244 research outputs found

    Virulence of newcastle disease virus: what is known so far?

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    In the last decade many studies have been performed on the virulence of Newcastle disease virus (NDV). This is mainly due to the development of reverse genetics systems which made it possible to genetically modify NDV and to investigate the contribution of individual genes and genome regions to its virulence. However, the available information is scattered and a comprehensive overview of the factors and conditions determining NDV virulence is lacking. This review summarises, compares and discusses the available literature and shows that virulence of NDV is a complex trait determined by multiple genetic factors

    Type I interferon receptor-independent and -dependent host transcriptional responses to mouse hepatitis coronavirus infection in vivo

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    <p>Abstract</p> <p>Background</p> <p>The role of type I IFNs in protecting against coronavirus (CoV) infections is not fully understood. While CoVs are poor inducers of type I IFNs in tissue culture, several studies have demonstrated the importance of the type I IFN response in controlling MHV infection in animals. The protective effectors against MHV infection are, however, still unknown.</p> <p>Results</p> <p>In order to get more insight into the antiviral gene expression induced in the brains of MHV-infected mice, we performed whole-genome expression profiling. Three different mouse strains, differing in their susceptibility to infection with MHV, were used. In BALB/c mice, which display high viral loads but are able to control the infection, 57 and 121 genes were significantly differentially expressed (≥ 1.5 fold change) upon infection at 2 and 5 days post infection, respectively. Functional association network analyses demonstrated a strong type I IFN response, with Irf1 and Irf7 as the central players. At 5 days post infection, a type II IFN response also becomes apparent. Both the type I and II IFN response, which were more pronounced in mice with a higher viral load, were not observed in 129SvEv mice, which are much less susceptible to infection with MHV. 129SvEv mice lacking the type I interferon receptor (IFNAR-/-), however, were not able to control the infection. Gene expression profiling of these mice identified type I IFN-independent responses to infection, with IFN-γ as the central player. As the BALB/c and the IFNAR-/- 129SvEv mice demonstrated very similar viral loads in their brains, we also compared their gene expression profiles upon infection with MHV in order to identify type I IFN-dependent transcriptional responses. Many known IFN-inducible genes were detected, several of which have previously been shown to play an important protective role against virus infections. We speculate that the additional type I IFN-dependent genes that we discovered may also be important for protection against MHV infection.</p> <p>Conclusion</p> <p>Transcriptional profiling of mice infected with MHV demonstrated the induction of a robust IFN response, which correlated with the viral load. Profiling of IFNAR-/- mice allowed us to identify type I IFN-independent and -dependent responses. Overall, this study broadens our present knowledge of the type I and II IFN-mediated effector responses during CoV infection <it>in vivo</it>.</p

    SynGEM: An intranasal prefusion-like RSV F subunit vaccine

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    Respiratory syncytial virus (RSV) is an important cause of respiratory tract disease in (naive) young infants, older infants, the elderly and immune-compromised. Despite the medical need and the market potential, no licensed vaccine is available. Mucosis B.V. is a Dutch biotech company developing innovative mucosal vaccines, based on the Bacterium-Like Particles (BLP) technology. Such vaccines can be administered needle-free, e.g. through the nasal mucosa. In support of the mucosal approach, there is accumulating evidence that RSV F-specific local S-IgA antibodies secreted in the upper respiratory tract of humans correlate well with protection. Because of its ability to induce broadly neutralizing antibodies the RSV F protein is the most attractive antigen. The current view is that in particular serum antibodies directed against the prefusion form of RSV F belong to the most potent neutralizing antibodies and the ability to elicit these is a pivotal attribute for a successful RSV vaccine. We studied different variants of F with respect to their conformation using neutralizing monoclonal antibodies (mAbs), following the view that F proteins mimicking the meta-stable prefusion form of F expose a more extensive and relevant epitope repertoire than F proteins corresponding to the postfusion F structure. Both addition of a trimerization motif and mutation of the furin cleavage sites increased the reactivity of F with the prefusion-specific mAb D25, with the highest reactivity being observed for F proteins in which both these features were combined. This candidate antigen, called Flys-GCN, is suitable in the development of mucosal as well as intramuscular RSV vaccines. Here we describe the development of our intranasal candidate RSV vaccine, SynGEM, which is based on the validated BLP technology. The non-living BLPs allow for presentation of stable, trimeric prefusion-like RSV F proteins bound to the particle surface. Intranasal vaccination of naïve and convalescent mice with SynGEM induced long-lasting virus neutralizing RSV-specific serum IgG and robust levels of local IgA. Cotton rats immunized intranasally with SynGEM were protected upon RSV challenge, as represented by a low viral load in the lungs. Enhanced levels of pre- versus postfusion specific antibodies were observed in individual animals, confirming the ability of the SynGEM vaccine to induce potent neutralizing antibodies. A clinical Phase I study with intranasal SynGEM is planned for 2016

    Central ions and lateral asparagine/glutamine zippers stabilize the post-fusion hairpin conformation of the SARS coronavirus spike glycoprotein

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    AbstractThe coronavirus spike glycoprotein is a class I membrane fusion protein with two characteristic heptad repeat regions (HR1 and HR2) in its ectodomain. Here, we report the X-ray structure of a previously characterized HR1/HR2 complex of the severe acute respiratory syndrome coronavirus spike protein. As expected, the HR1 and HR2 segments are organized in antiparallel orientations within a rod-like molecule. The HR1 helices form an exceptionally long (120 Å) internal coiled coil stabilized by hydrophobic and polar interactions. A striking arrangement of conserved asparagine and glutamine residues of HR1 propagates from two central chloride ions, providing hydrogen-bonding “zippers” that strongly constrain the path of the HR2 main chain, forcing it to adopt an extended conformation at either end of a short HR2 α-helix

    Improved microarray gene expression profiling of virus-infected cells after removal of viral RNA

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    <p>Abstract</p> <p>Background</p> <p>Sensitivity and accuracy are key points when using microarrays to detect alterations in gene expression under different conditions. Critical to the acquisition of reliable results is the preparation of the RNA. In the field of virology, when analyzing the host cell's reaction to infection, the often high representation of viral RNA (vRNA) within total RNA preparations from infected cells is likely to interfere with microarray analysis. Yet, this effect has not been investigated despite the many reports that describe gene expression profiling of virus-infected cells using microarrays.</p> <p>Results</p> <p>In this study we used coronaviruses as a model to show that vRNA indeed interferes with microarray analysis, decreasing both sensitivity and accuracy. We also demonstrate that the removal of vRNA from total RNA samples, by means of virus-specific oligonucleotide capturing, significantly reduced the number of false-positive hits and increased the sensitivity of the method as tested on different array platforms.</p> <p>Conclusion</p> <p>We therefore recommend the specific removal of vRNA, or of any other abundant 'contaminating' RNAs, from total RNA samples to improve the quality and reliability of microarray analyses.</p

    Електрофізичні властивості системи політетрафторетилен – вуглецеві нанотрубки

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    Проведено дослідження комплексної діелектричної проникності та електропровідності в надвисокочастотному діапазоні (9 ГГц) і на низьких частотах (0,1; 1 та 10 кГц) двох систем політетрафторетилен – багатошарові вуглецеві нанотрубки з вихідними та диспергованими у водному середовищі . Введення диспергованих нанотрубок в полімер знижує поріг перколяції з 4,5 % до 2,6 % (мас.) за рахунок рівномірного розподілу наповнювача у полімері, що призводить до зростання міжфазної поверхні взаємодії полімер – вуглецеві нанотрубки, яка проявляється в збільшенні значень дійсної та уявної складової комплексної діелектричної проникності.Проведены исследования комплексной диэлектрической проницаемости и электропроводности в сверхвысокочастотном диапазоне (9 ГГц) и на низких частотах (0,1; 1; 10 кГц) двух систем политетрафторэтилен–многослойные углеродные нанотрубки с исходными и диспергированными у водной среде. Введение диспергированных нанотрубок в полимер снижает порог перколяции с 4,5% до 2,6 % (масс.) за счет равномерного распределения наполнителя в полимере, что приводит к возрастанию межфазной поверхности взаимодействия полимер – углеродные нанотрубки, которая проявляется в увеличении значений действительной и мнимой составляющей комплексной диэлектрической проницаемости.Complex dielectric permeability and conductivity of two systems, namely polytetrafluorethylene – intact carbon nanotubes and polytetrafluorethylene – carbon nanotubes dispersed in aqueous media, has been studied in super high-frequency range (9 GHz) and at low frequencies (0,1; 1 and 10 kHz). Doping of the polymer with the dispersed nanotubes decreases percolation threshold (limit ) from 4,5 wt. % to 2,6 wt. % due to uniform distribution of the filler in the polymer. This results to increase of interface interaction polymer - carbon nanotubes that is demonstrated by increase of value of real and imaginary component of complex dielectric permeability

    Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway

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    Influenza A virus (IAV) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. Whereas dynamin-dependent, clathrin-mediated endocytosis (CME) is generally considered as the IAV infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. By manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and -independent entry of IAV. Whereas entry of IAV in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. This finding was confirmed with the use of small interfering RNAs targeting dynamin-2. In the presence of serum, both IAV entry pathways were operational. Under these conditions entry could be fully blocked by combined treatment with dynasore and the amiloride derivative EIPA, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. The sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in IAV entry. Consistently, IAV particles and soluble FITC-dextran were shown to co-localize in cells in the same vesicles. Thus, in addition to the classical dynamin-dependent, clathrin-mediated endocytosis pathway, IAV enters host cells by a dynamin-independent route that has all the characteristics of macropinocytosis

    Mouse model for the lysosomal disorder galactosialidosis and correction of the phenotype with overexpressing erythroid precursor cells

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    The lysosomal storage disorder galactosialidosis results from a primary deficiency of the protective protein/cathepsin A (PPCA), which in turn affects the activities of β-galactosidase and neuraminidase. Mice homozygous for a null mutation at the PPCA locus present with signs of the disease shortly after birth and develop a phenotype closely resembling human patients with galactosialidosis. Most of their tissues show characteristic vacuolation of specific cells, attributable to lysosomal storage. Excessive excretion of sialyloligosaccharides in urine is diagnostic of the disease. Affected mice progressively deteriorate as a consequence of severe organ dysfunction, especially of the kidney. The deficient phenotype can be corrected by transplanting null mutants with bone marrow from a transgenic line overexpressing human PPCA in erythroid precursor cells. The transgenic bone marrow gives a more efficient and complete correction of the visceral organs than normal bone marrow. Our data demonstrate the usefulness of this animal model, very similar to the human disease, for experimenting therapeutic strategies aimed to deliver the functional protein or gene to affected organs. Furthermore, they suggest the feasibility of gene therapy for galactosialidosis and other disorders, using bone marrow cells engineered to overexpress and secrete the correcting lysosomal protein.</p

    Mouse model for the lysosomal disorder galactosialidosis and correction of the phenotype with overexpressing erythroid precursor cells

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    The lysosomal storage disorder galactosialidosis results from a primary deficiency of the protective protein/cathepsin A (PPCA), which in turn affects the activities of β-galactosidase and neuraminidase. Mice homozygous for a null mutation at the PPCA locus present with signs of the disease shortly after birth and develop a phenotype closely resembling human patients with galactosialidosis. Most of their tissues show characteristic vacuolation of specific cells, attributable to lysosomal storage. Excessive excretion of sialyloligosaccharides in urine is diagnostic of the disease. Affected mice progressively deteriorate as a consequence of severe organ dysfunction, especially of the kidney. The deficient phenotype can be corrected by transplanting null mutants with bone marrow from a transgenic line overexpressing human PPCA in erythroid precursor cells. The transgenic bone marrow gives a more efficient and complete correction of the visceral organs than normal bone marrow. Our data demonstrate the usefulness of this animal model, very similar to the human disease, for experimenting therapeutic strategies aimed to deliver the functional protein or gene to affected organs. Furthermore, they suggest the feasibility of gene therapy for galactosialidosis and other disorders, using bone marrow cells engineered to overexpress and secrete the correcting lysosomal protein.</p
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