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900 | Marine Organism Cell Biology and Regulatory Sequence Discoveryin Comparative Functional Genomics | The use of bioinformatics to integrate phenotypic and genomic data from mammalian models is well established as a means of understanding human biology and disease. Beyond direct biomedical applications of these approaches in predicting structure–function relationships between coding sequences and protein activities, comparative studies also promote understanding of molecular evolution and the relationship between genomic sequence and morphological and physiological specialization. Recently recognized is the potential of comparative studies to identify functionally significant regulatory regions and to generate experimentally testable hypotheses that contribute to understanding mechanisms that regulate gene expression, including transcriptional activity, alternative splicing and transcript stability. Functional tests of hypotheses generated by computational approaches require experimentally tractable in vitro systems, including cell cultures. Comparative sequence analysis strategies that use genomic sequences from a variety of evolutionarily diverse organisms are critical for identifying conserved regulatory motifs in the 5′-upstream, 3′-downstream and introns of genes. Genomic sequences and gene orthologues in the first aquatic vertebrate and protovertebrate organisms to be fully sequenced (Fugu rubripes, Ciona intestinalis, Tetraodon nigroviridis, Danio rerio) as well as in the elasmobranchs, spiny dogfish shark (Squalus acanthias) and little skate (Raja erinacea), and marine invertebrate models such as the sea urchin (Strongylocentrotus purpuratus) are valuable in the prediction of putative genomic regulatory regions. Cell cultures have been derived for these and other model species. Data and tools resulting from these kinds of studies will contribute to understanding transcriptional regulation of biomedically important genes and provide new avenues for medical therapeutics and disease prevention. |
901 | Regulated multicistronic expression technology for mammalian metabolic engineering | Contemporary basic research is rapidly revealing increasingly complex molecular regulatory networks which are often interconnected via key signal integrators. These connections among regulatory and catalytic networks often frustrate bioengineers as promising metabolic engineering strategies are bypassed by compensatory metabolic responses or cause unexpected, undesired outcomes such as apoptosis, product protein degradation or inappropriate post- translational modification. Therefore, for metabolic engineering to achieve greater success in mammalian cell culture processes and to become important for future applications such as gene therapy and tissue engineering, this technology must be enhanced to allow simultaneous, in cases conditional, reshaping of metabolic pathways to access difficult-to-attain cell states. Recent advances in this new territory of multigene metabolic engineering are intimately linked to the development of multicistronic expression technology which allows the simultaneous, and in some cases, regulated expression of several genes in mammalian cells. Here we review recent achievements in multicistronic expression technology in view of multigene metabolic engineering. |
902 | Production of high titre disabled infectious single cycle (DISC) HSV from a microcarrier culture | Disabled Infectious Single Cycle (DISC) HSV-2 has been cultured in the complimentary cell line CR2 to provide high titre bulk material suitable for the purification of the virus as a live viral vaccine. CR2 cells are cultured on the microcarrier Cytodex-1 at 5 g l-1 in small scale (1 l) and larger scale (15 l) reactors. The cells are infected at an MOI of 0.01 pfu cell-1 and the culture harvested 60–72 h later. The infected cells are removed from the microcarriers by the addition of a hypotonic saline and the virus released by low-pressure disruption techniques. Virus titres achieved are compared to the standard roller bottle process. The resulting material is the starting point for the purification of the DISC-HSV virus. |
903 | Diagnosis of influenza viruses with special reference to novel H1N1 2009 influenza virus | On 15 April and 17 April 2009, novel swineorigin influenza A (H1N1) virus was identifi ed in specimens obtained from two epidemiologically unlinked patients in the United States. The ongoing outbreak of novel H1N1 2009 influenza (swine influenza) has caused more than 3,99,232 laboratory confi rmed cases of pandemic influenza H1N1 and over 4735 deaths globally. This novel 2009 influenza virus designated as H1N1 A/swine/California/04/2009 virus is not zoonotic swine flu and is transmitted from person to person and has higher transmissibility then that of seasonal influenza viruses. In India the novel H1N1 virus infection has been reported from all over the country. A total of 68,919 samples from clinically suspected persons have been tested for influenza A H1N1 across the country and 13,330 (18.9%) of them have been found positive with 427 deaths. At the All India Institute of Medical Sciences, New Delhi India, we tested 1096 clinical samples for the presence of novel H1N1 influenza virus and seasonal influenza viruses. Of these 1096 samples, 194 samples (17.7%) were positive for novel H1N1 influenza virus and 197 samples (18%) were positive for seasonal influenza viruses. During outbreaks of emerging infectious diseases accurate and rapid diagnosis is critical for minimizing further spread through timely implementation of appropriate vaccines and antiviral treatment. Since the symptoms of novel H1N1 influenza infection are not specifi c, laboratory confi rmation of suspected cases is of prime importance. |
904 | Influenza A: From highly pathogenic H5N1 to pandemic 2009 H1N1. Epidemiology and clinical features | The last decade has seen the emergence of two new influenza A subtypes and they have become a cause of concern for the global community. These are the highly pathogenic H5N1 influenza A virus (H5N1) and the Pandemic 2009 influenza H1N1 virus. Since 2003 the H5N1 virus has caused widespread disease and death in poultry, mainly in south East Asia and Africa. In humans the number of cases infected with this virus is few but the mortality has been about 60%. Most patients have presented with severe pneumonia and acute respiratory distress syndrome. The second influenza virus, the pandemic H1N1 2009, emerged in Mexico in March this year. This virus acquired the ability for sustained human to human spread and within a few months spread throughout the world and infected over 4 lakh individuals. The symptoms of infection with this virus are similar to seasonal influenza but it currently affecting younger individuals more often. Fortunately the mortality has been low. Both these new influenza viruses are currently circulating and have different clinical and epidemiological characteristics. |
905 | Lessons learned from the 1918–1919 influenza pandemic | The 1918 influenza pandemic was one of the most virulent strains of influenza in history. Phylogenic evidence of the novel H1N1 strain of influenza discovered in Mexico last spring (2009) links it to the 1918 influenza strain. With information gained from analyzing viral genetics, public health records and advances in medical science we can confront the 2009 H1N1 influenza on a global scale. The paper analyses the causes and characteristics of a pandemic, and major issues in controlling the spread of the disease. Wide public vaccination and open communication between government and health sciences professionals will be an essential and vital component in managing the 2009 H1N1 pandemic and any future pandemics. |
906 | Differential Effects of IL-12 on Tregs and Non-Treg T Cells: Roles of IFN-γ, IL-2 and IL-2R | Complex interactions between effector T cells and Foxp3(+) regulatory T cells (Treg) contribute to clinical outcomes in cancer, and autoimmune and infectious diseases. Previous work showed that IL-12 reversed Treg-mediated suppression of CD4(+)Foxp3(−) T cell (Tconv) proliferation. We and others have also shown that Tregs express T-bet and IFN-γ at sites of Th1 inflammation and that IL-12 induces IFN-γ production by Tregs in vitro. To investigate whether loss of immunosuppression occurs when IFN-γ is expressed by Tregs we treated mouse lymphocyte cultures with IL-12. IFN-γ expression did not decrease the ability of Tregs to suppress Tconv proliferation. Rather, IL-12 treatment decreased Treg frequency and Foxp3 levels in Tregs. We further showed that IL-12 increased IL-2R expression on Tconv and CD8 T cells, diminished its expression on Tregs and decreased IL-2 production by Tconv and CD8 T cells. Together, these IL-12 mediated changes favored the outgrowth of non-Tregs. Additionally, we showed that treatment with a second cytokine, IL-27, decreased IL-2 expression without augmenting Tconv and CD8 T cell proliferation. Notably, IL-27 only slightly modified levels of IL-2R on non-Treg T cells. Together, these results show that IL-12 has multiple effects that modify the balance between Tregs and non-Tregs and support an important role for relative levels of IL-2R but not for IFN-γ expression in IL-12-mediated reversal of Treg immunosuppression. |
907 | Genetic Variation in the TNF Gene Is Associated with Susceptibility to Severe Sepsis, but Not with Mortality | BACKGROUND: Tumor necrosis factor (TNF) and TNF receptor superfamily (TNFR)-mediated immune response play an essential role in the pathogenesis of severe sepsis. Studies examining associations of TNF and lymphotoxin-α (LTA) single nucleotide polymorphisms (SNPs) with severe sepsis have produced conflicting results. The objective of this study was to investigate whether genetic variation in TNF, LTA, TNFRSF1A and TNFRSF1B was associated with susceptibility to or death from severe sepsis in Chinese Han population. METHODOLOGY/PRINCIPAL FINDINGS: Ten SNPs in TNF, LTA, TNFRSF1A and TNFRSF1B were genotyped in samples of patients with severe sepsis (n = 432), sepsis (n = 384) and healthy controls (n = 624). Our results showed that rs1800629, a SNP in the promoter region of TNF, was significantly associated with risk for severe sepsis. The minor allele frequency of rs1800629 was significantly higher in severe sepsis patients than that in both healthy controls (P(adj) = 0.00046, odds ratio (OR)(adj) = 1.92) and sepsis patients (P(adj) = 0.002, OR(adj) = 1.56). Further, we investigated the correlation between rs1800629 genotypes and TNF-α concentrations in peripheral blood mononuclear cells (PBMCs) of healthy volunteers exposed to lipopolysaccharides (LPS) ex vivo, and the association between rs1800629 and TNF-α serum levels in severe sepsis patients. After exposure to LPS, the TNF-α concentration in culture supernatants of PBMCs was significantly higher in the subjects with AA+AG genotypes than that with GG genotype (P = 0.007). Moreover, in patients with severe sepsis, individuals with AA+AG genotypes had significantly higher TNF-α serum concentrations than those with GG genotype (P(adj) = 0.02). However, there were no significant associations between SNPs in the four candidate genes and 30 day mortality for patients with severe sepsis. CONCLUSIONS/SIGNIFICANCE: Our findings suggested that the functional TNF gene SNP rs1800629 was strongly associated with susceptibility to severe sepsis, but not with lethality in Chinese Han population. |
908 | Identification, Characterization and Application of a G-Quadruplex Structured DNA Aptamer against Cancer Biomarker Protein Anterior Gradient Homolog 2 | BACKGROUND: Anterior gradient homolog 2 (AGR2) is a functional protein with critical roles in a diverse range of biological systems, including vertebrate tissue development, inflammatory tissue injury responses, and cancer progression. Clinical studies have shown that the AGR2 protein is overexpressed in a wide range of human cancers, including carcinomas of the esophagus, pancreas, breast, prostate, and lung, making the protein as a potential cancer biomarker. However, the general biochemical functions of AGR2 in human cells remain undefined, and the signaling mechanisms that drive AGR2 to inhibit p53 are still not clearly illustrated. Therefore, it is of great interest to develop molecular probes specifically recognizing AGR2 for its detection and for the elucidation of AGR2-associated molecular mechanism. METHODOLOGY/PRINCIPAL FINDINGS: Through a bead-based and flow cytometry monitored SELEX technology, we have identified a group of DNA aptamers that can specifically bind to AGR2 with K(d) values in the nanomolar range after 14 rounds of selections. Aptamer C14B was chosen to further study, due to its high binding affinity and specificity. The optimized and shortened C14B1 has special G-rich characteristics, and the G-rich region of this binding motif was further characterized to reveal an intramolecular parallel G-quadruplex by CD spectroscopy and UV spectroscopy. Our experiments confirmed that the stability of the G-quadruplex structure was strongly dependent on the nature of the monovalent ions and the formation of G-quadruplex structure was also important for the binding capacity of C14B1 to the target. Furthermore, we have designed a kind of allosteric molecule beacon (aMB) probe for selective and sensitive detection of AGR2. CONCLUSION/SIGNIFICANCE: In this work, we have developed new aptamer probes for specific recognition of the AGR2. Structural study have identified that the binding motif of aptamer is an intramolecular parallel G-quadruplex structure and its structure and binding affinity are strongly dependent on the nature of the monovalent ion. Furthermore, with our design of AGR2-aMB, AGR2 could be sensitively and selectively detected. This aptamer probe has great potential to serve as a useful tool for early diagnosis and prognosis of cancer and for fundamental research to elucidate the biochemical functions of AGR2. |
909 | Flt3L Combined with Rapamycin Promotes Cardiac Allograft Tolerance by Inducing Regulatory Dendritic Cells and Allograft Autophagy in Mice | The induction of immune tolerance is still a formidable challenge in organ transplantation. Dendritic cells (DCs) play an important role in orchestrating immune responses by either mediating protective immune responses or inducing antigen specific tolerance. Previous studies demonstrated that the fms-like tyrosine kinase 3 receptor (Flt3) and its ligand (Flt3L) play an essential role in the regulation of DC commitment and development. Here, we report a synergic effect between Flt3L and low-dose rapamycin (Rapa) in the protection of allograft rejction. It was found that Flt3L combined with Rapa significantly prolonged murine cardiac allograft survival time as compared with that of untreated recipients or recipients treated with Rapa or Flt3L alone. Mechanistic studies revealed that Flt3L combined with low-dose of Rapa induced the generation of tolerogenic DCs along with the production of CD25(+) Foxp3(+) regulatory T cells and IL-10 secretion. We also observed enhanced autophagy in the cardiac allograft, which could be another asset contributing to the enhanced allograft survival. All together, these data suggest that Flt3L combined with low-dose of Rapa could be an effective therapeutic approach to induce tolerance in clinical setting of transplantation. |
910 | Renalase's Expression and Distribution in Renal Tissue and Cells | To study renalase's expression and distribution in renal tissues and cells, renalase coded DNA vaccine was constructed, and anti-renalase monoclonal antibodies were produced using DNA immunization and hybridoma technique, followed by further investigation with immunological testing and western blotting to detect the expression and distribution of renalase among the renal tissue and cells. Anti-renalase monoclonal antibodies were successfully prepared by using DNA immunization technique. Further studies with anti-renalase monoclonal antibody showed that renalase expressed in glomeruli, tubule, mesangial cells, podocytes, renal tubule epithelial cells and its cells supernatant. Renalase is wildly expressed in kidney, including glomeruli, tubule, mesangial cells, podocytes and tubule epithelial cells, and may be secreted by tubule epithelial cells primarily. |
911 | Virus contaminations of cell cultures – A biotechnological view | In contrast to contamination by microbes and mycoplasma, which can be relatively easily detected, viral contamination present a serious threat because of the difficulty in detecting some viruses and the lack of effective methods of treating infected cell cultures. While some viruses are capable of causing morphological changes to infected cells (e.g. cytopathic effect)which are detectable by microscopy some viral contaminations result in the integration of the viral genome as provirus, this causes no visual evidence, by means of modification of the cellular morphology. Virus production from such cell lines, are potentially dangerous for other cell cultures (in research labs)by cross contaminations, or for operators and patients (in the case of the production of injectable biologicals) because of potential infection. The only way to keep cell cultures for research, development, and the biotech industry virus-free is the prevention of such contaminations. Cell cultures can become contaminated by the following means: firstly, they may already be contaminated as primary cultures (because the source of the cells was already infected), secondly, they were contaminated due to the use of contaminated raw materials, or thirdly, they were contaminated via an animal passage. This overview describes the problems and risks associated with viral contaminations in animal cell culture, describes the origins of these contaminations as well as the most important virsuses associated with viral contaminations in cell culture. In addition, ways to prevent viral contaminations as well as measures undertaken to avoid and assess risks for viral contaminations as performed in the biotech industry are briefly described. |
912 | DENV Inhibits Type I IFN Production in Infected Cells by Cleaving Human STING | Dengue virus (DENV) is a pathogen with a high impact on human health. It replicates in a wide range of cells involved in the immune response. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response. DENV circumvents the host immune response by expressing proteins that antagonize the cellular innate immunity. We have recently documented the inhibition of type I IFN production by the proteolytic activity of DENV NS2B3 protease complex in human monocyte derived dendritic cells (MDDCs). In the present report we identify the human adaptor molecule STING as a target of the NS2B3 protease complex. We characterize the mechanism of inhibition of type I IFN production in primary human MDDCs by this viral factor. Using different human and mouse primary cells lacking STING, we show enhanced DENV replication. Conversely, mutated versions of STING that cannot be cleaved by the DENV NS2B3 protease induced higher levels of type I IFN after infection with DENV. Additionally, we show that DENV NS2B3 is not able to degrade the mouse version of STING, a phenomenon that severely restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition of DENV infection and spread in mice. |
913 | Acute Reactogenicity after Intramuscular Immunization with Recombinant Vesicular Stomatitis Virus Is Linked to Production of IL-1β | Vaccines based on live viruses are attractive because they are immunogenic, cost-effective, and can be delivered by multiple routes. However, live virus vaccines also cause reactogenic side effects such as fever, myalgia, and injection site pain that have reduced their acceptance in the clinic. Several recent studies have linked vaccine-induced reactogenic side effects to production of the pro-inflammatory cytokine interleukin-1β (IL-1β) in humans. Our objective was therefore to determine whether IL-1β contributed to pathology after immunization with recombinant vesicular stomatitis virus (rVSV) vaccine vectors, and if so, to identify strategies by which IL-1β mediated pathology might be reduced without compromising immunogenicity. We found that an rVSV vaccine induced local and systemic production of IL-1β in vivo, and that accumulation of IL-1β correlated with acute pathology after rVSV immunization. rVSV-induced pathology was reduced in mice deficient in the IL-1 receptor Type I, but the IL-1R−/− mice were fully protected from lethal rechallenge with a high dose of VSV. This result demonstrated that IL-1 contributed to reactogenicity of the rVSV, but was dispensable for induction of protective immunity. The amount of IL-1β detected in mice deficient in either caspase-1 or the inflammasome adaptor molecule ASC after rVSV immunization was not significantly different than that produced by wild type animals, and caspase-1−/− and ASC−/− mice were only partially protected from rVSV-induced pathology. Those data support the idea that some of the IL-1β expressed in vivo in response to VSV may be activated by a caspase-1 and ASC-independent mechanism. Together these results suggest that rVSV vectors engineered to suppress the induction of IL-1β, or signaling through the IL-1R would be less reactogenic in vivo, but would retain their immunogenicity and protective capacity. Such rVSV would be highly desirable as either vaccine vectors or oncolytic therapies, and would likely be better tolerated in human vaccinees. |
914 | Competition between Influenza A Virus Genome Segments | Influenza A virus (IAV) contains a segmented negative-strand RNA genome. How IAV balances the replication and transcription of its multiple genome segments is not understood. We developed a dual competition assay based on the co-transfection of firefly or Gaussia luciferase-encoding genome segments together with plasmids encoding IAV polymerase subunits and nucleoprotein. At limiting amounts of polymerase subunits, expression of the firefly luciferase segment was negatively affected by the presence of its Gaussia luciferase counterpart, indicative of competition between reporter genome segments. This competition could be relieved by increasing or decreasing the relative amounts of firefly or Gaussia reporter segment, respectively. The balance between the luciferase expression levels was also affected by the identity of the untranslated regions (UTRs) as well as segment length. In general it appeared that genome segments displaying inherent higher expression levels were more efficient competitors of another segment. When natural genome segments were tested for their ability to suppress reporter gene expression, shorter genome segments generally reduced firefly luciferase expression to a larger extent, with the M and NS segments having the largest effect. The balance between different reporter segments was most dramatically affected by the introduction of UTR panhandle-stabilizing mutations. Furthermore, only reporter genome segments carrying these mutations were able to efficiently compete with the natural genome segments in infected cells. Our data indicate that IAV genome segments compete for available polymerases. Competition is affected by segment length, coding region, and UTRs. This competition is probably most apparent early during infection, when limiting amounts of polymerases are present, and may contribute to the regulation of segment-specific replication and transcription. |
915 | Oral health in China – trends and challenges | For a long time, oral disease is one of the major problems of the public health for its high prevalence and incidence throughout the world, which is especially true for low-income populations. Since China's economic reform in 1978, great changes have taken place in China. These changes have significant impact on and have been reflected in oral disease trends in China. This paper provides an overview and assessment of the oral health status in China. It focuses on changes in the nation's demographic profile, in the marketplace, the oral disease status and trends. The paper also suggests some possible measures and strategies for bettering oral health in future China. |
916 | National intensive care unit bed capacity and ICU patient characteristics in a low income country | BACKGROUND: Primary health care delivery in the developing world faces many challenges. Priority setting favours HIV, TB and malaria interventions. Little is known about the challenges faced in this setting with regard to critical care medicine. The aim of this study was to analyse and categorise the diagnosis and outcomes of 1,774 patients admitted to a hospital intensive care unit (ICU) in a low-income country over a 7-year period. We also assessed the country’s ICU bed capacity and described the challenges faced in dealing with critically ill patients in this setting. FINDINGS: A retrospective audit was conducted in a general ICU in a university hospital in Uganda. Demographic data, admission diagnosis, and ICU length of stay were recorded for the 1,774 patients who presented to the ICU in the period January 2003 to December 2009. Their mean age was 35.5 years. Males accounted for 56.5% of the study population; 92.8% were indigenous, and 42.9% were referrals from upcountry units. The average mortality rate over the study period was 40.1% (n = 715). The highest mortality rate (44%) was recorded in 2004 and the lowest (33.2%) in 2005. Children accounted for 11.6% of admissions (40.1% mortality). Sepsis, ARDS, traumatic brain injuries and HIV related conditions were the most frequent admission diagnoses. A telephonic survey determined that there are 33 adult ICU beds in the whole country. CONCLUSIONS: Mortality was 40.1%, with sepsis, head injury, acute lung injury and HIV/AIDS the most common admission diagnoses. The country has a very low ICU bed capacity. Prioritising infectious diseases poses a challenge to ensuring that critical care is an essential part of the health care package in Uganda. |
917 | Influenza Virus Infection in Nonhuman Primates | To determine whether nonhuman primates are infected with influenza viruses in nature, we conducted serologic and swab studies among macaques from several parts of the world. Our detection of influenza virus and antibodies to influenza virus raises questions about the role of nonhuman primates in the ecology of influenza. |
918 | Label-Free Electrochemical Diagnosis of Viral Antigens with Genetically Engineered Fusion Protein | We have developed a simple electrochemical biosensing strategy for the label-free diagnosis of hepatitis B virus (HBV) on a gold electrode surface. Gold-binding polypeptide (GBP) fused with single-chain antibody (ScFv) against HBV surface antigen (HBsAg), in forms of genetically engineered protein, was utilized. This GBP-ScFv fusion protein can directly bind onto the gold substrate with the strong binding affinity between the GBP and the gold surface, while the recognition site orients toward the sample for target binding at the same time. Furthermore, this one-step immobilization strategy greatly simplifies a fabrication process without any chemical modification as well as maintaining activity of biological recognition elements. This system allows specific immobilization of proteins and sensitive detection of targets, which were verified by surface plasmon resonance analysis and successfully applied to electrochemical cyclic voltammetry and impedance spectroscopy upto 0.14 ng/mL HBsAg. |
919 | Development of a Plastic-Based Microfluidic Immunosensor Chip for Detection of H1N1 Influenza | Lab-on-a-chip can provide convenient and accurate diagnosis tools. In this paper, a plastic-based microfluidic immunosensor chip for the diagnosis of swine flu (H1N1) was developed by immobilizing hemagglutinin antigen on a gold surface using a genetically engineered polypeptide. A fluorescent dye-labeled antibody (Ab) was used for quantifying the concentration of Ab in the immunosensor chip using a fluorescent technique. For increasing the detection efficiency and reducing the errors, three chambers and three microchannels were designed in one microfluidic chip. This protocol could be applied to the diagnosis of other infectious diseases in a microfluidic device. |
920 | Hepatitis C VLPs Delivered to Dendritic Cells by a TLR2 Targeting Lipopeptide Results in Enhanced Antibody and Cell-Mediated Responses | Although many studies provide strong evidence supporting the development of HCV virus-like particle (VLP)-based vaccines, the fact that heterologous viral vectors and/or multiple dosing regimes are required to induce protective immunity indicates that it is necessary to improve their immunogenicity. In this study, we have evaluated the use of an anionic self-adjuvanting lipopeptide containing the TLR2 agonist Pam(2)Cys (E(8)Pam(2)Cys) to enhance the immunogenicity of VLPs containing the HCV structural proteins (core, E1 and E2) of genotype 1a. While co-formulation of this lipopeptide with VLPs only resulted in marginal improvements in dendritic cell (DC) uptake, its ability to concomitantly induce DC maturation at very small doses is a feature not observed using VLPs alone or in the presence of an aluminium hydroxide-based adjuvant (Alum). Dramatically improved VLP and E2-specific antibody responses were observed in VLP+E(8)Pam(2)Cys vaccinated mice where up to 3 doses of non-adjuvanted or traditionally alum-adjuvanted VLPs was required to match the antibody titres obtained with a single dose of VLPs formulated with this lipopeptide. This result also correlated with significantly higher numbers of specific antibody secreting cells that was detected in the spleens of VLP+E(8)Pam(2)Cys vaccinated mice and greater ability of sera from these mice to neutralise the binding and uptake of VLPs by Huh7 cells. Moreover, vaccination of HLA-A2 transgenic mice with this formulation also induced better VLP-specific IFN-γ-mediated responses compared to non-adjuvanted VLPs but comparable levels to that achieved when coadministered with complete freund’s adjuvant. These results suggest overall that the immunogenicity of HCV VLPs can be significantly improved by the addition of this novel adjuvant by targeting their delivery to DCs and could therefore constitute a viable vaccine strategy for the treatment of HCV. |
921 | Viral metagenomic analysis of bushpigs (Potamochoerus larvatus) in Uganda identifies novel variants of Porcine parvovirus 4 and Torque teno sus virus 1 and 2 | BACKGROUND: As a result of rapidly growing human populations, intensification of livestock production and increasing exploitation of wildlife habitats for animal agriculture, the interface between wildlife, livestock and humans is expanding, with potential impacts on both domestic animal and human health. Wild animals serve as reservoirs for many viruses, which may occasionally result in novel infections of domestic animals and/or the human population. Given this background, we used metagenomics to investigate the presence of viral pathogens in sera collected from bushpigs (Potamochoerus larvatus), a nocturnal species of wild Suid known to move between national parks and farmland, in Uganda. RESULTS: Application of 454 pyrosequencing demonstrated the presence of Torque teno sus virus (TTSuV), porcine parvovirus 4 (PPV4), porcine endogenous retrovirus (PERV), a GB Hepatitis C–like virus, and a Sclerotinia hypovirulence-associated-like virus in sera from the bushpigs. PCR assays for each specific virus combined with Sanger sequencing revealed two TTSuV-1 variants, one TTSuV-2 variant as well as PPV4 in the serum samples and thereby confirming the findings from the 454 sequencing. CONCLUSIONS: Using a viral metagenomic approach we have made an initial analysis of viruses present in bushpig sera and demonstrated for the first time the presence of PPV4 in a wild African Suid. In addition we identified novel variants of TTSuV-1 and 2 in bushpigs. |
922 | A Three-Dimensional Comparison of Tick-Borne Flavivirus Infection in Mammalian and Tick Cell Lines | Tick-borne flaviviruses (TBFV) are sustained in nature through cycling between mammalian and tick hosts. In this study, we used African green monkey kidney cells (Vero) and Ixodes scapularis tick cells (ISE6) to compare virus-induced changes in mammalian and arthropod cells. Using confocal microscopy, transmission electron microscopy (TEM), and electron tomography (ET), we examined viral protein distribution and the ultrastructural changes that occur during TBFV infection. Within host cells, flaviviruses cause complex rearrangement of cellular membranes for the purpose of virus replication. Virus infection was accompanied by a marked expansion in endoplasmic reticulum (ER) staining and markers for TBFV replication were localized mainly to the ER in both cell lines. TEM of Vero cells showed membrane-bound vesicles enclosed in a network of dilated, anastomosing ER cisternae. Virions were seen within the ER and were sometimes in paracrystalline arrays. Tubular structures or elongated vesicles were occasionally noted. In acutely and persistently infected ISE6 cells, membrane proliferation and vesicles were also noted; however, the extent of membrane expansion and the abundance of vesicles were lower and no viral particles were observed. Tubular profiles were far more prevalent in persistently infected ISE6 cells than in acutely infected cells. By ET, tubular profiles, in persistently infected tick cells, had a cross-sectional diameter of 60–100 nm, reached up to 800 nm in length, were closed at the ends, and were often arranged in fascicle-like bundles, shrouded with ER membrane. Our experiments provide analysis of viral protein localization within the context of both mammalian and arthropod cell lines as well as both acute and persistent arthropod cell infection. Additionally, we show for the first time 3D flavivirus infection in a vector cell line and the first ET of persistent flavivirus infection. |
923 | SEED Servers: High-Performance Access to the SEED Genomes, Annotations, and Metabolic Models | The remarkable advance in sequencing technology and the rising interest in medical and environmental microbiology, biotechnology, and synthetic biology resulted in a deluge of published microbial genomes. Yet, genome annotation, comparison, and modeling remain a major bottleneck to the translation of sequence information into biological knowledge, hence computational analysis tools are continuously being developed for rapid genome annotation and interpretation. Among the earliest, most comprehensive resources for prokaryotic genome analysis, the SEED project, initiated in 2003 as an integration of genomic data and analysis tools, now contains >5,000 complete genomes, a constantly updated set of curated annotations embodied in a large and growing collection of encoded subsystems, a derived set of protein families, and hundreds of genome-scale metabolic models. Until recently, however, maintaining current copies of the SEED code and data at remote locations has been a pressing issue. To allow high-performance remote access to the SEED database, we developed the SEED Servers (http://www.theseed.org/servers): four network-based servers intended to expose the data in the underlying relational database, support basic annotation services, offer programmatic access to the capabilities of the RAST annotation server, and provide access to a growing collection of metabolic models that support flux balance analysis. The SEED servers offer open access to regularly updated data, the ability to annotate prokaryotic genomes, the ability to create metabolic reconstructions and detailed models of metabolism, and access to hundreds of existing metabolic models. This work offers and supports a framework upon which other groups can build independent research efforts. Large integrations of genomic data represent one of the major intellectual resources driving research in biology, and programmatic access to the SEED data will provide significant utility to a broad collection of potential users. |
924 | Discovery and Targeted LC-MS/MS of Purified Polerovirus Reveals Differences in the Virus-Host Interactome Associated with Altered Aphid Transmission | Circulative transmission of viruses in the Luteoviridae, such as cereal yellow dwarf virus (CYDV), requires a series of precisely orchestrated interactions between virus, plant, and aphid proteins. Natural selection has favored these viruses to be retained in the phloem to facilitate acquisition and transmission by aphids. We show that treatment of infected oat tissue homogenate with sodium sulfite reduces transmission of the purified virus by aphids. Transmission electron microscopy data indicated no gross change in virion morphology due to treatments. However, treated virions were not acquired by aphids through the hindgut epithelial cells and were not transmitted when injected directly into the hemocoel. Analysis of virus preparations using nanoflow liquid chromatography coupled to tandem mass spectrometry revealed a number of host plant proteins co-purifying with viruses, some of which were lost following sodium sulfite treatment. Using targeted mass spectrometry, we show data suggesting that several of the virus-associated host plant proteins accumulated to higher levels in aphids that were fed on CYDV-infected plants compared to healthy plants. We propose two hypotheses to explain these observations, and these are not mutually exclusive: (a) that sodium sulfite treatment disrupts critical virion-host protein interactions required for aphid transmission, or (b) that host infection with CYDV modulates phloem protein expression in a way that is favorable for virus uptake by aphids. Importantly, the genes coding for the plant proteins associated with virus may be examined as targets in breeding cereal crops for new modes of virus resistance that disrupt phloem-virus or aphid-virus interactions. |
925 | Mortality, Severe Acute Respiratory Infection, and Influenza-Like Illness Associated with Influenza A(H1N1)pdm09 in Argentina, 2009 | INTRODUCTION: While there is much information about the burden of influenza A(H1N1)pdm09 in North America, little data exist on its burden in South America. METHODS: During April to December 2009, we actively searched for persons with severe acute respiratory infection and influenza-like illness (ILI) in three sentinel cities. A proportion of case-patients provided swabs for influenza testing. We estimated the number of case-patients that would have tested positive for influenza by multiplying the number of untested case-patients by the proportion who tested positive. We estimated rates by dividing the estimated number of case-patients by the census population after adjusting for the proportion of case-patients with missing illness onset information and ILI case-patients who visited physicians multiple times for one illness event. RESULTS: We estimated that the influenza A(H1N1)pdm09 mortality rate per 100,000 person-years (py) ranged from 1.5 among persons aged 5–44 years to 5.6 among persons aged ≥65 years. A(H1N1)pdm09 hospitalization rates per 100,000 py ranged between 26.9 among children aged <5 years to 41.8 among persons aged ≥65 years. Influenza A(H1N1)pdm09 ILI rates per 100 py ranged between 1.6 among children aged <5 to 17.1 among persons aged 45–64 years. While 9 (53%) of 17 influenza A(H1N1)pdm09 decedents with available data had obesity and 7 (17%) of 40 had diabetes, less than 4% of surviving influenza A(H1N1)pdm09 case-patients had these pre-existing conditions (p≤0.001). CONCLUSION: Influenza A(H1N1)pdm09 caused a similar burden of disease in Argentina as in other countries. Such disease burden suggests the potential value of timely influenza vaccinations. |
926 | C. difficile 630Δerm Spo0A Regulates Sporulation, but Does Not Contribute to Toxin Production, by Direct High-Affinity Binding to Target DNA | Clostridium difficile is a Gram positive, anaerobic bacterium that can form highly resistant endospores. The bacterium is the causative agent of C. difficile infection (CDI), for which the symptoms can range from a mild diarrhea to potentially fatal pseudomembranous colitis and toxic megacolon. Endospore formation in Firmicutes, including C. difficile, is governed by the key regulator for sporulation, Spo0A. In Bacillus subtilis, this transcription factor is also directly or indirectly involved in various other cellular processes. Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630. In vitro binding assays using purified C-terminal DNA binding domain of the C. difficile Spo0A protein demonstrate direct binding to DNA upstream of spo0A and sigH, early sporulation genes and several other putative targets. In vitro binding assays suggest that the gene encoding the major clostridial toxin TcdB may be a direct target of Spo0A, but supernatant derived from a spo0A negative strain was no less toxic towards Vero cells than that obtained from a wild type strain, in contrast to previous reports. These results identify for the first time direct (putative) targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely. |
927 | Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus | Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157–165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV. DOI: http://dx.doi.org/10.7554/eLife.00049.001 |
928 | The Evolutionary Pattern of Glycosylation Sites in Influenza Virus (H5N1) Hemagglutinin and Neuraminidase | Two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), on the surface of influenza viruses play crucial roles in transfaunation, membrane fusion and the release of progeny virions. To explore the distribution of N-glycosylation sites (glycosites) in these two glycoproteins, we collected and aligned the amino acid sequences of all the HA and NA subtypes. Two glycosites were located at HA0 cleavage sites and fusion peptides and were strikingly conserved in all HA subtypes, while the remaining glycosites were unique to their subtypes. Two to four conserved glycosites were found in the stalk domain of NA, but these are affected by the deletion of specific stalk domain sequences. Another highly conserved glycosite appeared at the top center of tetrameric global domain, while the others glycosites were distributed around the global domain. Here we present a detailed investigation of the distribution and the evolutionary pattern of the glycosites in the envelope glycoproteins of IVs, and further focus on the H5N1 virus and conclude that the glycosites in H5N1 have become more complicated in HA and less influential in NA in the last five years. |
929 | Niclosamide Is a Proton Carrier and Targets Acidic Endosomes with Broad Antiviral Effects | Viruses use a limited set of host pathways for infection. These pathways represent bona fide antiviral targets with low likelihood of viral resistance. We identified the salicylanilide niclosamide as a broad range antiviral agent targeting acidified endosomes. Niclosamide is approved for human use against helminthic infections, and has anti-neoplastic and antiviral effects. Its mode of action is unknown. Here, we show that niclosamide, which is a weak lipophilic acid inhibited infection with pH-dependent human rhinoviruses (HRV) and influenza virus. Structure-activity studies showed that antiviral efficacy and endolysosomal pH neutralization co-tracked, and acidification of the extracellular medium bypassed the virus entry block. Niclosamide did not affect the vacuolar H(+)-ATPase, but neutralized coated vesicles or synthetic liposomes, indicating a proton carrier mode-of-action independent of any protein target. This report demonstrates that physico-chemical interference with host pathways has broad range antiviral effects, and provides a proof of concept for the development of host-directed antivirals. |
930 | A Single Native Ganglioside GM(1)-Binding Site Is Sufficient for Cholera Toxin To Bind to Cells and Complete the Intoxication Pathway | Cholera toxin (CT) from Vibrio cholerae is responsible for the majority of the symptoms of the diarrheal disease cholera. CT is a heterohexameric protein complex with a 240-residue A subunit and a pentameric B subunit of identical 103-residue B polypeptides. The A subunit is proteolytically cleaved within a disulfide-linked loop to generate the A1 and A2 fragments. The B subunit of wild-type (wt) CT binds 5 cell surface ganglioside GM(1) (GM(1)) molecules, and the toxin-GM(1) complex traffics from the plasma membrane (PM) retrograde through endosomes and the Golgi apparatus to the endoplasmic reticulum (ER). From the ER, the enzymatic A1 fragment retrotranslocates to the cytosol to cause disease. Clustering of GM(1) by multivalent toxin binding can structurally remodel cell membranes in ways that may assist toxin uptake and retrograde trafficking. We have recently found, however, that CT may traffic from the PM to the ER by exploiting an endogenous glycosphingolipid pathway (A. A. Wolf et al., Infect. Immun. 76:1476–1484, 2008, and D. J. F. Chinnapen et al., Dev. Cell 23:573–586, 2012), suggesting that multivalent binding to GM(1) is dispensable. Here we formally tested this idea by creating homogenous chimeric holotoxins with defined numbers of native GM(1) binding sites from zero (nonbinding) to five (wild type). We found that a single GM(1) binding site is sufficient for activity of the holotoxin. Therefore, remodeling of cell membranes by mechanisms that involve multivalent binding of toxin to GM(1) receptors is not essential for toxicity of CT. |
931 | A Single Residue Substitution in the Receptor-Binding Domain of H5N1 Hemagglutinin Is Critical for Packaging into Pseudotyped Lentiviral Particles | BACKGROUND: Serological studies for influenza infection and vaccine response often involve microneutralization and hemagglutination inhibition assays to evaluate neutralizing antibodies against human and avian influenza viruses, including H5N1. We have previously characterized lentiviral particles pseudotyped with H5-HA (H5pp) and validated an H5pp-based assay as a safe alternative for high-throughput serological studies in BSL-2 facilities. Here we show that H5-HAs from different clades do not always give rise to efficient production of H5pp and the underlying mechanisms are addressed. METHODOLOGY/FINDINGS: We have carried out mutational analysis to delineate the molecular determinants responsible for efficient packaging of HA from A/Cambodia/40808/2005 (H5Cam) and A/Anhui/1/2005 (H5Anh) into H5pp. Our results demonstrate that a single A134V mutation in the 130-loop of the receptor binding domain is sufficient to render H5Anh the ability to generate H5Anh-pp efficiently, whereas the reverse V134A mutation greatly hampers production of H5Cam-pp. Although protein expression in total cell lysates is similar for H5Anh and H5Cam, cell surface expression of H5Cam is detected at a significantly higher level than that of H5Anh. We further demonstrate by several independent lines of evidence that the behaviour of H5Anh can be explained by a stronger binding to sialic acid receptors implicating residue 134. CONCLUSIONS: We have identified a single A134V mutation as the molecular determinant in H5-HA for efficient incorporation into H5pp envelope and delineated the underlying mechanism. The reduced binding to sialic acid receptors as a result of the A134V mutation not only exerts a critical influence in pseudotyping efficiency of H5-HA, but has also an impact at the whole virus level. Because A134V substitution has been reported as a naturally occurring mutation in human host, our results may have implications for the understanding of human host adaptation of avian influenza H5N1 viruses. |
932 | Particulate matter air pollution disrupts endothelial cell barrier via calpain-mediated tight junction protein degradation | BACKGROUND: Exposure to particulate matter (PM) is a significant risk factor for increased cardiopulmonary morbidity and mortality. The mechanism of PM-mediated pathophysiology remains unknown. However, PM is proinflammatory to the endothelium and increases vascular permeability in vitro and in vivo via ROS generation. OBJECTIVES: We explored the role of tight junction proteins as targets for PM-induced loss of lung endothelial cell (EC) barrier integrity and enhanced cardiopulmonary dysfunction. METHODS: Changes in human lung EC monolayer permeability were assessed by Transendothelial Electrical Resistance (TER) in response to PM challenge (collected from Ft. McHenry Tunnel, Baltimore, MD, particle size >0.1 μm). Biochemical assessment of ROS generation and Ca(2+) mobilization were also measured. RESULTS: PM exposure induced tight junction protein Zona occludens-1 (ZO-1) relocation from the cell periphery, which was accompanied by significant reductions in ZO-1 protein levels but not in adherens junction proteins (VE-cadherin and β-catenin). N-acetyl-cysteine (NAC, 5 mM) reduced PM-induced ROS generation in ECs, which further prevented TER decreases and atteneuated ZO-1 degradation. PM also mediated intracellular calcium mobilization via the transient receptor potential cation channel M2 (TRPM2), in a ROS-dependent manner with subsequent activation of the Ca(2+)-dependent protease calpain. PM-activated calpain is responsible for ZO-1 degradation and EC barrier disruption. Overexpression of ZO-1 attenuated PM-induced endothelial barrier disruption and vascular hyperpermeability in vivo and in vitro. CONCLUSIONS: These results demonstrate that PM induces marked increases in vascular permeability via ROS-mediated calcium leakage via activated TRPM2, and via ZO-1 degradation by activated calpain. These findings support a novel mechanism for PM-induced lung damage and adverse cardiovascular outcomes. |
933 | The expression of nicotinic receptor alpha7 during cochlear development | Nicotinic acetylcholine receptor alpha7 expression was examined in the developing and adult auditory system using mice that were modified through homologous recombination to coexpress either GFP (alpha7GFP) or Cre (alpha7Cre), respectively. The expression of alpha7GFP is first detected at embryonic (E) day E13.5 in cells of the spiral prominence. By E14.5, sensory regions including the putative outer hair cells and Deiters' cells express alpha7GFP as do solitary efferent fibers. This pattern diminishes after E16.5 in a basal to apex progression, as Hensen's cells and cells of the spiral ligament acquire alpha7GFP expression. At birth and thereafter alpha7GFP also identifies a subset of spiral ganglion cells whose processes terminate on inner hair cells. Efferent fibers identified by peripherin or calcitonin gene-related protein do not coexpress alpha7GFP. In addition to cochlear structures, there is strong expression of alpha7GFP by cells of the central auditory pathways including the ventral posterior cochlear nucleus, lateral lemniscus, central inferior colliculus, and the medial geniculate nucleus. Our findings suggest that alpha7 expression by both neuronal and non-neuronal cells has the potential to impact multiple auditory functions through mechanisms that are not traditionally attributed to this receptor. |
934 | Identification of serum proteomic biomarkers for early porcine reproductive and respiratory syndrome (PRRS) infection | BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine diseases worldwide. Despite its relevance, serum biomarkers associated with early-onset viral infection, when clinical signs are not detectable and the disease is characterized by a weak anti-viral response and persistent infection, have not yet been identified. Surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) is a reproducible, accurate, and simple method for the identification of biomarker proteins related to disease in serum. This work describes the SELDI-TOF MS analyses of sera of 60 PRRSV-positive and 60 PRRSV-negative, as measured by PCR, asymptomatic Large White piglets at weaning. Sera with comparable and low content of hemoglobin (< 4.52 μg/mL) were fractionated in 6 different fractions by anion-exchange chromatography and protein profiles in the mass range 1–200 kDa were obtained with the CM10, IMAC30, and H50 surfaces. RESULTS: A total of 200 significant peaks (p < 0.05) were identified in the initial discovery phase of the study and 47 of them were confirmed in the validation phase. The majority of peaks (42) were up-regulated in PRRSV-positive piglets, while 5 were down-regulated. A panel of 14 discriminatory peaks identified in fraction 1 (pH = 9), on the surface CM10, and acquired at low focus mass provided a serum protein profile diagnostic pattern that enabled to discriminate between PRRSV-positive and -negative piglets with a sensitivity and specificity of 77% and 73%, respectively. CONCLUSIONS: SELDI-TOF MS profiling of sera from PRRSV-positive and PRRSV-negative asymptomatic piglets provided a proteomic signature with large scale diagnostic potential for early identification of PRRSV infection in weaning piglets. Furthermore, SELDI-TOF protein markers represent a refined phenotype of PRRSV infection that might be useful for whole genome association studies. |
935 | Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors | BACKGROUND: Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3) is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5) binding and presence of epitopes recognized by broadly neutralizing antibodies. METHODS: Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20–57 years (median = 33 years) were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB) fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. RESULTS: We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL), suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. CONCLUSIONS: Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with non-clade C viruses, in context of V3 region. |
936 | Toxicology, biodistribution and shedding profile of a recombinant measles vaccine vector expressing HIV-1 antigens, in cynomolgus macaques | As a new human immunodeficiency virus type 1 (HIV-1) vaccine approach, the live-attenuated measles virus (MV) Schwarz vaccine strain was genetically engineered to express the F4 antigen (MV1-F4). F4 is a fusion protein comprising HIV-1 antigens p17 and p24, reverse transcriptase and Nef. This study assessed the toxicity, biodistribution and shedding profiles of MV1-F4. Cynomolgus macaques were intramuscularly immunized one or three times with the highest dose of MV1-F4 intended for clinical use, the reference (Schwarz) measles vaccine or saline, and monitored clinically for 11 or 85 days. Toxicological parameters included local and systemic clinical signs, organ weights, haematology, clinical and gross pathology and histopathology. Both vaccines were well tolerated, with no morbidity, clinical signs or gross pathological findings observed. Mean spleen weights were increased after three doses of either vaccine, which corresponded with increased numbers and/or sizes of germinal centers. This was likely a result of the immune response to the vaccines. Either vaccine virus replicated preferentially in secondary lymphoid organs and to a lesser extent in epithelium-rich tissues (e.g., intestine, urinary bladder and trachea) and the liver. At the expected peak of viremia, viral RNA was detected in some biological fluid samples from few animals immunized with either vaccine, but none of these samples contained infectious virus. In conclusion, no shedding of infectious viral particles was identified in cynomolgus monkeys after injection of MV1-F4 or Schwarz measles vaccines. Furthermore, no toxic effect in relation to the MV vaccination was found with these vaccines in this study. |
937 | Forty-Five Years of Marburg Virus Research | In 1967, the first reported filovirus hemorrhagic fever outbreak took place in Germany and the former Yugoslavia. The causative agent that was identified during this outbreak, Marburg virus, is one of the most deadly human pathogens. This article provides a comprehensive overview of our current knowledge about Marburg virus disease ranging from ecology to pathogenesis and molecular biology. |
938 | The Human Cytomegalovirus DNA Polymerase Processivity Factor UL44 Is Modified by SUMO in a DNA-Dependent Manner | During the replication of human cytomegalovirus (HCMV) genome, the viral DNA polymerase subunit UL44 plays a key role, as by binding both DNA and the polymerase catalytic subunit it confers processivity to the holoenzyme. However, several lines of evidence suggest that UL44 might have additional roles during virus life cycle. To shed light on this, we searched for cellular partners of UL44 by yeast two-hybrid screenings. Intriguingly, we discovered the interaction of UL44 with Ubc9, an enzyme involved in the covalent conjugation of SUMO (Small Ubiquitin-related MOdifier) to cellular and viral proteins. We found that UL44 can be extensively sumoylated not only in a cell-free system and in transfected cells, but also in HCMV-infected cells, in which about 50% of the protein resulted to be modified at late times post-infection, when viral genome replication is accomplished. Mass spectrometry studies revealed that UL44 possesses multiple SUMO target sites, located throughout the protein. Remarkably, we observed that binding of UL44 to DNA greatly stimulates its sumoylation both in vitro and in vivo. In addition, we showed that overexpression of SUMO alters the intranuclear distribution of UL44 in HCMV-infected cells, and enhances both virus production and DNA replication, arguing for an important role for sumoylation in HCMV life cycle and UL44 function(s). These data report for the first time the sumoylation of a viral processivity factor and show that there is a functional interplay between the HCMV UL44 protein and the cellular sumoylation system. |
939 | Fas-deficient mice have impaired alveolar neutrophil recruitment and decreased expression of anti-KC autoantibody:KC complexes in a model of acute lung injury | BACKGROUND: Exposure to mechanical ventilation enhances lung injury in response to various stimuli, such as bacterial endotoxin (LPS). The Fas/FasL system is a receptor ligand system that has dual pro-apoptotic and pro-inflammatory functions and has been implicated in the pathogenesis of lung injury. In this study we test the hypothesis that a functioning Fas/FasL system is required for the development of lung injury in mechanically ventilated mice. METHODS: C57BL/6 (B6) and Fas-deficient lpr mice were exposed to either intra-tracheal PBS followed by spontaneous breathing or intra-tracheal LPS followed by four hours mechanical ventilation with tidal volumes of 10 mL/kg, respiratory rate of 150 breaths per minute, inspired oxygen 0.21 and positive end expiratory pressure (PEEP) of 3 cm of water. RESULTS: Compared with the B6 mice, the lpr mice showed attenuation of the neutrophilic response as measured by decreased numbers of BAL neutrophils and lung myeloperoxidase activity. Interestingly, the B6 and lpr mice had similar concentrations of pro-inflammatory cytokines, including CXCL1 (KC), and similar measurements of permeability and apoptosis. However, the B6 mice showed greater deposition of anti-KC:KC immune complexes in the lungs, as compared with the lpr mice. CONCLUSIONS: We conclude that a functioning Fas/FasL system is required for full neutrophilic response to LPS in mechanically ventilated mice. |
940 | Evolutionary Dynamics of the Interferon-Induced Transmembrane Gene Family in Vertebrates | Vertebrate interferon-induced transmembrane (IFITM) genes have been demonstrated to have extensive and diverse functions, playing important roles in the evolution of vertebrates. Despite observance of their functionality, the evolutionary dynamics of this gene family are complex and currently unknown. Here, we performed detailed evolutionary analyses to unravel the evolutionary history of the vertebrate IFITM family. A total of 174 IFITM orthologous genes and 112 pseudogenes were identified from 27 vertebrate genome sequences. The vertebrate IFITM family can be divided into immunity-related IFITM (IR-IFITM), IFITM5 and IFITM10 sub-families in phylogeny, implying origins from three different progenitors. In general, vertebrate IFITM genes are located in two loci, one containing the IFITM10 gene, and the other locus containing IFITM5 and various numbers of IR-IFITM genes. Conservation of evolutionary synteny was observed in these IFITM genes. Significant functional divergence was detected among the three IFITM sub-families. No gene duplication or positive selection was found in IFITM5 sub-family, implying the functional conservation of IFITM5 in vertebrate evolution, which is involved in bone formation. No IFITM5 locus was identified in the marmoset genome, suggesting a potential association with the tiny size of this monkey. The IFITM10 sub-family was divided into two groups: aquatic and terrestrial types. Functional divergence was detected between the two groups, and five IFITM10-like genes from frog were dispersed into the two groups. Both gene duplication and positive selection were observed in aquatic vertebrate IFITM10-like genes, indicating that IFITM10 might be associated with the adaptation to aquatic environments. A large number of lineage- and species-specific gene duplications were observed in IR-IFITM sub-family and positive selection was detected in IR-IFITM of primates and rodents. Because primates have experienced a long history of viral infection, such rapid expansion and positive selection suggests that the evolution of primate IR-IFITM genes is associated with broad-spectrum antiviral activity. |
941 | Filovirus Research in Gabon and Equatorial Africa: The Experience of a Research Center in the Heart of Africa | Health research programs targeting the population of Gabon and Equatorial Africa at the International Center for Medical Research in Franceville (CIRMF), Gabon, have evolved during the years since its inception in 1979 in accordance with emerging diseases. Since the reemergence of Ebola virus in Central Africa, the CIRMF “Emerging Viral Disease Unit” developed diagnostic tools and epidemiologic strategies and transfers of such technology to support the response of the National Public Health System and the World Health Organization to epidemics of Ebola virus disease. The Unit carries out a unique investigation program on the natural history of the filoviruses, emergence of epidemics, and Ebola virus pathogenesis. In addition, academic training is provided at all levels to regional and international students covering emerging conditions (host factors, molecular biology, genetics) that favor the spread of viral diseases. |
942 | Reproductive Number and Serial Interval of the First Wave of Influenza A(H1N1)pdm09 Virus in South Africa | BACKGROUND/OBJECTIVE: Describing transmissibility parameters of past pandemics from diverse geographic sites remains critical to planning responses to future outbreaks. We characterize the transmissibility of influenza A(H1N1)pdm09 (hereafter pH1N1) in South Africa during 2009 by estimating the serial interval (SI), the initial effective reproductive number (initial R(t)) and the temporal variation of R(t). METHODS: We make use of data from a central registry of all pH1N1 laboratory-confirmed cases detected throughout South Africa. Whenever date of symptom onset is missing, we estimate it from the date of specimen collection using a multiple imputation approach repeated 100 times for each missing value. We apply a likelihood-based method (method 1) for simultaneous estimation of initial R(t) and the SI; estimate initial R(t) from SI distributions established from prior field studies (method 2); and the Wallinga and Teunis method (method 3) to model the temporal variation of R(t). RESULTS: 12,360 confirmed pH1N1 cases were reported in the central registry. During the period of exponential growth of the epidemic (June 21 to August 3, 2009), we simultaneously estimate a mean R(t) of 1.47 (95% CI: 1.30–1.72) and mean SI of 2.78 days (95% CI: 1.80–3.75) (method 1). Field studies found a mean SI of 2.3 days between primary cases and laboratory-confirmed secondary cases, and 2.7 days when considering both suspected and confirmed secondary cases. Incorporating the SI estimate from field studies using laboratory-confirmed cases, we found an initial R(t) of 1.43 (95% CI: 1.38–1.49) (method 2). The mean R(t) peaked at 2.91 (95% CI: 0.85–2.91) on June 21, as the epidemic commenced, and R(t)>1 was sustained until August 22 (method 3). CONCLUSIONS: Transmissibility characteristics of pH1N1 in South Africa are similar to estimates reported by countries outside of Africa. Estimations using the likelihood-based method are in agreement with field findings. |
943 | Measuring healthcare preparedness: an all-hazards approach | In a paper appearing in this issue, Adini, et al. describe a struggle familiar to many emergency planners—the challenge of planning for all scenarios. The authors contend that all-hazards, or capabilities-based planning, in which a set of core capabilities applicable to numerous types of events is developed, is a more efficient way to achieve general health care system emergency preparedness than scenario-based planning. Essentially, the core of what is necessary to plan for and respond to one kind of disaster (e.g. a biologic event) is also necessary for planning and responding to other types of disasters, allowing for improvements in planning and maximizing efficiencies. While Adini, et al. have advanced the science of health care emergency preparedness through their consideration of 490 measures to assess preparedness, a shorter set of validated preparedness measures would support the dual goals of accountability and improved outcomes and could provide the basis for determining which actions in the name of preparedness really matter. |
944 | Presentation of hemophagocytic lymphohistiocytosis due to a novel MUNC 13–4 mutation masked by partial therapeutic immunosuppression | Hemophagocytic lymphohistiocytosis is a potentially fatal disease characterized by excessive macrophage and lymphocyte activity. Patients can be affected following immune activation after an oncologic, autoimmune or infectious trigger. An associated gene mutation may be found which impairs cytolytic lymphocyte function. We describe a pediatric case of hemophagocytic lymphohistiocytosis with a novel mutation of MUNC 13–4 whose diagnosis was confounded by concurrent immunosuppression. Clinical reassessment for hemophagocytic lymphohistiocytosis is necessary in persistently febrile patients with laboratory derangements in the setting of immunosuppressive agent exposure. |
945 | Relative cost and outcomes in the intensive care unit of acute lung injury (ALI) due to pandemic influenza compared with other etiologies: a single-center study | BACKGROUND: Critical illness due to 2009 H1N1 influenza has been characterized by respiratory complications, including acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), and associated with high mortality. We studied the severity, outcomes, and hospital charges of patients with ALI/ARDS secondary to pandemic influenza A infection compared with ALI and ARDS from other etiologies. METHODS: A retrospective review was conducted that included patients admitted to the Cleveland Clinic MICU with ALI/ARDS and confirmed influenza A infection, and all patients admitted with ALI/ARDS from any other etiology from September 2009 to March 2010. An itemized list of individual hospital charges was obtained for each patient from the hospital billing office and organized by billing code into a database. Continuous data that were normally distributed are presented as the mean ± SD and were analyzed by the Student’s t test. The chi-square and Fisher exact tests were used to evaluate differences in proportions between patient subgroups. Data that were not normally distributed were compared with the Wilcoxon rank-sum test. RESULTS: Forty-five patients were studied: 23 in the H1N1 group and 22 in the noninfluenza group. Mean ± SD age was similar (44 ± 13 and 51 ± 17 years, respectively, p = 0.15). H1N1 patients had lower APACHE III scores (66 ± 20 vs. 89 ± 32, p = 0.015) and had higher Pplat and PEEP on days 1, 3, and 14. Hospital and ICU length of stay and duration of mechanical ventilation were comparable. SOFA scores over the first 2 weeks in the ICU indicate more severe organ failure in the noninfluenza group (p = 0.017). Hospital mortality was significantly higher in the noninfluenza group (77 vs. 39%, p = 0.016). The noninfluenza group tended to have higher overall charges, including significantly higher cost of blood products in the ICU. CONCLUSIONS: ALI/ARDS secondary to pandemic influenza infection is associated with more severe respiratory compromise but has lower overall acuity and better survival rates than ALI/ARDS due to other causes. Higher absolute charges in the noninfluenza group are likely due to underlying comorbid medical conditions. |
946 | Identification of a Conserved B-cell Epitope on Reticuloendotheliosis Virus Envelope Protein by Screening a Phage-displayed Random Peptide Library | BACKGROUND: The gp90 protein of avian reticuloendotheliosis-associated virus (REV-A) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported. METHODS AND RESULTS: This study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb) A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched (213)SVQYHPL(219) of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that (213)SVQYHPL(219) was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group. CONCLUSIONS AND SIGNIFICANCE: We identified (213)SVQYHPL(219) as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group. |
947 | Lower Respiratory Tract Infection in a Renal Transplant Recipient: Do not Forget Metapneumovirus | Human metapneumovirus (hMPV) is emerging as a cause of a severe respiratory tract infection in immunocompromised patients. hMPV pneumonia has only been seldom reported in nonpulmonary solid organ transplanted patients, such as renal transplant recipients. We report here a case of a 39-year-old patient presenting with fever, cough, and interstitial opacities on CT scan diagnosed as a nonsevere hMPV pneumonia 11 years after a renal transplantation. Infection resolved spontaneously. Differential diagnosis with Pneumocystis pneumonia was discussed. We review the medical literature and discuss clinical presentation and detection methods that can be proposed in solid organ transplant recipients. |
948 | What was the primary mode of smallpox transmission? Implications for biodefense | The mode of infection transmission has profound implications for effective containment by public health interventions. The mode of smallpox transmission was never conclusively established. Although, “respiratory droplet” transmission was generally regarded as the primary mode of transmission, the relative importance of large ballistic droplets and fine particle aerosols that remain suspended in air for more than a few seconds was never resolved. This review examines evidence from the history of variolation, data on mucosal infection collected in the last decades of smallpox transmission, aerosol measurements, animal models, reports of smallpox lung among healthcare workers, and the epidemiology of smallpox regarding the potential importance of fine particle aerosol mediated transmission. I introduce briefly the term anisotropic infection to describe the behavior of Variola major in which route of infection appears to have altered the severity of disease. |
949 | Chitinase Dependent Control of Protozoan Cyst Burden in the Brain | Chronic infections represent a continuous battle between the host's immune system and pathogen replication. Many protozoan parasites have evolved a cyst lifecycle stage that provides it with increased protection from environmental degradation as well as endogenous host mechanisms of attack. In the case of Toxoplasma gondii, these cysts are predominantly found in the immune protected brain making clearance of the parasite more difficult and resulting in a lifelong infection. Currently, little is known about the nature of the immune response stimulated by the presence of these cysts or how they are able to propagate. Here we establish a novel chitinase-dependent mechanism of cyst control in the infected brain. Despite a dominant Th1 immune response during Toxoplasma infection there exists a population of alternatively activated macrophages (AAMØ) in the infected CNS. These cells are capable of cyst lysis via the production of AMCase as revealed by live imaging, and this chitinase is necessary for protective immunity within the CNS. These data demonstrate chitinase activity in the brain in response to a protozoan pathogen and provide a novel mechanism to facilitate cyst clearance during chronic infections. |
950 | Automatic Detection and Quantification of Tree-in-Bud (TIB) Opacities from CT Scans | This study presents a novel computer-assisted detection (CAD) system for automatically detecting and precisely quantifying abnormal nodular branching opacities in chest computed tomography (CT), termed tree-in-bud (TIB) opacities by radiology literature. The developed CAD system in this study is based on 1) fast localization of candidate imaging patterns using local scale information of the images, and 2) Möbius invariant feature extraction method based on learned local shape and texture properties of TIB patterns. For fast localization of candidate imaging patterns, we use ball-scale filtering and, based on the observation of the pattern of interest, a suitable scale selection is used to retain only small size patterns. Once candidate abnormality patterns are identified, we extract proposed shape features from regions where at least one candidate pattern occupies. The comparative evaluation of the proposed method with commonly used CAD methods is presented with a dataset of 60 chest CTs (laboratory confirmed 39 viral bronchiolitis human parainfluenza CTs and 21 normal chest CTs). The quantitative results are presented as the area under the receiver operator characteristics curves and a computer score (volume affected by TIB) provided as an output of the CAD system. In addition, a visual grading scheme is applied to the patient data by three well-trained radiologists. Interobserver and observer–computer agreements are obtained by the relevant statistical methods over different lung zones. Experimental results demonstrate that the proposed CAD system can achieve high detection rates with an overall accuracy of 90.96%. Moreover, correlations of observer–observer [Formula: see text] , [Formula: see text] and observer–CAD agreements [Formula: see text] , [Formula: see text] validate the feasibility of the use of the proposed CAD system in detecting and quantifying TIB patterns. |
951 | Mannose-binding lectin deficiency and acute exacerbations of chronic obstructive pulmonary disease | BACKGROUND: Mannose-binding lectin is a collectin involved in host defense against infection. Whether mannose-binding lectin deficiency is associated with acute exacerbations of chronic obstructive pulmonary disease is debated. METHODS: Participants in a study designed to determine if azithromycin taken daily for one year decreased acute exacerbations had serum mannose-binding lectin concentrations measured at the time of enrollment. RESULTS: Samples were obtained from 1037 subjects (91%) in the trial. The prevalence of mannose-binding lectin deficiency ranged from 0.5% to 52.2%, depending on how deficiency was defined. No differences in the prevalence of deficiency were observed with respect to any demographic variable assessed, and no differences were observed in time to first exacerbation, rate of exacerbations, or percentage of subjects requiring hospitalization for exacerbations in those with deficiency versus those without, regardless of how deficiency was defined. CONCLUSION: In a large sample of subjects with chronic obstructive pulmonary disease selected for having an increased risk of experiencing an acute exacerbation of chronic obstructive pulmonary disease, only 1.9% had mannose-binding lectin concentrations below the normal range and we found no association between mannose-binding lectin concentrations and time to first acute exacerbation or frequency of acute exacerbations during one year of prospective follow-up. |
952 | The changing phenotype of microglia from homeostasis to disease | It has been nearly a century since the early description of microglia by Rio-Hortega; since then many more biological and pathological features of microglia have been recognized. Today, microglia are generally considered to be beneficial to homeostasis at the resting state through their abilities to survey the environment and phagocytose debris. However, when activated microglia assume diverse phenotypes ranging from fully inflamed, which involves the release of many pro-inflammatory cytokines, to alternatively activated, releasing anti-inflammatory cytokines or neurotrophins, the consequences to neurons can range from detrimental to supportive. Due to the different experimental sets and conditions, contradictory results have been obtained regarding the controversial question of whether microglia are “good” or “bad.” While it is well understood that the dual roles of activated microglia depend on specific situations, the underlying mechanisms have remained largely unclear, and the interpretation of certain findings related to diverse microglial phenotypes continues to be problematic. In this review we discuss the functions of microglia in neuronal survival and neurogenesis, the crosstalk between microglia and surrounding cells, and the potential factors that could influence the eventual manifestation of microglia. |
953 | Diversity of Salmonella spp. serovars isolated from the intestines of water buffalo calves with gastroenteritis | BACKGROUND: Salmonellosis in water buffalo (Bubalus bubalis) calves is a widespread disease characterized by severe gastrointestinal lesions, profuse diarrhea and severe dehydration, occasionally exhibiting a systemic course. Several Salmonella serovars seem to be able to infect water buffalo, but Salmonella isolates collected from this animal species have been poorly characterized. In the present study, the prevalence of Salmonella spp. in water buffalo calves affected by lethal gastroenteritis was assessed, and a polyphasic characterization of isolated strains of S. Typhimurium was performed. RESULTS: The microbiological analysis of the intestinal contents obtained from 248 water buffalo calves affected by lethal gastroenteritis exhibited a significant prevalence of Salmonella spp. (25%), characterized by different serovars, most frequently Typhimurium (21%), Muenster (11%), and Give (11%). The 13 S. Typhimurium isolates were all associated with enterocolitis characterized by severe damage of the intestine, and only sporadically isolated with another possible causative agent responsible for gastroenteritis, such as Cryptosporidium spp., Rotavirus or Clostridium perfringens. Other Salmonella isolates were mostly isolated from minor intestinal lesions, and often (78% of cases) isolated with other microorganisms, mainly toxinogenic Escherichia coli (35%), Cryptosporidium spp. (20%) and Rotavirus (10%). The S. Typhimurium strains were characterized by phage typing and further genotyped by polymerase chain reaction (PCR) detection of 24 virulence genes. The isolates exhibited nine different phage types and 10 different genetic profiles. Three monophasic S. Typhimurium (B:4,12:i:-) isolates were also found and characterized, displaying three different phage types and three different virulotypes. The molecular characterization was extended to the 7 S. Muenster and 7 S. Give isolates collected, indicating the existence of different virulotypes also within these serovars. Three representative strains of S. Typhimurium were tested in vivo in a mouse model of mixed infection. The most pathogenic strain was characterized by a high number of virulence factors and the presence of the locus agfA, coding for a thin aggregative fimbria. CONCLUSIONS: These results provide evidence that Salmonella is frequently associated with gastroenteritis in water buffalo calves, particularly S. Typhimurium. Moreover, the variety in the number and distribution of different virulence markers among the collected S. Typhimurium strains suggests that within this serovar there are different pathotypes potentially responsible for different clinical syndromes. |
954 | Severe Childhood Malaria Syndromes Defined by Plasma Proteome Profiles | BACKGROUND: Cerebral malaria (CM) and severe malarial anemia (SMA) are the most serious life-threatening clinical syndromes of Plasmodium falciparum infection in childhood. Therefore it is important to understand the pathology underlying the development of CM and SMA, as opposed to uncomplicated malaria (UM). Different host responses to infection are likely to be reflected in plasma proteome-patterns that associate with clinical status and therefore provide indicators of the pathogenesis of these syndromes. METHODS AND FINDINGS: Plasma and comprehensive clinical data for discovery and validation cohorts were obtained as part of a prospective case-control study of severe childhood malaria at the main tertiary hospital of the city of Ibadan, an urban and densely populated holoendemic malaria area in Nigeria. A total of 946 children participated in this study. Plasma was subjected to high-throughput proteomic profiling. Statistical pattern-recognition methods were used to find proteome-patterns that defined disease groups. Plasma proteome-patterns accurately distinguished children with CM and with SMA from those with UM, and from healthy or severely ill malaria-negative children. CONCLUSIONS: We report that an accurate definition of the major childhood malaria syndromes can be achieved using plasma proteome-patterns. Our proteomic data can be exploited to understand the pathogenesis of the different childhood severe malaria syndromes. |
955 | Predicting pseudoknotted structures across two RNA sequences | Motivation: Laboratory RNA structure determination is demanding and costly and thus, computational structure prediction is an important task. Single sequence methods for RNA secondary structure prediction are limited by the accuracy of the underlying folding model, if a structure is supported by a family of evolutionarily related sequences, one can be more confident that the prediction is accurate. RNA pseudoknots are functional elements, which have highly conserved structures. However, few comparative structure prediction methods can handle pseudoknots due to the computational complexity. Results: A comparative pseudoknot prediction method called DotKnot-PW is introduced based on structural comparison of secondary structure elements and H-type pseudoknot candidates. DotKnot-PW outperforms other methods from the literature on a hand-curated test set of RNA structures with experimental support. Availability: DotKnot-PW and the RNA structure test set are available at the web site http://dotknot.csse.uwa.edu.au/pw. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online. |
956 | Horizontal Transposon Transfer in Eukarya: Detection, Bias, and Perspectives | The genetic similarity observed among species is normally attributed to the existence of a common ancestor. However, a growing body of evidence suggests that the exchange of genetic material is not limited to the transfer from parent to offspring but can also occur through horizontal transfer (HT). Transposable elements (TEs) are DNA fragments with an innate propensity for HT; they are mobile and possess parasitic characteristics that allow them to exist and proliferate within host genomes. However, horizontal transposon transfer (HTT) is not easily detected, primarily because the complex TE life cycle can generate phylogenetic patterns similar to those expected for HTT events. The increasingly large number of new genome projects, in all branches of life, has provided an unprecedented opportunity to evaluate the TE content and HTT events in these species, although a standardized method of HTT detection is required before trends in the HTT rates can be evaluated in a wide range of eukaryotic taxa and predictions about these events can be made. Thus, we propose a straightforward hypothesis test that can be used by TE specialists and nonspecialists alike to discriminate between HTT events and natural TE life cycle patterns. We also discuss several plausible explanations and predictions for the distribution and frequency of HTT and for the inherent biases of HTT detection. Finally, we discuss some of the methodological concerns for HTT detection that may result in the underestimation and overestimation of HTT rates during eukaryotic genome evolution. |
957 | Mannosylated Lipid Nano-emulsions Loaded with Lycorine-oleic Acid Ionic Complex for Tumor Cell-specific Delivery | This study was to prepare a mannosylated lycorine lipid nano-emulsion formulation (M-LYC-OA-LNEs) for the aim of achieving tumor targeting delivery of lycorine (LYC) . The low lipophilicity of LYC made it hard to be dispersed into lipid nano-emulsions (LNEs). In order to increase its lipophilicity, lycorine-oleic acid ionic complex (LYC-OA) was made. M-LYC-OA-LNEs and uncoated lycorine-oleic acid loaded lipid nano-emulsions (LYC-OA-LNEs) were prepared by solvent injection method and characterized by transmission electron microscopy (TEM), particle size, polydispersity index, zeta-potential and entrapment efficiency analysis. The in vitro cellular uptake and growth inhibition activity studies were performed on A549 cell lines. The entrapment efficiency of M-LYC-OA-LNEs was 82.7 ± 1.6 %. The cellular uptake study showed that coated LNEs were preferably taken up by A549 cells than uncoated LNEs. The effective test by MTT assay showed better growth inhibition activity of M-LYC-OA-LNEs on A549 cell lines when compared with LYC-OA-LNEs and blank LNEs. These results demonstrated that M-LYC-OA-LNEs could be a promising formulation for tumor targeting delivery of LYC with the potential of being applied in the diagnosis and treatment of cancer. |
958 | The influence of climatic conditions on the transmission dynamics of the 2009 A/H1N1 influenza pandemic in Chile | BACKGROUND: The role of demographic factors, climatic conditions, school cycles, and connectivity patterns in shaping the spatio-temporal dynamics of pandemic influenza is not clearly understood. Here we analyzed the spatial, age and temporal evolution of the 2009 A/H1N1 influenza pandemic in Chile, a southern hemisphere country covering a long and narrow strip comprising latitudes 17°S to 56°S. METHODS: We analyzed the dissemination patterns of the 2009 A/H1N1 pandemic across 15 regions of Chile based on daily hospitalizations for severe acute respiratory disease and laboratory confirmed A/H1N1 influenza infection from 01-May to 31-December, 2009. We explored the association between timing of pandemic onset and peak pandemic activity and several geographical and demographic indicators, school vacations, climatic factors, and international passengers. We also estimated the reproduction number (R) based on the growth rate of the exponential pandemic phase by date of symptoms onset, estimated using maximum likelihood methods. RESULTS: While earlier pandemic onset was associated with larger population size, there was no association with connectivity, demographic, school or climatic factors. In contrast, there was a latitudinal gradient in peak pandemic timing, representing a 16-39-day lag in disease activity from the southern regions relative to the northernmost region (P < 0.001). Geographical differences in latitude of Chilean regions, maximum temperature and specific humidity explained 68.5% of the variability in peak timing (P = 0.01). In addition, there was a decreasing gradient in reproduction number from south to north Chile (P < 0.0001). The regional mean R estimates were 1.6-2.0, 1.3-1.5, and 1.2-1.3 for southern, central and northern regions, respectively, which were not affected by the winter vacation period. CONCLUSIONS: There was a lag in the period of most intense 2009 pandemic influenza activity following a South to North traveling pattern across regions of Chile, significantly associated with geographical differences in minimum temperature and specific humidity. The latitudinal gradient in timing of pandemic activity was accompanied by a gradient in reproduction number (P < 0.0001). Intensified surveillance strategies in colder and drier southern regions could lead to earlier detection of pandemic influenza viruses and improved control outcomes. |
959 | A MultiSite Gateway(TM )vector set for the functional analysis of genes in the model Saccharomyces cerevisiae | BACKGROUND: Recombinatorial cloning using the Gateway(TM) technology has been the method of choice for high-throughput omics projects, resulting in the availability of entire ORFeomes in Gateway(TM) compatible vectors. The MultiSite Gateway(TM) system allows combining multiple genetic fragments such as promoter, ORF and epitope tag in one single reaction. To date, this technology has not been accessible in the yeast Saccharomyces cerevisiae, one of the most widely used experimental systems in molecular biology, due to the lack of appropriate destination vectors. RESULTS: Here, we present a set of three-fragment MultiSite Gateway(TM) destination vectors that have been developed for gene expression in S. cerevisiae and that allow the assembly of any promoter, open reading frame, epitope tag arrangement in combination with any of four auxotrophic markers and three distinct replication mechanisms. As an example of its applicability, we used yeast three-hybrid to provide evidence for the assembly of a ternary complex of plant proteins involved in jasmonate signalling and consisting of the JAZ, NINJA and TOPLESS proteins. CONCLUSION: Our vectors make MultiSite Gateway(TM) cloning accessible in S. cerevisiae and implement a fast and versatile cloning method for the high-throughput functional analysis of (heterologous) proteins in one of the most widely used model organisms for molecular biology research. |
960 | Antagonistic Pleiotropy and Fitness Trade-Offs Reveal Specialist and Generalist Traits in Strains of Canine Distemper Virus | Theoretically, homogeneous environments favor the evolution of specialists whereas heterogeneous environments favor generalists. Canine distemper is a multi-host carnivore disease caused by canine distemper virus (CDV). The described cell receptor of CDV is SLAM (CD150). Attachment of CDV hemagglutinin protein (CDV-H) to this receptor facilitates fusion and virus entry in cooperation with the fusion protein (CDV-F). We investigated whether CDV strains co-evolved in the large, homogeneous domestic dog population exhibited specialist traits, and strains adapted to the heterogeneous environment of smaller populations of different carnivores exhibited generalist traits. Comparison of amino acid sequences of the SLAM binding region revealed higher similarity between sequences from Canidae species than to sequences from other carnivore families. Using an in vitro assay, we quantified syncytia formation mediated by CDV-H proteins from dog and non-dog CDV strains in cells expressing dog, lion or cat SLAM. CDV-H proteins from dog strains produced significantly higher values with cells expressing dog SLAM than with cells expressing lion or cat SLAM. CDV-H proteins from strains of non-dog species produced similar values in all three cell types, but lower values in cells expressing dog SLAM than the values obtained for CDV-H proteins from dog strains. By experimentally changing one amino acid (Y549H) in the CDV-H protein of one dog strain we decreased expression of specialist traits and increased expression of generalist traits, thereby confirming its functional importance. A virus titer assay demonstrated that dog strains produced higher titers in cells expressing dog SLAM than cells expressing SLAM of non-dog hosts, which suggested possible fitness benefits of specialization post-cell entry. We provide in vitro evidence for the expression of specialist and generalist traits by CDV strains, and fitness trade-offs across carnivore host environments caused by antagonistic pleiotropy. These findings extend knowledge on CDV molecular epidemiology of particular relevance to wild carnivores. |
961 | Host-protective effect of circulating pentraxin 3 (PTX3) and complex formation with neutrophil extracellular traps | Pentraxin 3 (PTX3) is a soluble pattern recognition receptor which is classified as a long-pentraxin in the pentraxin family. It is known to play an important role in innate immunity, inflammatory regulation, and female fertility. PTX3 is synthesized by specific cells, primarily in response to inflammatory signals. Among these various cells, neutrophils have a unique PTX3 production system. Neutrophils store PTX3 in neutrophil-specific granules and then the stored PTX3 is released and localizes in neutrophil extracellular traps (NETs). Although certain NET components have been identified, such as histones and anti-microbial proteins, the detailed mechanisms by which NETs localize, as well as capture and kill microbes, have not been fully elucidated. PTX3 is a candidate diagnostic marker of infection and vascular damage. In severe infectious diseases such as sepsis, the circulating PTX3 concentration increases greatly (up to 100 ng/mL, i.e., up to 100-fold of the normal level). Even though it is clearly implied that PTX3 plays a protective role in sepsis and certain other disorders, the detailed mechanisms by which it does so remain unclear. A proteomic study of PTX3 ligands in septic patients revealed that PTX3 forms a complex with certain NET component proteins. This suggests a role for PTX3 in which it facilitates the efficiency of anti-microbial protein pathogen clearance by interacting with both pathogens and anti-microbial proteins. We discuss the possible relationships between PTX3 and NET component proteins in the host protection afforded by the innate immune response. The PTX3 complex has the potential to be a highly useful diagnostic marker of sepsis and other inflammatory diseases. |
962 | Antigenic Subversion: A Novel Mechanism of Host Immune Evasion by Ebola Virus | In addition to its surface glycoprotein (GP(1,2)), Ebola virus (EBOV) directs the production of large quantities of a truncated glycoprotein isoform (sGP) that is secreted into the extracellular space. The generation of secreted antigens has been studied in several viruses and suggested as a mechanism of host immune evasion through absorption of antibodies and interference with antibody-mediated clearance. However such a role has not been conclusively determined for the Ebola virus sGP. In this study, we immunized mice with DNA constructs expressing GP(1,2) and/or sGP, and demonstrate that sGP can efficiently compete for anti-GP(12) antibodies, but only from mice that have been immunized by sGP. We term this phenomenon “antigenic subversion”, and propose a model whereby sGP redirects the host antibody response to focus on epitopes which it shares with membrane-bound GP(1,2), thereby allowing it to absorb anti-GP(1,2) antibodies. Unexpectedly, we found that sGP can also subvert a previously immunized host's anti-GP(1,2) response resulting in strong cross-reactivity with sGP. This finding is particularly relevant to EBOV vaccinology since it underscores the importance of eliciting robust immunity that is sufficient to rapidly clear an infection before antigenic subversion can occur. Antigenic subversion represents a novel virus escape strategy that likely helps EBOV evade host immunity, and may represent an important obstacle to EBOV vaccine design. |
963 | Application of Molecular Diagnostic Techniques for Viral Testing | Nucleic acid amplification techniques are commonly used currently to diagnose viral diseases and manage patients with this kind of illnesses. These techniques have had a rapid but unconventional route of development during the last 30 years, with the discovery and introduction of several assays in clinical diagnosis. The increase in the number of commercially available methods has facilitated the use of this technology in the majority of laboratories worldwide. This technology has reduced the use of some other techniques such as viral culture based methods and serological assays in the clinical virology laboratory. Moreover, nucleic acid amplification techniques are now the methods of reference and also the most useful assays for the diagnosis in several diseases. The introduction of these techniques and their automation provides new opportunities for the clinical laboratory to affect patient care. The main objectives in performing nucleic acid tests in this field are to provide timely results useful for high-quality patient care at a reasonable cost, because rapid results are associated with improvements in patients care. The use of amplification techniques such as polymerase chain reaction, real-time polymerase chain reaction or nucleic acid sequence-based amplification for virus detection, genotyping and quantification have some advantages like high sensitivity and reproducibility, as well as a broad dynamic range. This review is an up-to-date of the main nucleic acid techniques and their clinical applications, and special challenges and opportunities that these techniques currently provide for the clinical virology laboratory. |
964 | Imaging Findings in Patients With H1N1 Influenza A Infection | BACKGROUND: Swine influenza (H1N1) is a very contagious respiratory infection and World Health Organization (WHO) has raised the alert level to phase 6 (pandemic). The study of clinical and laboratory manifestations as well as radiologic imaging findings helps in its early diagnosis. OBJECTIVES: The aim of this study was to evaluate the imaging findings of patients with documented H1N1 infection referred to our center. PATIENTS AND METHODS: Thirty-one patients (16 men) with documented H1N1 infection were included in our study. The initial radiography obtained from the patients was reviewed regarding pattern (consolidation, ground glass, nodules and reticulation), distribution (focal, multifocal, and diffuse) and the lung zones involved. Computed tomography (CT) scans were also reviewed for the same abnormalities. The patient files were studied for their possible underlying diseases. RESULTS: The mean age was 37.97 ± 13.9 years. Seventeen (54.8%) patients had co-existing condition (eight respiratory, five cardiovascular, two immunodeficiency, two cancer, four others). Twelve (38.7%) patients required intensive care unit (ICU) admission. Five (16.1%) patients died. (25.8%) had normal initial radiographs. The most common abnormality was consolidation (12/31; 38.7%) in the peripheral region (11/31; 35.5%) followed by peribronchovascular areas (10/31; 32.3%) which was most commonly observed in the lower zone. The patients admitted to the ICU were more likely to have two or more lung zones involved (P = 0.005). CONCLUSIONS: In patients with the novel swine flu infection, the most common radiographic abnormality observed was consolidation in the lower lung zones. Patients admitted to ICU were more likely to have two or more lung zones involved. |
965 | Retrovirus Entry by Endocytosis and Cathepsin Proteases | Retroviruses include infectious agents inducing severe diseases in humans and animals. In addition, retroviruses are widely used as tools to transfer genes of interest to target cells. Understanding the entry mechanism of retroviruses contributes to developments of novel therapeutic approaches against retrovirus-induced diseases and efficient exploitation of retroviral vectors. Entry of enveloped viruses into host cell cytoplasm is achieved by fusion between the viral envelope and host cell membranes at either the cell surface or intracellular vesicles. Many animal retroviruses enter host cells through endosomes and require endosome acidification. Ecotropic murine leukemia virus entry requires cathepsin proteases activated by the endosome acidification. CD4-dependent human immunodeficiency virus (HIV) infection is thought to occur via endosomes, but endosome acidification is not necessary for the entry whereas entry of CD4-independent HIVs, which are thought to be prototypes of CD4-dependent viruses, is low pH dependent. There are several controversial results on the retroviral entry pathways. Because endocytosis and endosome acidification are complicatedly controlled by cellular mechanisms, the retrovirus entry pathways may be different in different cell lines. |
966 | Immunization with a Recombinant Vaccinia Virus That Encodes Nonstructural Proteins of the Hepatitis C Virus Suppresses Viral Protein Levels in Mouse Liver | Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid–polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/−))/MxCre((+/−)) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine. |
967 | Brain Transcriptome-Wide Screen for HIV-1 Nef Protein Interaction Partners Reveals Various Membrane-Associated Proteins | HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases. |
968 | Infant Pertussis and Household Transmission in Korea | A recent resurgence of pertussis has raised public health concerns even in developed countries with high vaccination coverage. The aim of this study was to describe the clinical characteristics of infant pertussis, and to determine the relative importance of household transmission in Korea. The multicenter study was prospectively conducted from January 2009 to September 2011. We identified the demographic and clinical data from these patients and performed the diagnostic tests for pertussis in their household contacts. Twenty-one patients with confirmed pertussis were included in the analysis. All infections occurred in infants younger than 6 months of age (mean age, 2.5 months) who had not completed the primary DTaP vaccination except for one patient. Infants without immunization history had a significant higher lymphocytosis and longer duration of hospital stay compared to those with immunization. All were diagnosed with PCR (100%), however, culture tests showed the lowest sensitivity (42.9%). Presumed source of infection in household contacts was documented in 85.7%, mainly parents (52.6%). Pertussis had a major morbidity in young infants who were not fully immunized. Household members were responsible for pertussis transmission of infants in whom a source could be identified. The control of pertussis through booster vaccination with Tdap in family who is taking care of young infants is necessary in Korea. |
969 | LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets | The study of cell-population heterogeneity in a range of biological systems, from viruses to bacterial isolates to tumor samples, has been transformed by recent advances in sequencing throughput. While the high-coverage afforded can be used, in principle, to identify very rare variants in a population, existing ad hoc approaches frequently fail to distinguish true variants from sequencing errors. We report a method (LoFreq) that models sequencing run-specific error rates to accurately call variants occurring in <0.05% of a population. Using simulated and real datasets (viral, bacterial and human), we show that LoFreq has near-perfect specificity, with significantly improved sensitivity compared with existing methods and can efficiently analyze deep Illumina sequencing datasets without resorting to approximations or heuristics. We also present experimental validation for LoFreq on two different platforms (Fluidigm and Sequenom) and its application to call rare somatic variants from exome sequencing datasets for gastric cancer. Source code and executables for LoFreq are freely available at http://sourceforge.net/projects/lofreq/. |
970 | BUHO: A MATLAB Script for the Study of Stress Granules and Processing Bodies by High-Throughput Image Analysis | The spontaneous and reversible formation of foci and filaments that contain proteins involved in different metabolic processes is common in both the nucleus and the cytoplasm. Stress granules (SGs) and processing bodies (PBs) belong to a novel family of cellular structures collectively known as mRNA silencing foci that harbour repressed mRNAs and their associated proteins. SGs and PBs are highly dynamic and they form upon stress and dissolve thus releasing the repressed mRNAs according to changes in cell physiology. In addition, aggregates containing abnormal proteins are frequent in neurodegenerative disorders. In spite of the growing relevance of these supramolecular aggregates to diverse cellular functions a reliable automated tool for their systematic analysis is lacking. Here we report a MATLAB Script termed BUHO for the high-throughput image analysis of cellular foci. We used BUHO to assess the number, size and distribution of distinct objects with minimal deviation from manually obtained parameters. BUHO successfully addressed the induction of both SGs and PBs in mammalian and insect cells exposed to different stress stimuli. We also used BUHO to assess the dynamics of specific mRNA-silencing foci termed Smaug 1 foci (S-foci) in primary neurons upon synaptic stimulation. Finally, we used BUHO to analyze the role of candidate genes on SG formation in an RNAi-based experiment. We found that FAK56D, GCN2 and PP1 govern SG formation. The role of PP1 is conserved in mammalian cells as judged by the effect of the PP1 inhibitor salubrinal, and involves dephosphorylation of the translation factor eIF2α. All these experiments were analyzed manually and by BUHO and the results differed in less than 5% of the average value. The automated analysis by this user-friendly method will allow high-throughput image processing in short times by providing a robust, flexible and reliable alternative to the laborious and sometimes unfeasible visual scrutiny. |
971 | Adjuvant Activity of Sargassum pallidum Polysaccharides against Combined Newcastle Disease, Infectious Bronchitis and Avian Influenza Inactivated Vaccines | This study evaluates the effects of Sargassum pallidum polysaccharides (SPP) on the immune responses in a chicken model. The adjuvanticity of Sargassum pallidum polysaccharides in Newcastle disease (ND), infectious bronchitis (IB) and avian influenza (AI) was investigated by examining the antibody titers and lymphocyte proliferation following immunization in chickens. The chickens were administrated combined ND, IB and AI inactivated vaccines containing SPP at 10, 30 and 50 mg/mL, using an oil adjuvant vaccine as a control. The ND, IB and AI antibody titers and the lymphocyte proliferation were enhanced at 30 mg/mL SPP. In conclusion, an appropriate dose of SPP may be a safe and efficacious immune stimulator candidate that is suitable for vaccines to produce early and persistent prophylaxis. |
972 | Existing health inequalities in India: informing preparedness planning for an influenza pandemic | On 11 June 2009, the World Health Organization (WHO) declared that the world was in phase 6 of an influenza pandemic. In India, the first case of 2009 H1N1 influenza was reported on 16 May 2009 and by August 2010 (when the pandemic was declared over), 38 730 cases of 2009 H1N1 had been confirmed of which there were 2024 deaths. Here, we propose a conceptual model of the sources of health disparities in an influenza pandemic in India. Guided by a published model of the plausible sources of such disparities in the United States, we reviewed the literature for the determinants of the plausible sources of health disparities during a pandemic in India. We find that factors at multiple social levels could determine inequalities in the risk of exposure and susceptibility to influenza, as well as access to treatment once infected: (1) religion, caste and indigenous identity, as well as education and gender at the individual level; (2) wealth at the household level; and (3) the type of location, ratio of health care practitioners to population served, access to transportation and public spending on health care in the geographic area of residence. Such inequalities could lead to unequal levels of disease and death. Whereas causal factors can only be determined by testing the model when incidence and mortality data, collected in conjunction with socio-economic and geographic factors, become available, we put forth recommendations that policy makers can undertake to ensure that the pandemic preparedness plan includes a focus on social inequalities in India in order to prevent their exacerbation in a pandemic. |
973 | Phylodynamic Inference and Model Assessment with Approximate Bayesian Computation: Influenza as a Case Study | A key priority in infectious disease research is to understand the ecological and evolutionary drivers of viral diseases from data on disease incidence as well as viral genetic and antigenic variation. We propose using a simulation-based, Bayesian method known as Approximate Bayesian Computation (ABC) to fit and assess phylodynamic models that simulate pathogen evolution and ecology against summaries of these data. We illustrate the versatility of the method by analyzing two spatial models describing the phylodynamics of interpandemic human influenza virus subtype A(H3N2). The first model captures antigenic drift phenomenologically with continuously waning immunity, and the second epochal evolution model describes the replacement of major, relatively long-lived antigenic clusters. Combining features of long-term surveillance data from the Netherlands with features of influenza A (H3N2) hemagglutinin gene sequences sampled in northern Europe, key phylodynamic parameters can be estimated with ABC. Goodness-of-fit analyses reveal that the irregularity in interannual incidence and H3N2's ladder-like hemagglutinin phylogeny are quantitatively only reproduced under the epochal evolution model within a spatial context. However, the concomitant incidence dynamics result in a very large reproductive number and are not consistent with empirical estimates of H3N2's population level attack rate. These results demonstrate that the interactions between the evolutionary and ecological processes impose multiple quantitative constraints on the phylodynamic trajectories of influenza A(H3N2), so that sequence and surveillance data can be used synergistically. ABC, one of several data synthesis approaches, can easily interface a broad class of phylodynamic models with various types of data but requires careful calibration of the summaries and tolerance parameters. |
974 | First Discovery and Stucture-Activity Relationship Study of Phenanthroquinolizidines as Novel Antiviral Agents against Tobacco Mosaic Virus (TMV) | A series of phenanthroquinolizidine alkaloids 1–24 were prepared and first evaluated for their antiviral activity against tobacco mosaic virus (TMV). The bioassay results showed that most of these compounds exhibited good to excellent in vivo anti-TMV activity, of which compounds 1, 2, 15 and 16 displayed significantly higher activity than (R)-antofine and commercial Ningnanmycin at the same test condition. The substituents on the phenanthrene moiety play an important role for maintaining high in vivo antiviral activity. The introduction of 6-hydroxyl, which is proposed to interact with TMV RNA, did increased anti-TMV activity. The 14aR-configuration was confirmed to be the preferred antiviral configuration for phenanthroquinolizidine alkaloids. Introduction of hydroxy group at 15-position of phenanthroquinolizidine alkaloids increased activity for S-configuration but decreased activity for R-configuration. Present study provides fundamental support for development and optimization of phenanthroquinolizidine alkaloids as potential inhibitors of plant virus. |
975 | CD4(+) T-Cell Expansion Predicts Neutralizing Antibody Responses to Monovalent, Inactivated 2009 Pandemic Influenza A(H1N1) Virus Subtype H1N1 Vaccine | Background. The ability of influenza vaccines to elicit CD4(+) T cells and the relationship between induction of CD4(+) T cells and vaccine-induced neutralizing antibody responses has been controversial. The emergence of swine-origin 2009 pandemic influenza A virus subtype H1N1 (A[H1N1]pdm09) provided a unique opportunity to examine responses to an influenza vaccine composed of both novel and previously encountered antigens and to probe the relationship between B-cell and T-cell responses to vaccination. Methods. We tracked CD4(+) T-cell and antibody responses of human subjects vaccinated with monovalent subunit A(H1N1)pdm09 vaccine. The specificity and magnitude of the CD4(+) T-cell response was evaluated using cytokine enzyme-linked immunosorbent spot assays in conjugation with peptide pools representing distinct influenza virus proteins. Results. Our studies revealed that vaccination induced readily detectable CD4(+) T cells specific for conserved portions of hemagglutinin (HA) and the internal viral proteins. Interestingly, expansion of HA-specific CD4(+) T cells was most tightly correlated with the antibody response. Conclusions. These results indicate that CD4(+) T-cell expansion may be a limiting factor in development of neutralizing antibody responses to pandemic influenza vaccines and suggest that approaches to facilitate CD4(+) T-cell recruitment may increase the neutralizing antibody produced in response to vaccines against novel influenza strains. |
976 | Leave entitlements, time off work and the household financial impacts of quarantine compliance during an H1N1 outbreak | BACKGROUND: The Australian state of Victoria, with 5.2 million residents, enforced home quarantine during a H1N1 pandemic in 2009. The strategy was targeted at school children. The objective of this study was to investigate the extent to which parents’ access to paid sick leave or paid carer’s leave was associated with (a) time taken off work to care for quarantined children, (b) household finances, and (c) compliance with quarantine recommendations. METHODS: We conducted an online and telephone survey of households recruited through 33 schools (85% of eligible schools), received 314 responses (27%), and analysed the subsample of 133 households in which all resident parents were employed. RESULTS: In 52% of households, parents took time off work to care for quarantined children. Households in which no resident parent had access to leave appeared to be less likely to take time off work (42% vs 58%, p=0.08) although this difference had only borderline significance. Among parents who did take time off work, those in households without access to leave were more likely to lose pay (73% vs 21%, p<0.001). Of the 26 households in which a parent lost pay due to taking time off work, 42% experienced further financial consequences such as being unable to pay a bill. Access to leave did not predict compliance with quarantine recommendations. CONCLUSIONS: Future pandemic plans should consider the economic costs borne by households and options for compensating quarantined families for income losses. |
977 | Effectiveness of Integrated HIV Prevention Interventions among Chinese Men Who Have Sex with Men: Evaluation of a 16-City Public Health Program | To examine the impacts of a multi-city HIV prevention public health program (China Global Fund Round 5 Project) on condom use and HIV infection, we analyzed four yearly cross-sectional surveys from 2006 through 2009 among 20,843 men who have sex with men (MSM) in 16 Chinese cities. Self-reported condom use at last sex with a male partner increased from 58% in 2006 to 81% in 2009 (trend test, P<0.001). HIV prevalence increased from 2.3% in 2006 to 5.3% in 2009 (P<0.001). Multivariable logistic regression analysis showed that self-reported receipt of interventions was an independent predictor of increased condom use at last sex with a male partner over time (adjusted odds ratio [aOR], 1.63 in 2006 to 2.33 in 2009; P<0.001), and lower HIV prevalence (aOR, 1.08 in 2006 to 0.45 in 2009; P<0.001). HIV prevalence increased from 2006–2009 for participants with no self-reported receipt of interventions (2.1% in 2006 to 10.3% in 2009) and less so for those with interventions (2.4% to 4.7%). This Chinese public health program had positive impacts on both behaviors and disease rate among MSM population. Escalation of the coverage and intensity of effective interventions is needed for further increasing condom use and for reversing the rising trend of HIV epidemic. |
978 | Pandemic influenza A/H1N1 virus infection and TNF, LTA, IL1B, IL6, IL8, and CCL polymorphisms in Mexican population: a case–control study | BACKGROUND: Some patients have a greater response to viral infection than do others having a similar level of viral replication. Hypercytokinemia is the principal immunopathological mechanism that contributes to a severer clinical course in cases of influenza A/H1N1. The benefit produced, or damage caused, by these cytokines in severe disease is not known. The genes that code for these molecules are polymorphic and certain alleles have been associated with susceptibility to various diseases. The objective of the present study was to determine whether there was an association between polymorphisms of TNF, LTA, IL1B, IL6, IL8, and CCL1 and the infection and severity of the illness caused by the pandemic A/H1N1 in Mexico in 2009. METHODS: Case–control study. The cases were patients confirmed with real time PCR with infection by the A/H1N1 pandemic virus. The controls were patients with infection like to influenza and non-familial healthy contacts of the patients with influenza. Medical history and outcome of the disease was registered. The DNA samples were genotyped for polymorphisms TNF rs361525, rs1800629, and rs1800750; LTA rs909253; IL1B rs16944; IL6 rs1818879; IL8 rs4073; and CCL1 rs2282691. Odds ratio (OR) and the 95% confidence interval (95% CI) were calculated. The logistic regression model was adjusted by age and severity of the illness in cases. RESULTS: Infection with the pandemic A/H1N1 virus was associated with the following genotypes: TNF rs361525 AA, OR = 27.00; 95% CI = 3.07–1248.77); LTA rs909253 AG (OR = 4.33, 95% CI = 1.82–10.32); TNF rs1800750 AA (OR = 4.33, 95% CI = 1.48–12.64); additionally, LTA rs909253 AG showed a limited statistically significant association with mortality (p = 0.06, OR = 3.13). Carriers of the TNF rs1800629 GA genotype were associated with high levels of blood urea nitrogen (p = 0.05); those of the TNF rs1800750 AA genotype, with high levels of creatine phosphokinase (p=0.05). The IL1B rs16944 AA genotype was associated with an elevated number of leukocytes (p <0.001) and the IL8 rs4073 AA genotype, with a higher value for P(a)O(2) mm Hg. CONCLUSION: The polymorphisms of genes involved in the inflammatory process contributed to the severity of the clinical behavior of infection by the pandemic influenza A/H1N1 virus. |
979 | Complement in Action: An Analysis of Patent Trends from 1976 Through 2011 | Complement is an essential part of the innate immune response. It interacts with diverse endogenous pathways and contributes to the maintenance of homeostasis, the modulation of adaptive immune responses, and the development of various pathologies. The potential usefulness, in both research and clinical settings, of compounds that detect or modulate complement activity has resulted in thousands of publications on complement-related innovations in fields such as drug discovery, disease diagnosis and treatment, and immunoassays, among others. This study highlights the distribution and publication trends of patents related to the complement system that were granted by the United States Patent and Trademark Office from 1976 to the present day. A comparison to complement-related documents published by the World Intellectual Property Organization is also included. Statistical analyses revealed increasing diversity in complement-related research interests over time. More than half of the patents were found to focus on the discovery of inhibitors; interest in various inhibitor classes exhibited a remarkable transformation from chemical compounds early on to proteins and antibodies in more recent years. Among clinical applications, complement proteins and their modulators have been extensively patented for the diagnosis and treatment of eye diseases (especially age-related macular degeneration), graft rejection, cancer, sepsis, and a variety of other inflammatory and immune diseases. All of the patents discussed in this chapter, as well as those from other databases, are available from our newly constructed complement patent database: www.innateimmunity.us/patent. |
980 | A novel strategy for efficient production of anti-V3 human scFvs against HIV-1 clade C | BACKGROUND: Production of human monoclonal antibodies that exhibit broadly neutralizing activity is needed for preventing HIV-1 infection, however only a few such antibodies have been generated till date. Isolation of antibodies by the hybridoma technology is a cumbersome process with fewer yields. Further, the loss of unstable or slowly growing clones which may have unique binding specificities often occurs during cloning and propagation and the strongly positive clones are often lost. This has been avoided by the process described in this paper, wherein, by combining the strategy of EBV transformation and recombinant DNA technology, we constructed human single chain variable fragments (scFvs) against the third variable region (V3) of the clade C HIV-1 envelope. RESULTS: An antigen specific phage library of 7000 clones was constructed from the enriched V3- positive antibody secreting EBV transformed cells. By ligation of the digested scFv DNA into phagemid vector and bio panning against the HIV-1 consensus C and B V3 peptides followed by random selection of 40 clones, we identified 15 clones that showed V3 reactivity in phage ELISA. DNA fingerprinting analysis and sequencing showed that 13 out of the 15 clones were distinct. Expression of the positive clones was tested by SDS-PAGE and Western blot. All the 13 anti-V3 scFvs showed cross-reactivity against both the clade C and B V3 peptides and did not show any reactivity against other unrelated peptides in ELISA. Preliminary neutralization assays indicated varying degrees of neutralization of clade C and B viruses. EBV transformation, followed by antigen selection of lines to identify specific binders, enabled the selection of phage from un-cloned lines for scFv generation, thus avoiding the problems of hybridoma technology. Moreover, as the clones were pretested for antigen binding, a comparatively small library sufficed for the selection of a considerable number of unique antigen binding phage. After selection, the phage clones were propagated in a clonal manner. CONCLUSIONS: This strategy can be efficiently used and is cost effective for the generation of diverse recombinant antibodies. This is the first study to generate anti-V3 scFvs against HIV-1 Clade C. |
981 | Wearing face masks in public during the influenza season may reflect other positive hygiene practices in Japan | BACKGROUND: Although the wearing of face masks in public has not been recommended for preventing influenza, these devices are often worn in many Asian countries during the influenza season. In Japan, it is thought that such behavior may be an indicator of other positive hygiene practices. The aim of this study, therefore, was to determine if wearing a face mask in public is associated with other positive hygiene practices and health behaviors among Japanese adults. METHODS: We initially recruited around 3,000 Japanese individuals ranging from 20 to 69 years of age who were registered with a web survey company. Participants were asked to recall their personal hygiene practices during the influenza season of the previous year. Logistic regression analysis was then used to examine the associations between wearing a face mask in public and personal hygiene practices and health behaviors. RESULTS: A total of 3,129 persons responded to the survey, among whom 38% reported that they had worn a face mask in public during the previous influenza season. Wearing a face mask in public was associated with various self-reported hygiene practices including: frequent hand washing (adjusted Odds Ratio [OR]: 1.67; 95% Confidence Interval [95%CI]: 1.34-1.96), occasional hand washing (OR: 1.43; 95%CI: 1.10-1.75), frequently avoiding crowds (OR: 1.85; 95%CI: 1.70-1.98), occasionally avoiding crowds (OR: 1.65; 95%CI: 1.53-1.76), frequent gargling (OR: 1.68; 95%CI: 1.51-1.84), occasional gargling (OR: 1.46; 95%CI: 1.29-1.62), regularly avoiding close contact with an infected person (OR: 1.50; 95%CI: 1.33-1.67), occasionally avoiding close contact with an infected person (OR: 1.31; 95%CI: 1.16-1.46), and being vaccinated of influenza in the last season (OR: 1.31; 95%CI: 1.17-1.45). CONCLUSIONS: Overall, this study suggests that wearing a face mask in public may be associated with other personal hygiene practices and health behaviors among Japanese adults. Rather than preventing influenza itself, face mask use might instead be a marker of additional, positive hygiene practices and other favorable health behaviors in the same individuals. |
982 | Inhibitory Influence of Enterococcus faecium on the Propagation of Swine Influenza A Virus In Vitro | The control of infectious diseases such as swine influenza viruses (SwIV) plays an important role in food production both from the animal health and from the public health point of view. Probiotic microorganisms and other health improving food supplements have been given increasing attention in recent years, but, no information on the effects of probiotics on swine influenza virus is available. Here we address this question by assessing the inhibitory potential of the probiotic Enterococcus faecium NCIMB 10415 (E. faecium) on the replication of two porcine strains of influenza virus (H1N1 and H3N2 strain) in a continuous porcine macrophage cell line (3D4/21) and in MDBK cells. Cell cultures were treated with E. faecium at the non-toxic concentration of 1×10(6) CFU/ml in growth medium for 60 to 90 min before, during and after SwIV infection. After further incubation of cultures in probiotic-free growth medium, cell viability and virus propagation were determined at 48 h or 96 h post infection. The results obtained reveal an almost complete recovery of viability of SwIV infected cells and an inhibition of virus multiplication by up to four log units in the E. faecium treated cells. In both 3D4/21- and MDBK-cells a 60 min treatment with E. faecium stimulated nitric oxide (NO) release which is in line with published evidence for an antiviral function of NO. Furthermore, E. faecium caused a modified cellular expression of selected mediators of defence in 3D4-cells: while the expression of TNF-α, TLR-3 and IL-6 were decreased in the SwIV-infected and probiotic treated cells, IL-10 was found to be increased. Since we obtained experimental evidence for the direct adsorptive trapping of SwIV through E. faecium, this probiotic microorganism inhibits influenza viruses by at least two mechanisms, direct physical interaction and strengthening of innate defence at the cellular level. |
983 | Natural killer cells act as rheostats modulating anti-viral T cells | Anti-viral T cells are thought to regulate whether hepatitis C virus (HCV) and HIV infections result in viral control, asymptomatic persistence, or severe disease, though the reasons for these different outcomes remain unclear. Recent genetic evidence, however, has indicated a correlation between certain natural killer (NK) cell receptors and progression of both HIV and HCV infection(1–3), implying that NK cells are playing a role in these T cell-associated diseases. While direct NK cell-mediated lysis of virus-infected cells may contribute to anti-viral defense during some virus infections, especially murine cytomegalovirus (MCMV) infections in mice and perhaps HIV in humans(4–5), NK cells have also been suspected as having immunoregulatory functions. For instance, NK cells may indirectly regulate T cell responses by lysing MCMV-infected antigen-presenting cells(6–7). In contrast to MCMV, lymphocytic choromeningitis virus (LCMV) infection in mice seems resistant to any direct anti-viral effects of NK cells(5,8). Here the roles of NK cells in regulating T cell-dependent viral persistence and immunopathology were examined in mice infected with LCMV, an established model for HIV and HCV infections in humans. We describe a three-way interaction, whereby activated NK cells cytolytically eliminate activated CD4 T cells that affect CD8 T-cell function and exhaustion. At high virus dose NK cells prevented fatal pathology while enabling T-cell exhaustion and viral persistence, but at a medium dose NK cells paradoxically facilitated lethal T cell-mediated pathology. Thus, NK cells can act as rheostats, regulating CD4 T cell-mediated support for the anti-viral CD8 T cells that control viral pathogenesis and persistence. |
984 | The impact of influenza A(H1N1)pdm09 compared with seasonal influenza on intensive care admissions in New South Wales, Australia, 2007 to 2010: a time series analysis | BACKGROUND: In Australia, the 2009 epidemic of influenza A(H1N1)pdm09 resulted in increased admissions to intensive care. The annual contribution of influenza to use of intensive care is difficult to estimate, as many people with influenza present without a classic influenza syndrome and laboratory testing may not be performed. We used a population-based approach to estimate and compare the impact of recent epidemics of seasonal and pandemic influenza. METHODS: For 2007 to 2010, time series describing health outcomes in various population groups were prepared from a database of all intensive care unit (ICU) admissions in the state of New South Wales, Australia. The Serfling approach, a time series method, was used to estimate seasonal patterns in health outcomes in the absence of influenza epidemics. The contribution of influenza was estimated by subtracting expected seasonal use from observed use during each epidemic period. RESULTS: The estimated excess rate of influenza-associated respiratory ICU admissions per 100,000 inhabitants was more than three times higher in 2007 (2.6/100,000, 95% CI 2.0 to 3.1) than the pandemic year, 2009 (0.76/100,000, 95% CI 0.04 to 1.48). In 2009, the highest excess respiratory ICU admission rate was in 17 to 64 year olds (2.9/100,000, 95% CI 2.2 to 3.6), while in 2007, the highest excess rate was in those aged 65 years or older (9.5/100,000, 95% CI 6.2 to 12.8). In 2009, the excess rate was 17/100,000 (95% CI 14 to 20) in Aboriginal people and 14/100,000 (95% CI 13 to 16) in pregnant women. CONCLUSION: While influenza was diagnosed more frequently and peak use of intensive care was higher during the epidemic of pandemic influenza in 2009, overall excess admissions to intensive care for respiratory illness was much greater during the influenza season in 2007. Thus, the impact of seasonal influenza on intensive care use may have previously been under-recognised. In 2009, high ICU use among young to middle aged adults was offset by relatively low use among older adults, and Aboriginal people and pregnant women were substantially over-represented in ICUs. Greater emphasis on prevention of serious illness in Aboriginal people and pregnant women should be a priority in pandemic planning. |
985 | Activation of the Cellular Unfolded Protein Response by Recombinant Adeno-Associated Virus Vectors | The unfolded protein response (UPR) is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER). In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold) and PERK (up to 8 fold) genes 12–48 hours after infection with self-complementary (sc)AAV2 but less prominent with single-stranded (ss)AAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold) while AAV6 vectors induced a significant increase on all the three major UPR pathways [6–16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5–2 fold) in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively). However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin) during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer. |
986 | Hypoxia-regulated target genes implicated in tumor metastasis | Hypoxia is an important microenvironmental factor that induces cancer metastasis. Hypoxia/hypoxia-inducible factor-1α (HIF-1α) regulates many important steps of the metastatic processes, especially epithelial-mesenchymal transition (EMT) that is one of the crucial mechanisms to cause early stage of tumor metastasis. To have a better understanding of the mechanism of hypoxia-regulated metastasis, various hypoxia/HIF-1α-regulated target genes are categorized into different classes including transcription factors, histone modifiers, enzymes, receptors, kinases, small GTPases, transporters, adhesion molecules, surface molecules, membrane proteins, and microRNAs. Different roles of these target genes are described with regards to their relationship to hypoxia-induced metastasis. We hope that this review will provide a framework for further exploration of hypoxia/HIF-1α-regulated target genes and a comprehensive view of the metastatic picture induced by hypoxia. |
987 | Targeting Herpetic Keratitis by Gene Therapy | Ocular gene therapy is rapidly becoming a reality. By November 2012, approximately 28 clinical trials were approved to assess novel gene therapy agents. Viral infections such as herpetic keratitis caused by herpes simplex virus 1 (HSV-1) can cause serious complications that may lead to blindness. Recurrence of the disease is likely and cornea transplantation, therefore, might not be the ideal therapeutic solution. This paper will focus on the current situation of ocular gene therapy research against herpetic keratitis, including the use of viral and nonviral vectors, routes of delivery of therapeutic genes, new techniques, and key research strategies. Whereas the correction of inherited diseases was the initial goal of the field of gene therapy, here we discuss transgene expression, gene replacement, silencing, or clipping. Gene therapy of herpetic keratitis previously reported in the literature is screened emphasizing candidate gene therapy targets. Commonly adopted strategies are discussed to assess the relative advantages of the protective therapy using antiviral drugs and the common gene therapy against long-term HSV-1 ocular infections signs, inflammation and neovascularization. Successful gene therapy can provide innovative physiological and pharmaceutical solutions against herpetic keratitis. |
988 | Indigenous populations health protection: A Canadian perspective | The disproportionate effects of the 2009 H1N1 pandemic on many Canadian Aboriginal communities have drawn attention to the vulnerability of these communities in terms of health outcomes in the face of emerging and reemerging infectious diseases. Exploring the particular challenges facing these communities is essential to improving public health planning. In alignment with the objectives of the Pandemic Influenza Outbreak Research Modelling (Pan-InfORM) team, a Canadian public health workshop was held at the Centre for Disease Modelling (CDM) to: (i) evaluate post-pandemic research findings; (ii) identify existing gaps in knowledge that have yet to be addressed through ongoing research and collaborative activities; and (iii) build upon existing partnerships within the research community to forge new collaborative links with Aboriginal health organizations. The workshop achieved its objectives in identifying main research findings and emerging information post pandemic, and highlighting key challenges that pose significant impediments to the health protection and promotion of Canadian Aboriginal populations. The health challenges faced by Canadian indigenous populations are unique and complex, and can only be addressed through active engagement with affected communities. The academic research community will need to develop a new interdisciplinary framework, building upon concepts from ‘Communities of Practice’, to ensure that the research priorities are identified and targeted, and the outcomes are translated into the context of community health to improve policy and practice. |
989 | Schmallenberg Virus Pathogenesis, Tropism and Interaction with the Innate Immune System of the Host | Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and “synthetic” SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV. |
990 | Myometrial cavernous hemangioma with pulmonary thromboembolism in a post-partum woman: a case report and review of the literature | INTRODUCTION: Cavernous hemangiomas of the uterus are rare benign vascular lesions. Nine cases of diffuse cavernous hemangioma of the gravid uterus have been reported, most of which diffusely involved the myometrium. These vascular malformations are clinically significant, and may cause pronounced bleeding resulting in maternal or fetal demise. Thrombosis of cavernous hemangiomas of the uterus has been previously reported. We here report the first case in which a thrombosed cavernous hemangioma of the myometrium resulted in a fatal pulmonary embolism in a post-partum woman. CASE PRESENTATION: A 25-year-old obese African-American woman who had one pregnancy and was delivered of twins by cesarean section was admitted 1 week after the successful delivery. The 12-day clinical course included ventilator-dependent respiratory failure, systemic hypertension, methicillin-resistant Staphylococcus aureus in the sputum, leukocytosis and asystole. A transabdominal ultrasound examination showed heterogeneous thickened and irregular products in the endometrial canal. The laboratory values were relevant for an increased prothrombin time, activated partial thromboplastin time, ferritin and a decrease in hemoglobin. The clinical cause of death was cited as acute respiratory distress syndrome. At autopsy, a 400g spongy, hemorrhagic uterus with multiple cystic spaces measuring approximately 0.5 × 0.4cm filled with thrombi within the myometrium was identified. Immunohistological examination with a CD31 stain for vascular endothelium associated antigen confirmed several endothelium-lined vessels, some of which contained thrombi. These histological features were consistent with cavernous hemangioma of the myometrium. A histological examination of the lungs revealed multiple fresh thromboemboli in small- and medium-sized pulmonary arteries in the right upper and lower lobes without organization, but with adjacent areas of fresh hemorrhagic infarction. CONCLUSION: This case underscores the importance of a high index of suspicion in a pregnant or post-partum woman presenting with respiratory symptoms. Thrombosis of the cavernous hemangiomas of the gravid or post-partum uterus is a rare entity. This case is of interest because it indicates that this condition can be fatally complicated by embolization of the thrombi in the cavernous myometrial hemangiomas. Although delivery by conservative methods, as well as cesarean section, is possible without resorting to hysterectomy, occasionally, the consequences could be fatal as in this case. |
991 | Pandemic influenza A virus codon usage revisited: biases, adaptation and implications for vaccine strain development | BACKGROUND: Influenza A virus (IAV) is a member of the family Orthomyxoviridae and contains eight segments of a single-stranded RNA genome with negative polarity. The first influenza pandemic of this century was declared in April of 2009, with the emergence of a novel H1N1 IAV strain (H1N1pdm) in Mexico and USA. Understanding the extent and causes of biases in codon usage is essential to the understanding of viral evolution. A comprehensive study to investigate the effect of selection pressure imposed by the human host on the codon usage of an emerging, pandemic IAV strain and the trends in viral codon usage involved over the pandemic time period is much needed. RESULTS: We performed a comprehensive codon usage analysis of 310 IAV strains from the pandemic of 2009. Highly biased codon usage for Ala, Arg, Pro, Thr and Ser were found. Codon usage is strongly influenced by underlying biases in base composition. When correspondence analysis (COA) on relative synonymous codon usage (RSCU) is applied, the distribution of IAV ORFs in the plane defined by the first two major dimensional factors showed that different strains are located at different places, suggesting that IAV codon usage also reflects an evolutionary process. CONCLUSIONS: A general association between codon usage bias, base composition and poor adaptation of the virus to the respective host tRNA pool, suggests that mutational pressure is the main force shaping H1N1 pdm IAV codon usage. A dynamic process is observed in the variation of codon usage of the strains enrolled in these studies. These results suggest a balance of mutational bias and natural selection, which allow the virus to explore and re-adapt its codon usage to different environments. Recoding of IAV taking into account codon bias, base composition and adaptation to host tRNA may provide important clues to develop new and appropriate vaccines. |
992 | Acute care utilization due to hospitalizations for pediatric lower respiratory tract infections in British Columbia, Canada | BACKGROUND: Pediatric LRTI hospitalizations are a significant burden on patients, families, and healthcare systems. This study determined the burden of pediatric LRTIs on hospital settings in British Columbia and the benefits of prevention strategies as they relate to healthcare resource demand. METHODS: LRTI inpatient episodes for patients <19 years of age during 2008–2010 were extracted from the BC Discharge Abstract Database. The annual number of acute care beds required to treat pediatric LRTIs was estimated. Sub-analyses determined the burden due to infants <1 year of age and high-risk infants. Population projections were used to forecast LRTI hospitalizations and the effectiveness of public health initiatives to reduce the incidence of LRTIs to 2020 and 2030. RESULTS: During 2008–2010, LRTI as the primary diagnosis accounted for 32.0 and 75.9% hospitalizations for diseases of the respiratory system in children <19 years of age and infants <1 year of age, respectively. Infants <1 year of age accounted for 47 and 77% hospitalizations due to pediatric LRTIs and pediatric LRTI hospitalizations specifically due to respiratory syncytial virus (RSV), respectively. The average length of stay was 3.1 days for otherwise healthy infants <1 year of age and 9.1 days for high-risk infants (P <0.0001). 73.1% pediatric LRTI hospitalizations occurred between November and April. Over the study timeframe, 19.6 acute care beds were required on average to care for pediatric LRTIs which increased to 64.0 beds at the peak of LRTI hospitalizations. Increases in LRTI bed-days of 5.5 and 16.2% among <19 year olds by 2020 and 2030, respectively, were predicted. Implementation of appropriate prevention strategies could cause 307 and 338 less LRTI hospitalizations in <19 year olds in 2020 and 2030, respectively. CONCLUSION: Pediatric LRTI hospitalizations require significant use of acute care infrastructure particularly between November and April. Population projections show the burden may increase in the next 20 years, but implementation of effective public health prevention strategies may contribute to reducing the acute care demand and to supporting efforts for overall pediatric healthcare sustainability. |
993 | Mutations in the Fusion Protein Cleavage Site of Avian Paramyxovirus Serotype 4 Confer Increased Replication and Syncytium Formation In Vitro but Not Increased Replication and Pathogenicity in Chickens and Ducks | To evaluate the role of the F protein cleavage site in the replication and pathogenicity of avian paramyxoviruses (APMVs), we constructed a reverse genetics system for recovery of infectious recombinant APMV-4 from cloned cDNA. The recovered recombinant APMV-4 resembled the biological virus in growth characteristics in vitro and in pathogenicity in vivo. The F cleavage site sequence of APMV-4 (DIQPR↓F) contains a single basic amino acid, at the -1 position. Six mutant APMV-4 viruses were recovered in which the F protein cleavage site was mutated to contain increased numbers of basic amino acids or to mimic the naturally occurring cleavage sites of several paramyxoviruses, including neurovirulent and avirulent strains of NDV. The presence of a glutamine residue at the -3 position was found to be important for mutant virus recovery. In addition, cleavage sites containing the furin protease motif conferred increased replication and syncytium formation in vitro. However, analysis of viral pathogenicity in 9-day-old embryonated chicken eggs, 1-day-old and 2-week-old chickens, and 3-week-old ducks showed that none the F protein cleavage site mutations altered the replication, tropism, and pathogenicity of APMV-4, and no significant differences were observed among the parental and mutant APMV-4 viruses in vivo. Although parental and mutant viruses replicated somewhat better in ducks than in chickens, they all were highly restricted and avirulent in both species. These results suggested that the cleavage site sequence of the F protein is not a limiting determinant of APMV-4 pathogenicity in chickens and ducks. |
994 | Influenza surveillance in the Pacific Island countries and territories during the 2009 pandemic: an observational study | BACKGROUND: Historically, Pacific island countries and territories (PICTs) have been more severely affected by influenza pandemics than any other part of the world. We herein describe the emergence and epidemiologic characteristics of pandemic influenza H1N1 in PICTs from 2009 to 2010. METHODS: The World Health Organization gathered reports of influenza-like-illness and laboratory-confirmed pandemic H1N1 cases from all 23 Pacific island countries and territories, from April 2009 through August 2010. Data were gathered through weekly email reports from Pacific island countries and territories and through email or telephone follow-up. RESULTS: Pacific island countries and territories started detecting pandemic H1N1 cases in June 2009, firstly in French Polynesia, with the last new detection occurring in August 2009 in Tuvalu. Nineteen Pacific island countries and territories reported 1,972 confirmed cases, peaking in August 2009. No confirmed pandemic H1N1 cases were identified in Niue, Pitcairn and Tokelau; the latter instituted strict maritime quarantine. Influenza-like-illness surveillance showed trends similar to surveillance of confirmed cases. Seven Pacific island countries and territories reported 21 deaths of confirmed pandemic H1N1. Case-patients died of acute respiratory distress syndrome or multi-organ failure, or both. The most reported pre-existing conditions were obesity, lung disease, heart disease, and pregnancy. Pacific island countries and territories instituted a variety of mitigation measures, including arrival health screening. Multiple partners facilitated influenza preparedness planning and outbreak response. CONCLUSIONS: Pandemic influenza spread rapidly throughout the Pacific despite enormous distances and relative isolation. Tokelau and Pitcairn may be the only jurisdictions to have remained pandemic-free. Despite being well-prepared, Pacific island countries and territories experienced significant morbidity and mortality, consistent with other indigenous and low-resource settings. For the first time, regional influenza-like-illness surveillance was conducted in the Pacific, allowing health authorities to monitor the pandemic’s spread and severity in real-time. Future regional outbreak responses will likely benefit from the lessons learned during this outbreak. |
995 | Observational Research in Childhood Infectious Diseases (ORChID): a dynamic birth cohort study | INTRODUCTION: Even in developed economies infectious diseases remain the most common cause of illness in early childhood. Our current understanding of the epidemiology of these infections is limited by reliance on data from decades ago performed using low-sensitivity laboratory methods, and recent studies reporting severe, hospital-managed disease. METHODS AND ANALYSIS: The Observational Research in Childhood Infectious Diseases (ORChID) study is an ongoing study enrolling a dynamic birth cohort to document the community-based epidemiology of viral respiratory and gastrointestinal infections in early childhood. Women are recruited antenatally, and their healthy newborn is followed for the first 2 years of life. Parents keep a daily symptom diary for the study child, collect a weekly anterior nose swab and dirty nappy swab and complete a burden diary when a child meets pre-defined illness criteria. Specimens will be tested for a wide range of viruses by real-time PCR assays. Primary analyses involves calculating incidence rates for acute respiratory illness (ARI) and acute gastroenteritis (AGE) for the cohort by age and seasonality. Control material from children when they are without symptoms will allow us to determine what proportion of ARIs and AGE can be attributed to specific pathogens. Secondary analyses will assess the incidence and shedding duration of specific respiratory and gastrointestinal pathogens. ETHICS AND DISSEMINATION: This study is approved by The Human Research Ethics Committees of the Children's Health Queensland Hospital and Health Service, the Royal Brisbane and Women's Hospital and The University of Queensland. TRIAL REGISTRATION: clinicaltrials.gov NCT01304914. |
996 | Identification of a novel Getah virus by Virus-Discovery-cDNA random amplified polymorphic DNA (RAPD) | BACKGROUND: The identification of new virus strains is important for the study of infectious disease, but current (or existing) molecular biology methods are limited since the target sequence must be known to design genome-specific PCR primers. Thus, we developed a new method for the discovery of unknown viruses based on the cDNA - random amplified polymorphic DNA (cDNA-RAPD) technique. Getah virus, belonging to the family Togaviridae in the genus Alphavirus, is a mosquito-borne enveloped RNA virus that was identified using the Virus-Discovery-cDNA RAPD (VIDISCR) method. RESULTS: A novel Getah virus was identified by VIDISCR from suckling mice exposed to mosquitoes (Aedes albopictus) collected in Yunnan Province, China. The non-structural protein gene, nsP3, the structural protein gene, the capsid protein gene, and the 3'-untranslated region (UTR) of the novel Getah virus isolate were cloned and sequenced. Nucleotide sequence identities of each gene were determined to be 97.1–99.3%, 94.9–99.4%, and 93.6–99.9%, respectively, when compared with the genomes of 10 other representative strains of Getah virus. CONCLUSIONS: The VIDISCR method was able to identify known virus isolates and a novel isolate of Getah virus from infected mice. Phylogenetic analysis indicated that the YN08 isolate was more closely related to the Hebei HB0234 strain than the YN0540 strain, and more genetically distinct from the MM2021 Malaysia primitive strain. |
997 | The Immunosuppressive Agent Mizoribine Monophosphate Is an Inhibitor of the Human RNA Capping Enzyme | Mizoribine monophosphate (MZP) is a specific inhibitor of the cellular inosine-5′-monophosphate dehydrogenase (IMPDH), the enzyme catalyzing the rate-limiting step of de novo guanine nucleotide biosynthesis. MZP is a highly potent antagonistic inhibitor of IMPDH that blocks the proliferation of T and B lymphocytes that use the de novo pathway of guanine nucleotide synthesis almost exclusively. In the present study, we investigated the ability of MZP to directly inhibit the human RNA capping enzyme (HCE), a protein harboring both RNA 5′-triphosphatase and RNA guanylyltransferase activities. HCE is involved in the synthesis of the cap structure found at the 5′ end of eukaryotic mRNAs, which is critical for the splicing of the cap-proximal intron, the transport of mRNAs from the nucleus to the cytoplasm, and for both the stability and translation of mRNAs. Our biochemical studies provide the first insight that MZP can inhibit the formation of the RNA cap structure catalyzed by HCE. In the presence of MZP, the RNA 5′-triphosphatase activity appears to be relatively unaffected while the RNA guanylyltransferase activity is inhibited, indicating that the RNA guanylyltransferase activity is the main target of MZP inhibition. Kinetic studies reveal that MZP is a non-competitive inhibitor that likely targets an allosteric site on HCE. Mizoribine also impairs mRNA capping in living cells, which could account for the global mechanism of action of this therapeutic agent. Together, our study clearly demonstrates that mizoribine monophosphate inhibits the human RNA guanylyltransferase in vitro and impair mRNA capping in cellulo. |
998 | Clinical use of exhaled volatile organic compounds in pulmonary diseases: a systematic review | There is an increasing interest in the potential of exhaled biomarkers, such as volatile organic compounds (VOCs), to improve accurate diagnoses and management decisions in pulmonary diseases. The objective of this manuscript is to systematically review the current knowledge on exhaled VOCs with respect to their potential clinical use in asthma, lung cancer, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and respiratory tract infections. A systematic literature search was performed in PubMed, EMBASE, Cochrane database, and reference lists of retrieved studies. Controlled, clinical, English-language studies exploring the diagnostic and monitoring value of VOCs in asthma, COPD, CF, lung cancer and respiratory tract infections were included. Data on study design, setting, participant characteristics, VOCs techniques, and outcome measures were extracted. Seventy-three studies were included, counting in total 3,952 patients and 2,973 healthy controls. The collection and analysis of exhaled VOCs is non-invasive and could be easily applied in the broad range of patients, including subjects with severe disease and children. Various research groups demonstrated that VOCs profiles could accurately distinguish patients with a pulmonary disease from healthy controls. Pulmonary diseases seem to be characterized by a disease specific breath-print, as distinct profiles were found in patients with dissimilar diseases. The heterogeneity of studies challenged the inter-laboratory comparability. In conclusion, profiles of VOCs are potentially able to accurately diagnose various pulmonary diseases. Despite these promising findings, multiple challenges such as further standardization and validation of the diverse techniques need to be mastered before VOCs can be applied into clinical practice. |
999 | HIV-1 Fusion Is Blocked through Binding of GB Virus C E2D Peptides to the HIV-1 gp41 Disulfide Loop | A strategy for antiviral drug discovery is the elucidation and imitation of viral interference mechanisms. HIV-1 patients benefit from a coinfection with GB Virus C (GBV-C), since HIV-positive individuals with long-term GBV-C viraemia show better survival rates than HIV-1 patients without persisting GBV-C. A direct influence of GBV-C on HIV-1 replication has been shown in coinfection experiments. GBV-C is a human non-pathogenic member of the flaviviridae family that can replicate in T and B cells. Therefore, GBV-C shares partly the same ecological niche with HIV-1. In earlier work we have demonstrated that recombinant glycoprotein E2 of GBV-C and peptides derived from the E2 N-terminus interfere with HIV entry. In this study we investigated the underlying mechanism. Performing a virus-cell fusion assay and temperature-arrested HIV-infection kinetics, we provide evidence that the HIV-inhibitory E2 peptides interfere with late HIV-1 entry steps after the engagement of gp120 with CD4 receptor and coreceptor. Binding and competition experiments revealed that the N-terminal E2 peptides bind to the disulfide loop region of HIV-1 transmembrane protein gp41. In conjunction with computational analyses, we identified sequence similarities between the N-termini of GBV-C E2 and the HIV-1 glycoprotein gp120. This similarity appears to enable the GBV-C E2 N-terminus to interact with the HIV-1 gp41 disulfide loop, a crucial domain involved in the gp120-gp41 interface. Furthermore, the results of the present study provide initial proof of concept that peptides targeted to the gp41 disulfide loop are able to inhibit HIV fusion and should inspire the development of this new class of HIV-1 entry inhibitors. |
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