneumonia with successive development of severe acute respiratory distress syndrome. During his stay in intensive care the clinical and radiological situation of the previously observed acute respiratory distress syndrome unexpectedly worsened due to acute pulmonary eosinophilic infiltrate of iatrogenic origin. CONCLUSION: Levofloxacin treatment caused the occurrence of acute eosinophilic infiltrate. Diagnosis was possible following bronchoscopic examination using bronchoaspirate and transbronchial biopsy.

Functional analysis of the SRV-1 RNA frameshifting pseudoknot

Simian retrovirus type-1 uses programmed ribosomal frameshifting to control expression of the Gag-Pol polyprotein from overlapping gag and pol open-reading frames. The frameshifting signal consists of a heptanucleotide slippery sequence and a downstream-located 12-base pair pseudoknot. The solution structure of this pseudoknot, previously solved by NMR [Michiels,P.J., Versleijen,A.A., Verlaan,P.W., Pleij,C.W., Hilbers,C.W. and Heus,H.A. (2001) Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting. J. Mol. Biol., 310, 1109–1123] has a classical H-type fold and forms an extended triple helix by interactions between loop 2 and the minor groove of stem 1 involving base–base and base–sugar contacts. A mutational analysis was performed to test the functional importance of the triple helix for −1 frameshifting in vitro. Changing bases in L2 or base pairs in S1 involved in a base triple resulted in a 2- to 5-fold decrease in frameshifting efficiency. Alterations in the length of L2 had adverse effects on frameshifting. The in vitro effects were well reproduced in vivo, although the effect of enlarging L2 was more dramatic in vivo. The putative role of refolding kinetics of frameshifter pseudoknots is discussed. Overall, the data emphasize the role of the triple helix in −1 frameshifting.

A molecular clamp ensures allosteric coordination of peptidyltransfer and ligand binding to the ribosomal A-site

Although the ribosome is mainly comprised of rRNA and many of its critical functions occur through RNA–RNA interactions, distinct domains of ribosomal proteins also participate in switching the ribosome between different conformational/functional states. Prior studies demonstrated that two extended domains of ribosomal protein L3 form an allosteric switch between the pre- and post-translocational states. Missing was an explanation for how the movements of these domains are communicated among the ribosome's functional centers. Here, a third domain of L3 called the basic thumb, that protrudes roughly perpendicular from the W-finger and is nestled in the center of a cagelike structure formed by elements from three separate domains of the large subunit rRNA is investigated. Mutagenesis of basically charged amino acids of the basic thumb to alanines followed by detailed analyses suggests that it acts as a molecular clamp, playing a role in allosterically communicating the ribosome's tRNA occupancy status to the elongation factor binding region and the peptidyltransferase center, facilitating coordination of their functions through the elongation cycle. The observation that these mutations affected translational fidelity, virus propagation and cell growth demonstrates how small structural changes at the atomic scale can propagate outward to broadly impact the biology of cell.

Hybridization properties of long nucleic acid probes for detection of variable target sequences, and development of a hybridization prediction algorithm

One of the main problems in nucleic acid-based techniques for detection of infectious agents, such as influenza viruses, is that of nucleic acid sequence variation. DNA probes, 70-nt long, some including the nucleotide analog deoxyribose-Inosine (dInosine), were analyzed for hybridization tolerance to different amounts and distributions of mismatching bases, e.g. synonymous mutations, in target DNA. Microsphere-linked 70-mer probes were hybridized in 3M TMAC buffer to biotinylated single-stranded (ss) DNA for subsequent analysis in a Luminex® system. When mismatches interrupted contiguous matching stretches of 6 nt or longer, it had a strong impact on hybridization. Contiguous matching stretches are more important than the same number of matching nucleotides separated by mismatches into several regions. dInosine, but not 5-nitroindole, substitutions at mismatching positions stabilized hybridization remarkably well, comparable to N (4-fold) wobbles in the same positions. In contrast to shorter probes, 70-nt probes with judiciously placed dInosine substitutions and/or wobble positions were remarkably mismatch tolerant, with preserved specificity. An algorithm, NucZip, was constructed to model the nucleation and zipping phases of hybridization, integrating both local and distant binding contributions. It predicted hybridization more exactly than previous algorithms, and has the potential to guide the design of variation-tolerant yet specific probes.

Asymmetry in the Presence of Migration Stabilizes Multistrain Disease Outbreaks

We study the effect of migration between coupled populations, or patches, on the stability properties of multistrain disease dynamics. The epidemic model used in this work displays a Hopf bifurcation to oscillations in a single, well-mixed population. It is shown numerically that migration between two non-identical patches stabilizes the endemic steady state, delaying the onset of large amplitude outbreaks and reducing the total number of infections. This result is motivated by analyzing generic Hopf bifurcations with different frequencies and with diffusive coupling between them. Stabilization of the steady state is again seen, indicating that our observation in the full multistrain model is based on qualitative characteristics of the dynamics rather than on details of the disease model.

Clinical factors associated with severity in hospitalized children infected with avian influenza (H5N1)

OBJECTIVE: The World Health organization received reports of 478 laboratory-confirmed cases of influenza A (H5N1) from 15 countries between November 2003 and February 2010. More than 50% of these cases involved patients <20 years of age. Determining an association between the clinical factors at the time of hospital admission and prognosis may be useful for timely and adequate consultation and treatment. It has been difficult to obtain these clinical factors adjusted with other confounding factors, such as age and sex, as published studies of H5N1 virus infection usually reported only a few cases. So, we performed a pooled analysis of the reported cases. METHODS: Five case reports (36 patients <18 years of age) of H5N1 infection from four countries published between 2004 and 2009 were assessed based on available individual clinical data. Using the pooled data for all patients, we investigated the associations between patients’ prognosis and available laboratory findings, such as white blood cell (WBC) counts, platelet (PLT) counts, and serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) by adjusting for age and/or sex. RESULTS: The linear regression analysis revealed that mortality was negatively associated with WBC and PLT counts adjusted with age and sex. Increased log AST tended to be associated with a poor prognosis (p = 0.054), but there was no significant association between survival and log ALT level. CONCLUSIONS: Both decreased WBC and PLT counts can be considered to be common predictors of poor prognosis in H5N1 influenza patients <18 years of age. Further studies are needed for clarification.

Human monoclonal IgG selection of Plasmodium falciparum for the expression of placental malaria-specific variant surface antigens

Pregnancy-associatedPlasmodium falciparum malaria (PAM) is a major cause of morbidity and mortality in African women and their offspring. PAM is characterized by accumulation of infected erythrocytes (IEs) that adhere to chondroitin sulphate A (CSA) in the placental intervillous space. We show here that human monoclonal IgG antibodies with specificity for variant surface antigens (VSA) specifically expressed by CSA-adhering IEs (VSA(PAM)) can be used in vitro to select parasites from nonpregnant donors to express VSA(PAM) and that this selection for VSA(PAM) expression results in preferential transcription of var2csa. The results corroborate current efforts to develop PAM-specific vaccines based on VAR2CSA.

Stimulation of ribosomal frameshifting by antisense LNA

Programmed ribosomal frameshifting is a translational recoding mechanism commonly used by RNA viruses to express two or more proteins from a single mRNA at a fixed ratio. An essential element in this process is the presence of an RNA secondary structure, such as a pseudoknot or a hairpin, located downstream of the slippery sequence. Here, we have tested the efficiency of RNA oligonucleotides annealing downstream of the slippery sequence to induce frameshifting in vitro. Maximal frameshifting was observed with oligonucleotides of 12–18 nt. Antisense oligonucleotides bearing locked nucleid acid (LNA) modifications also proved to be efficient frameshift-stimulators in contrast to DNA oligonucleotides. The number, sequence and location of LNA bases in an otherwise DNA oligonucleotide have to be carefully manipulated to obtain optimal levels of frameshifting. Our data favor a model in which RNA stability at the entrance of the ribosomal tunnel is the major determinant of stimulating slippage rather than a specific three-dimensional structure of the stimulating RNA element.

A flexible loop in yeast ribosomal protein L11 coordinates P-site tRNA binding

High-resolution structures reveal that yeast ribosomal protein L11 and its bacterial/archael homologs called L5 contain a highly conserved, basically charged internal loop that interacts with the peptidyl-transfer RNA (tRNA) T-loop. We call this the L11 ‘P-site loop’. Chemical protection of wild-type ribosome shows that that the P-site loop is inherently flexible, i.e. it is extended into the ribosomal P-site when this is unoccupied by tRNA, while it is retracted into the terminal loop of 25S rRNA Helix 84 when the P-site is occupied. To further analyze the function of this structure, a series of mutants within the P-site loop were created and analyzed. A mutant that favors interaction of the P-site loop with the terminal loop of Helix 84 promoted increased affinity for peptidyl-tRNA, while another that favors its extension into the ribosomal P-site had the opposite effect. The two mutants also had opposing effects on binding of aa-tRNA to the ribosomal A-site, and downstream functional effects were observed on translational fidelity, drug resistance/hypersensitivity, virus maintenance and overall cell growth. These analyses suggest that the L11 P-site loop normally helps to optimize ribosome function by monitoring the occupancy status of the ribosomal P-site.

Involvement of microRNAs in physiological and pathological processes in the lung

To date, at least 900 different microRNA (miRNA) genes have been discovered in the human genome. These short, single-stranded RNA molecules originate from larger precursor molecules that fold to produce hairpin structures, which are subsequently processed by ribonucleases Drosha/Pasha and Dicer to form mature miRNAs. MiRNAs play role in the posttranscriptional regulation of about one third of human genes, mainly via degradation of target mRNAs. Whereas the target mRNAs are often involved in the regulation of diverse physiological processes ranging from developmental timing to apoptosis, miRNAs have a strong potential to regulate fundamental biological processes also in the lung compartment. However, the knowledge of the role of miRNAs in physiological and pathological conditions in the lung is still limited. This review, therefore, summarizes current knowledge of the mechanism, function of miRNAs and their contribution to lung development and homeostasis. Besides the involvement of miRNAs in pulmonary physiological conditions, there is evidence that abnormal miRNA expression may lead to pathological processes and development of various pulmonary diseases. Next, the review describes current state-of-art on the miRNA expression profiles in smoking-related diseases including lung cancerogenesis, in immune system mediated pulmonary diseases and fibrotic processes in the lung. From the current research it is evident that miRNAs may play role in the posttranscriptional regulation of key genes in human pulmonary diseases. Further studies are, therefore, necessary to explore miRNA expression profiles and their association with target mRNAs in human pulmonary diseases.

Rapid detection of pandemic influenza in the presence of seasonal influenza

BACKGROUND: Key to the control of pandemic influenza are surveillance systems that raise alarms rapidly and sensitively. In addition, they must minimise false alarms during a normal influenza season. We develop a method that uses historical syndromic influenza data from the existing surveillance system 'SERVIS' (Scottish Enhanced Respiratory Virus Infection Surveillance) for influenza-like illness (ILI) in Scotland. METHODS: We develop an algorithm based on the weekly case ratio (WCR) of reported ILI cases to generate an alarm for pandemic influenza. From the seasonal influenza data from 13 Scottish health boards, we estimate the joint probability distribution of the country-level WCR and the number of health boards showing synchronous increases in reported influenza cases over the previous week. Pandemic cases are sampled with various case reporting rates from simulated pandemic influenza infections and overlaid with seasonal SERVIS data from 2001 to 2007. Using this combined time series we test our method for speed of detection, sensitivity and specificity. Also, the 2008-09 SERVIS ILI cases are used for testing detection performances of the three methods with a real pandemic data. RESULTS: We compare our method, based on our simulation study, to the moving-average Cumulative Sums (Mov-Avg Cusum) and ILI rate threshold methods and find it to be more sensitive and rapid. For 1% case reporting and detection specificity of 95%, our method is 100% sensitive and has median detection time (MDT) of 4 weeks while the Mov-Avg Cusum and ILI rate threshold methods are, respectively, 97% and 100% sensitive with MDT of 5 weeks. At 99% specificity, our method remains 100% sensitive with MDT of 5 weeks. Although the threshold method maintains its sensitivity of 100% with MDT of 5 weeks, sensitivity of Mov-Avg Cusum declines to 92% with increased MDT of 6 weeks. For a two-fold decrease in the case reporting rate (0.5%) and 99% specificity, the WCR and threshold methods, respectively, have MDT of 5 and 6 weeks with both having sensitivity close to 100% while the Mov-Avg Cusum method can only manage sensitivity of 77% with MDT of 6 weeks. However, the WCR and Mov-Avg Cusum methods outperform the ILI threshold method by 1 week in retrospective detection of the 2009 pandemic in Scotland. CONCLUSIONS: While computationally and statistically simple to implement, the WCR algorithm is capable of raising alarms, rapidly and sensitively, for influenza pandemics against a background of seasonal influenza. Although the algorithm was developed using the SERVIS data, it has the capacity to be used at other geographic scales and for different disease systems where buying some early extra time is critical.

Responses of Human Endothelial Cells to Pathogenic and Non-Pathogenic Leptospira Species

Leptospirosis is a widespread zoonotic infection that primarily affects residents of tropical regions, but causes infections in animals and humans in temperate regions as well. The agents of leptospirosis comprise several members of the genus Leptospira, which also includes non-pathogenic, saprophytic species. Leptospirosis can vary in severity from a mild, non-specific illness to severe disease that includes multi-organ failure and widespread endothelial damage and hemorrhage. To begin to investigate how pathogenic leptospires affect endothelial cells, we compared the responses of two endothelial cell lines to infection by pathogenic versus non-pathogenic leptospires. Microarray analyses suggested that pathogenic L. interrogans and non-pathogenic L. biflexa triggered changes in expression of genes whose products are involved in cellular architecture and interactions with the matrix, but that the changes were in opposite directions, with infection by L. biflexa primarily predicted to increase or maintain cell layer integrity, while L. interrogans lead primarily to changes predicted to disrupt cell layer integrity. Neither bacterial strain caused necrosis or apoptosis of the cells even after prolonged incubation. The pathogenic L. interrogans, however, did result in significant disruption of endothelial cell layers as assessed by microscopy and the ability of the bacteria to cross the cell layers. This disruption of endothelial layer integrity was abrogated by addition of the endothelial protective drug lisinopril at physiologically relevant concentrations. These results suggest that, through adhesion of L. interrogans to endothelial cells, the bacteria may disrupt endothelial barrier function, promoting dissemination of the bacteria and contributing to severe disease manifestations. In addition, supplementing antibiotic therapy with lisinopril or derivatives with endothelial protective activities may decrease the severity of leptospirosis.

A Porcine Adenovirus with Low Human Seroprevalence Is a Promising Alternative Vaccine Vector to Human Adenovirus 5 in an H5N1 Virus Disease Model

Human adenovirus 5 (AdHu5) vectors are robust vaccine platforms however the presence of naturally-acquired neutralizing antibodies may reduce vector efficacy and potential for re-administration. This study evaluates immune responses and protection following vaccination with a replication-incompetent porcine adenovirus 3 (PAV3) vector as an alternative vaccine to AdHu5 using an avian influenza H5N1 disease model. Vaccine efficacy was evaluated in BALB/c mice following vaccination with different doses of the PAV3 vector expressing an optimized A/Hanoi/30408/2005 H5N1 hemagglutinin antigen (PAV3-HA) and compared with an AdHu5-HA control. PAV3-HA rapidly generated antibody responses, with significant neutralizing antibody titers on day 21, and stronger cellular immune responses detected on day 8, compared to AdHu5-HA. The PAV3-HA vaccine, administered 8 days before challenge, demonstrated improved survival and lower virus load. Evaluation of long-term vaccine efficacy at 12 months post-vaccination showed better protection with the PAV3-HA than with the AdHu5-HA vaccine. Importantly, as opposed to AdHu5, PAV3 vector was not significantly neutralized by human antibodies pooled from over 10,000 individuals. Overall, PAV3-based vector is capable of mediating swift, strong immune responses and offer a promising alternative to AdHu5.

Liposome-Coupled Antigens Are Internalized by Antigen-Presenting Cells via Pinocytosis and Cross-Presented to CD8(+) T Cells

We have previously demonstrated that antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were cross-presented by antigen-presenting cells (APCs) to CD8(+) T cells, and that this process resulted in the induction of antigen-specific cytotoxic T lymphocytes. In the present study, the mechanism by which the liposome-coupled antigens were cross-presented to CD8(+) T cells by APCs was investigated. Confocal laser scanning microscopic analysis demonstrated that antigens coupled to the surface of unsaturated-fatty-acid-based liposomes received processing at both MHC class I and class II compartments, while most of the antigens coupled to the surface of saturated-fatty-acid-based liposomes received processing at the class II compartment. In addition, flow cytometric analysis demonstrated that antigens coupled to the surface of unsaturated-fatty-acid-liposomes were taken up by APCs even in a 4°C environment; this was not true of saturated-fatty-acid-liposomes. When two kinds of inhibitors, dimethylamiloride (DMA) and cytochalasin B, which inhibit pinocytosis and phagocytosis by APCs, respectively, were added to the culture of APCs prior to the antigen pulse, DMA but not cytochalasin B significantly reduced uptake of liposome-coupled antigens. Further analysis of intracellular trafficking of liposomal antigens using confocal laser scanning microscopy revealed that a portion of liposome-coupled antigens taken up by APCs were delivered to the lysosome compartment. In agreement with the reduction of antigen uptake by APCs, antigen presentation by APCs was significantly inhibited by DMA, and resulted in the reduction of IFN-γ production by antigen-specific CD8(+) T cells. These results suggest that antigens coupled to the surface of liposomes consisting of unsaturated fatty acids might be pinocytosed by APCs, loaded onto the class I MHC processing pathway, and presented to CD8(+) T cells. Thus, these liposome-coupled antigens are expected to be applicable for the development of vaccines that induce cellular immunity.

Therapeutic Vaccination in Chronic Hepatitis B: Preclinical Studies in the Woodchuck

Recommended treatment of chronic hepatitis B with interferon-α and/or nucleos(t)ide analogues does not lead to a satisfactory result. Induction of HBV-specific T cells by therapeutic vaccination or immunotherapies may be an innovative strategy to overcome virus persistence. Vaccination with commercially available HBV vaccines in patients did not result in effective control of HBV infection, suggesting that new formulations of therapeutic vaccines are needed. The woodchuck (Marmota monax) is a useful preclinical model for developing the new therapeutic approaches in chronic hepadnaviral infections. Several innovative approaches combining antiviral treatments with nucleos(t)ide analogues, DNA vaccines, and protein vaccines were tested in the woodchuck model. In this paper we summarize the available data concerning therapeutic immunization and gene therapy using recombinant viral vectors approaches in woodchucks, which show encouraging results. In addition, we present potential innovations in immunomodulatory strategies to be evaluated in this animal model.

Human immunome, bioinformatic analyses using HLA supermotifs and the parasite genome, binding assays, studies of human T cell responses, and immunization of HLA-A*1101 transgenic mice including novel adjuvants provide a foundation for HLA-A03 restricted CD8(+)T cell epitope based, adjuvanted vaccine protective against Toxoplasma gondii

BACKGROUND: Toxoplasmosis causes loss of life, cognitive and motor function, and sight. A vaccine is greatly needed to prevent this disease. The purpose of this study was to use an immmunosense approach to develop a foundation for development of vaccines to protect humans with the HLA-A03 supertype. Three peptides had been identified with high binding scores for HLA-A03 supertypes using bioinformatic algorhythms, high measured binding affinity for HLA-A03 supertype molecules, and ability to elicit IFN-γ production by human HLA-A03 supertype peripheral blood CD8(+ )T cells from seropositive but not seronegative persons. RESULTS: Herein, when these peptides were administered with the universal CD4(+)T cell epitope PADRE (AKFVAAWTLKAAA) and formulated as lipopeptides, or administered with GLA-SE either alone, or with Pam(2)Cys added, we found we successfully created preparations that induced IFN-γ and reduced parasite burden in HLA-A*1101(an HLA-A03 supertype allele) transgenic mice. GLA-SE is a novel emulsified synthetic TLR4 ligand that is known to facilitate development of T Helper 1 cell (TH1) responses. Then, so our peptides would include those expressed in tachyzoites, bradyzoites and sporozoites from both Type I and II parasites, we used our approaches which had identified the initial peptides. We identified additional peptides using bioinformatics, binding affinity assays, and study of responses of HLA-A03 human cells. Lastly, we found that immunization of HLA-A*1101 transgenic mice with all the pooled peptides administered with PADRE, GLA-SE, and Pam(2)Cys is an effective way to elicit IFN-γ producing CD8(+ )splenic T cells and protection. Immunizations included the following peptides together: KSFKDILPK (SAG1(224-232)); AMLTAFFLR (GRA6(164-172)); RSFKDLLKK (GRA7(134-142)); STFWPCLLR (SAG2C(13-21)); SSAYVFSVK((SPA250-258)); and AVVSLLRLLK(SPA(89-98)). This immunization elicited robust protection, measured as reduced parasite burden using a luciferase transfected parasite, luciferin, this novel, HLA transgenic mouse model, and imaging with a Xenogen camera. CONCLUSIONS: Toxoplasma gondii peptides elicit HLA-A03 restricted, IFN-γ producing, CD8(+ )T cells in humans and mice. These peptides administered with adjuvants reduce parasite burden in HLA-A*1101 transgenic mice. This work provides a foundation for immunosense based vaccines. It also defines novel adjuvants for newly identified peptides for vaccines to prevent toxoplasmosis in those with HLA-A03 supertype alleles.

Conserved epitopes of influenza A virus inducing protective immunity and their prospects for universal vaccine development

Influenza A viruses belong to the best studied viruses, however no effective prevention against influenza infection has been developed. The emerging of still new escape variants of influenza A viruses causing epidemics and periodic worldwide pandemics represents a threat for human population. Therefore, current, hot task of influenza virus research is to look for a way how to get us closer to a universal vaccine. Combination of chosen conserved antigens inducing cross-protective antibody response with epitopes activating also cross-protective cytotoxic T-cells would offer an attractive strategy for improving protection against drift variants of seasonal influenza viruses and reduces the impact of future pandemic strains. Antigenically conserved fusion-active subunit of hemagglutinin (HA2 gp) and ectodomain of matrix protein 2 (eM2) are promising candidates for preparation of broadly protective HA2- or eM2-based vaccine that may aid in pandemic preparedness. Overall protective effect could be achieved by contribution of epitopes recognized by cytotoxic T-lymphocytes (CTL) that have been studied extensively to reach much broader control of influenza infection. In this review we present the state-of-art in this field. We describe known adaptive immune mechanisms mediated by influenza specific B- and T-cells involved in the anti-influenza immune defense together with the contribution of innate immunity. We discuss the mechanisms of neutralization of influenza infection mediated by antibodies, the role of CTL in viral elimination and new approaches to develop epitope based vaccine inducing cross-protective influenza virus-specific immune response.

The intracellular dynamic of protein palmitoylation

S-palmitoylation describes the reversible attachment of fatty acids (predominantly palmitate) onto cysteine residues via a labile thioester bond. This posttranslational modification impacts protein functionality by regulating membrane interactions, intracellular sorting, stability, and membrane micropatterning. Several recent findings have provided a tantalizing insight into the regulation and spatiotemporal dynamics of protein palmitoylation. In mammalian cells, the Golgi has emerged as a possible super-reaction center for the palmitoylation of peripheral membrane proteins, whereas palmitoylation reactions on post-Golgi compartments contribute to the regulation of specific substrates. In addition to palmitoylating and depalmitoylating enzymes, intracellular palmitoylation dynamics may also be controlled through interplay with distinct posttranslational modifications, such as phosphorylation and nitrosylation.

The impact of sex, gender and pregnancy on 2009 H1N1 disease

Children and young adults of reproductive age have emerged as groups that are highly vulnerable to the current 2009 H1N1 pandemic. The sex of an individual is a fundamental factor that can influence exposure, susceptibility and immune responses to influenza. Worldwide, the incidence, disease burden, morbidity and mortality rates following exposure to the 2009 H1N1 influenza virus differ between males and females and are often age-dependent. Pregnancy and differences in the presentation of various risk factors contribute to the worse outcome of infection in women. Vaccination and antiviral treatment efficacy also vary in a sex-dependent manner. Finally, sex-specific genetic and hormonal differences may contribute to the severity of influenza and the clearance of viral infection. The contribution of sex and gender to influenza can only be determined by a greater consideration of these factors in clinical and epidemiological studies and increased research into the biological basis underlying these differences.

PhEVER: a database for the global exploration of virus–host evolutionary relationships

Fast viral adaptation and the implication of this rapid evolution in the emergence of several new infectious diseases have turned this issue into a major challenge for various research domains. Indeed, viruses are involved in the development of a wide range of pathologies and understanding how viruses and host cells interact in the context of adaptation remains an open question. In order to provide insights into the complex interactions between viruses and their host organisms and namely in the acquisition of novel functions through exchanges of genetic material, we developed the PhEVER database. This database aims at providing accurate evolutionary and phylogenetic information to analyse the nature of virus–virus and virus–host lateral gene transfers. PhEVER (http://pbil.univ-lyon1.fr/databases/phever) is a unique database of homologous families both (i) between sequences from different viruses and (ii) between viral sequences and sequences from cellular organisms. PhEVER integrates extensive data from up-to-date completely sequenced genomes (2426 non-redundant viral genomes, 1007 non-redundant prokaryotic genomes, 43 eukaryotic genomes ranging from plants to vertebrates) and offers a clustering of proteins into homologous families containing at least one viral sequences, as well as alignments and phylogenies for each of these families. Public access to PhEVER is available through its webpage and through all dedicated ACNUC retrieval systems.

Reporting errors in infectious disease outbreaks, with an application to Pandemic Influenza A/H1N1

BACKGROUND: Effectively responding to infectious disease outbreaks requires a well-informed response. Quantitative methods for analyzing outbreak data and estimating key parameters to characterize the spread of the outbreak, including the reproductive number and the serial interval, often assume that the data collected is complete. In reality reporting delays, undetected cases or lack of sensitive and specific tests to diagnose disease lead to reporting errors in the case counts. Here we provide insight on the impact that such reporting errors might have on the estimation of these key parameters. RESULTS: We show that when the proportion of cases reported is changing through the study period, the estimates of key epidemiological parameters are biased. Using data from the Influenza A/H1N1 outbreak in La Gloria, Mexico, we provide estimates of these parameters, accounting for possible reporting errors, and show that they can be biased by as much as 33%, if reporting issues are not accounted for. CONCLUSIONS: Failure to account for missing data can lead to misleading and inaccurate estimates of epidemic parameters.

Pathological and ultrastructural analysis of surgical lung biopsies in patients with swine‐origin influenza type A/H1N1 and acute respiratory failure

BACKGROUND: Cases of H1N1 and other pulmonary infections evolve to acute respiratory failure and death when co‐infections or lung injury predominate over the immune response, thus requiring early diagnosis to improve treatment. OBJECTIVE: To perform a detailed histopathological analysis of the open lung biopsy specimens from five patients with ARDS with confirmed H1N1. METHODS: Lung specimens underwent microbiologic analysis, and examination by optical and electron microscopy. Immunophenotyping was used to characterize macrophages, natural killer, T and B cells, and expression of cytokines and iNOS. RESULTS: The pathological features observed were necrotizing bronchiolitis, diffuse alveolar damage, alveolar hemorrhage and abnormal immune response. Ultrastructural analysis showed viral‐like particles in all cases. CONCLUSIONS: Viral‐like particles can be successfully demonstrated in lung tissue by ultrastructural examination, without confirmation of the virus by RT‐PCR on nasopharyngeal aspirates. Bronchioles and epithelium, rather than endothelium, are probably the primary target of infection, and diffuse alveolar damage the consequence of the effect of airways obliteration and dysfunction on innate immunity, suggesting that treatment should be focused on epithelial repair.

Ventilatory and ECMO treatment of H1N1-induced severe respiratory failure: results of an Italian referral ECMO center

BACKGROUND: Since the first outbreak of a respiratory illness caused by H1N1 virus in Mexico, several reports have described the need of intensive care or extracorporeal membrane oxygenation (ECMO) assistance in young and often healthy patients. Here we describe our experience in H1N1-induced ARDS using both ventilation strategy and ECMO assistance. METHODS: Following Italian Ministry of Health instructions, an Emergency Service was established at the Careggi Teaching Hospital (Florence, Italy) for the novel pandemic influenza. From Sept 09 to Jan 10, all patients admitted to our Intensive Care Unit (ICU) of the Emergency Department with ARDS due to H1N1 infection were studied. All ECMO treatments were veno-venous. H1N1 infection was confirmed by PCR assayed on pharyngeal swab, subglottic aspiration and bronchoalveolar lavage. Lung pathology was evaluated daily by lung ultrasound (LUS) examination. RESULTS: A total of 12 patients were studied: 7 underwent ECMO treatment, and 5 responded to protective mechanical ventilation. Two patients had co-infection by Legionella Pneumophila. One woman was pregnant. In our series, PCR from bronchoalveolar lavage had a 100% sensitivity compared to 75% from pharyngeal swab samples. The routine use of LUS limited the number of chest X-ray examinations and decreased transportation to radiology for CT-scan, increasing patient safety and avoiding the transitory disconnection from ventilator. No major complications occurred during ECMO treatments. In three cases, bleeding from vascular access sites due to heparin infusion required blood transfusions. Overall mortality rate was 8.3%. CONCLUSIONS: In our experience, early ECMO assistance resulted safe and feasible, considering the life threatening condition, in H1N1-induced ARDS. Lung ultrasound is an effective mean for daily assessment of ARDS patients.

Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection

The 2009 pandemic H1N1 influenza pandemic demonstrated the global health threat of reassortant influenza strains. Herein, we report a detailed analysis of plasmablast and monoclonal antibody responses induced by pandemic H1N1 infection in humans. Unlike antibodies elicited by annual influenza vaccinations, most neutralizing antibodies induced by pandemic H1N1 infection were broadly cross-reactive against epitopes in the hemagglutinin (HA) stalk and head domain of multiple influenza strains. The antibodies were from cells that had undergone extensive affinity maturation. Based on these observations, we postulate that the plasmablasts producing these broadly neutralizing antibodies were predominantly derived from activated memory B cells specific for epitopes conserved in several influenza strains. Consequently, most neutralizing antibodies were broadly reactive against divergent H1N1 and H5N1 influenza strains. This suggests that a pan-influenza vaccine may be possible, given the right immunogen. Antibodies generated potently protected and rescued mice from lethal challenge with pandemic H1N1 or antigenically distinct influenza strains, making them excellent therapeutic candidates.

Assessment of Virally Vectored Autoimmunity as a Biocontrol Strategy for Cane Toads

BACKGROUND: The cane toad, Bufo (Chaunus) marinus, is one of the most notorious vertebrate pests introduced into Australia over the last 200 years and, so far, efforts to identify a naturally occurring B. marinus-specific pathogen for use as a biological control agent have been unsuccessful. We explored an alternative approach that entailed genetically modifying a pathogen with broad host specificity so that it no longer caused disease, but carried a gene to disrupt the cane toad life cycle in a species specific manner. METHODOLOGY/PRINCIPAL FINDINGS: The adult beta globin gene was selected as the model gene for proof of concept of autoimmunity as a biocontrol method for cane toads. A previous report showed injection of bullfrog tadpoles with adult beta globin resulted in an alteration in the form of beta globin expressed in metamorphs as well as reduced survival. In B. marinus we established for the first time that the switch from tadpole to adult globin exists. The effect of injecting B. marinus tadpoles with purified recombinant adult globin protein was then assessed using behavioural (swim speed in tadpoles and jump length in metamorphs), developmental (time to metamorphosis, weight and length at various developmental stages, protein profile of adult globin) and genetic (adult globin mRNA levels) measures. However, we were unable to detect any differences between treated and control animals. Further, globin delivery using Bohle iridovirus, an Australian ranavirus isolate belonging to the Iridovirus family, did not reduce the survival of metamorphs or alter the form of beta globin expressed in metamorphs. CONCLUSIONS/SIGNIFICANCE: While we were able to show for the first time that the switch from tadpole to adult globin does occur in B. marinus, we were not able to induce autoimmunity and disrupt metamorphosis. The short development time of B. marinus tadpoles may preclude this approach.

Analysing the eosinophil cationic protein - a clue to the function of the eosinophil granulocyte

Eosinophil granulocytes reside in respiratory mucosa including lungs, in the gastro-intestinal tract, and in lymphocyte associated organs, the thymus, lymph nodes and the spleen. In parasitic infections, atopic diseases such as atopic dermatitis and asthma, the numbers of the circulating eosinophils are frequently elevated. In conditions such as Hypereosinophilic Syndrome (HES) circulating eosinophil levels are even further raised. Although, eosinophils were identified more than hundred years ago, their roles in homeostasis and in disease still remain unclear. The most prominent feature of the eosinophils are their large secondary granules, each containing four basic proteins, the best known being the eosinophil cationic protein (ECP). This protein has been developed as a marker for eosinophilic disease and quantified in biological fluids including serum, bronchoalveolar lavage and nasal secretions. Elevated ECP levels are found in T helper lymphocyte type 2 (atopic) diseases such as allergic asthma and allergic rhinitis but also occasionally in other diseases such as bacterial sinusitis. ECP is a ribonuclease which has been attributed with cytotoxic, neurotoxic, fibrosis promoting and immune-regulatory functions. ECP regulates mucosal and immune cells and may directly act against helminth, bacterial and viral infections. The levels of ECP measured in disease in combination with the catalogue of known functions of the protein and its polymorphisms presented here will build a foundation for further speculations of the role of ECP, and ultimately the role of the eosinophil.

Excess healthcare burden during 1918-1920 influenza pandemic in Taiwan: implications for post-pandemic preparedness

BACKGROUND: It is speculated that the 2009 pandemic H1N1 influenza virus might fall into a seasonal pattern during the current post-pandemic period with more severe clinical presentation for high-risk groups identified during the 2009 pandemic. Hence the extent of likely excess healthcare needs during this period must be fully considered. We will make use of the historical healthcare record in Taiwan during and after the 1918 influenza pandemic to ascertain the scope of potential excess healthcare burden during the post-pandemic period. METHODS: To establish the healthcare needs after the initial wave in 1918, the yearly healthcare records (hospitalizations, outpatients, etc.) in Taiwan during 1918-1920 are compared with the corresponding data from the adjacent "baseline" years of 1916, 1917, 1921, and 1922 to estimate the excess healthcare burden during the initial outbreak in 1918 and in the years immediately after. RESULTS: In 1918 the number of public hospital outpatients exceeded the yearly average of the baseline years by 20.11% (95% CI: 16.43, 25.90), and the number of hospitalizations exceeded the corresponding yearly average of the baseline years by 12.20% (10.59, 14.38), while the excess number of patients treated by the public medics was statistically significant at 32.21% (28.48, 39.82) more than the yearly average of the baseline years. For 1920, only the excess number of hospitalizations was statistically significant at 19.83% (95% CI: 17.21, 23.38) more than the yearly average of the baseline years. CONCLUSIONS: Considerable extra burden with significant loss of lives was reported in 1918 by both the public medics system and the public hospitals. In comparison, only a substantial number of excess hospitalizations in the public hospitals was reported in 1920, indicating that the population was relatively unprepared for the first wave in 1918 and did not fully utilize the public hospitals. Moreover, comparatively low mortality was reported by the public hospitals and the public medics during the second wave in 1920 even though significantly more patients were hospitalized, suggesting that there had been substantially less fatal illnesses among the hospitalized patients during the second wave. Our results provide viable parameters for assessing healthcare needs for post-pandemic preparedness.

Multifunctional nanoparticles as simulants for a gravimetric immunoassay

Immunoassays are important tools for the rapid detection and identification of pathogens, both clinically and in the research laboratory. An immunoassay with the potential for the detection of influenza was developed and tested using hemagglutinin (HA), a commonly studied glycoprotein found on the surface of influenza virions. Gold nanoparticles were synthesized, which present multiple peptide epitopes, including the HA epitope, in order to increase the gravimetric response achieved with the use of a QCM immunosensor for influenza. Specifically, epitopes associated with HA and FLAG peptides were affixed to gold nanoparticles by a six-mer PEG spacer between the epitope and the terminal cysteine. The PEG spacer was shown to enhance the probability for interaction with antibodies by increasing the distance the epitope extends from the gold surface. These nanoparticles were characterized using thermogravimetric analysis, transmission electron microscopy, matrix-assisted laser desorption/ionization-time of flight, and (1)H nuclear magnetic resonance analysis. Anti-FLAG and anti-HA antibodies were adhered to the surface of a QCM, and the response of each antibody upon exposure to HA, FLAG, and dual functionalized nanoparticles was compared with binding of Au–tiopronin nanoparticles and H5 HA proteins from influenza virus (H5N1). Results demonstrate that the immunoassay was capable of differentiating between nanoparticles presenting orthogonal epitopes in real-time with minimal nonspecific binding. The detection of H5 HA protein demonstrates the logical extension of using these nanoparticle mimics as a safe positive control in the detection of influenza, making this a vital step in improving influenza detection methodology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00216-010-4419-8) contains supplementary material, which is available to authorized users.

Hepatitis G Virus associated aplastic anemia: A recent case from Pakistan

BACKGROUND: Aplastic anemia (AA) is a serious and rare disorder characterized by a hypocellular bone marrow. Hepatitis associated aplastic anemia (HAAA) is a variant of aplastic anemia in which aplastic anemia follows an acute attack of hepatitis. Several reports have noted an association between HGV and hepatitis-associated aplastic anemia besides other hepatitis causing viruses. CASE PRESENTATION: A female girl of age 11 year with a history of loose motion for one month, vomiting for last 15 days and poor oral intake for last few days is reported here. The physical examination presents fever, pallor whereas bleeding, hepatomegaly, Splenomegaly and bruising were absent, abdominal ultrasonography confirmed the absence of hepatomegaly, Splenomegaly and lymphodenopathy. The laboratory investigation parameters were: haemoglobin 6.2 g/L, total leucocytes count 1.51, neutrophils 0.47%, absolute reticulocyte count 0.5%, Monocytes 0.16%, red cell count 3.2 mil/uL, Picked cell volume (PCV) 30.13%, Mean Corpuscular Volume (MCV) 78 fL, Mean Corpuscular Hemoglobin (MCH) 26.3 pg. The liver enzymes were alanine aminotransferease (ALT) 98 IU/L, aspartate aminotransferase (AST) 114 IU/L. Serologic and molecular tests for hepatitis A, B, C, D, E, TTV, B19 were negative, whereas HGV RNA PCR test was found positive for hepatitis G virus. The bone marrow aspirate and trephine biopsy examination revealed hypo- cellularity, erythropoiesis, myelopoiesis and megakaryopoiesis. CONCLUSION: HAAA is an uncommon but severe condition, which may occur following idiopathic cases of acute hepatitis. Our finding suggests the involvement of HGV in the development of aplastic anemia. In patients presenting with pancytopenia after an episode of acute hepatitis, the definitive diagnosis should be considered and confirmed by RT-PCR and if possible by bone marrow biopsy.

Travel Patterns in China

The spread of infectious disease epidemics is mediated by human travel. Yet human mobility patterns vary substantially between countries and regions. Quantifying the frequency of travel and length of journeys in well-defined population is therefore critical for predicting the likely speed and pattern of spread of emerging infectious diseases, such as a new influenza pandemic. Here we present the results of a large population survey undertaken in 2007 in two areas of China: Shenzhen city in Guangdong province, and Huangshan city in Anhui province. In each area, 10,000 randomly selected individuals were interviewed, and data on regular and occasional journeys collected. Travel behaviour was examined as a function of age, sex, economic status and home location. Women and children were generally found to travel shorter distances than men. Travel patterns in the economically developed Shenzhen region are shown to resemble those in developed and economically advanced middle income countries with a significant fraction of the population commuting over distances in excess of 50 km. Conversely, in the less developed rural region of Anhui, travel was much more local, with very few journeys over 30 km. Travel patterns in both populations were well-fitted by a gravity model with a lognormal kernel function. The results provide the first quantitative information on human travel patterns in modern China, and suggest that a pandemic emerging in a less developed area of rural China might spread geographically sufficiently slowly for containment to be feasible, while spatial spread in the more economically developed areas might be expected to be much more rapid, making containment more difficult.

Spatial dynamics of the 1918 influenza pandemic in England, Wales and the United States

There is still limited understanding of key determinants of spatial spread of influenza. The 1918 pandemic provides an opportunity to elucidate spatial determinants of spread on a large scale. To better characterize the spread of the 1918 major wave, we fitted a range of city-to-city transmission models to mortality data collected for 246 population centres in England and Wales and 47 cities in the US. Using a gravity model for city-to-city contacts, we explored the effect of population size and distance on the spread of disease and tested assumptions regarding density dependence in connectivity between cities. We employed Bayesian Markov Chain Monte Carlo methods to estimate parameters of the model for population, infectivity, distance and density dependence. We inferred the most likely transmission trees for both countries. For England and Wales, a model that estimated the degree of density dependence in connectivity between cities was preferable by deviance information criterion comparison. Early in the major wave, long distance infective interactions predominated, with local infection events more likely as the epidemic became widespread. For the US, with fewer more widely dispersed cities, statistical power was lacking to estimate population size dependence or the degree of density dependence, with the preferred model depending on distance only. We find that parameters estimated from the England and Wales dataset can be applied to the US data with no likelihood penalty.

Charge-Surrounded Pockets and Electrostatic Interactions with Small Ions Modulate the Activity of Retroviral Fusion Proteins

Refolding of viral class-1 membrane fusion proteins from a native state to a trimer-of-hairpins structure promotes entry of viruses into cells. Here we present the structure of the bovine leukaemia virus transmembrane glycoprotein (TM) and identify a group of asparagine residues at the membrane-distal end of the trimer-of-hairpins that is strikingly conserved among divergent viruses. These asparagines are not essential for surface display of pre-fusogenic envelope. Instead, substitution of these residues dramatically disrupts membrane fusion. Our data indicate that, through electrostatic interactions with a chloride ion, the asparagine residues promote assembly and profoundly stabilize the fusion-active structures that are required for viral envelope-mediated membrane fusion. Moreover, the BLV TM structure also reveals a charge-surrounded hydrophobic pocket on the central coiled coil and interactions with basic residues that cluster around this pocket are critical to membrane fusion and form a target for peptide inhibitors of envelope function. Charge-surrounded pockets and electrostatic interactions with small ions are common among class-1 fusion proteins, suggesting that small molecules that specifically target such motifs should prevent assembly of the trimer-of-hairpins and be of value as therapeutic inhibitors of viral entry.

Tracheostomy and mechanical ventilation weaning in children affected by respiratory virus according to a weaning protocol in a pediatric intensive care unit in Argentina: an observational restrospective trial

We describe difficult weaning after prolonged mechanical ventilation in three tracheostomized children affected by respiratory virus infection. Although the spontaneous breathing trials were successful, the patients failed all extubations. Therefore a tracheostomy was performed and the weaning plan was begun. The strategy for weaning was the decrease of ventilation support combining pressure control ventilation (PCV) with increasing periods of continuous positive airway pressure + pressure support ventilation (CPAP + PSV) and then CPAP + PSV with increasing intervals of T-piece. They presented acute respiratory distress syndrome on admission with high requirements of mechanical ventilation (MV). Intervening factors in the capabilities and loads of the respiratory system were considered and optimized. The average MV time was 69 days and weaning time 31 days. We report satisfactory results within the context of a directed weaning protocol.

The activity of the HIV-1 IRES is stimulated by oxidative stress and controlled by a negative regulatory element

Initiation of translation of the full-length messenger RNA of HIV-1, which generates the viral structural proteins and enzymes, is cap-dependent but can also use an internal ribosome entry site (IRES) located in the 5′ untranslated region. Our aim was to define, through a mutational analysis, regions of HIV-1 IRES that are important for its activity. A dual-luciferase reporter construct where the Renilla luciferase (Rluc) translation is cap-dependent while the firefly luciferase (Fluc) translation depends on HIV-1 IRES was used. The Fluc/Rluc ratio was measured in lysates of Jurkat T cells transfected with the dual-luciferase plasmid bearing either the wild-type or a mutated IRES. Deletions or mutations in three regions decreased the IRES activity but deletion or mutations of a stem-loop preceding the primer binding site increased the IRES activity. The wild-type IRES activity, but not that of an IRES with a mutated stem-loop, was increased when cells were treated with agents that induce oxidative stress. Such stress is known to be caused by HIV-1 infection and we propose that this stem-loop is involved in a switch that stimulates the IRES activity in cells infected with HIV-1, supporting the suggestion that the IRES activity is up-regulated in the course of HIV-1 replication cycle.

Further Characterisation of the Translational Termination-Reinitiation Signal of the Influenza B Virus Segment 7 RNA

Termination-dependent reinitiation is used to co-ordinately regulate expression of the M1 and BM2 open-reading frames (ORFs) of the dicistronic influenza B segment 7 RNA. The start codon of the BM2 ORF overlaps the stop codon of the M1 ORF in the pentanucleotide UAA UG and ∼10% of ribosomes terminating at the M1 stop codon reinitiate translation at the overlapping AUG. BM2 synthesis requires the presence of, and translation through, 45 nt of RNA immediately upstream of the UAA UG, known as the ‘termination upstream ribosome binding site’ (TURBS). This region may tether ribosomal 40S subunits to the mRNA following termination and a short region of the TURBS, motif 1, with complementarity to helix 26 of 18S rRNA has been implicated in this process. Here, we provide further evidence for a direct interaction between mRNA and rRNA using antisense oligonucleotide targeting and functional analysis in yeast cells. The TURBS also binds initiation factor eIF3 and we show here that this protein stimulates reinitiation from both wild-type and defective TURBS when added exogenously, perhaps by stabilising ribosome-mRNA interactions. Further, we show that the position of the TURBS with respect to the UAA UG overlap is crucial, and that termination too far downstream of the 18S complementary sequence inhibits the process, probably due to reduced 40S tethering. However, in reporter mRNAs where the restart codon alone is moved downstream, termination-reinitiation is inhibited but not abolished, thus the site of reinitiation is somewhat flexible. Reinitiation on distant AUGs is not inhibited in eIF4G-depleted RRL, suggesting that the tethered 40S subunit can move some distance without a requirement for linear scanning.

RNA and DNA Bacteriophages as Molecular Diagnosis Controls in Clinical Virology: A Comprehensive Study of More than 45,000 Routine PCR Tests

Real-time PCR techniques are now commonly used for the detection of viral genomes in various human specimens and require for validation both external and internal controls (ECs and ICs). In particular, ICs added to clinical samples are necessary to monitor the extraction, reverse transcription, and amplification steps in order to detect false-negative results resulting from PCR-inhibition or errors in the technical procedure. Here, we performed a large scale evaluation of the use of bacteriophages as ICs in routine molecular diagnosis. This allowed to propose simple standardized procedures (i) to design specific ECs for both DNA and RNA viruses and (ii) to use T4 (DNA) or MS2 (RNA) phages as ICs in routine diagnosis. Various technical formats for using phages as ICs were optimised and validated. Subsequently, T4 and MS2 ICs were evaluated in routine real-time PCR or RT-PCR virological diagnostic tests, using a series of 8,950 clinical samples (representing 36 distinct specimen types) sent to our laboratory for the detection of a variety of DNA and RNA viruses. The frequency of inefficient detection of ICs was analyzed according to the nature of the sample. Inhibitors of enzymatic reactions were detected at high frequency in specific sample types such as heparinized blood and bone marrow (>70%), broncho-alveolar liquid (41%) and stools (36%). The use of T4 and MS2 phages as ICs proved to be cost-effective, flexible and adaptable to various technical procedures of real-time PCR detection in virology. It represents a valuable strategy for enhancing the quality of routine molecular diagnosis in laboratories that use in-house designed diagnostic systems, which can conveniently be associated to the use of specific synthetic ECs. The high rate of inhibitors observed in a variety of specimen types should stimulate the elaboration of improved technical protocols for the extraction and amplification of nucleic acids.

Pandemic H1N1 2009 influenza virus with the H275Y oseltamivir resistance neuraminidase mutation shows a small compromise in enzyme activity and viral fitness

BACKGROUND: Resistance to the neuraminidase inhibitor oseltamivir can be conferred by a well-characterized mutation in the neuraminidase gene, H275Y. In human H1N1 viruses that circulated in the first years of the 21st century, this mutation carried a fitness cost and resistant viruses were rare. During the 2007–08 influenza season, oseltamivir-resistant viruses of H1N1 phenotype emerged and predominated. March 2009 saw the emergence of a novel H1N1 influenza pandemic. We examined whether the H275Y mutation affected neuraminidase enzyme activity or replication of the pandemic influenza virus. METHODS: Using reverse genetics we engineered the H275Y mutation into the neuraminidase of a 2009 pandemic H1N1 virus and assessed the ability of this enzyme to desialylate mono- and multivalent substrates. The growth kinetics of wild-type and mutant viruses were assessed in Madin–Darby canine kidney (MDCK) and fully differentiated human airway epithelial (HAE) cells. RESULTS: The presence of H275Y was associated with a 1.3-fold decrease in the affinity of the neuraminidase for a monovalent substrate and a 4-fold compromise in desialylation of multivalent substrate. This was associated with a fitness cost to viral replication in vitro, which only became apparent during competitive replication in the mucus-rich HAE culture system. CONCLUSIONS: The neuraminidase protein of pandemic influenza isolates tolerates the H275Y mutation and this mutation confers resistance to oseltamivir. However, unlike seasonal H1N1 viruses isolated since 2007, the mutation is not associated with any fitness advantage and thus is unlikely to predominate without further antigenic drift, compensating mutations or intense selection pressure.

Ketamine inhibits tumor necrosis factor secretion by RAW264.7 murine macrophages stimulated with antibiotic-exposed strains of community-associated, methicillin-resistant Staphylococcus aureus

BACKGROUND: Infections caused by community-associated strains of methicillin-resistant Staphylococcus aureus (CA-MRSA) are associated with a marked and prolonged host inflammatory response. In a sepsis simulation model, we tested whether the anesthetic ketamine inhibits the macrophage TNF response to antibiotic-exposed CA-MRSA bacteria via its antagonism of N-methyl-D-aspartate (NMDA) receptors. RAW264.7 cells were stimulated for 18 hrs with 10(5 )to 10(7 )CFU/mL inocula of either of two prototypical CA-MRSA isolates, USA300 strain LAC and USA400 strain MW2, in the presence of either vancomycin or daptomycin. One hour before bacterial stimulation, ketamine was added with or without MK-801 (dizocilpine, a chemically unrelated non-competitive NMDA receptor antagonist), APV (D-2-amino-5-phosphono-valerate, a competitive NMDA receptor antagonist), NMDA, or combinations of these agents. Supernatants were collected and assayed for TNF concentration by ELISA. RESULTS: RAW264.7 cells exposed to either LAC or MW2 in the presence of daptomycin secreted less TNF than in the presence of vancomycin. The addition of ketamine inhibited macrophage TNF secretion after stimulation with either of the CA-MRSA isolates (LAC, MW2) in the presence of either antibiotic. The NMDA inhibitors, MK-801 and APV, also suppressed macrophage TNF secretion after stimulation with either of the antibiotic-exposed CA-MRSA isolates, and the effect was not additive or synergistic with ketamine. The addition of NMDA substrate augmented TNF secretion in response to the CA-MRSA bacteria, and the addition of APV suppressed the effect of NMDA in a dose-dependent fashion. CONCLUSIONS: Ketamine inhibits TNF secretion by MRSA-stimulated RAW264.7 macrophages and the mechanism likely involves NMDA receptor antagonism. These findings may have therapeutic significance in MRSA sepsis.

Avipoxviruses: infection biology and their use as vaccine vectors

Avipoxviruses (APVs) belong to the Chordopoxvirinae subfamily of the Poxviridae family. APVs are distributed worldwide and cause disease in domestic, pet and wild birds of many species. APVs are transmitted by aerosols and biting insects, particularly mosquitoes and arthropods and are usually named after the bird species from which they were originally isolated. The virus species Fowlpox virus (FWPV) causes disease in poultry and associated mortality is usually low, but in flocks under stress (other diseases, high production) mortality can reach up to 50%. APVs are also major players in viral vaccine vector development for diseases in human and veterinary medicine. Abortive infection in mammalian cells (no production of progeny viruses) and their ability to accommodate multiple gene inserts are some of the characteristics that make APVs promising vaccine vectors. Although abortive infection in mammalian cells conceivably represents a major vaccine bio-safety advantage, molecular mechanisms restricting APVs to certain hosts are not yet fully understood. This review summarizes the current knowledge relating to APVs, including classification, morphogenesis, host-virus interactions, diagnostics and disease, and also highlights the use of APVs as recombinant vaccine vectors.

Apolipoprotein M Gene (APOM) Polymorphism Modifies Metabolic and Disease Traits in Type 2 Diabetes

This study aimed at substantiating the associations of the apolipoproein M gene (APOM) with type 2 diabetes (T2D) as well as with metabolic traits in Hong Kong Chinese. In addition, APOM gene function was further characterized to elucidate its activity in cholesterol metabolism. Seventeen APOM SNPs documented in the NCBI database were genotyped. Five SNPs were confirmed in our study cohort of 1234 T2D and 606 control participants. Three of the five SNPs rs707921(C+1871A), rs707922(G+1837T) and rs805264(G+203A) were in linkage disequilibrium (LD). We chose rs707922 to tag this LD region for down stream association analyses and characterized the function of this SNP at molecular level. No association between APOM and T2D susceptibility was detected in our Hong Kong Chinese cohort. Interestingly, the C allele of rs805297 was significantly associated with T2D duration of longer than 10 years (OR = 1.245, p = 0.015). The rs707922 TT genotype was significantly associated with elevated plasma total- and LDL- cholesterol levels (p = 0.006 and p = 0.009, respectively) in T2D patients. Molecular analyses of rs707922 lead to the discoveries of a novel transcript APOM5 as well as the cryptic nature of exon 5 of the gene. Ectopic expression of APOM5 transcript confirmed rs707922 allele-dependent activity of the transcript in modifying cholesterol homeostasis in vitro. In conclusion, the results here did not support APOM as a T2D susceptibility gene in Hong Kong Chinese. However, in T2D patients, a subset of APOM SNPs was associated with disease duration and metabolic traits. Further molecular analysis proved the functional activity of rs707922 in APOM expression and in regulation of cellular cholesterol content.

Non-Invasive Microstructure and Morphology Investigation of the Mouse Lung: Qualitative Description and Quantitative Measurement

BACKGROUND: Early detection of lung cancer is known to improve the chances of successful treatment. However, lungs are soft tissues with complex three-dimensional configuration. Conventional X-ray imaging is based purely on absorption resulting in very low contrast when imaging soft tissues without contrast agents. It is difficult to obtain adequate information of lung lesions from conventional X-ray imaging. METHODS: In this study, a recently emerged imaging technique, in-line X-ray phase contrast imaging (IL-XPCI) was used. This powerful technique enabled high-resolution investigations of soft tissues without contrast agents. We applied IL-XPCI to observe the lungs in an intact mouse for the purpose of defining quantitatively the micro-structures in lung. FINDINGS: The three-dimensional model of the lung was successfully established, which provided an excellent view of lung airways. We highlighted the use of IL-XPCI in the visualization and assessment of alveoli which had rarely been studied in three dimensions (3D). The precise view of individual alveolus was achieved. The morphological parameters, such as diameter and alveolar surface area were measured. These parameters were of great importance in the diagnosis of diseases related to alveolus and alveolar scar. CONCLUSION: Our results indicated that IL-XPCI had the ability to represent complex anatomical structures in lung. This offered a new perspective on the diagnosis of respiratory disease and may guide future work in the study of respiratory mechanism on the alveoli level.

Evolution of size and pattern in the social amoebas

A fundamental goal of biology is to understand how novel phenotypes evolved through changes in existing genes. The Dictyostelia or social amoebas represent a simple form of multicellularity, where starving cells aggregate to build fruiting structures. This review summarizes efforts to provide a framework for investigating the genetic changes that generated novel morphologies in the Dictyostelia. The foundation is a recently constructed molecular phylogeny of the Dictyostelia, which was used to examine trends in the evolution of novel forms and in the divergence of genes that shape these forms. There is a major trend towards the formation of large unbranched fruiting bodies, which is correlated with the use of cyclic AMP (cAMP) as a secreted signal to coordinate cell aggregation. The role of cAMP in aggregation arose through co‐option of a pathway that originally acted to coordinate fruiting body formation. The genotypic changes that caused this innovation and the role of dynamic cAMP signaling in defining fruiting body size and pattern throughout social amoeba evolution are discussed. BioEssays 29:635–644, 2007. © 2007 Wiley Periodicals, Inc.

Plant Plastid Engineering

Genetic material in plants is distributed into nucleus, plastids and mitochondria. Plastid has a central role of carrying out photosynthesis in plant cells. Plastid transformation is becoming more popular and an alternative to nuclear gene transformation because of various advantages like high protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs, and gene containment through the lack of pollen transmission. Recently, much progress in plastid engineering has been made. In addition to model plant tobacco, many transplastomic crop plants have been generated which possess higher resistance to biotic and abiotic stresses and molecular pharming. In this mini review, we will discuss the features of the plastid DNA and advantages of plastid transformation. We will also present some examples of transplastomic plants developed so far through plastid engineering, and the various applications of plastid transformation.

Role of glutathione in immunity and inflammation in the lung

Reactive oxygen species and thiol antioxidants, including glutathione (GSH), regulate innate immunity at various levels. This review outlines the redox-sensitive steps of the cellular mechanisms implicated in inflammation and host defense against infection, and describes how GSH is not only important as an antioxidant but also as a signaling molecule. There is an extensive literature of the role of GSH in immunity. Most reviews are biased by an oversimplified picture where “bad” free radicals cause all sorts of diseases and “good” antioxidants protect from them and prevent oxidative stress. While this may be the case in certain fields (eg, toxicology), the role of thiols (the topic of this review) in immunity certainly requires wearing scientist’s goggles and being prepared to accept a more complex picture. This review aims at describing the role of GSH in the lung in the context of immunity and inflammation. The first part summarizes the history and basic concepts of this picture. The second part focuses on GSH metabolism/levels in pathology, the third on the role of GSH in innate immunity and inflammation, and the fourth gives 4 examples describing the importance of GSH in the response to infections.

We should not be complacent about our population-based public health response to the first influenza pandemic of the 21(st )century

BACKGROUND: More than a year after an influenza pandemic was declared in June 2009, the World Health Organization declared the pandemic to be over. Evaluations of the pandemic response are beginning to appear in the public domain. DISCUSSION: We argue that, despite the enormous effort made to control the pandemic, it is now time to acknowledge that many of the population-based public health interventions may not have been well considered. Prior to the pandemic, there was limited scientific evidence to support border control measures. In particular no border screening measures would have detected prodromal or asymptomatic infections, and asymptomatic infections with pandemic influenza were common. School closures, when they were partial or of short duration, would not have interrupted spread of the virus in school-aged children, the group with the highest rate of infection worldwide. In most countries where they were available, neuraminidase inhibitors were not distributed quickly enough to have had an effect at the population level, although they will have benefited individuals, and prophylaxis within closed communities will have been effective. A pandemic specific vaccine will have protected the people who received it, although in most countries only a small minority was vaccinated, and often a small minority of those most at risk. The pandemic vaccine was generally not available early enough to have influenced the shape of the first pandemic wave and it is likely that any future pandemic vaccine manufactured using current technology will also be available too late, at least in one hemisphere. SUMMARY: Border screening, school closure, widespread anti-viral prophylaxis and a pandemic-specific vaccine were unlikely to have been effective during a pandemic which was less severe than anticipated in the pandemic plans of many countries. These were cornerstones of the population-based public health response. Similar responses would be even less likely to be effective in a more severe pandemic. We agree with the recommendation from the World Health Organisation that pandemic preparedness plans need review.

The GLEaMviz computational tool, a publicly available software to explore realistic epidemic spreading scenarios at the global scale

BACKGROUND: Computational models play an increasingly important role in the assessment and control of public health crises, as demonstrated during the 2009 H1N1 influenza pandemic. Much research has been done in recent years in the development of sophisticated data-driven models for realistic computer-based simulations of infectious disease spreading. However, only a few computational tools are presently available for assessing scenarios, predicting epidemic evolutions, and managing health emergencies that can benefit a broad audience of users including policy makers and health institutions. RESULTS: We present "GLEaMviz", a publicly available software system that simulates the spread of emerging human-to-human infectious diseases across the world. The GLEaMviz tool comprises three components: the client application, the proxy middleware, and the simulation engine. The latter two components constitute the GLEaMviz server. The simulation engine leverages on the Global Epidemic and Mobility (GLEaM) framework, a stochastic computational scheme that integrates worldwide high-resolution demographic and mobility data to simulate disease spread on the global scale. The GLEaMviz design aims at maximizing flexibility in defining the disease compartmental model and configuring the simulation scenario; it allows the user to set a variety of parameters including: compartment-specific features, transition values, and environmental effects. The output is a dynamic map and a corresponding set of charts that quantitatively describe the geo-temporal evolution of the disease. The software is designed as a client-server system. The multi-platform client, which can be installed on the user's local machine, is used to set up simulations that will be executed on the server, thus avoiding specific requirements for large computational capabilities on the user side. CONCLUSIONS: The user-friendly graphical interface of the GLEaMviz tool, along with its high level of detail and the realism of its embedded modeling approach, opens up the platform to simulate realistic epidemic scenarios. These features make the GLEaMviz computational tool a convenient teaching/training tool as well as a first step toward the development of a computational tool aimed at facilitating the use and exploitation of computational models for the policy making and scenario analysis of infectious disease outbreaks.

Nursing heroism in the 21(st )Century'

BACKGROUND: The Vivian Bullwinkel Oration honours the life and work of an extraordinary nurse. Given her story and that of her World War II colleagues, the topic of nursing heroism in the 21(st )century could not be more germane. DISCUSSION: Is heroism a legitimate part of nursing, or are nurses simply 'just doing their job' even when facing extreme personal danger? In this paper I explore the place and relevance of heroism in contemporary nursing. I propose that nursing heroism deserves a broader appreciation and that within the term lie many hidden, 'unsung' or 'unrecorded' heroisms. I also challenge the critiques of heroism that would condemn it as part of a 'militarisation' of nursing. Finally, I argue that nursing needs to be more open in celebrating our heroes and the transformative power of nursing achievements. SUMMARY: The language of heroism may sound quaint by 21(st )Century standards but nursing heroism is alive and well in the best of our contemporary nursing ethos and practice.

Implications of copy number variation in people with chromosomal abnormalities: potential for greater variation in copy number state may contribute to variability of phenotype

Copy number variation is common in the human genome with many regions, overlapping thousands of genes, now known to be deleted or amplified. Aneuploidies and other forms of chromosomal imbalance have a wide range of adverse phenotypes and are a common cause of birth defects resulting in significant morbidity and mortality. “Normal” copy number variants (CNVs) embedded within the regions of chromosome imbalance may affect the clinical outcomes by altering the local copy number of important genes or regulatory regions: this could alleviate or exacerbate certain phenotypes. In this way CNVs may contribute to the clinical variability seen in many disorders caused by chromosomal abnormalities, such as the congenital heart defects (CHD) seen in ~40% of Down’s syndrome (DS) patients. Investigation of CNVs may therefore help to pinpoint critical genes or regulatory elements, elucidating the molecular mechanisms underlying these conditions, also shedding light on the aetiology of such phenotypes in people without major chromosome imbalances, and ultimately leading to their improved detection and treatment.

Hepatitis Associated Aplastic Anemia: A review

Hepatitis-associated aplastic anemia (HAAA) is an uncommon but distinct variant of aplastic anemia in which pancytopenia appears two to three months after an acute attack of hepatitis. HAAA occurs most frequently in young male children and is lethal if leave untreated. The etiology of this syndrome is proposed to be attributed to various hepatitis and non hepatitis viruses. Several hepatitis viruses such as HAV, HBV, HCV, HDV, HEV and HGV have been associated with this set of symptoms. Viruses other than the hepatitis viruses such as parvovirus B19, Cytomegalovirus, Epstein bar virus, Transfusion Transmitted virus (TTV) and non-A-E hepatitis virus (unknown viruses) has also been documented to develop the syndrome. Considerable evidences including the clinical features, severe imbalance of the T cell immune system and effective response to immunosuppressive therapy strongly present HAAA as an immune mediated mechanism. However, no association of HAAA has been found with blood transfusions, drugs and toxins. Besides hepatitis and non hepatitis viruses and immunopathogenesis phenomenon as causative agents of the disorder, telomerase mutation, a genetic factor has also been predisposed for the development of aplastic anemia. Diagnosis includes clinical manifestations, blood profiling, viral serological markers testing, immune functioning and bone marrow hypocellularity examination. Patients presenting the features of HAAA have been mostly treated with bone marrow or hematopoietic cell transplantation from HLA matched donor, and if not available then by immunosuppressive therapy. New therapeutic approaches involve the administration of steroids especially the glucocorticoids to augment the immunosuppressive therapy response. Pancytopenia following an episode of acute hepatitis response better to hematopoietic cell transplantation than immunosuppressive therapy.

Factors Affecting Intention to Receive and Self-Reported Receipt of 2009 Pandemic (H1N1) Vaccine in Hong Kong: A Longitudinal Study

BACKGROUND: Vaccination was a core component for mitigating the 2009 influenza pandemic (pH1N1). However, a vaccination program's efficacy largely depends on population compliance. We examined general population decision-making for pH1N1 vaccination using a modified Theory of Planned Behaviour (TBP). METHODOLOGY: We conducted a longitudinal study, collecting data before and after the introduction of pH1N1 vaccine in Hong Kong. Structural equation modeling (SEM) tested if a modified TPB had explanatory utility for vaccine uptake among adults. PRINCIPAL FINDINGS: Among 896 subjects who completed both the baseline and the follow-up surveys, 7% (67/896) reported being “likely/very likely/certain” to be vaccinated (intent) but two months later only 0.8% (7/896) reported having received pH1N1 vaccination. Perception of low risk from pH1N1 (60%) and concerns regarding adverse effects of the vaccine (37%) were primary justifications for avoiding pH1N1 vaccination. Greater perceived vaccine benefits (β = 0.15), less concerns regarding vaccine side-effects (β = −0.20), greater adherence to social norms of vaccination (β = 0.39), anticipated higher regret if not vaccinated (β = 0.47), perceived higher self-efficacy for vaccination (β = 0.12) and history of seasonal influenza vaccination (β = 0.12) were associated with higher intention to receive the pH1N1 vaccine, which in turn predicted self-reported vaccination uptake (β = 0.30). Social norm (β = 0.70), anticipated regret (β = 0.19) and vaccination intention (β = 0.31) were positively associated with, and accounted for 70% of variance in vaccination planning, which, in turn subsequently predicted self-reported vaccination uptake (β = 0.36) accounting for 36% of variance in reported vaccination behaviour. CONCLUSIONS/SIGNIFICANCE: Perceived low risk from pH1N1 and perceived high risk from pH1N1 vaccine inhibited pH1N1 vaccine uptake. Both the TPB and the additional components contributed to intended vaccination uptake but social norms and anticipated regret predominantly associated with vaccination intention and planning. Vaccination planning is a more significant proximal determinant of uptake of pH1N1 vaccine than is intention. Intention alone is an unreliable predictor of future vaccine uptake.

Dengue Virus Virulence and Transmission Determinants

The mechanisms of dengue virus (DENV) pathogenesis are little understood because we have no models of disease; only humans develop symptoms (dengue fever, DF, or dengue hemorrhagic fever, DHF) and research has been limited to studies involving patients. DENV is very diverse: there are four antigenic groups (serotypes) and three to five genetic groups (genotypes) within each serotype. Thus, it has been difficult to evaluate the relative virulence or transmissibility of each DENV genotype; both of these factors are important determinants of epidemiology and their measurement is complex because the natural cycle of this disease involves human-mosquito-human transmission. Although epidemiological and evolutionary studies have pointed to viral factors in determining disease outcome, only recently developed models could prove the importance of specific viral genotypes in causing severe epidemics and their potential to spread to other continents. These new models involve infection of primary human cell cultures, “humanized” mice and field-collected mosquitoes; also, new mathematical models can estimate the impact of viral replication, human immunity and mosquito transmission on epidemic behavior. DENV evolution does not seem to be rapid and the transmission and dispersal of stable, replication-fit genotypes has been more important in the causation of more severe epidemics. Controversy regarding viral determinants of DENV pathogenesis and epidemiology will continue until virulence and transmissibility can be measured under various conditions.

Tylosema esculentum (Marama) Tuber and Bean Extracts Are Strong Antiviral Agents against Rotavirus Infection

Tylosema esculentum (marama) beans and tubers are used as food, and traditional medicine against diarrhoea in Southern Africa. Rotaviruses (RVs) are a major cause of diarrhoea among infants, young children, immunocompromised people, and domesticated animals. Our work is first to determine anti-RV activity of marama bean and tuber ethanol and water extracts; in this case on intestinal enterocyte cells of human infant (H4), adult pig (CLAB) and adult bovine (CIEB) origin. Marama cotyledon ethanolic extract (MCE) and cotyledon water extract (MCW) without RV were not cytotoxic to all cells tested, while seed coat and tuber extracts showed variable levels of cytotoxicity. Marama cotyledon ethanolic and water extracts (MCE and MCW, resp.) (≥0.1 mg/mL), seed coat extract (MSCE) and seed coat water extract (MSCW) (0.01 to 0.001 mg/mL), especially ethanolic, significantly increased cell survival and enhanced survival to cytopathic effects of RV by at least 100% after in vitro co- and pre-incubation treatments. All marama extracts used significantly enhanced nitric oxide release from H4 cells and enhanced TER (Ω/cm(2)) of enterocyte barriers after coincubation with RV. Marama cotyledon and seed coat extracts inhibited virion infectivity possibly through interference with replication due to accumulation of nitric oxide. Marama extracts are therefore promising microbicides against RV.

Sphaeranthus indicus Linn.: A phytopharmacological review

Sphaeranthus indicus Linn. (Asteraceae) is widely used in Ayurvedic system of medicine to treat vitiated conditions of epilepsy, mental illness, hemicrania, jaundice, hepatopathy, diabetes, leprosy, fever, pectoralgia, cough, gastropathy, hernia, hemorrhoids, helminthiasis, dyspepsia and skin diseases. There are reports providing scientific evidences for hypotensive, anxiolytic, neuroleptic, hypolipidemic, immunomodulatory, antioxidant, anti-inflammatory, bronchodialatory, antihyperglycemic and hepatoprotective activities of this plant. A wide range of phytochemical constituents have been isolated from this plant including sesquiterpene lactones, eudesmenolides, flavanoids and essential oil. A comprehensive account of the morphology, phytochemical constituents, ethnobotanical uses and pharmacological activities reported are included in this review for exploring the immense medicinal potential of this plant.

Autonomous Targeting of Infectious Superspreaders Using Engineered Transmissible Therapies

Infectious disease treatments, both pharmaceutical and vaccine, face three universal challenges: the difficulty of targeting treatments to high-risk ‘superspreader’ populations who drive the great majority of disease spread, behavioral barriers in the host population (such as poor compliance and risk disinhibition), and the evolution of pathogen resistance. Here, we describe a proposed intervention that would overcome these challenges by capitalizing upon Therapeutic Interfering Particles (TIPs) that are engineered to replicate conditionally in the presence of the pathogen and spread between individuals — analogous to ‘transmissible immunization’ that occurs with live-attenuated vaccines (but without the potential for reversion to virulence). Building on analyses of HIV field data from sub-Saharan Africa, we construct a multi-scale model, beginning at the single-cell level, to predict the effect of TIPs on individual patient viral loads and ultimately population-level disease prevalence. Our results show that a TIP, engineered with properties based on a recent HIV gene-therapy trial, could stably lower HIV/AIDS prevalence by ∼30-fold within 50 years and could complement current therapies. In contrast, optimistic antiretroviral therapy or vaccination campaigns alone could only lower HIV/AIDS prevalence by <2-fold over 50 years. The TIP's efficacy arises from its exploitation of the same risk factors as the pathogen, allowing it to autonomously penetrate superspreader populations, maintain efficacy despite behavioral disinhibition, and limit viral resistance. While demonstrated here for HIV, the TIP concept could apply broadly to many viral infectious diseases and would represent a new paradigm for disease control, away from pathogen eradication but toward robust disease suppression.

Screening of Random Peptide Library of Hemagglutinin from Pandemic 2009 A(H1N1) Influenza Virus Reveals Unexpected Antigenically Important Regions

The antigenic structure of the membrane protein hemagglutinin (HA) from the 2009 A(H1N1) influenza virus was dissected with a high-throughput screening method using complex antisera. The approach involves generating yeast cell libraries displaying a pool of random peptides of controllable lengths on the cell surface, followed by one round of fluorescence-activated cell sorting (FACS) against antisera from mouse, goat and human, respectively. The amino acid residue frequency appearing in the antigenic peptides at both the primary sequence and structural level was determined and used to identify “hot spots” or antigenically important regions. Unexpectedly, different antigenic structures were seen for different antisera. Moreover, five antigenic regions were identified, of which all but one are located in the conserved HA stem region that is responsible for membrane fusion. Our findings are corroborated by several recent studies on cross-neutralizing H1 subtype antibodies that recognize the HA stem region. The antigenic peptides identified may provide clues for creating peptide vaccines with better accessibility to memory B cells and better induction of cross-neutralizing antibodies than the whole HA protein. The scheme used in this study enables a direct mapping of the antigenic regions of viral proteins recognized by antisera, and may be useful for dissecting the antigenic structures of other viral proteins.

New Insights of an Old Defense System: Structure, Function, and Clinical Relevance of the Complement System

The complement system was discovered a century ago as a potent defense cascade of innate immunity. After its first description, continuous experimental and clinical research was performed, and three canonical pathways of activation were established. Upon activation by traumatic or surgical tissue damage, complement reveals beneficial functions of pathogen and danger defense by sensing and clearing injured cells. However, the latest research efforts have provided a more distinct insight into the complement system and its clinical subsequences. Complement has been shown to play a significant role in the pathogenesis of various inflammatory processes such as sepsis, multiorgan dysfunction, ischemia/reperfusion, cardiovascular diseases and many others. The three well-known activation pathways of the complement system have been challenged by newer findings that demonstrate direct production of central complement effectors (for example, C5a) by serine proteases of the coagulation cascade. In particular, thrombin is capable of producing C5a, which not only plays a decisive role on pathogens and infected/damaged tissues, but also acts systemically. In the case of uncontrolled complement activation, “friendly fire” is generated, resulting in the destruction of healthy host tissue. Therefore, the traditional research that focuses on a mainly positive-acting cascade has now shifted to the negative effects and how tissue damage originated by the activation of the complement can be contained. In a translational approach including structure-function relations of this ancient defense system, this review provides new insights of complement-mediated clinical relevant diseases and the development of complement modulation strategies and current research aspects.

Targeting vaccination against novel infections: risk, age and spatial structure for pandemic influenza in Great Britain

The emergence of a novel strain of H1N1 influenza virus in Mexico in 2009, and its subsequent worldwide spread, has focused attention to the question of optimal deployment of mass vaccination campaigns. Here, we use three relatively simple models to address three issues of primary concern in the targeting of any vaccine. The advantages of such simple models are that the underlying assumptions and effects of individual parameters are relatively clear, and the impact of uncertainty in the parametrization can be readily assessed in the early stages of an outbreak. In particular, we examine whether targeting risk-groups, age-groups or spatial regions could be optimal in terms of reducing the predicted number of cases or severe effects; and how these targeted strategies vary as the epidemic progresses. We examine the conditions under which it is optimal to initially target vaccination towards those individuals within the population who are most at risk of severe effects of infection. Using age-structured mixing matrices, we show that targeting vaccination towards the more epidemiologically important age groups (5–14 year olds and then 15–24 year olds) leads to the greatest reduction in the epidemic growth and hence reduces the total number of cases. Finally, we consider how spatially targeting the vaccine towards regions of country worst affected could provide an advantage. We discuss how all three of these priorities change as both the speed at which vaccination can be deployed and the start of the vaccination programme is varied.

Networks and the Epidemiology of Infectious Disease

The science of networks has revolutionised research into the dynamics of interacting elements. It could be argued that epidemiology in particular has embraced the potential of network theory more than any other discipline. Here we review the growing body of research concerning the spread of infectious diseases on networks, focusing on the interplay between network theory and epidemiology. The review is split into four main sections, which examine: the types of network relevant to epidemiology; the multitude of ways these networks can be characterised; the statistical methods that can be applied to infer the epidemiological parameters on a realised network; and finally simulation and analytical methods to determine epidemic dynamics on a given network. Given the breadth of areas covered and the ever-expanding number of publications, a comprehensive review of all work is impossible. Instead, we provide a personalised overview into the areas of network epidemiology that have seen the greatest progress in recent years or have the greatest potential to provide novel insights. As such, considerable importance is placed on analytical approaches and statistical methods which are both rapidly expanding fields. Throughout this review we restrict our attention to epidemiological issues.