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Studies are limited on evaluating the potential of influenza viruses for egg-borne dissemination. In our previous studies, experimental infection of breeder turkeys with A/turkey/Ohio/313053/04 resulted in drastic declines in egg ...
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Studies are limited on evaluating the potential of influenza viruses for egg-borne dissemination. In our previous studies, experimental infection of breeder turkeys with A/turkey/Ohio/313053/04 resulted in drastic declines in egg production, and we confirmed high levels of virus replication and an abundant distribution of avian-specific alpha 2,3 sialic acid-gal receptors in the oviduct of these turkeys. In the present study, following experimental inoculation of A/turkey/Ohio/313053/04 in breeder turkeys, we detected these viruses in the albumin of eggs using real-time RT-PCR (RRT-PCR) and virus isolation in embryonated chicken eggs. Swabs from egg shells were also found positive by RRT-PCR. This is the first report of the detection of low pathogenic influenza viruses from internal egg contents following experimental infection. The possibility of hatchery contamination by egg-borne influenza viruses, and the spread of virus during movement of contaminated cracked eggs and egg flats, pose concerns regarding viral dissemination of influenza.
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The HIV-1 transactivator of transcription (Tat) is a protein essential for virus replication. Tat is an intrinsically disordered RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase...
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The HIV-1 transactivator of transcription (Tat) is a protein essential for virus replication. Tat is an intrinsically disordered RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase 9, regulates transcription at the level of elongation. Tat also interacts with numerous other intracellular and extracellular proteins, and is implicated in a number of pathogenic processes. The physico-chemical properties of Tat make it a particularly challenging target for structural studies: Tat contains seven Cys residues, six of which are essential for transactivation, and is highly susceptible to oxidative cross-linking and aggregation. In addition, a basic segment (residues 48-57) gives the protein a high net positive charge of +12 at pH 7, endowing it with a high affinity for anionic polymers and surfaces. In order to study the structure of Tat, both alone and in complex with partner molecules, we have developed a system for the bacterial expression and purification of 6x Histidine-tagged and isotopically enriched (in N-15 and C-13) recombinant HIV-1 Tat(1-72) (BH10 isolate) that yields large amounts of protein. These preparations have facilitated the assignment of 95% of the backbone NMR resonances. Analysis by mass spectrometry and NMR demonstrate that the cysteine-rich Tat protein is unambiguously reduced, monomeric, and unfolded in aqueous solution at pH 4.
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We recently reported that p28, one of the two turnip crinkle virus (TCV) replication proteins, trans-complemented a defective TCV lacking p28, yet repressed the replication of another TCV replicon encoding wild type p28 (Zhang et ...
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We recently reported that p28, one of the two turnip crinkle virus (TCV) replication proteins, trans-complemented a defective TCV lacking p28, yet repressed the replication of another TCV replicon encoding wild type p28 (Zhang et al., 2017). Here we show that p88, the TCV-encoded RNA-dependent RNA polymerase, likewise trans-complemented a p88-defective TCV replicon, but repressed one encoding wild-type p88. Surprisingly, lowering p88 protein levels enhanced trans-complementation, but weakened repression. Repression by p88 was not simply due to protein over-expression, as deletion mutants missing 127 or 224 N-terminal amino acids accumulated to higher levels but were poor repressors. Finally, both trans-complementation and repression by p88 were accompanied by preferential accumulation of subgenomic RNA2, and a novel class of small TCV RNAs. Our results suggest that repression of TCV replication by p88 may manifest a viral mechanism that regulates the ratio of genomic and subgenomic RNAs based on p88 abundance.
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The replication of coronaviruses, as in other positive-strand RNA viruses, is closely tied to the formation of membrane-bound replicative organelles inside infected cells. The proteins responsible for rearranging cellular membrane...
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The replication of coronaviruses, as in other positive-strand RNA viruses, is closely tied to the formation of membrane-bound replicative organelles inside infected cells. The proteins responsible for rearranging cellular membranes to form the organelles are conserved not just among the Coronaviridae family members, but across the order Nidovirales. Taken together, these observations suggest that the coronavirus replicative organelle plays an important role in viral replication, perhaps facilitating the production or protection of viral RNA. However, the exact nature of this role, and the specific contexts under which it is important have not been fully elucidated. Here, we collect and interpret the recent experimental evidence about the role and importance of membrane-bound organelles in coronavirus replication. (C) 2015 Elsevier B.V. All rights reserved.
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The orthoreoviruses are segmented double strand RNA viruses and are the most abundant viruses in nature. Three main serotypes are known, named 1, 2 and 3. The designation "reovirus" is the acronym for "respiratory enteric orphan v...
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The orthoreoviruses are segmented double strand RNA viruses and are the most abundant viruses in nature. Three main serotypes are known, named 1, 2 and 3. The designation "reovirus" is the acronym for "respiratory enteric orphan virus", expression underlining their respiratory and enteric origin and the fact that they are not associated with well defined clinical disease. Nevertheless. strains of orthoreoviruses have been isolated from several cases of symptomatic diseases in human, namely diseases of the central nervous system such as encephalitis and meningitis sometimes leading to patient death. These different cases show that orthoreoviruses could be pathogenic, causing fatal diseases. Orthoreoviruses infection in animals induces also several diseases. Indeed, according to the inoculation route and the serotype of inoculated strain, encephalitis or hepatitis can be observed. The RNA segments M2 and S1 seem to be involved in this neurovirulence property and are oil the basis of cellular mechanisms, Such its virus entry, virus replication and apoptosis. However, the mechanisms of virulence remain complex.
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The West Nile virus strain Kunjin virus (WNV.(KUN)) NS4A protein is a multifunctional protein involved in many aspects of the virus life-cycle and is a major component of the WNVKUN replication complex (RC). Previously we identifi...
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The West Nile virus strain Kunjin virus (WNV.(KUN)) NS4A protein is a multifunctional protein involved in many aspects of the virus life-cycle and is a major component of the WNVKUN replication complex (RC). Previously we identified a conserved region in the C-terminus of NS4A regulating proteolytic processing and RC assembly, and now investigate key conserved residues in the N-terminus of NS4A and their contribution to WNVKUN replication. Mutation of P13 completely ablated replication, whereas, mutation of P48 and D49, near the first transmembrane helix, and G66 within the helix, showed variable defects in replication, virion secretion and membrane proliferation. Intriguingly, the P48 and G66 NS4A mutants resulted in specific proteasome depletion of NS4A that could in part be rescued with a proteasome inhibitor. Our results suggest that the N-terminus of NS4A contributes to correct folding and stability, essential for facilitating the essential roles of NS4A during replication. (C) 2015 Elsevier Inc. All rights reserved.
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Phosphoinositides (PIs) play an essential role in mediating key signaling pathways on biological membranes. Hepatitis C virus (HCV) replicates its RNA genome by establishing a viral replication complex (RC) on host cell membranes....
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Phosphoinositides (PIs) play an essential role in mediating key signaling pathways on biological membranes. Hepatitis C virus (HCV) replicates its RNA genome by establishing a viral replication complex (RC) on host cell membranes. Recently, an increasing body of literature reported that not only PIs themselves but also several PIs-specific kinases are required for efficient replication of HCV RNA genome. Especially, PI 4-kinases type III alpha, beta as well as their enzymatic products including phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are consistently identified to be host factors essential for HCV replication. In this article, the current state of our knowledge of PIs and PIs-specific kinases together with their roles in modulating HCV replication is reviewed. The effects of various PIsspecific kinases inhibitors on HCV replication are also highlighted, proposing them as promising candidate targets to which a new class of anti-HCV therapeutics can be envisaged.
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Phosphoinositides (PIs) play an essential role in mediating key signaling pathways on biological membranes. Hepatitis C virus (HCV) replicates its RNA genome by establishing a viral replication complex (RC) on host cell membranes....
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Phosphoinositides (PIs) play an essential role in mediating key signaling pathways on biological membranes. Hepatitis C virus (HCV) replicates its RNA genome by establishing a viral replication complex (RC) on host cell membranes. Recently, an increasing body of literature reported that not only PIs themselves but also several PIs-specific kinases are required for efficient replication of HCV RNA genome. Especially, PI 4-kinases type III alpha, beta as well as their enzymatic products including phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are consistently identified to be host factors essential for HCV replication. In this article, the current state of our knowledge of PIs and PIs-specific kinases together with their roles in modulating HCV replication is reviewed. The effects of various PIsspecific kinases inhibitors on HCV replication are also highlighted, proposing them as promising candidate targets to which a new class of anti-HCV therapeutics can be envisaged.
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In this report we describe foamy virus vectors with conditional expression of short interfering RNAs (siRNAs) in HIV infected cells. Short hairpin RNAs (shRNAs) based on two targets in the 5' end of the untranslated region and one...
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In this report we describe foamy virus vectors with conditional expression of short interfering RNAs (siRNAs) in HIV infected cells. Short hairpin RNAs (shRNAs) based on two targets in the 5' end of the untranslated region and one in the rev gene flanked with 5' and 3' microRNA 30 (miR30) sequences were synthesized and placed under the control of an HIV promoter for Tat-mediated expression. HIV permissive cells were transduced with foamy virus vectors containing each hybrid shRNA expression cassette and tested for their efficacy on the inhibition of HIV replication. Effective Tat dependent expression of the shRNAs, as well as GFP placed downstream each shRNA was evident. In addition the results show inhibition of HIV replication by greater than 98%. Interestingly, transduction of cells with a vector lacking an shRNA also revealed GFP expression in the presence of Tat with similar levels of inhibition of virus replication. When the TAR region was removed from this vector there was neither reduction in virus replication nor Tat-induced GFP expression. These results suggest that TAR in the vector, which Tat interacts to promote expression of the shRNA, is a potent inhibitor of virus replication. Previous studies with TAR regulated expression of antiviral genes ignore the contribution of TAR in the repression of virus replication. Interpretation of effective inhibition of HIV replication by antiviral genes located downstream of TAR while neglecting the efficacy of a potent repression by TAR is misleading.
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Despite intensive efforts, no safe and effective vaccine has been developed for the prophylaxis of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). Studies with the simian immunodeficiency virus (SIV)/ma...
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Despite intensive efforts, no safe and effective vaccine has been developed for the prophylaxis of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). Studies with the simian immunodeficiency virus (SIV)/macaque model demonstrated that live attenuated viruses are the most effective vaccines tested thus far. However, due to ongoing low-level replication of the attenuated virus and the error-prone replication machinery, the attenuated virus may regain replication capacity and become pathogenic. We therefore designed a novel vaccine strategy with an HIV-1 virus that replicates exclusively in the presence of the nontoxic effector doxycycline (dox). This was achieved by replacement of the viral TAR-Tat system for transcriptional activation by the Escherichia coli-derived Tet system for inducible gene expression. This designer HIV-rtTA virus replicates in a strictly dox-dependent manner and may represent an improved vaccine strain because its replication can be turned on and off at will. Spontaneous virus evolution resulted in optimization of the components of the Tet system for their new function to support virus replication in human cells. The optimised Tet system may be of particular use in other applications such as inducible expression of gene therapy vectors in the brain.
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