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The epidemiological and public health research response to 2009 pandemic influenza A(H1N1): experiences from Hong Kong

In recent years, Hong Kong has invested in research infrastructure to appropriately respond to novel infectious disease epidemics. Research from Hong Kong made a strong contribution to the international response to the 2009 influenza A (H1N1) pandemic (pH1N1). Summarizing, describing, and reviewing Hong Kong’s response to the 2009 pandemic, this article aimed to identify key elements of a real‐time research response. A systematic search in PubMed and EMBASE for research into the infection dynamics and natural history, impact, or control of pH1N1 in Hong Kong. Eligible articles were analyzed according to their scope. Fifty‐five articles were included in the review. Transmissibility of pH1N1 was similar in Hong Kong to elsewhere, and only a small fraction of infections were associated with severe disease. School closures were effective in reducing pH1N1 transmission, oseltamivir was effective for treatment of severe cases while convalescent plasma therapy has the potential to mitigate future pandemics. There was a rapid and comprehensive research response to pH1N1 in Hong Kong, providing important information on the epidemiology of the novel virus with relevance internationally as well as locally. The scientific knowledge gained through these detailed studies of pH1N1 is now being used to revise and update pandemic plans. The experiences of the research response in Hong Kong could provide a template for the research response to future emerging and reemerging disease epidemics.

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Microglia Play a Major Role in Direct Viral-Induced Demyelination

Microglia are the resident macrophage-like populations in the central nervous system (CNS). Microglia remain quiescent, unable to perform effector and antigen presentation (APC) functions until activated by injury or infection, and have been suggested to represent the first line of defence for the CNS. Previous studies demonstrated that microglia can be persistently infected by neurotropic mouse hepatitis virus (MHV) which causes meningoencephalitis, myelitis with subsequent axonal loss, and demyelination and serve as a virus-induced model of human neurological disease multiple sclerosis (MS). Current studies revealed that MHV infection is associated with the pronounced activation of microglia during acute inflammation, as evidenced by characteristic changes in cellular morphology and increased expression of microglia-specific proteins, Iba1 (ionized calcium-binding adaptor molecule 1), which is a macrophage/microglia-specific novel calcium-binding protein and involved in membrane ruffling and phagocytosis. During chronic inflammation (day 30 postinfection), microglia were still present within areas of demyelination. Experiments performed in ex vivo spinal cord slice culture and in vitro neonatal microglial culture confirmed direct microglial infection. Our results suggest that MHV can directly infect and activate microglia during acute inflammation, which in turn during chronic inflammation stage causes phagocytosis of myelin sheath leading to chronic inflammatory demyelination.

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Hepatitis C Virus Non-Structural Protein 3 Interacts with Cytosolic 5′(3′)-Deoxyribonucleotidase and Partially Inhibits Its Activity

Infection with hepatitis C virus (HCV) is etiologically involved in liver cirrhosis, hepatocellular carcinoma and B-cell lymphomas. It has been demonstrated previously that HCV non-structural protein 3 (NS3) is involved in cell transformation. In this study, a yeast two-hybrid screening experiment was conducted to identify cellular proteins interacting with HCV NS3 protein. Cytosolic 5′(3′)-deoxyribonucleotidase (cdN, dNT-1) was found to interact with HCV NS3 protein. Binding domains of HCV NS3 and cellular cdN proteins were also determined using the yeast two-hybrid system. Interactions between HCV NS3 and cdN proteins were further demonstrated by co-immunoprecipitation and confocal analysis in cultured cells. The cellular cdN activity was partially repressed by NS3 protein in both the transiently-transfected and the stably-transfected systems. Furthermore, HCV partially repressed the cdN activity while had no effect on its protein expression in the systems of HCV sub-genomic replicons and infectious HCV virions. Deoxyribonucleotidases are present in most mammalian cells and involve in the regulation of intracellular deoxyribonucleotides pools by substrate cycles. Control of DNA precursor concentration is essential for the maintenance of genetic stability. Reduction of cdN activity would result in the imbalance of DNA precursor concentrations. Thus, our results suggested that HCV partially reduced the cdN activity via its NS3 protein and this may in turn cause diseases.

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Dendritic Cell Immunoreceptor Is a New Target for Anti-AIDS Drug Development: Identification of DCIR/HIV-1 Inhibitors

The HIV-1 pandemic continues to expand while no effective vaccine or cure is yet available. Existing therapies have managed to limit mortality and control viral proliferation, but are associated with side effects, do not cure the disease and are subject to development of resistance. Finding new therapeutic targets and drugs is therefore crucial. We have previously shown that the dendritic cell immunoreceptor (DCIR), a C-type lectin receptor expressed on dendritic cells (DCs), acts as an attachment factor for HIV-1 to DCs and contributes to HIV-1 transmission to CD4(+) T lymphocytes (CD4TL). Directly involved in HIV-1 infection, DCIR is expressed in apoptotic or infected CD4TL and promotes trans-infection to bystander cells. Here we report the 3D modelling of the extracellular domain of DCIR. Based on this structure, two surface accessible pockets containing the carbohydrate recognition domain and the EPS binding motif, respectively, were targeted for screening of chemicals that will disrupt normal interaction with HIV-1 particle. Preliminary screening using Raji-CD4-DCIR cells allowed identification of two inhibitors that decreased HIV-1 attachment and propagation. The impact of these inhibitors on infection of DCs and CD4TL was evaluated as well. The results of this study thus identify novel molecules capable of blocking HIV-1 transmission by DCs and CD4TL.

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Adipose-derived stem cells weigh in as novel therapeutics for acute lung injury

Acute lung injury is characterized by intense neutrophilic lung inflammation and increased alveolar-capillary barrier permeability leading to severe hypoxemia, and is associated with high mortality despite improvements in supportive care. There is an urgent need for effective therapies for acute lung injury. Zhang and colleagues tested the efficacy of adipose-derived stem cells in acute lung injury in mice. When adipose-derived stem cells were delivered to mice that had been challenged with lipopolysaccharide, they potently limited acute lung inflammation and injury in the mice, indicating that adipose-derived stem cells have therapeutic potential in acute lung injury in humans. Herein, we discuss the advantages and potential limitations of using adipose-derived stem cells as therapeutics for human acute lung injury.

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A Dual-Mode Surface Display System for the Maturation and Production of Monoclonal Antibodies in Glyco-Engineered Pichia pastoris

State-of-the-art monoclonal antibody (mAb) discovery methods that utilize surface display techniques in prokaryotic and eukaryotic cells require multiple steps of reformatting and switching of hosts to transition from display to expression. This results in a separation between antibody affinity maturation and full-length mAb production platforms. Here, we report for the first time, a method in Glyco-engineered Pichia pastoris that enables simultaneous surface display and secretion of full-length mAb molecules with human-like N-glycans using the same yeast cell. This paradigm takes advantage of homo-dimerization of the Fc portion of an IgG molecule to a surface-anchored "bait" Fc, which results in targeting functional “half” IgGs to the cell wall of Pichia pastoris without interfering with the secretion of full length mAb. We show the utility of this method in isolating high affinity, well-expressed anti-PCSK9 leads from a designed library that was created by mating yeasts containing either light chain or heavy chain IgG libraries. Coupled with Glyco-engineered Pichia pastoris , this method provides a powerful tool for the discovery and production of therapeutic human mAbs in the same host thus improving drug developability and potentially shortening the discovery time cycle.

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Picornavirus uncoating intermediate captured in atomic detail

It remains largely mysterious how the genomes of non-enveloped eukaryotic viruses are transferred across a membrane into the host cell. Picornaviruses are simple models for such viruses, and initiate this uncoating process through particle expansion, which reveals channels through which internal capsid proteins and the viral genome presumably exit the particle, although this has not been clearly seen until now. Here we present the atomic structure of an uncoating intermediate for the major human picornavirus pathogen CAV16, which reveals VP1 partly extruded from the capsid, poised to embed in the host membrane. Together with previous low-resolution results, we are able to propose a detailed hypothesis for the ordered egress of the internal proteins, using two distinct sets of channels through the capsid, and suggest a structural link to the condensed RNA within the particle, which may be involved in triggering RNA release.

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China’s biggest, most neglected health challenge: Non-communicable diseases

BACKGROUND: Over the past two decades, international health policies focusing on the fight against the human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), tuberculosis (TB), malaria, and those diseases that address maternal and child health problems, among others, have skewed disease control priorities in China and other Asian countries. Although these are important health problems, an epidemic of chronic, non-communicable diseases (NCDs) in China has accounted for a much greater burden of disease due to the ongoing rapid socioeconomic and demographic transition. DISCUSSION: Although NCDs currently account for more than 80% of the overall disease burden in China, they remain very low on the nation’s disease control priorities, attracting marginal investment from central and local governments. This leaves the majority of patients with chronic conditions without effective treatment. International organizations and national governments have recognized the devastating social and economic consequences caused by NCDs in low- and middle-income countries, including China. Yet, few donor-funded projects that address NCDs have been implemented in these countries over the past decade. Due to a lack of strong support from international organizations and national governments for fighting against NCDs, affected persons in China, especially the poor and those who live in rural and less developed regions, continue to have limited access to the needed care. Costs associated with frequent health facility visits and regular treatment have become a major factor in medical impoverishment in China. This article argues that although China's ongoing health system reform would provide a unique opportunity to tackle current public health problems, it may not be sufficient to address the emerging threat of NCDs unless targeted steps are taken to assure that adequate financial and human resources are mapped for effective control and management of NCDs in the country. SUMMARY: The Chinese government needs to develop a domestically-driven and evidence-based disease control policy and funding priorities that respond appropriately to the country’s current epidemiological transition, and rapid sociodemographic and lifestyle changes.

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Peptide-Based Vaccinology: Experimental and Computational Approaches to Target Hypervariable Viruses through the Fine Characterization of Protective Epitopes Recognized by Monoclonal Antibodies and the Identification of T-Cell-Activating Peptides

Defining immunogenic domains of viral proteins capable of eliciting a protective immune response is crucial in the development of novel epitope-based prophylactic strategies. This is particularly important for the selective targeting of conserved regions shared among hypervariable viruses. Studying postinfection and postimmunization sera, as well as cloning and characterization of monoclonal antibodies (mAbs), still represents the best approach to identify protective epitopes. In particular, a protective mAb directed against conserved regions can play a key role in immunogen design and in human therapy as well. Experimental approaches aiming to characterize protective mAb epitopes or to identify T-cell-activating peptides are often burdened by technical limitations and can require long time to be correctly addressed. Thus, in the last decade many epitope predictive algorithms have been developed. These algorithms are continually evolving, and their use to address the empirical research is widely increasing. Here, we review several strategies based on experimental techniques alone or addressed by in silico analysis that are frequently used to predict immunogens to be included in novel epitope-based vaccine approaches. We will list the main strategies aiming to design a new vaccine preparation conferring the protection of a neutralizing mAb combined with an effective cell-mediated response.

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Systemic varicella-zoster virus infection in two critically ill patients in an intensive care unit

Varicella-zoster virus (VZV) usually causes localized zoster in adults. However, in immunocompromised patients, it can cause systemic infection accompanied by complications such as pneumonia, encephalitis, and hepatitis. Although most of critically ill patients in intensive care unit (ICU) are immunologically compromised, they are usually not considered to be at risk for systemic VZV infection. We report two cases of systemic VZV infection occurring in critically ill patients in an ICU. One patient was a 69-year-old man with Streptococcus pneumoniae-induced purpurafulminans, and the other was a 75-year-old woman with severe acute pancreatitis. During the clinical course in the ICU, characteristic vesicles with umbilical fossa appeared diffusely and bilaterally on their face, trunk, and extremities. VZV-specific IgG levels were confirmed to be elevated compared to that of the pre-onset, and a diagnosis of recurrent VZV infection was made in both patients. The patients were treated at the same ICU but did not coincide with each other; therefore a cross-infection was unlikely. They were treated with intravenous acyclovir, but the latter patient eventually died of respiratory failure. VZV infection can cause a number of serious complications, and can lead to death in some patients. Early detection and proper treatment are needed to prevent the infection from spreading out and save the patients. It might be necessary to consider antiviral prophylaxis against VZV infection for a part of critically ill patients in ICU, although the effectiveness of this approach is yet to be established.

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Abstracts from the 12th International Symposium on NeuroVirology: October 29–November 2, 2013 Washington, D.C., USA

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Human mobility and the worldwide impact of intentional localized highly pathogenic virus release

The threat of bioterrorism and the possibility of accidental release have spawned a growth of interest in modeling the course of the release of a highly pathogenic agent. Studies focused on strategies to contain local outbreaks after their detection show that timely interventions with vaccination and contact tracing are able to halt transmission. However, such studies do not consider the effects of human mobility patterns. Using a large-scale structured metapopulation model to simulate the global spread of smallpox after an intentional release event, we show that index cases and potential outbreaks can occur in different continents even before the detection of the pathogen release. These results have two major implications: i) intentional release of a highly pathogenic agent within a country will have global effects; ii) the release event may trigger outbreaks in countries lacking the health infrastructure necessary for effective containment. The presented study provides data with potential uses in defining contingency plans at the National and International level.

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Active Surveillance for Influenza A Virus among Swine, Midwestern United States, 2009–2011

Veterinary diagnostic laboratories identify and characterize influenza A viruses primarily through passive surveillance. However, additional surveillance programs are needed. To meet this need, an active surveillance program was conducted at pig farms throughout the midwestern United States. From June 2009 through December 2011, nasal swab samples were collected monthly from among 540 groups of growing pigs and tested for influenza A virus by real-time reverse transcription PCR. Of 16,170 samples, 746 were positive for influenza A virus; of these, 18.0% were subtype H1N1, 16.0% H1N2, 7.6% H3N2, and 14.5% (H1N1)pdm09. An influenza (H3N2) and (H1N1)pdm09 virus were identified simultaneously in 8 groups. This active influenza A virus surveillance program provided quality data and increased the understanding of the current situation of circulating viruses in the midwestern US pig population.

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Transmission Potential of Rift Valley Fever Virus over the Course of the 2010 Epidemic in South Africa

A Rift Valley fever (RVF) epidemic affecting animals on domestic livestock farms was reported in South Africa during January–August 2010. The first cases occurred after heavy rainfall, and the virus subsequently spread countrywide. To determine the possible effect of environmental conditions and vaccination on RVF virus transmissibility, we estimated the effective reproduction number (R(e)) for the virus over the course of the epidemic by extending the Wallinga and Teunis algorithm with spatial information. R(e) reached its highest value in mid-February and fell below unity around mid-March, when vaccination coverage was 7.5%–45.7% and vector-suitable environmental conditions were maintained. The epidemic fade-out likely resulted first from the immunization of animals following natural infection or vaccination. The decline in vector-suitable environmental conditions from April onwards and further vaccination helped maintain R(e) below unity. Increased availability of vaccine use data would enable evaluation of the effect of RVF vaccination campaigns.

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Cosmopolitanism and foreign policy for health: ethics for and beyond the state

BACKGROUND: Foreign policy holds great potential to improve the health of a global citizenship. Our contemporary political order is, in part, characterized by sovereign states acting either in opposition or cooperation with other sovereign states. This order is also characterized by transnational efforts to address transnational issues such as those featured so prominently in the area of global health, such as the spread of infectious disease, health worker migration and the movement of health-harming products. These two features of the current order understandably create tension for truly global initiatives. DISCUSSION: National security has become the dominant ethical frame underlying the health-based foreign policy of many states, despite the transnational nature of many contemporary health challenges. This ethical approach engages global health as a means to achieving national security objectives. Implicit in this ethical frame is the version of humanity that dichotomizes between “us” and “them”. What has been left out of this discourse, for the most part, is the role that foreign policy can play in extending the responsibility of states to protect and promote health of the other, for the sake of the other. SUMMARY: The principal purpose of this paper is to review arguments for a cosmopolitan ethics of health-based foreign policy. I will argue that health-based foreign policy that is motivated by security interests is lacking both morally and practically to further global health goals. In other words, a cosmopolitan ethic is not only intrinsically superior as a moral ideal, but also has potential to contribute to utilitarian ends. This paper draws on the cosmopolitanism literature to build robust support for foreign policies that contribute to sustainable systems of global health governance.

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Insights on conducting research in low-resource settings: examples from Vietnam and Uganda

This commentary describes key observations and strategies, based on the author's experiences in Vietnam and Uganda, for tailoring evidence-based behavioral medicine research in low-resource settings.

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Population-Level Antibody Estimates to Novel Influenza A/H7N9

There are no contemporary data available describing human immunity to novel influenza A/H7N9. Using 1723 prospectively collected serum samples in southern Vietnam, we tested for antibodies to 5 avian influenza virus antigens, using a protein microarray. General-population antibody titers against subtype H7 virus are higher than antibody titers against subtype H5 and lower than titers against H9. The highest titers were observed for human influenza virus subtypes. Titers to avian influenza virus antigens increased with age and with geometric mean antibody titer to human influenza virus antigens. There were no titer differences between the urban and the rural location in our study.

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Development of an Aerosol Model of Cryptococcus Reveals Humidity as an Important Factor Affecting the Viability of Cryptococcus during Aerosolization

Cryptococcus is an emerging global health threat that is annually responsible for over 1,000,000 infections and one third of all AIDS patient deaths. There is an ongoing outbreak of cryptococcosis in the western United States and Canada. Cryptococcosis is a disease resulting from the inhalation of the infectious propagules from the environment. The current and most frequently used animal infection models initiate infection via liquid suspension through intranasal instillation or intravenous injection. These models do not replicate the typically dry nature of aerosol exposure and may hinder our ability to decipher the initial events that lead to clearance or the establishment of infection. We have established a standardized aerosol model of murine infection for the human fungal pathogen Cryptococcus. Aerosolized cells were generated utilizing a Collison nebulizer in a whole-body Madison Chamber at different humidity conditions. The aerosols inside the chamber were sampled using a BioSampler to determine viable aerosol concentration and spray factor (ratio of viable aerosol concentration to total inoculum concentration). We have effectively delivered yeast and yeast-spore mixtures to the lungs of mice and observed the establishment of disease. We observed that growth conditions prior to exposure and humidity within the Madison Chamber during exposure can alter Cryptococcus survival and dose retained in mice.

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Can informal social distancing interventions minimize demand for antiviral treatment during a severe pandemic?

BACKGROUND: In the case of a pandemic, individuals may alter their behaviour. A dynamic model incorporating social distancing can provide a mechanism to consider complex scenarios to support decisions regarding antiviral stockpile size while considering uncertainty around behavioural interventions. We have examined the impact of social distancing measures on the demand for limited healthcare resources such as antiviral drugs from a central stockpile during a severe pandemic. METHODS: We used an existing age-structured model for pandemic influenza in Canada and biologically plausible scenarios for severe influenza transmission within the population. We incorporated data from published reports regarding stated intentions to change behaviour during a pandemic as well as the magnitude and duration of time that individuals expected to maintain the behavioural change. We ran simulations for all combinations of parameter values to identify the projected antiviral requirements in each scenario. RESULTS: With 12 weeks of distancing, the effect is relatively small for the lowest R0 of 1.6 with a projected stockpile to treat 25.6% being required (IQR = 21.7 – 28.7%) unless the proportion of people involved (81%) and magnitude of the behaviour change is large (69% reduction in contacts). If 24 weeks of distancing occurs, with only a low to moderate reduction in contacts (38% or less), it is not possible to bring treatment requirements below 20% regardless of what proportion of the population engages in distancing measures when transmissibility is high (R0 = 2.0; stockpile size = 31%, IQR = 29.2 – 33.5%). CONCLUSIONS: Our results demonstrate that the magnitude and duration of social distancing behaviours during a severe pandemic have an impact on the need for antiviral drugs. However, significant investments over a long period of time (>16 weeks) are required to decrease the need for antiviral treatment to below 10% of the total population for a highly transmissible viral strain (R0 > 1.8). Encouraging individuals to adopt behaviours that decrease their daily contact rate can help to control the spread of the virus until a vaccine becomes available however; relying on these measures to justify stockpiling fewer courses of treatment will not be sufficient in the case of a severe pandemic.

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Expression of Recombinant Antibodies

Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

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Evaluation of Mucosal and Systemic Immune Responses Elicited by GPI-0100- Adjuvanted Influenza Vaccine Delivered by Different Immunization Strategies

Vaccines for protection against respiratory infections should optimally induce a mucosal immune response in the respiratory tract in addition to a systemic immune response. However, current parenteral immunization modalities generally fail to induce mucosal immunity, while mucosal vaccine delivery often results in poor systemic immunity. In order to find an immunization strategy which satisfies the need for induction of both mucosal and systemic immunity, we compared local and systemic immune responses elicited by two mucosal immunizations, given either by the intranasal (IN) or the intrapulmonary (IPL) route, with responses elicited by a mucosal prime followed by a systemic boost immunization. The study was conducted in BALB/c mice and the vaccine formulation was an influenza subunit vaccine supplemented with GPI-0100, a saponin-derived adjuvant. While optimal mucosal antibody titers were obtained after two intrapulmonary vaccinations, optimal systemic antibody responses were achieved by intranasal prime followed by intramuscular boost. The latter strategy also resulted in the best T cell response, yet, it was ineffective in inducing nose or lung IgA. Successful induction of secretory IgA, IgG and T cell responses was only achieved with prime-boost strategies involving intrapulmonary immunization and was optimal when both immunizations were given via the intrapulmonary route. Our results underline that immunization via the lungs is particularly effective for priming as well as boosting of local and systemic immune responses.

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The role of pattern recognition receptors in intestinal inflammation

Recognition of microorganisms by pattern-recognition receptors (PRRs) is the primary component of innate immunity that is responsible for the maintenance of host–microbial interactions in intestinal mucosa. Dysregulation in host–commensal interactions has been implicated as the central pathogenesis of inflammatory bowel disease (IBD), which predisposes to developing colorectal cancer. Recent animal studies have begun to outline some unique physiology and pathology involving each PRR signaling in the intestine. The major roles played by PRRs in the gut appear to be the regulation of the number and the composition of commensal bacteria, epithelial proliferation, and mucosal permeability in response to epithelial injury. In addition, PRR signaling in lamina propria immune cells may be involved in induction of inflammation in response to invasion of pathogens. Because some PRR-deficient mice have shown variable susceptibility to colitis, the outcome of intestinal inflammation may be modified depending on PRR signaling in epithelial cells, immune cells, and the composition of commensal flora. Through recent findings in animal models of IBD, this review will discuss how abnormal PRR signaling may contribute to the pathogenesis of inflammation and inflammation-associated tumorigenesis in the intestine. SUPPLEMENTARY INFORMATION: The online version of this article (doi:10.1038/mi.2013.13) contains supplementary material, which is available to authorized users.

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Steps to a Sustainable Public Health Surveillance Enterprise A Commentary from the International Society for Disease Surveillance

More than a decade into the 21(st) century, the ability to effectively monitor community health status, as well as forecast, detect, and respond to disease outbreaks and other events of public health significance, remains a major challenge. As an issue that affects population health, economic stability, and global security, the public health surveillance enterprise warrants the attention of decision makers at all levels. Public health practitioners responsible for surveillance functions are best positioned to identify the key elements needed for creating and maintaining effective and sustainable surveillance systems. This paper presents the recommendations of the Sustainable Surveillance Workgroup convened by the International Society for Disease Surveillance (ISDS) to identify strategies for building, strengthening, and maintaining surveillance systems that are equipped to provide data continuity and to handle both established and new data sources and public health surveillance practices.

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TNF-α Acts as an Immunoregulator in the Mouse Brain by Reducing the Incidence of Severe Disease Following Japanese Encephalitis Virus Infection

Japanese encephalitis virus (JEV) causes acute central nervous system (CNS) disease in humans, in whom the clinical symptoms vary from febrile illness to meningitis and encephalitis. However, the mechanism of severe encephalitis has not been fully elucidated. In this study, using a mouse model, we investigated the pathogenetic mechanisms that correlate with fatal JEV infection. Following extraneural infection with the JaOArS982 strain of JEV, infected mice exhibited clinical signs ranging from mild to fatal outcome. Comparison of the pathogenetic response between severe and mild cases of JaOArS982-infected mice revealed increased levels of TNF-α in the brains of severe cases. However, unexpectedly, the mortality rate of TNF-α KO mice was significantly increased compared with that of WT mice, indicating that TNF-α plays a protective role against fatal infection. Interestingly, there were no significant differences of viral load in the CNS between WT and TNF-α KO mice. However, exaggerated inflammatory responses were observed in the CNS of TNF-α KO mice. Although these observations were also obtained in IL-10 KO mice, the mortality and enhanced inflammatory responses were more pronounced in TNF-α KO mice. Our findings therefore provide the first evidence that TNF-α has an immunoregulatory effect on pro-inflammatory cytokines in the CNS during JEV infection and consequently protects the animals from fatal disease. Thus, we propose that the increased level of TNF-α in severe cases was the result of severe disease, and secondly that immunopathological effects contribute to severe neuronal degeneration resulting in fatal disease. In future, further elucidation of the immunoregulatory mechanism of TNF-α will be an important priority to enable the development of effective treatment strategies for Japanese encephalitis.

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Design, Synthesis, Evaluation and Thermodynamics of 1-Substituted Pyridylimidazo[1,5-a]Pyridine Derivatives as Cysteine Protease Inhibitors

Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their K(i) and IC(50) values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC(50) values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a).

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Estimating the reproductive number in the presence of spatial heterogeneity of transmission patterns

BACKGROUND: Estimates of parameters for disease transmission in large-scale infectious disease outbreaks are often obtained to represent large groups of people, providing an average over a potentially very diverse area. For control measures to be more effective, a measure of the heterogeneity of the parameters is desirable. METHODS: We propose a novel extension of a network-based approach to estimating the reproductive number. With this we can incorporate spatial and/or demographic information through a similarity matrix. We apply this to the 2009 Influenza pandemic in South Africa to understand the spatial variability across provinces. We explore the use of five similarity matrices to illustrate their impact on the subsequent epidemic parameter estimates. RESULTS: When treating South Africa as a single entity with homogeneous transmission characteristics across the country, the basic reproductive number, R(0), (and imputation range) is 1.33 (1.31, 1.36). When fitting a new model for each province with no inter-province connections this estimate varies little (1.23-1.37). Using the proposed method with any of the four similarity measures yields an overall R(0) that varies little across the four new models (1.33 to 1.34). However, when allowed to vary across provinces, the estimated R(0) is greater than one consistently in only two of the nine provinces, the most densely populated provinces of Gauteng and Western Cape. CONCLUSIONS: Our results suggest that the spatial heterogeneity of influenza transmission was compelling in South Africa during the 2009 pandemic. This variability makes a qualitative difference in our understanding of the epidemic. While the cause of this fluctuation might be partially due to reporting differences, there is substantial evidence to warrant further investigation.

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Complete Genome Sequence of Porcine Epidemic Diarrhea Virus Strain USA/Colorado/2013 from the United States

Porcine epidemic diarrhea virus (PEDV) is newly emerging in the United States. PEDV strain USA/Colorado/2013 (CO/13) was obtained from a 7-day-old piglet with severe diarrhea, and the complete genome was sequenced to further study the PEDV outbreak in the United States.

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A Prospective Evaluation of Real-Time PCR Assays for the Detection of Orientia tsutsugamushi and Rickettsia spp. for Early Diagnosis of Rickettsial Infections during the Acute Phase of Undifferentiated Febrile Illness

One hundred and eighty febrile patients were analyzed in a prospective evaluation of Orientia tsutsugamushi and Rickettsia spp. real-time polymerase chain reaction (PCR) assays for early diagnosis of rickettsial infections. By paired serology, 3.9% (7 of 180) and 6.1% (11 of 180) of patients were confirmed to have acute scrub or murine typhus, respectively. The PCR assays for the detection of O. tsutsugamushi and Rickettsia spp. had high specificity (99.4% [95% confidence interval (CI): 96.8–100] and 100% [95% CI: 97.8–100], respectively). The PCR results were also compared with immunoglobulin M (IgM) immunofluorescence assay (IFA) on acute sera. For O. tsutsugamushi, PCR sensitivity was twice that of acute specimen IgM IFA (28.6% versus 14.3%; McNemar's P = 0.3). For Rickettsia spp., PCR was four times as sensitive as acute specimen IgM IFA (36.4% versus 9.1%; P = 0.08), although this was not statistically significant. Whole blood and buffy coat, but not serum, were acceptable specimens for these PCRs. Further evaluation of these assays in a larger prospective study is warranted.

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A Protective and Safe Intranasal RSV Vaccine Based on a Recombinant Prefusion-Like Form of the F Protein Bound to Bacterium-Like Particles

Respiratory syncytial virus (RSV) is an important cause of respiratory tract disease in infants and the elderly. Currently, no licensed vaccine against RSV is available. Here we describe the development of a safe and effective intranasal subunit vaccine that is based on recombinant fusion (F) protein bound to the surface of immunostimulatory bacterium-like particles (BLPs) derived from the food-grade bacterium Lactococcus lactis. Different variants of F were analyzed with respect to their conformation and reactivity with neutralizing antibodies, assuming that F proteins mimicking the metastable prefusion form of RSV F expose a more extensive and relevant epitope repertoire than F proteins corresponding to the postfusion structure. Our results indicate that the recombinant soluble ectodomain of RSV F readily adopts a postfusion conformation, generation of which cannot be prevented by C-terminal addition of a trimerization motif, but whose formation is prevented by mutation of the two furin cleavage sites in F. While the putative postfusion form of F is recognized well by the monoclonal antibody Palivizumab, this is much less so for the more potently neutralizing, prefusion-specific antibodies D25 and AM22. Both addition of the trimerization motif and mutation of the furin cleavage sites increased the reactivity of F with D25 and AM22, with the highest reactivity being observed for F proteins in which both these features were combined. Intranasal vaccination of mice or cotton rats with BLPs loaded with this latter prefusion-like F protein (BLP-F), resulted in the potent induction of F-specific immunoglobulins and in significantly decreased virus titers in the lungs upon RSV challenge. Moreover, and in contrast to animals vaccinated with formalin-inactivated RSV, animals that received BLP-F exhibited high levels of F-specific secretory IgA in the nose and RSV-neutralizing antibodies in sera, but did not show symptoms of enhanced disease after challenge with RSV.

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Using the Electronic Medical Record to Identify Community-Acquired Pneumonia: Toward a Replicable Automated Strategy

BACKGROUND: Timely information about disease severity can be central to the detection and management of outbreaks of acute respiratory infections (ARI), including influenza. We asked if two resources: 1) free text, and 2) structured data from an electronic medical record (EMR) could complement each other to identify patients with pneumonia, an ARI severity landmark. METHODS: A manual EMR review of 2747 outpatient ARI visits with associated chest imaging identified x-ray reports that could support the diagnosis of pneumonia (kappa score = 0.88 (95% CI 0.82∶0.93)), along with attendant cases with Possible Pneumonia (adds either cough, sputum, fever/chills/night sweats, dyspnea or pleuritic chest pain) or with Pneumonia-in-Plan (adds pneumonia stated as a likely diagnosis by the provider). The x-ray reports served as a reference to develop a text classifier using machine-learning software that did not require custom coding. To identify pneumonia cases, the classifier was combined with EMR-based structured data and with text analyses aimed at ARI symptoms in clinical notes. RESULTS: 370 reference cases with Possible Pneumonia and 250 with Pneumonia-in-Plan were identified. The x-ray report text classifier increased the positive predictive value of otherwise identical EMR-based case-detection algorithms by 20–70%, while retaining sensitivities of 58–75%. These performance gains were independent of the case definitions and of whether patients were admitted to the hospital or sent home. Text analyses seeking ARI symptoms in clinical notes did not add further value. CONCLUSION: Specialized software development is not required for automated text analyses to help identify pneumonia patients. These results begin to map an efficient, replicable strategy through which EMR data can be used to stratify ARI severity.

49u2onq0

A Compact Viral Processing Proteinase/Ubiquitin Hydrolase from the OTU Family

Turnip yellow mosaic virus (TYMV) - a member of the alphavirus-like supergroup of viruses - serves as a model system for positive-stranded RNA virus membrane-bound replication. TYMV encodes a precursor replication polyprotein that is processed by the endoproteolytic activity of its internal cysteine proteinase domain (PRO). We recently reported that PRO is actually a multifunctional enzyme with a specific ubiquitin hydrolase (DUB) activity that contributes to viral infectivity. Here, we report the crystal structure of the 150-residue PRO. Strikingly, PRO displays no homology to other processing proteinases from positive-stranded RNA viruses, including that of alphaviruses. Instead, the closest structural homologs of PRO are DUBs from the Ovarian tumor (OTU) family. In the crystal, one molecule's C-terminus inserts into the catalytic cleft of the next, providing a view of the N-terminal product complex in replication polyprotein processing. This allows us to locate the specificity determinants of PRO for its proteinase substrates. In addition to the catalytic cleft, at the exit of which the active site is unusually pared down and solvent-exposed, a key element in molecular recognition by PRO is a lobe N-terminal to the catalytic domain. Docking models and the activities of PRO and PRO mutants in a deubiquitylating assay suggest that this N-terminal lobe is also likely involved in PRO's DUB function. Our data thus establish that DUBs can evolve to specifically hydrolyze both iso- and endopeptide bonds with different sequences. This is achieved by the use of multiple specificity determinants, as recognition of substrate patches distant from the cleavage sites allows a relaxed specificity of PRO at the sites themselves. Our results thus shed light on how such a compact protein achieves a diversity of key functions in viral genome replication and host-pathogen interaction.

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Enhanced Nasal Mucosal Delivery and Immunogenicity of Anti-Caries DNA Vaccine through Incorporation of Anionic Liposomes in Chitosan/DNA Complexes

The design of optimized nanoparticles offers a promising strategy to enable DNA vaccines to cross various physiological barriers for eliciting a specific and protective mucosal immunity via intranasal administration. Here, we reported a new designed nanoparticle system through incorporating anionic liposomes (AL) into chitosan/DNA (CS/DNA) complexes. With enhanced cellular uptake, the constructed AL/CS/DNA nanoparticles can deliver the anti-caries DNA vaccine pGJA-P/VAX into nasal mucosa. TEM results showed the AL/CS/DNA had a spherical structure. High DNA loading ability and effective DNA protection against nuclease were proved by gel electrophoresis. The surface charge of the AL/CS/DNA depended strongly on pH environment, enabling the intracellular release of loaded DNA via a pH-mediated manner. In comparison to the traditional CS/DNA system, our new design rendered a higher transfection efficiency and longer residence time of the AL/CS/DNA at nasal mucosal surface. These outstanding features enable the AL/CS/DNA to induce a significantly (p<0.01) higher level of secretory IgA (SIgA) than the CS/DNA in animal study, and a longer-term mucosal immunity. On the other hand, the AL/CS/DNA exhibited minimal cytotoxicity. These results suggest that the developed nanoparticles offer a potential platform for DNA vaccine packaging and delivery for more efficient elicitation of mucosal immunity.

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Proposals for the classification of human rhinovirus species A, B and C into genotypically assigned types

Human rhinoviruses (HRVs) frequently cause mild upper respiratory tract infections and more severe disease manifestations such as bronchiolitis and asthma exacerbations. HRV is classified into three species within the genus Enterovirus of the family Picornaviridae. HRV species A and B contain 75 and 25 serotypes identified by cross-neutralization assays, although the use of such assays for routine HRV typing is hampered by the large number of serotypes, replacement of virus isolation by molecular methods in HRV diagnosis and the poor or absent replication of HRV species C in cell culture. To address these problems, we propose an alternative, genotypic classification of HRV-based genetic relatedness analogous to that used for enteroviruses. Nucleotide distances between 384 complete VP1 sequences of currently assigned HRV (sero)types identified divergence thresholds of 13, 12 and 13 % for species A, B and C, respectively, that divided inter- and intra-type comparisons. These were paralleled by 10, 9.5 and 10 % thresholds in the larger dataset of >3800 VP4 region sequences. Assignments based on VP1 sequences led to minor revisions of existing type designations (such as the reclassification of serotype pairs, e.g. A8/A95 and A29/A44, as single serotypes) and the designation of new HRV types A101–106, B101–103 and C34–C51. A protocol for assignment and numbering of new HRV types using VP1 sequences and the restriction of VP4 sequence comparisons to type identification and provisional type assignments is proposed. Genotypic assignment and identification of HRV types will be of considerable value in the future investigation of type-associated differences in disease outcomes, transmission and epidemiology.

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Identification of MicroRNA-Like RNAs in Mycelial and Yeast Phases of the Thermal Dimorphic Fungus Penicillium marneffei

BACKGROUND: Penicillium marneffei is the most important thermal dimorphic fungus causing systemic mycosis in China and Southeast Asia. While miRNAs are increasingly recognized for their roles in post-transcriptional regulation of gene expression in animals and plants, miRNAs in fungi were less well studied and their potential roles in fungal dimorphism were largely unknown. Based on P. marneffei genome sequence, we hypothesize that miRNA-like RNAs (milRNAs) may be expressed in the dimorphic fungus. METHODOLOGY/PRINCIPAL FINDINGS: We attempted to identify milRNAs in P. marneffei in both mycelial and yeast phase using high-throughput sequencing technology. Small RNAs were more abundantly expressed in mycelial than yeast phase. Sequence analysis revealed 24 potential milRNA candidates, including 17 candidates in mycelial and seven in yeast phase. Two genes, dcl-1 and dcl-2, encoding putative Dicer-like proteins and the gene, qde-2, encoding Argonaute-like protein, were identified in P. marneffei. Phylogenetic analysis showed that dcl-2 of P. marneffei was more closely related to the homologues in other thermal dimorphic pathogenic fungi than to Penicillium chrysogenum and Aspergillus spp., suggesting the co-evolution of dcl-2 among the thermal dimorphic fungi. Moreover, dcl-2 demonstrated higher mRNA expression levels in mycelial than yeast phase by 7 folds (P<0.001). Northern blot analysis confirmed the expression of two milRNAs, PM-milR-M1 and PM-milR-M2, only in mycelial phase. Using dcl-1(KO), dcl-2(KO), dcl(DKO) and qde-2(KO) deletion mutants, we showed that the biogenesis of both milRNAs were dependent on dcl-2 but not dcl-1 or qde-2. The mRNA expression levels of three predicted targets of PM-milR-M1 were upregulated in knockdown strain PM-milR-M1 (KD), supporting regulatory function of milRNAs. CONCLUSIONS/SIGNIFICANCE: Our findings provided the first evidence for differential expression of milRNAs in different growth phases of thermal dimorphic fungi and shed light on the evolution of fungal proteins involved in milRNA biogenesis and possible role of post-transcriptional control in governing thermal dimorphism.

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Severe leukopenia in Staphylococcus aureus-necrotizing, community-acquired pneumonia: risk factors and impact on survival

BACKGROUND: Necrotizing pneumonia attributed to Panton-Valentine leukocidin-positive Staphylococcus aureus has mainly been reported in otherwise healthy children and young adults, with a high mortality rate. Erythroderma, airway bleeding, and leukopenia have been shown to be predictive of mortality. The objectives of this study were to define the characteristics of patients with severe leukopenia at 48-h hospitalization and to update our data regarding mortality predicting factors in a larger population than we had previously described. METHODS: It was designed as a case-case study nested in a cohort study. A total of 148 cases of community-acquired, necrotizing pneumonia were included. The following data were collected: basic demographic information, medical history, signs and symptoms, radiological findings and laboratory results during the first 48 h of hospitalization. The study population was divided into 2 groups: (1) with severe leukopenia (leukocyte count ≤3,000 leukocytes/mL, n=62) and (2) without severe leukopenia (>3,000 leukocytes/mL, n=86). RESULTS: Median age was 22 years, and the male-to-female gender ratio was 1.5. The overall in-hospital mortality rate was 41.2%. Death occurred in 75.8% of severe leukopenia cases with median survival time of 4 days, and in 16.3% of cases with leukocyte count >3,000/mL (P<0.001). Multivariate analysis indicated that the factors associated with severe leukopenia were influenza-like illness (adjusted odds ratio (aOR) 4.45, 95% CI (95% confidence interval) 1.67-11.88, P=0.003), airway bleeding (aOR 4.53, 95% CI 1.85-11.13, P=0.001) and age over 30 years (aOR 2.69, 95% CI 1.08-6.68, P=0.033). A personal history of furuncles appeared to be protective (OR 0.11, 95% CI 0.01-0.96, P=0.046). CONCLUSION: S. aureus-necrotizing pneumonia is still an extremely severe disease in patients with severe leukopenia. Some factors could distinguish these patients, allowing better initial identification to initiate adapted, rapid administration of appropriate therapy.

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The mouse and ferret models for studying the novel avian-origin human influenza A (H7N9) virus

BACKGROUND: The current study was conducted to establish animal models (including mouse and ferret) for the novel avian-origin H7N9 influenza virus. FINDINGS: A/Anhui/1/2013 (H7N9) virus was administered by intranasal instillation to groups of mice and ferrets, and animals developed typical clinical signs including body weight loss (mice and ferrets), ruffled fur (mice), sneezing (ferrets), and death (mice). Peak virus shedding from respiratory tract was observed on 2 days post inoculation (d.p.i.) for mice and 3–5 d.p.i. for ferrets. Virus could also be detected in brain, liver, spleen, kidney, and intestine from inoculated mice, and in heart, liver, and olfactory bulb from inoculated ferrets. The inoculation of H7N9 could elicit seroconversion titers up to 1280 in ferrets and 160 in mice. Leukopenia, significantly reduced lymphocytes but increased neutrophils were also observed in mouse and ferret models. CONCLUSIONS: The mouse and ferret model enables detailed studies of the pathogenesis of this illness and lay the foundation for drug or vaccine evaluation.

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The role of helium gas in medicine

The noble gas helium has many applications owing to its distinct physical and chemical characteristics, namely: its low density, low solubility, and high thermal conductivity. Chiefly, the abundance of studies in medicine relating to helium are concentrated in its possibility of being used as an adjunct therapy in a number of respiratory ailments such as asthma exacerbation, COPD, ARDS, croup, and bronchiolitis. Helium gas, once believed to be biologically inert, has been recently shown to be beneficial in protecting the myocardium from ischemia by various mechanisms. Though neuroprotection of brain tissue has been documented, the mechanism by which it does so has yet to be made clear. Surgeons are exploring using helium instead of carbon dioxide to insufflate the abdomen of patients undergoing laparoscopic abdominal procedures due to its superiority in preventing respiratory acidosis in patients with comorbid conditions that cause carbon dioxide retention. Newly discovered applications in Pulmonary MRI radiology and imaging of organs in very fine detail using Helium Ion Microscopy has opened exciting new possibilities for the use of helium gas in technologically advanced fields of medicine.

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Co-expression of RNA–protein complexes in Escherichia coli and applications to RNA biology

RNA has emerged as a major player in many cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. In this work, we have extended this method to RNA/protein co-expression. We have engineered several plasmids that allow overexpression of RNA–protein complexes in E. coli. We have investigated the potential of these tools in many applications, including the production of nuclease-sensitive RNAs encapsulated in viral protein pseudo-particles, the co-production of non-coding RNAs with chaperone proteins, the incorporation of a post-transcriptional RNA modification by co-production with the appropriate modifying enzyme and finally the production and purification of an RNA–His-tagged protein complex by nickel affinity chromatography. We show that this last application easily provides pure material for crystallographic studies. The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins.

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Duplex Molecular Assay Intended for Point-of-Care Diagnosis of Influenza A/B Virus Infection

Early diagnosis and management of influenza virus infection directly correlates with the effectiveness in disease control. Current molecular influenza virus tests were designed for use in diagnostic testing facilities, where sophisticated equipment and highly trained technicians are available. A longer turnaround time for the centralized testing than when testing near the sample source could delay the initiation of medical intervention, thereby reducing the efficacy of antiviral treatment. The new assay, the SAMBA (simple amplification-based assay) Flu duplex test, is a dipstick-based molecular assay developed to provide a simple, accurate, and cost-effective solution for the diagnosis of influenza A/B viruses intended for near-patient testing. The test presents an alternative format of influenza virus molecular testing that utilizes isothermal amplification and visual detection of nucleic acid on a test strip. The entire test procedure (extraction, amplification, and detection) is integrated into an enclosed semiautomated system. Analytically, the SAMBA Flu duplex test detects 95 and 85 copies of viral genomes for influenza A and B viruses, respectively, with no cross-reactivity observed against other common respiratory pathogens. The clinical performance was established by blind testing of 328 nasal/throat and nasopharyngeal swab specimens from the United Kingdom and Belgium and comparing the results with the quantitative reverse transcription-PCR method routinely used in two public health laboratories. The SAMBA Flu duplex test showed a clinical sensitivity and specificity of 100% and 97.9% for influenza virus A and 100% and 100% for influenza virus B. The test provides a new technology that could facilitate simple and timely identification of influenza virus infection, potentially resulting in more efficient control measures.

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iGPCR-Drug: A Web Server for Predicting Interaction between GPCRs and Drugs in Cellular Networking

Involved in many diseases such as cancer, diabetes, neurodegenerative, inflammatory and respiratory disorders, G-protein-coupled receptors (GPCRs) are among the most frequent targets of therapeutic drugs. It is time-consuming and expensive to determine whether a drug and a GPCR are to interact with each other in a cellular network purely by means of experimental techniques. Although some computational methods were developed in this regard based on the knowledge of the 3D (dimensional) structure of protein, unfortunately their usage is quite limited because the 3D structures for most GPCRs are still unknown. To overcome the situation, a sequence-based classifier, called “iGPCR-drug”, was developed to predict the interactions between GPCRs and drugs in cellular networking. In the predictor, the drug compound is formulated by a 2D (dimensional) fingerprint via a 256D vector, GPCR by the PseAAC (pseudo amino acid composition) generated with the grey model theory, and the prediction engine is operated by the fuzzy K-nearest neighbour algorithm. Moreover, a user-friendly web-server for iGPCR-drug was established at http://www.jci-bioinfo.cn/iGPCR-Drug/. For the convenience of most experimental scientists, a step-by-step guide is provided on how to use the web-server to get the desired results without the need to follow the complicated math equations presented in this paper just for its integrity. The overall success rate achieved by iGPCR-drug via the jackknife test was 85.5%, which is remarkably higher than the rate by the existing peer method developed in 2010 although no web server was ever established for it. It is anticipated that iGPCR-Drug may become a useful high throughput tool for both basic research and drug development, and that the approach presented here can also be extended to study other drug – target interaction networks.

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Deubiquitylating Enzymes and DNA Damage Response Pathways

Covalent post-translational modification of proteins by ubiquitin and ubiquitin-like factors has emerged as a general mechanism to regulate myriad intra-cellular processes. The addition and removal of ubiquitin or ubiquitin-like proteins from factors has recently been demonstrated as a key mechanism to modulate DNA damage response (DDR) pathways. It is thus, timely to evaluate the potential for ubiquitin pathway enzymes as DDR drug targets for therapeutic intervention. The synthetic lethal approach provides exciting opportunities for the development of targeted therapies to treat cancer: most tumours have lost critical DDR pathways, and thus rely more heavily on the remaining pathways, while normal tissues are still equipped with all DDR pathways. Here, we review key deubiquitylating enzymes (DUBs) involved in DDR pathways, and describe how targeting DUBs may lead to selective therapies to treat cancer patients.

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The Emergent Discipline of Health Web Science

The transformative power of the Internet on all aspects of daily life, including health care, has been widely recognized both in the scientific literature and in public discourse. Viewed through the various lenses of diverse academic disciplines, these transformations reveal opportunities realized, the promise of future advances, and even potential problems created by the penetration of the World Wide Web for both individuals and for society at large. Discussions about the clinical and health research implications of the widespread adoption of information technologies, including the Internet, have been subsumed under the disciplinary label of Medicine 2.0. More recently, however, multi-disciplinary research has emerged that is focused on the achievement and promise of the Web itself, as it relates to healthcare issues. In this paper, we explore and interrogate the contributions of the burgeoning field of Web Science in relation to health maintenance, health care, and health policy. From this, we introduce Health Web Science as a subdiscipline of Web Science, distinct from but overlapping with Medicine 2.0. This paper builds on the presentations and subsequent interdisciplinary dialogue that developed among Web-oriented investigators present at the 2012 Medicine 2.0 Conference in Boston, Massachusetts.

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Estimating a Markovian Epidemic Model Using Household Serial Interval Data from the Early Phase of an Epidemic

The clinical serial interval of an infectious disease is the time between date of symptom onset in an index case and the date of symptom onset in one of its secondary cases. It is a quantity which is commonly collected during a pandemic and is of fundamental importance to public health policy and mathematical modelling. In this paper we present a novel method for calculating the serial interval distribution for a Markovian model of household transmission dynamics. This allows the use of Bayesian MCMC methods, with explicit evaluation of the likelihood, to fit to serial interval data and infer parameters of the underlying model. We use simulated and real data to verify the accuracy of our methodology and illustrate the importance of accounting for household size. The output of our approach can be used to produce posterior distributions of population level epidemic characteristics.

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H7N9 Influenza: The Emerging Infectious Disease

Influenza virus infection is a common respiratory pathogen. Emerging of new atypical influenza is usually a big public health threat. H7N9 bird flu is the newest atypical influenza virus infection that has just been reported since early 2013. The emerging of this new disease occurred in China and becomes the present focus for possible worldwide pandemic. In this specific article, the author will discus and describe on epidemiology, symptomatology, pathology, diagnosis, treatment, and prevention of this new bird flu. The literature researching by PubMed and Google is used for data gathering in this collective review.

9g8rwsm1

Assessment of the Evolution of Cancer Treatment Therapies

Cancer therapy has been characterized throughout history by ups and downs, not only due to the ineffectiveness of treatments and side effects, but also by hope and the reality of complete remission and cure in many cases. Within the therapeutic arsenal, alongside surgery in the case of solid tumors, are the antitumor drugs and radiation that have been the treatment of choice in some instances. In recent years, immunotherapy has become an important therapeutic alternative, and is now the first choice in many cases. Nanotechnology has recently arrived on the scene, offering nanostructures as new therapeutic alternatives for controlled drug delivery, for combining imaging and treatment, applying hyperthermia, and providing directed target therapy, among others. These therapies can be applied either alone or in combination with other components (antibodies, peptides, folic acid, etc.). In addition, gene therapy is also offering promising new methods for treatment. Here, we present a review of the evolution of cancer treatments, starting with chemotherapy, surgery, radiation and immunotherapy, and moving on to the most promising cutting-edge therapies (gene therapy and nanomedicine). We offer an historical point of view that covers the arrival of these therapies to clinical practice and the market, and the promises and challenges they present.

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Asymmetry of Cell Division in CFSE-Based Lymphocyte Proliferation Analysis

Flow cytometry-based analysis of lymphocyte division using carboxyfluorescein succinimidyl ester (CFSE) dye dilution permits acquisition of data describing cellular proliferation and differentiation. For example, CFSE histogram data enable quantitative insight into cellular turnover rates by applying mathematical models and parameter estimation techniques. Several mathematical models have been developed using different types of deterministic or stochastic approaches. However, analysis of CFSE proliferation assays is based on the premise that the label is halved in the two daughter cells. Importantly, asymmetry of protein distribution in lymphocyte division is a basic biological feature of cell division with the degree of the asymmetry depending on various factors. Here, we review the recent literature on asymmetric lymphocyte division and CFSE-based lymphocyte proliferation analysis. We suggest that division- and label-structured mathematical models describing CFSE-based cell proliferation should take into account asymmetry and time-lag in cell proliferation. Utilization of improved modeling algorithms will permit straightforward quantification of essential parameters describing the performance of activated lymphocytes.

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A Strategy To Estimate Unknown Viral Diversity in Mammals

The majority of emerging zoonoses originate in wildlife, and many are caused by viruses. However, there are no rigorous estimates of total viral diversity (here termed “virodiversity”) for any wildlife species, despite the utility of this to future surveillance and control of emerging zoonoses. In this case study, we repeatedly sampled a mammalian wildlife host known to harbor emerging zoonotic pathogens (the Indian Flying Fox, Pteropus giganteus) and used PCR with degenerate viral family-level primers to discover and analyze the occurrence patterns of 55 viruses from nine viral families. We then adapted statistical techniques used to estimate biodiversity in vertebrates and plants and estimated the total viral richness of these nine families in P. giganteus to be 58 viruses. Our analyses demonstrate proof-of-concept of a strategy for estimating viral richness and provide the first statistically supported estimate of the number of undiscovered viruses in a mammalian host. We used a simple extrapolation to estimate that there are a minimum of 320,000 mammalian viruses awaiting discovery within these nine families, assuming all species harbor a similar number of viruses, with minimal turnover between host species. We estimate the cost of discovering these viruses to be ~$6.3 billion (or ~$1.4 billion for 85% of the total diversity), which if annualized over a 10-year study time frame would represent a small fraction of the cost of many pandemic zoonoses.

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The Viruses of Wild Pigeon Droppings

Birds are frequent sources of emerging human infectious diseases. Viral particles were enriched from the feces of 51 wild urban pigeons (Columba livia) from Hong Kong and Hungary, their nucleic acids randomly amplified and then sequenced. We identified sequences from known and novel species from the viral families Circoviridae, Parvoviridae, Picornaviridae, Reoviridae, Adenovirus, Astroviridae, and Caliciviridae (listed in decreasing number of reads), as well as plant and insect viruses likely originating from consumed food. The near full genome of a new species of a proposed parvovirus genus provisionally called Aviparvovirus contained an unusually long middle ORF showing weak similarity to an ORF of unknown function from a fowl adenovirus. Picornaviruses found in both Asia and Europe that are distantly related to the turkey megrivirus and contained a highly divergent 2A1 region were named mesiviruses. All eleven segments of a novel rotavirus subgroup related to a chicken rotavirus in group G were sequenced and phylogenetically analyzed. This study provides an initial assessment of the enteric virome in the droppings of pigeons, a feral urban species with frequent human contact.

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Problem- and Case-Based Learning in Science: An Introduction to Distinctions, Values, and Outcomes

Case-based learning and problem-based learning have demonstrated great promise in reforming science education. Yet an instructor, in newly considering this suite of interrelated pedagogical strategies, faces a number of important instructional choices. Different features and their related values and learning outcomes are profiled here, including: the level of student autonomy; instructional focus on content, skills development, or nature-of-science understanding; the role of history, or known outcomes; scope, clarity, and authenticity of problems provided to students; extent of collaboration; complexity, in terms of number of interpretive perspectives; and, perhaps most importantly, the role of applying versus generating knowledge.

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Human Health Risk Assessment (HHRA) for Environmental Development and Transfer of Antibiotic Resistance

Background: Only recently has the environment been clearly implicated in the risk of antibiotic resistance to clinical outcome, but to date there have been few documented approaches to formally assess these risks. Objective: We examined possible approaches and sought to identify research needs to enable human health risk assessments (HHRA) that focus on the role of the environment in the failure of antibiotic treatment caused by antibiotic-resistant pathogens. Methods: The authors participated in a workshop held 4–8 March 2012 in Québec, Canada, to define the scope and objectives of an environmental assessment of antibiotic-resistance risks to human health. We focused on key elements of environmental-resistance-development “hot spots,” exposure assessment (unrelated to food), and dose response to characterize risks that may improve antibiotic-resistance management options. Discussion: Various novel aspects to traditional risk assessments were identified to enable an assessment of environmental antibiotic resistance. These include a) accounting for an added selective pressure on the environmental resistome that, over time, allows for development of antibiotic-resistant bacteria (ARB); b) identifying and describing rates of horizontal gene transfer (HGT) in the relevant environmental “hot spot” compartments; and c) modifying traditional dose–response approaches to address doses of ARB for various health outcomes and pathways. Conclusions: We propose that environmental aspects of antibiotic-resistance development be included in the processes of any HHRA addressing ARB. Because of limited available data, a multicriteria decision analysis approach would be a useful way to undertake an HHRA of environmental antibiotic resistance that informs risk managers. Citation: Ashbolt NJ, Amézquita A, Backhaus T, Borriello P, Brandt KK, Collignon P, Coors A, Finley R, Gaze WH, Heberer T, Lawrence JR, Larsson DG, McEwen SA, Ryan JJ, Schönfeld J, Silley P, Snape JR, Van den Eede C, Topp E. 2013. Human health risk assessment (HHRA) for environmental development and transfer of antibiotic resistance. Environ Health Perspect 121:993–1001; http://dx.doi.org/10.1289/ehp.1206316

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In Vitro Infection of Pupae with Israeli Acute Paralysis Virus Suggests Disturbance of Transcriptional Homeostasis in Honey Bees (Apis mellifera)

The ongoing decline of honey bee health worldwide is a serious economic and ecological concern. One major contributor to the decline are pathogens, including several honey bee viruses. However, information is limited on the biology of bee viruses and molecular interactions with their hosts. An experimental protocol to test these systems was developed, using injections of Israeli Acute Paralysis Virus (IAPV) into honey bee pupae reared ex-situ under laboratory conditions. The infected pupae developed pronounced but variable patterns of disease. Symptoms varied from complete cessation of development with no visual evidence of disease to rapid darkening of a part or the entire body. Considerable differences in IAPV titer dynamics were observed, suggesting significant variation in resistance to IAPV among and possibly within honey bee colonies. Thus, selective breeding for virus resistance should be possible. Gene expression analyses of three separate experiments suggest IAPV disruption of transcriptional homeostasis of several fundamental cellular functions, including an up-regulation of the ribosomal biogenesis pathway. These results provide first insights into the mechanisms of IAPV pathogenicity. They mirror a transcriptional survey of honey bees afflicted with Colony Collapse Disorder and thus support the hypothesis that viruses play a critical role in declining honey bee health.

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First detection of canine parvovirus type 2c in Brazil

The presence of canine parvovirus type 2 (CPV-2), 2a and 2b has been described in Brazil, however, the type 2c had not been reported until now. In the current study, seven out of nine samples from dogs with diarrhea were characterized as CPV-2c, indicating that this virus is already circulating in the Brazilian canine population.

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Rhinovirus detection using different PCR-based strategies

Human rhinoviruses (HRVs) are the major cause of the common cold. HRVs were recently reclassified into the Enterovirus genus (HEV) in the Picornaviridae family. HRVs and other members of the HEV genus share many common features, including sense RNA genomes and partial nucleotide sequence identity. The aim of this study was to evaluate different HRV detection strategies. Samples from adults with acute respiratory infection (n = 291) who were treated in Sao Paulo Hospital (2001-2003) were tested using three assays. The first assay detected picornaviruses by RT-PCR and hybridization, the second detected rhinoviruses using RT-PCR/sequencing, and the third differentiated HRV from HEV using duplex semi-nested-RT-PCR. Analysis of the results obtained from the first two strategies revealed 83% concordance. Discordant samples were then evaluated by the third protocol, and 82% were negative. The picornavirus detection protocol was more sensitive but less specific than the rhinovirus detection protocols. The semi-nested protocol utilized in the present study was less sensitive and was not useful in differentiating HRV from HEV. Sequencing assays examining different genes would address the best strategy of confirming rhinovirus and enterovirus infections.

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Isolation and identification of feline calicivirus and feline herpesvirus in Southern Brazil

Feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1) are the two primary causes of upper respiratory tract disease in cats. The aim of this study was to demonstrate the distribution of FCV and FHV-1 among the feline population of several counties in Rio Grande do Sul State, Brazil. To this end, conjunctival and nasal swabs were collected from 302 cats from different locations, including households, breeding catteries, veterinary clinics, animal hospitals and experimental research facilities. The samples were collected between July 2006 to June 2009. The virus isolation was performed in CRFK cells and, subsequently, the identification was confirmed by PCR. FCV, FHV-1, or both were isolated from 55 cats from 28 different locations. FCV alone was isolated from 52.7% (29/55) of the animals that tested positively, FHV-1 alone was isolated from 38.2% (21/55) of the animals that tested positively, and co-infection were detected in 9.1% (5/55) of the animals that tested positively. Virus detection was more prevalent in cats that were less than 1 year old, among animals that shared a living space with other cats, and females. FCV and FHV-1 were isolated from vaccinated cats. In addition, both viruses were isolated from cats that showed no signs of disease. The results suggest that a carrier state is common for both viruses in the evaluated population. A search for other causes of respiratory disease in that population is necessary; and further studies relating to the molecular characterization of viruses and vaccine efficacy are also necessary.

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Seasonality of viral respiratory infections in southeast of Brazil: the influence of temperature and air humidity

Viruses are the major cause of lower respiratory tract infections in childhood and the main viruses involved are Human Respiratory Syncytial Virus (HRSV), Human Metapneumovirus (HMPV), Influenzavirus A and B (FLUA and FLUB), Human Parainfluenza Virus 1, 2 and 3 (HPIV1, 2 and 3) and Human Rhinovirus (HRV). The purposes of this study were to detect respiratory viruses in hospitalized children younger than six years and identify the influence of temperature and relative air humidity on the detected viruses. Samples of nasopharyngeal washes were collected from hospitalized children between May/2004 and September/2005. Methods of viral detection were RT-PCR, PCR and HRV amplicons were confirmed by hybridization. Results showed 54% (148/272) of viral positivity. HRSV was detected in 29% (79/272) of the samples; HRV in 23.1% (63/272); HPIV3 in 5.1% (14/272); HMPV in 3.3% (9/272); HPIV1 in 2.9% (8/272); FLUB in 1.4% (4/272), FLUA in 1.1% (3/272), and HPIV2 in 0.3% (1/272). The highest detection rates occurred mainly in the spring 2004 and in the autumn 2005. It was observed that viral respiratory infections tend to increase as the relative air humidity decreases, showing significant association with monthly averages of minimal temperature and minimal relative air humidity. In conclusion, viral respiratory infections vary according to temperature and relative air humidity and viral respiratory infections present major incidences it coldest and driest periods.

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Human rhinovirus infections in symptomatic and asymptomatic subjects

The role of rhinovirus asymptomatic infections in the transmission among close contacts subjects is unknown. We tested health care workers, a pair of one child and a family member and immunocompromised patients (n =191). HRV were detected on 22.9% symptomatic and 3.6% asymptomatic cases suggesting lower transmission among contacts.

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Estimating Potential Infection Transmission Routes in Hospital Wards Using Wearable Proximity Sensors

BACKGROUND: Contacts between patients, patients and health care workers (HCWs) and among HCWs represent one of the important routes of transmission of hospital-acquired infections (HAI). A detailed description and quantification of contacts in hospitals provides key information for HAIs epidemiology and for the design and validation of control measures. METHODS AND FINDINGS: We used wearable sensors to detect close-range interactions (“contacts”) between individuals in the geriatric unit of a university hospital. Contact events were measured with a spatial resolution of about 1.5 meters and a temporal resolution of 20 seconds. The study included 46 HCWs and 29 patients and lasted for 4 days and 4 nights. 14,037 contacts were recorded overall, 94.1% of which during daytime. The number and duration of contacts varied between mornings, afternoons and nights, and contact matrices describing the mixing patterns between HCW and patients were built for each time period. Contact patterns were qualitatively similar from one day to the next. 38% of the contacts occurred between pairs of HCWs and 6 HCWs accounted for 42% of all the contacts including at least one patient, suggesting a population of individuals who could potentially act as super-spreaders. CONCLUSIONS: Wearable sensors represent a novel tool for the measurement of contact patterns in hospitals. The collected data can provide information on important aspects that impact the spreading patterns of infectious diseases, such as the strong heterogeneity of contact numbers and durations across individuals, the variability in the number of contacts during a day, and the fraction of repeated contacts across days. This variability is however associated with a marked statistical stability of contact and mixing patterns across days. Our results highlight the need for such measurement efforts in order to correctly inform mathematical models of HAIs and use them to inform the design and evaluation of prevention strategies.

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Dissolving and biodegradable microneedle technologies for transdermal sustained delivery of drug and vaccine

Microneedles were first conceptualized for drug delivery many decades ago, overcoming the shortages and preserving the advantages of hypodermic needle and conventional transdermal drug-delivery systems to some extent. Dissolving and biodegradable microneedle technologies have been used for transdermal sustained deliveries of different drugs and vaccines. This review describes microneedle geometry and the representative dissolving and biodegradable microneedle delivery methods via the skin, followed by the fabricating methods. Finally, this review puts forward some perspectives that require further investigation.

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Transcriptome Analysis of Human Peripheral Blood Mononuclear Cells Exposed to Lassa Virus and to the Attenuated Mopeia/Lassa Reassortant 29 (ML29), a Vaccine Candidate

Lassa virus (LASV) is the causative agent of Lassa Fever and is responsible for several hundred thousand infections and thousands of deaths annually in West Africa. LASV and the non-pathogenic Mopeia virus (MOPV) are both rodent-borne African arenaviruses. A live attenuated reassortant of MOPV and LASV, designated ML29, protects rodents and primates from LASV challenge and appears to be more attenuated than MOPV. To gain better insight into LASV-induced pathology and mechanism of attenuation we performed gene expression profiling in human peripheral blood mononuclear cells (PBMC) exposed to LASV and the vaccine candidate ML29. PBMC from healthy human subjects were exposed to either LASV or ML29. Although most PBMC are non-permissive for virus replication, they remain susceptible to signal transduction by virus particles. Total RNA was extracted and global gene expression was evaluated during the first 24 hours using high-density microarrays. Results were validated using RT-PCR, flow cytometry and ELISA. LASV and ML29 elicited differential expression of interferon-stimulated genes (ISG), as well as genes involved in apoptosis, NF-kB signaling and the coagulation pathways. These genes could eventually serve as biomarkers to predict disease outcomes. The remarkable differential expression of thrombomodulin, a key regulator of inflammation and coagulation, suggests its involvement with vascular abnormalities and mortality in Lassa fever disease.

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A Neutralizing Monoclonal Antibody Targeting the Acid-Sensitive Region in Chikungunya Virus E2 Protects from Disease

The mosquito-borne alphavirus, chikungunya virus (CHIKV), has recently reemerged, producing the largest epidemic ever recorded for this virus, with up to 6.5 million cases of acute and chronic rheumatic disease. There are currently no licensed vaccines for CHIKV and current anti-inflammatory drug treatment is often inadequate. Here we describe the isolation and characterization of two human monoclonal antibodies, C9 and E8, from CHIKV infected and recovered individuals. C9 was determined to be a potent virus neutralizing antibody and a biosensor antibody binding study demonstrated it recognized residues on intact CHIKV VLPs. Shotgun mutagenesis alanine scanning of 98 percent of the residues in the E1 and E2 glycoproteins of CHIKV envelope showed that the epitope bound by C9 included amino-acid 162 in the acid-sensitive region (ASR) of the CHIKV E2 glycoprotein. The ASR is critical for the rearrangement of CHIKV E2 during fusion and viral entry into host cells, and we predict that C9 prevents these events from occurring. When used prophylactically in a CHIKV mouse model, C9 completely protected against CHIKV viremia and arthritis. We also observed that when administered therapeutically at 8 or 18 hours post-CHIKV challenge, C9 gave 100% protection in a pathogenic mouse model. Given that targeting this novel neutralizing epitope in E2 can potently protect both in vitro and in vivo, it is likely to be an important region both for future antibody and vaccine-based interventions against CHIKV.

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Low serum 25-hydroxyvitamin D levels are associated with increased risk of viral coinfections in wheezing children

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Pathogenic Mouse Hepatitis Virus or Poly(I:C) Induce IL-33 in Hepatocytes in Murine Models of Hepatitis

The IL-33/ST2 axis is known to be involved in liver pathologies. Although, the IL-33 levels increased in sera of viral hepatitis patients in human, the cellular sources of IL-33 in viral hepatitis remained obscure. Therefore, we aimed to investigate the expression of IL-33 in murine fulminant hepatitis induced by a Toll like receptor (TLR3) viral mimetic, poly(I:C) or by pathogenic mouse hepatitis virus (L2-MHV3). The administration of poly(I:C) plus D-galactosamine (D-GalN) in mice led to acute liver injury associated with the induction of IL-33 expression in liver sinusoidal endothelial cells (LSEC) and vascular endothelial cells (VEC), while the administration of poly(I:C) alone led to hepatocyte specific IL-33 expression in addition to vascular IL-33 expression. The hepatocyte-specific IL-33 expression was down-regulated in NK-depleted poly(I:C) treated mice suggesting a partial regulation of IL-33 by NK cells. The CD1d KO (NKT deficient) mice showed hepatoprotection against poly(I:C)-induced hepatitis in association with increased number of IL-33 expressing hepatocytes in CD1d KO mice than WT controls. These results suggest that hepatocyte-specific IL-33 expression in poly(I:C) induced liver injury was partially dependent of NK cells and with limited role of NKT cells. In parallel, the L2-MHV3 infection in mice induced fulminant hepatitis associated with up-regulated IL-33 expression as well as pro-inflammatory cytokine microenvironment in liver. The LSEC and VEC expressed inducible expression of IL-33 following L2-MHV3 infection but the hepatocyte-specific IL-33 expression was only evident between 24 to 32h of post infection. In conclusion, the alarmin cytokine IL-33 was over-expressed during fulminant hepatitis in mice with LSEC, VEC and hepatocytes as potential sources of IL-33.

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Emerging Influenza and Psychosis: An Important Story in Psychological Medicine

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Clinical Features and Factors Associated with Outcomes of Patients Infected with a Novel Influenza A (H7N9) Virus: A Preliminary Study

OBJECTIVE: The present study aimed to analyze clinical features and factors associated with treatment outcomes of H7N9 influenza A virus infection. METHODS: The clinical progress in 18 H7N9-infected patients was monitored and recorded. The clinical features of H7N9 infection were noted and factors associated with treatment outcomes were analyzed by univariate analyses. RESULTS: The average ages of patients in recovered and critical conditions were 67.0±10.83 years and 72.75±12.0 years, respectively. Renal insufficiency developed more frequently in critically ill patients (P = 0.023). The duration of traditional Chinese medicine (TCM) therapy was longer in recovered patients than in critically ill patients (P = 0.01). Laboratory tests showed that levels of C-reactive protein, serum creatinine, and myoglobin were significantly higher in critically ill patients than in recovered patients (P = 0.011, 0.04, and 0.016, respectively). Meanwhile, levels of all T cell subsets examined including total CD3(+), CD4(+), CD8(+), and CD45(+) T cells were lower in critically ill patients than in recovered patients (P = 0.033, 0.059, 0.015, and 0.039, respectively). Logistic regression analysis demonstrated that C-reactive protein level, myoglobin level and TCM therapy duration were likely associated with treatment outcomes of H7N9 infection (P = 0.032, 0.041 and 0.017, respectively). CONCLUSION: Elderly people may have increased risk for H7N9 virus infection. T cell-mediated responses play an important role in defense against the H7N9 virus. C-reactive protein level, myoglobin level and TCM duration may be associated with treatment outcomes of H7N9 infection.

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Therapy and prophylaxis of opportunistic infections in HIV-infected patients: a guideline by the German and Austrian AIDS societies (DAIG/ÖAG) (AWMF 055/066)

INTRODUCTION: There was a growing need for practical guidelines for the most common OIs in Germany and Austria under consideration of the local epidemiological conditions. MATERIALS AND METHODS: The German and Austrian AIDS societies developed these guidelines between March 2010 and November 2011. A structured Medline research was performed for 12 diseases, namely Immune reconstitution inflammatory syndrome, Pneumocystis jiroveci pneumonia, cerebral toxoplasmosis, cytomegalovirus manifestations, candidiasis, herpes simplex virus infections, varizella zoster virus infections, progressive multifocal leucencephalopathy, cryptosporidiosis, cryptococcosis, nontuberculosis mycobacteria infections and tuberculosis. Due to the lack of evidence by randomized controlled trials, part of the guidelines reflects expert opinions. The German version was accepted by the German and Austrian AIDS Societies and was previously published by the Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften (AWMF; German Association of the Scientific Medical Societies). CONCLUSION: The review presented here is a translation of a short version of the German–Austrian Guidelines of opportunistic infections in HIV patients. These guidelines are well-accepted in a clinical setting in both Germany and Austria. They lead to a similar treatment of a heterogeneous group of patients in these countries.

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Role of S-Palmitoylation on IFITM5 for the Interaction with FKBP11 in Osteoblast Cells

Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.

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Hepatitis C Virus Replication and Golgi Function in Brefeldin A-Resistant Hepatoma-Derived Cells

Recent reports indicate that the replication of hepatitis C virus (HCV) depends on the GBF1-Arf1-COP-I pathway. We generated Huh-7-derived cell lines resistant to brefeldin A (BFA), which is an inhibitor of this pathway. The resistant cell lines could be sorted into two phenotypes regarding BFA-induced toxicity, inhibition of albumin secretion, and inhibition of HCV infection. Two cell lines were more than 100 times more resistant to BFA than the parental Huh-7 cells in these 3 assays. This resistant phenotype was correlated with the presence of a point mutation in the Sec7 domain of GBF1, which is known to impair the binding of BFA. Surprisingly, the morphology of the cis-Golgi of these cells remained sensitive to BFA at concentrations of the drug that allowed albumin secretion, indicating a dichotomy between the phenotypes of secretion and Golgi morphology. Cells of the second group were about 10 times more resistant than parental Huh-7 cells to the BFA-induced toxicity. The EC(50) for albumin secretion was only 1.5–1.8 fold higher in these cells than in Huh-7 cells. However their level of secretion in the presence of inhibitory doses of BFA was 5 to 15 times higher. Despite this partially effective secretory pathway in the presence of BFA, the HCV infection was almost as sensitive to BFA as in Huh-7 cells. This suggests that the function of GBF1 in HCV replication does not simply reflect its role of regulator of the secretory pathway of the host cell. Thus, our results confirm the involvement of GBF1 in HCV replication, and suggest that GBF1 might fulfill another function, in addition to the regulation of the secretory pathway, during HCV replication.

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Vesicular Transport of Progeny Parvovirus Particles through ER and Golgi Regulates Maturation and Cytolysis

Progeny particles of non-enveloped lytic parvoviruses were previously shown to be actively transported to the cell periphery through vesicles in a gelsolin-dependent manner. This process involves rearrangement and destruction of actin filaments, while microtubules become protected throughout the infection. Here the focus is on the intracellular egress pathway, as well as its impact on the properties and release of progeny virions. By colocalization with cellular marker proteins and specific modulation of the pathways through over-expression of variant effector genes transduced by recombinant adeno-associated virus vectors, we show that progeny PV particles become engulfed into COPII-vesicles in the endoplasmic reticulum (ER) and are transported through the Golgi to the plasma membrane. Besides known factors like sar1, sec24, rab1, the ERM family proteins, radixin and moesin play (an) essential role(s) in the formation/loading and targeting of virus-containing COPII-vesicles. These proteins also contribute to the transport through ER and Golgi of the well described analogue of cellular proteins, the secreted Gaussia luciferase in absence of virus infection. It is therefore likely that radixin and moesin also serve for a more general function in cellular exocytosis. Finally, parvovirus egress via ER and Golgi appears to be necessary for virions to gain full infectivity through post-assembly modifications (e.g. phosphorylation). While not being absolutely required for cytolysis and progeny virus release, vesicular transport of parvoviruses through ER and Golgi significantly accelerates these processes pointing to a regulatory role of this transport pathway.

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Rab-GDI Complex Dissociation Factor Expressed through Translational Frameshifting in Filamentous Ascomycetes

In the model fungus Podospora anserina, the PaYIP3 gene encoding the orthologue of the Saccharomyces cerevisiae YIP3 Rab-GDI complex dissociation factor expresses two polypeptides, one of which, the long form, is produced through a programmed translation frameshift. Inactivation of PaYIP3 results in slightly delayed growth associated with modification in repartition of fruiting body on the thallus, along with reduced ascospore production on wood. Long and short forms of PaYIP3 are expressed in the mycelium, while only the short form appears expressed in the maturing fruiting body (perithecium). The frameshift has been conserved over the evolution of the Pezizomycotina, lasting for over 400 million years, suggesting that it has an important role in the wild.

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CD13 is essential for inflammatory trafficking and infarct healing following permanent coronary artery occlusion in mice

AIMS: To determine the role of CD13 as an adhesion molecule in trafficking of inflammatory cells to the site of injury in vivo and its function in wound healing following myocardial infarction induced by permanent coronary artery occlusion. METHODS AND RESULTS: Seven days post-permanent ligation, hearts from CD13 knockout (CD13(KO)) mice showed significant reductions in cardiac function, suggesting impaired healing in the absence of CD13. Mechanistically, CD13(KO) infarcts showed an increase in small, endothelial-lined luminal structures, but no increase in perfusion, arguing against an angiogenic defect in the absence of CD13. Cardiac myocytes of CD13(KO) mice showed normal basal contractile function, eliminating myocyte dysfunction as a mechanism of adverse remodelling. Conversely, immunohistochemical and flow cytometric analysis of CD13(KO) infarcts demonstrated a dramatic 65% reduction in infiltrating haematopoietic cells, including monocytes, macrophages, dendritic, and T cells, suggesting a critical role for CD13 adhesion in inflammatory trafficking. Accordingly, CD13(KO) infarcts also contained fewer myofibroblasts, consistent with attenuation of fibroblast differentiation resulting from the reduced inflammation, leading to adverse remodelling. CONCLUSION: In the ischaemic heart, while compensatory mechanisms apparently relieve potential angiogenic defects, CD13 is essential for proper trafficking of the inflammatory cells necessary to prime and sustain the reparative response, thus promoting optimal post-infarction healing.

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Different Mechanisms of Inflammation Induced in Virus and Autoimmune-Mediated Models of Multiple Sclerosis in C57BL6 Mice

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the human central nervous system (CNS). Neurotropic demyelinating strain of MHV (MHV-A59 or its isogenic recombinant strain RSA59) induces MS-like disease in mice mediated by microglia, along with a small population of T cells. The mechanism of demyelination is at least in part due to microglia-mediated myelin stripping, with some direct axonal injury. Immunization with myelin oligodendrocyte glycoprotein (MOG) induces experimental autoimmune encephalomyelitis (EAE), a mainly CD4(+) T-cell-mediated disease, although CD8(+) T cells may play a significant role in demyelination. It is possible that both autoimmune and nonimmune mechanisms such as direct viral toxicity may induce MS. Our study directly compares CNS pathology in autoimmune and viral-induced MS models. Mice with viral-induced and EAE demyelinating diseases demonstrated similar patterns and distributions of demyelination that accumulated over the course of the disease. However, significant differences in acute inflammation were noted. Inflammation was restricted mainly to white matter at all times in EAE, whereas inflammation initially largely involved gray matter in acute MHV-induced disease and then is subsequently localized only in white matter in the chronic disease phase. The presence of dual mechanisms of demyelination may be responsible for the failure of immunosuppression to promote long-term remission in many MS patients.

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Soluble Form of Canine Transferrin Receptor Inhibits Canine Parvovirus Infection In Vitro and In Vivo

Canine parvovirus (CPV) disease is an acute, highly infectious disease threatening the dog-raising industry. So far there are no effective therapeutic strategies to control this disease. Although the canine transferrin receptor (TfR) was identified as a receptor for CPV infection, whether extracellular domain of TfR (called soluble TfR (sTfR)) possesses anti-CPV activities remains elusive. Here, we used the recombinant sTfR prepared from HEK293T cells with codon-optimized gene structure to investigate its anti-CPV activity both in vitro and in vivo. Our results indicated that codon optimization could significantly improve sTfR expression in HEK293T cells. The prepared recombinant sTfR possessed a binding activity to both CPV and CPV VP2 capsid proteins and significantly inhibited CPV infection of cultured feline F81 cells and decreased the mortality of CPV-infected dogs, which indicates that the sTfR has the anti-CPV activity both in vitro and in vivo.

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The Battle Between Influenza and the Innate Immune Response in the Human Respiratory Tract

Influenza is a viral infection of the respiratory tract. Infection is normally confined to the upper respiratory tract but certain viral strains have evolved the ability to infect the lower respiratory tract, including the alveoli, leading to inflammation and a disease pattern of diffuse alveolar damage. Factors leading to this sequence of events are novel influenza strains, or strains that have viral proteins, in particular the NS1 protein that allow it to escape the innate immune system. There are three main barriers that prevent infection of pneumocytes - mucin, host defence lectins and cells such as macrophages. Viruses have developed strategies such as neuraminidase and glycosylation patterns that allow this evasion. Though there has been much investment in antiviral drugs, it is proposed that more attention should be directed towards developing or utilizing compounds that enhance the ability of the innate immune system to combat viral infection.

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Mucosal Vaccination with Recombinant Adenovirus Encoding Nucleoprotein Provides Potent Protection against Influenza Virus Infection

Influenza vaccines that target the highly variable surface glycoproteins hemagglutinin and neuraminidase cause inconvenience of having vaccination every year. For this reason, development of universal vaccines targeting conserved viral components is needed. In this study, we generated recombinant adenovirus (rAd) vaccine encoding nucleoprotein (NP) of A/PR/8/34 influenza virus, designated rAd/NP. BALB/c mice were immunized intranasally or sublingually with rAd/NP vaccine and subsequently challenged with lethal doses of heterologous as well as homologous influenza viruses. We found that intranasal immunization of rAd/NP elicited strong mucosal IgA responses as well as stronger CD8 T-cell responses toward immunodominant K(d)-restricted NP(147-155) epitope than sublingual immunization. Importantly, only single intranasal but not sublingual immunization of rAd/NP provides potent protection against both homologous and heterologous influenza virus challenges. These results suggest that recombinant rAd/NP could be a universal vaccine candidate for mucosal administration against influenza virus.

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Complete Genome Sequence of a Novel Feline Astrovirus from a Domestic Cat in Hong Kong

We report the first complete genome sequence of a feline astrovirus (FAstV), FAstV2 strain 1637F, identified from a domestic cat. The genome is 6,779 nucleotides (nt) in length and consists of three overlapping open reading frames (ORF1a-ORF1b-ORF2). Sequence analysis suggests that FAstV2 represents a new FAstV genotype that is closely related to human astroviruses.

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The Potential of Metatranscriptomics for Identifying Screening Targets for Bacterial Vaginosis

BACKGROUND: The ribosomal RNA content of a sample collected from a woman with bacterial vaginosis (BV) was analysed to determine the active microbial community, and to identify potential targets for further screening. METHODOLOGY/PRINCIPAL FINDINGS: The sample from the BV patient underwent total RNA extraction, followed by physical subtraction of human rRNA and whole transcriptome amplification. The metatranscriptome was sequenced using Roche 454 titanium chemistry. The bioinformatics pipeline MG-RAST and desktop DNA analysis platforms were utilised to analyse results. Bacteria of the genus Prevotella (predominately P. amnii) constituted 36% of the 16S rRNA reads, followed by Megasphaera (19%), Leptotrichia/Sneathia (8%) and Fusobacterium (8%). Comparison of the abundances of several bacteria to quantitative PCR (qPCR) screening of extracted DNA revealed comparable relative abundances. This suggests a correlation between what was present and transcriptionally active in this sample: however distinct differences were seen when compared to the microbiome determined by 16S rRNA gene amplicon sequencing. To assess the presence of P. amnii in a larger pool of samples, 90 sexually active women were screened using qPCR. This bacterium was found to be strongly associated with BV (P<0.001, OR 23.3 (95%CI:2.9–190.7)) among the 90 women. CONCLUSIONS/SIGNIFICANCE: This study highlighted the potential of metatranscriptomics as a tool for characterising metabolically active microbiota and identifying targets for further screening. Prevotella amnii was chosen as an example target, being the most metabolically active species present in the single patient with BV, and was found to be detected at a high concentration by qPCR in 31% of cohort with BV, with an association with both oral and penile-vaginal sex.

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Spatiotemporal Infectious Disease Modeling: A BME-SIR Approach

This paper is concerned with the modeling of infectious disease spread in a composite space-time domain under conditions of uncertainty. We focus on stochastic modeling that accounts for basic mechanisms of disease distribution and multi-sourced in situ uncertainties. Starting from the general formulation of population migration dynamics and the specification of transmission and recovery rates, the model studies the functional formulation of the evolution of the fractions of susceptible-infected-recovered individuals. The suggested approach is capable of: a) modeling population dynamics within and across localities, b) integrating the disease representation (i.e. susceptible-infected-recovered individuals) with observation time series at different geographical locations and other sources of information (e.g. hard and soft data, empirical relationships, secondary information), and c) generating predictions of disease spread and associated parameters in real time, while considering model and observation uncertainties. Key aspects of the proposed approach are illustrated by means of simulations (i.e. synthetic studies), and a real-world application using hand-foot-mouth disease (HFMD) data from China.

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Ligand Pose and Orientational Sampling in Molecular Docking

Molecular docking remains an important tool for structure-based screening to find new ligands and chemical probes. As docking ambitions grow to include new scoring function terms, and to address ever more targets, the reliability and extendability of the orientation sampling, and the throughput of the method, become pressing. Here we explore sampling techniques that eliminate stochastic behavior in DOCK3.6, allowing us to optimize the method for regularly variable sampling of orientations. This also enabled a focused effort to optimize the code for efficiency, with a three-fold increase in the speed of the program. This, in turn, facilitated extensive testing of the method on the 102 targets, 22,805 ligands and 1,411,214 decoys of the Directory of Useful Decoys - Enhanced (DUD-E) benchmarking set, at multiple levels of sampling. Encouragingly, we observe that as sampling increases from 50 to 500 to 2000 to 5000 to 20000 molecular orientations in the binding site (and so from about 1×10(10) to 4×10(10) to 1×10(11) to 2×10(11) to 5×10(11) mean atoms scored per target, since multiple conformations are sampled per orientation), the enrichment of ligands over decoys monotonically increases for most DUD-E targets. Meanwhile, including internal electrostatics in the evaluation ligand conformational energies, and restricting aromatic hydroxyls to low energy rotamers, further improved enrichment values. Several of the strategies used here to improve the efficiency of the code are broadly applicable in the field.

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Ligand Clouds around Protein Clouds: A Scenario of Ligand Binding with Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) were found to be widely associated with human diseases and may serve as potential drug design targets. However, drug design targeting IDPs is still in the very early stages. Progress in drug design is usually achieved using experimental screening; however, the structural disorder of IDPs makes it difficult to characterize their interaction with ligands using experiments alone. To better understand the structure of IDPs and their interactions with small molecule ligands, we performed extensive simulations on the c-Myc(370–409) peptide and its binding to a reported small molecule inhibitor, ligand 10074-A4. We found that the conformational space of the apo c-Myc(370–409) peptide was rather dispersed and that the conformations of the peptide were stabilized mainly by charge interactions and hydrogen bonds. Under the binding of the ligand, c-Myc(370–409) remained disordered. The ligand was found to bind to c-Myc(370–409) at different sites along the chain and behaved like a ‘ligand cloud’. In contrast to ligand binding to more rigid target proteins that usually results in a dominant bound structure, ligand binding to IDPs may better be described as ligand clouds around protein clouds. Nevertheless, the binding of the ligand and a non-ligand to the c-Myc(370–409) target could be clearly distinguished. The present study provides insights that will help improve rational drug design that targets IDPs.

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APOBEC3G Polymorphism as a Selective Barrier to Cross-Species Transmission and Emergence of Pathogenic SIV and AIDS in a Primate Host

Cellular restriction factors, which render cells intrinsically resistant to viruses, potentially impose genetic barriers to cross-species transmission and emergence of viral pathogens in nature. One such factor is APOBEC3G. To overcome APOBEC3G-mediated restriction, many lentiviruses encode Vif, a protein that targets APOBEC3G for degradation. As with many restriction factor genes, primate APOBEC3G displays strong signatures of positive selection. This is interpreted as evidence that the primate APOBEC3G locus reflects a long-term evolutionary “arms-race” between retroviruses and their primate hosts. Here, we provide direct evidence that APOBEC3G has functioned as a barrier to cross-species transmission, selecting for viral resistance during emergence of the AIDS-causing pathogen SIVmac in captive colonies of Asian macaques in the 1970s. Specifically, we found that rhesus macaques have multiple, functionally distinct APOBEC3G alleles, and that emergence of SIVmac and simian AIDS required adaptation of the virus to evade APOBEC3G-mediated restriction. Our evidence includes the first comparative analysis of APOBEC3G polymorphism and function in both a reservoir and recipient host species (sooty mangabeys and rhesus macaques, respectively), and identification of adaptations unique to Vif proteins of the SIVmac lineage that specifically antagonize rhesus APOBEC3G alleles. By demonstrating that interspecies variation in a known restriction factor selected for viral counter-adaptations in the context of a documented case of cross-species transmission, our results lend strong support to the evolutionary “arms-race” hypothesis. Importantly, our study confirms that APOBEC3G divergence can be a critical determinant of interspecies transmission and emergence of primate lentiviruses, including viruses with the potential to infect and spread in human populations.

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Characteristics of human infection with avian influenza viruses and development of new antiviral agents

Since 1997, several epizootic avian influenza viruses (AIVs) have been transmitted to humans, causing diseases and even deaths. The recent emergence of severe human infections with AIV (H7N9) in China has raised concerns about efficient interpersonal viral transmission, polygenic traits in viral pathogenicity and the management of newly emerging strains. The symptoms associated with viral infection are different in various AI strains: H5N1 and newly emerged H7N9 induce severe pneumonia and related complications in patients, while some H7 and H9 subtypes cause only conjunctivitis or mild respiratory symptoms. The virulence and tissue tropism of viruses as well as the host responses contribute to the pathogenesis of human AIV infection. Several preventive and therapeutic approaches have been proposed to combat AIV infection, including antiviral drugs such as M2 inhibitors, neuraminidase inhibitors, RNA polymerase inhibitors, attachment inhibitors and signal-transduction inhibitors etc. In this article, we summarize the recent progress in researches on the epidemiology, clinical features, pathogenicity determinants, and available or potential antivirals of AIV.

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Comparative Serological Assays for the Study of H5 and H7 Avian Influenza Viruses

The nature of influenza virus to randomly mutate and evolve into new types is an important challenge in the control of influenza infection. It is necessary to monitor virus evolution for a better understanding of the pandemic risk posed by certain variants as evidenced by the highly pathogenic avian influenza (HPAI) viruses. This has been clearly recognized in Egypt following the notification of the first HPAI H5N1 outbreak. The continuous circulation of the virus and the mass vaccination programme undertaken in poultry have resulted in a progressive genetic evolution and a significant antigenic drift near the major antigenic sites. In order to establish if vaccination is sufficient to provide significant intra- and interclade cross-protection, lentiviral pseudotypes derived from H5N1 HPAI viruses (A/Vietnam/1194/04, A/chicken/Egypt-1709-01/2007) and an antigenic drift variant (A/chicken/Egypt-1709-06-2008) were constructed and used in pseudotype-based neutralization assays (pp-NT). pp-NT data obtained was confirmed and correlated with HI and MN assays. A panel of pseudotypes belonging to influenza Groups 1 and 2, with a combination of reporter systems, was also employed for testing avian sera in order to support further application of pp-NT as an alternative valid assay that can improve avian vaccination efficacy testing, vaccine virus selection, and the reliability of reference sera.

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Revisiting Public Health Challenges in the New Millennium

Positive Health of the communities could only be brought out through the interrelationship between conventional health sector and other development sectors. It was a dream that came true when World Health Organization (WHO) accepted Primary Health Care (PHC) as the major tool to achieve its proposed goal of Health For All (HFA) by 2000 A.D., but we could not succeed as expected. Now we have the Millennium Development Goals (MDG), which place health at the heart of development but the achievements in health is still challenging. The literature search in this article has been conducted in Pub Med and Google scholar, with the aim to draw references to discuss the major health issues and ways to tackle them. The current article briefly narrates the burden and complexities of challenges faced by the present global health. Revisiting the concept of PHC and reaffirming our solidarity to this philosophy is the need of this hour.

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Evaluation of the Replication, Pathogenicity, and Immunogenicity of Avian Paramyxovirus (APMV) Serotypes 2, 3, 4, 5, 7, and 9 in Rhesus Macaques

Avian paramyxoviruses (APMV) serotypes 1–9 are frequently isolated from domestic and wild birds worldwide. APMV-1 (also called Newcastle disease virus, NDV) is attenuated in non-human primates and is being developed as a candidate human vaccine vector. The vector potential of the other serotypes was unknown. In the present study, we evaluated nine different biologically- or recombinantly-derived APMV strains for the ability to replicate and cause disease in rhesus macaque model. Five of the viruses were: biologically-derived wild type (wt) APMV-2, -3, -5, -7 and -9. Another virus was a recombinant (r) version of wt APMV-4. The remaining three viruses were versions of wt rAPMV-2, -4 and -7 in which the F cleavage site had been modified to be multi-basic. Rhesus macaques were inoculated intranasally and intratracheally and monitored for clinical disease, virus shedding from the upper and lower respiratory tract, and seroconversion. Virus shedding was not detected for wt APMV-5. Very limited shedding was detected for wt rAPMV-4 and modified rAPMV-4, and only in a subset of animals. Shedding by the other viruses was detected in every infected animal, and usually from both the upper and lower respiratory tract. In particular, shedding over a number of days in every animal was observed for modified rAPMV-2, wt APMV-7, and modified rAPMV-7. Modification of the F protein cleavage site appeared to increase shedding by wt rAPMV-2 and marginally by wt rAPMV-4. All APMVs except wt APMV-5 induced a virus-specific serum antibody response in all infected animals. None of the animals exhibited any clinical disease signs. These results indicate that APMVs 2, 3, 4, 7, and 9 are competent to infect non-human primates, but are moderately-to-highly restricted, depending on the serotype. This suggests that they are not likely to significantly infect primates in nature, and represent promising attenuated candidates for vector development.

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Caveolin-1 Associated Adenovirus Entry into Human Corneal Cells

The cellular entry of viruses represents a critical area of study, not only for viral tropism, but also because viral entry dictates the nature of the immune response elicited upon infection. Epidemic keratoconjunctivitis (EKC), caused by viruses within human adenovirus species D (HAdV-D), is a severe, ocular surface infection associated with corneal inflammation. Clathrin-mediated endocytosis has previously been shown to play a critical role in entry of other HAdV species into many host cell types. However, HAdV-D endocytosis into corneal cells has not been extensively studied. Herein, we show an essential role for cholesterol rich, lipid raft microdomains and caveolin-1, in the entry of HAdV-D37 into primary human corneal fibroblasts. Cholesterol depletion using methyl-β-cyclodextrin (MβCD) profoundly reduced viral infection. When replenished with soluble cholesterol, the effect of MβCD was reversed, allowing productive viral infection. HAdV-D37 DNA was identified in caveolin-1 rich endosomal fractions after infection. Src kinase activity was also increased in caveolin-1 rich endosomal fractions after infection, and Src phosphorylation and CXCL1 induction were both decreased in caveolin-1-/- mice corneas compared to wild type mice. siRNA knock down of caveolin-1 in corneal cells reduced chemokine induction upon viral infection, and caveolin-1-/- mouse corneas showed reduced cellular entry of HAdV-D37. As a control, HAdV-C2, a non-corneal pathogen, appeared to utilize the caveolar pathway for entry into A549 cells, but failed to infect corneal cells entirely, indicating virus and cell specific tropism. Immuno-electron microscopy confirmed the presence of caveolin-1 in HAdV-D37-containing vesicles during the earliest stages of viral entry. Collectively, these experiments indicate for the first time that HAdV-D37 uses a lipid raft mediated caveolin-1 associated pathway for entry into corneal cells, and connects the processes of viral entry with downstream proinflammatory cell signaling.

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Influenza A virus-mediated priming enhances cytokine secretion by human dendritic cells infected with Streptococcus pneumoniae

Secondary infections with Streptococcus pneumoniae (SP) are frequently observed following influenza A virus (IAV) infection and have a substantial impact on global health. Despite this, the basis for the disease progression is incompletely understood. To investigate the effect of co-infection on human monocyte-derived dendritic cells (MDDCs) we analysed the expression of clinically important pro-inflammatory and immune-modulatory cytokines. IAV infection or treatment with supernatants from IAV-infected cell cultures resulted in priming of the DCs which subsequently influenced the production of IL-12p70, as well as IL-6, following SP infection. Co-infection of the same cell was not required but this effect was dependent on the time, dose and duration of the infections, as well as pathogen viability, bacterial uptake and endosome acidification. Bacterially infected cells were characterized as the main producers of IL-12p70. Finally, we showed that type I interferons were primarily responsible for the priming of IL-12p70 that was observed by infection with IAV. These results provide a probable mechanism for the elevated levels of particular cytokines observed in IAV and SP co-infected cell cultures with implications for the pathogenic outcome observed during in vivo infection.

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Genetic Analysis of West Nile Virus Isolates from an Outbreak in Idaho, United States, 2006–2007

West Nile virus (WNV) appeared in the U.S. in 1999 and has since become endemic, with yearly summer epidemics causing tens of thousands of cases of serious disease over the past 14 years. Analysis of WNV strains isolated during the 2006–2007 epidemic seasons demonstrates that a new genetic variant had emerged coincidentally with an intense outbreak in Idaho during 2006. The isolates belonging to the new variant carry a 13 nt deletion, termed ID-Δ13, located at the variable region of the 3′UTR, and are genetically related. The analysis of deletions and insertions in the 3′UTR of two major lineages of WNV revealed the presence of conserved repeats and two indel motifs in the variable region of the 3′UTR. One human and two bird isolates from the Idaho 2006–2007 outbreaks were sequenced using Illumina technology and within-host variability was analyzed. Continued monitoring of new genetic variants is important for public health as WNV continues to evolve.

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The Serological and Virological Investigation of Canine Adenovirus Infection on the Dogs

Two types of Canine Adenovirus (CAVs), Canine Adenovirus type 1 (CAV-1), the virus which causes infectious canine hepatitis, and Canine Adenovirus type 2 (CAV-2), which causes canine infectious laryngotracheitis, have been found in dogs. In this study, blood samples taken from 111 dogs, which were admitted to the Internal Medicine Clinic of Selcuk University, Faculty of Veterinary Medicine, with clinical symptoms. Seventy-seven dogs were sampled from Isparta and Burdur dog shelters by random sampling, regardless of the clinical findings. Dogs showed a systemic disease, characterized by fever, diarrhea, vomiting, oculonasal discharge, conjunctivitis, severe moist cough, signs of pulmonary disease and dehydration. Two dogs had corneal opacity and photophobia. In serological studies, 188 serum samples were investigated on the presence of CAV antibodies by ELISA. Total 103 (103/188–54.7%) blood samples were detected to be positive for CAV antibodies by ELISA. However, 85 (85/188–45.2%) blood samples were negative. Blood leukocyte samples from dogs were processed and inoculated onto confluent monolayers of MDCK cells using standard virological techniques. After third passage, cells were examined by direct immunoflourescence test for virus isolation. But positive result was not detected. In conclusion, this study clearly demonstrates the high prevalence of CAV infection in dogs.

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Sociotechnical Challenges and Progress in Using Social Media for Health

Social media tools that connect patients, caregivers, and health providers offer great potential for helping people access health advice, receive and give social support, manage or cope with chronic conditions, and make day-to-day health decisions. These systems have seen widespread adoption, but often fail to support the goals as fully as designers and users would like. Through Ackerman’s lens of the “sociotechnical gap” and computer supported cooperative work (CSCW) as a science of the artificial, we review contemporary sociotechnical challenges and progress for using social media to support health. These challenges include a tension between privacy and sharing, policy information credibility, accessibility, and tailoring in social spaces. Those studying, building, deploying, and using social media systems to further health goals will benefit from approaching this work by borrowing from Ackerman’s framing of CSCW. In particular, this requires acknowledgment that technical systems will not fully meet our social goals, and then adopting design and educational approaches that are appropriate to fill this gap, building less-nuanced systems as partial solutions and tools for advancing our understanding, and by working with the CSCW research community to develop and pursue key lines of inquiry.

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Eukaryotic Initiation Factor 2α - a Downstream Effector of Mammalian Target of Rapamycin - Modulates DNA Repair and Cancer Response to Treatment

In an effort to circumvent resistance to rapamycin – an mTOR inhibitor - we searched for novel rapamycin-downstream-targets that may be key players in the response of cancer cells to therapy. We found that rapamycin, at nM concentrations, increased phosphorylation of eukaryotic initiation factor (eIF) 2α in rapamycin-sensitive and estrogen-dependent MCF-7 cells, but had only a minimal effect on eIF2α phosphorylation in the rapamycin-insensitive triple-negative MDA-MB-231 cells. Addition of salubrinal – an inhibitor of eIF2α dephosphorylation – decreased expression of a surface marker associated with capacity for self renewal, increased senescence and induced clonogenic cell death, suggesting that excessive phosphorylation of eIF2α is detrimental to the cells' survival. Treating cells with salubrinal enhanced radiation-induced increase in eIF2α phosphorylation and clonogenic death and showed that irradiated cells are more sensitive to increased eIF2α phosphorylation than non-irradiated ones. Similar to salubrinal - the phosphomimetic eIF2α variant - S51D - increased sensitivity to radiation, and both abrogated radiation-induced increase in breast cancer type 1 susceptibility gene, thus implicating enhanced phosphorylation of eIF2α in modulation of DNA repair. Indeed, salubrinal inhibited non-homologous end joining as well as homologous recombination repair of double strand breaks that were induced by I-SceI in green fluorescent protein reporter plasmids. In addition to its effect on radiation, salubrinal enhanced eIF2α phosphorylation and clonogenic death in response to the histone deacetylase inhibitor – vorinostat. Finally, the catalytic competitive inhibitor of mTOR - Ku-0063794 - increased phosphorylation of eIF2α demonstrating further the involvement of mTOR activity in modulating eIF2α phosphorylation. These experiments suggest that excessive phosphorylation of eIF2α decreases survival of cancer cells; making eIF2α a worthy target for drug development, with the potential to enhance the cytotoxic effects of established anti-neoplastic therapies and circumvent resistance to rapalogues and possibly to other drugs that inhibit upstream components of the mTOR pathway.

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Development of a risk assessment tool for contact tracing people after contact with infectious patients while travelling by bus or other public ground transport: a Delphi consensus approach

BACKGROUND: Tracing persons who have been in contact with an infectious patient may be very effective in preventing the spread of communicable diseases. However, criteria to decide when to conduct contact tracing are not well established. We have investigated the available evidence for contact tracing with a focus on public ground transport aiming to give guidance in what situations contact tracing should be considered. METHODS: Relevant infectious diseases suitable for contact tracing in ground transport and a set of disease-specific epidemiological criteria were defined through literature search and structured multistep expert consultations. We developed continuous scales for each criterion to be rated for its relevance to contact tracing in ground transport. We used the Delphi method with an international expert panel to position the values of criteria on the respective scales. RESULTS: The study led to the development of the ‘Contact Tracing-Risk Assessment Profile’ (CT-RAP), a decision-making instrument, taking into account pathogen-specific as well as situation-specific criteria. This report describes the methodology of this instrument and presents two examples of ready-to-use CT-RAP for tuberculosis and for meningococcal disease in public ground transport. DISCUSSION: The systematic and transparent use of the CT-RAP for tuberculosis and meningococcal disease is likely to facilitate reasonable, efficient and user-friendly decisions with respect to contact tracing. New CT-RAPs for additional pathogens and different settings such as schools and kindergartens are being planned.

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MicroRNA-based strategy to mitigate the risk of gain-of-function influenza studies

Recent gain-of-function studies in influenza A virus H5N1 strains revealed that as few as three amino-acid changes in the hemagglutinin protein confer the capacity for viral transmission between ferrets(1, 2). As transmission between ferrets is considered a surrogate indicator of transmissibility between humans, these studies raised concerns about the risks of gain-of-function influenza A virus research. Here we present an approach to strengthen the biosafety of gain-of-function influenza experiments. We exploit species-specific endogenous small RNAs to restrict influenza A virus tropism. In particular, we found that the microRNA miR-192 was expressed in primary human respiratory tract epithelial cells as well as mouse lungs but absent from the ferret respiratory tract. Incorporation of miR-192 target sites into influenza A virus did not prevent influenza replication and transmissibility in ferrets, but did attenuate influenza pathogenicity in mice. This molecular biocontainment approach should be applicable beyond influenza A virus to minimize the risk of experiments involving other pathogenic viruses.

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Diversity and Epidemiology of Mokola Virus

Mokola virus (MOKV) appears to be exclusive to Africa. Although the first isolates were from Nigeria and other Congo basin countries, all reports over the past 20 years have been from southern Africa. Previous phylogenetic studies analyzed few isolates or used partial gene sequence for analysis since limited sequence information is available for MOKV and the isolates were distributed among various laboratories. The complete nucleoprotein, phosphoprotein, matrix and glycoprotein genes of 18 MOKV isolates in various laboratories were sequenced either using partial or full genome sequencing using pyrosequencing and a phylogenetic analysis was undertaken. The results indicated that MOKV isolates from the Republic of South Africa, Zimbabwe, Central African Republic and Nigeria clustered according to geographic origin irrespective of the genes used for phylogenetic analysis, similar to that observed with Lagos bat virus. A Bayesian Markov-Chain-Monte-Carlo- (MCMC) analysis revealed the age of the most recent common ancestor (MRCA) of MOKV to be between 279 and 2034 years depending on the genes used. Generally, all MOKV isolates showed a similar pattern at the amino acid sites considered influential for viral properties.

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GenomeFingerprinter: The Genome Fingerprint and the Universal Genome Fingerprint Analysis for Systematic Comparative Genomics

BACKGROUND: No attention has been paid on comparing a set of genome sequences crossing genetic components and biological categories with far divergence over large size range. We define it as the systematic comparative genomics and aim to develop the methodology. RESULTS: First, we create a method, GenomeFingerprinter, to unambiguously produce a set of three-dimensional coordinates from a sequence, followed by one three-dimensional plot and six two-dimensional trajectory projections, to illustrate the genome fingerprint of a given genome sequence. Second, we develop a set of concepts and tools, and thereby establish a method called the universal genome fingerprint analysis (UGFA). Particularly, we define the total genetic component configuration (TGCC) (including chromosome, plasmid, and phage) for describing a strain as a systematic unit, the universal genome fingerprint map (UGFM) of TGCC for differentiating strains as a universal system, and the systematic comparative genomics (SCG) for comparing a set of genomes crossing genetic components and biological categories. Third, we construct a method of quantitative analysis to compare two genomes by using the outcome dataset of genome fingerprint analysis. Specifically, we define the geometric center and its geometric mean for a given genome fingerprint map, followed by the Euclidean distance, the differentiate rate, and the weighted differentiate rate to quantitatively describe the difference between two genomes of comparison. Moreover, we demonstrate the applications through case studies on various genome sequences, giving tremendous insights into the critical issues in microbial genomics and taxonomy. CONCLUSIONS: We have created a method, GenomeFingerprinter, for rapidly computing, geometrically visualizing, intuitively comparing a set of genomes at genome fingerprint level, and hence established a method called the universal genome fingerprint analysis, as well as developed a method of quantitative analysis of the outcome dataset. These have set up the methodology of systematic comparative genomics based on the genome fingerprint analysis.

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Defective Viral Genomes Arising In Vivo Provide Critical Danger Signals for the Triggering of Lung Antiviral Immunity

The innate immune response to viruses is initiated when specialized cellular sensors recognize viral danger signals. Here we show that truncated forms of viral genomes that accumulate in infected cells potently trigger the sustained activation of the transcription factors IRF3 and NF-κB and the production type I IFNs through a mechanism independent of IFN signaling. We demonstrate that these defective viral genomes (DVGs) are generated naturally during respiratory infections in vivo even in mice lacking the type I IFN receptor, and their appearance coincides with the production of cytokines during infections with Sendai virus (SeV) or influenza virus. Remarkably, the hallmark antiviral cytokine IFNβ is only expressed in lung epithelial cells containing DVGs, while cells within the lung that contain standard viral genomes alone do not express this cytokine. Together, our data indicate that DVGs generated during viral replication are a primary source of danger signals for the initiation of the host immune response to infection.

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Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC(50) values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity - inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis.

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Strategies for Designing Transgenic DNA Constructs

Generation and characterization of transgenic mice are important elements of biomedical research. In recent years, transgenic technology has become more versatile and sophisticated, mainly because of the incorporation of recombinase-mediated conditional expression and targeted insertion, site-specific endonuclease-mediated genome editing, siRNA-mediated gene knockdown, various inducible gene expression systems, and fluorescent protein marking and tracking techniques. Site-specific recombinases (such as PhiC31) and engineered endonucleases (such as ZFN and Talen) have significantly enhanced our ability to target transgenes into specific genomic loci, but currently a great majority of transgenic mouse lines are continuingly being created using the conventional random insertion method. A major challenge for using this conventional method is that the genomic environment at the integration site has a substantial influence on the expression of the transgene. Although our understanding of such chromosomal position effects and our means to combat them are still primitive, adhering to some general guidelines can significantly increase the odds of successful transgene expression. This chapter first discusses the major problems associated with transgene expression, and then describes some of the principles for using plasmid and bacterial artificial chromosomes (BACs) for generating transgenic constructs. Finally, the strategies for conducting each of the major types of transgenic research are discussed, including gene overexpression, promoter characterization, cell-lineage tracing, mutant complementation, expression of double or multiple transgenes, siRNA knockdown, and conditional and inducible systems.

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Vaccines for the future: learning from human immunology

Conventional vaccines have been extremely successful in preventing infections by pathogens expressing relatively conserved antigens through antibody‐mediated effector mechanisms. Thanks to vaccination some diseases have been eradicated and mortality due to infectious diseases has been significantly reduced. However, there are still many infections that are not preventable with vaccination, which represent a major cause of mortality worldwide. Some of these infections are caused by pathogens with a high degree of antigen variability that cannot be controlled only by antibodies, but require a mix of humoral and cellular immune responses. Novel technologies for antigen discovery, expression and formulation allow now for the development of vaccines that can better cope with pathogen diversity and trigger multifunctional immune responses. In addition, the application of new genomic assays and systems biology approaches in human immunology can help to better identify vaccine correlates of protection. The availability of novel vaccine technologies, together with the knowledge of the distinct human immune responses that are required to prevent different types of infection, should help to rationally design effective vaccines where conventional approaches have failed.

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Docking, virtual high throughput screening and in silico fragment-based drug design

The drug discovery process has been profoundly changed recently by the adoption of computational methods helping the design of new drug candidates more rapidly and at lower costs. In silico drug design consists of a collection of tools helping to make rational decisions at the different steps of the drug discovery process, such as the identification of a biomolecular target of therapeutical interest, the selection or the design of new lead compounds and their modification to obtain better affinities, as well as pharmacokinetic and pharmacodynamic properties. Among the different tools available, a particular emphasis is placed in this review on molecular docking, virtual high-throughput screening and fragment-based ligand design.

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Ubiquitin-Specific Proteases 25 Negatively Regulates Virus-Induced Type I Interferon Signaling

Ubiquitination and deubiquitination have emerged as critical regulatory processes in the virus-triggered type I interferon (IFN) induction pathway. In this study, we carried out a targeted siRNA screen of 54 ubiquitin-specific proteases (USPs) and identified USP25 as a negative regulator of the virus-triggered type I IFN signaling pathway. Overexpression of USP25 inhibited virus-induced activation of IFN-β, interferon regulation factor 3 (IRF3) and nuclear factor-kappa B (NF-κB), as well as the phosphorylation of IRF3 and NF-κB subunit p65. Furthermore, Knockdown of USP25 potentiated virus-induced induction of the IFN-β. In addition, detailed analysis demonstrated that USP25 cleaved lysine 48- and lysine 63-linked polyubiquitin chains in vitro and in vivo, and its deubiquitinating enzyme (DUB) activity, were dependent on a cysteine residue (Cys178) and a histidine residue (His607). USP25 mutants lacking DUB activity lost the ability to block virus-induced type I IFN to some degree. Mechanistically, USP25 deubiquitinated retinoic acid-inducible gene I (RIG-I), tumornecrosis factor (TNF) receptor-associated factor 2 (TRAF2), and TRAF6 to inhibit RIG-I-like receptor-mediated IFN signaling. Our findings suggest that USP25 is a novel DUB negatively regulating virus-induced type I IFN production.