Name: using reverse genetics to manipulate the nss gene of the rift valley fever virus mp-12 strain to improve vaccine safety and efficacy
Uploaded: 2011-11-01T12:00:00
Description: Watch this Scientific Journal Video about Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy at JoVE.com

इस काम अनुदान 5 नंबर U54-07 उत्कृष्टता के पश्चिमी क्षेत्रीय केंद्र (WRCE) के माध्यम से AI057156, नेशनल इंस्टीट्यूट ऑफ एलर्जी और संक्रामक रोगों से 1 AI08764301 - A1 R01, और विश्वविद्यालय में एक टीके के विकास के लिए Sealy केंद्र से आंतरिक धन के द्वारा वित्त पोषित किया गया था टेक्सास चिकित्सा शाखा है.

1. पुनः संयोजक सांसद 12 एन्कोडिंग एनएसएस उत्परिवर्तन (ओं) के प्लाज्मिड DNAs 2 से रिकवरी <ol><li> बिखरा हुआ बच्चा हम्सटर गुर्दे (BHK) / T7 9 15 कोशिकाओं, जो stably व्यक्त T7 शाही सेना पोलीमरेज़, न्यूनतम आवश्यक मध्यम में ...

<ol>
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G., Tang, W., Ravindranath, A. K., Clark, W. A., Croze, E., Fuchs, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCF(HOS)+ubiquitin+ligase+mediates+the+ligand-induced+down-regulation+of+the+interferon-alpha+receptor.">SCF(HOS) ubiquitin ligase mediates the ligand-induced down-regulation of the interferon-alpha receptor.</a> EMBO J. 22, 5480-5490 (2003).</li><li>Kakach, L. T., Suzich, J. A., Collett, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rift+Valley+fever+virus+M+segment:+phlebovirus+expression+strategy+and+protein+glycosylation.+Virology.">Rift Valley fever virus M segment: phlebovirus expression strategy and protein glycosylation. Virology.</a> 170, 505-510 (1989).</li><li>Kakach, L. T., Wasmoen, T. L., Collett, M. 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Virol. 62, 826-833 (1988).</li><li>Niwa, H., Yamamura, K., Miyazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+selection+for+high-expression+transfectants+with+a+novel+eukaryotic+vector.">Efficient selection for high-expression transfectants with a novel eukaryotic vector.</a> Gene. 108, 193-199 (1991).</li><li>Muller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+clone+13,+a+naturally+attenuated+avirulent+isolate+of+Rift+Valley+fever+virus,+which+is+altered+in+the+small+segment.">Characterization of clone 13, a naturally attenuated avirulent isolate of Rift Valley fever virus, which is altered in the small segment.</a> Am. J. Trop. Med. Hyg. 53, 405-411 (1995).</li><li>Le May, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+SAP30+complex+inhibits+IFN-beta+expression+in+Rift+Valley+fever+virus+infected+cells.">A SAP30 complex inhibits IFN-beta expression in Rift Valley fever virus infected cells.</a> PLoS Pathog. 4, e13-e13 (2008).</li><li>Kalveram, B., Lihoradova, O., Ikegami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NSs+Protein+of+Rift+Valley+Fever+Virus+Promotes+Post-Translational+Downregulation+of+the+TFIIH+Subunit+p62.">NSs Protein of Rift Valley Fever Virus Promotes Post-Translational Downregulation of the TFIIH Subunit p62.</a> J. 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Pathol. 37, 99-109 (1956).</li><li>Bouloy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+evidence+for+an+interferon-antagonistic+function+of+rift+valley+fever+virus+nonstructural+protein+NSs.">Genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein NSs.</a> J. Virol. 75, 1371-1377 (2001).</li><li>Bird, B. H., Albarino, C. G., Nichol, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rift+Valley+fever+virus+lacking+NSm+proteins+retains+high+virulence+in+vivo+and+may+provide+a+model+of+human+delayed+onset+neurologic+disease.">Rift Valley fever virus lacking NSm proteins retains high virulence in vivo and may provide a model of human delayed onset neurologic disease.</a> Virology. 362, 10-15 (2007).</li></ol>

RVFV के लिए आनुवंशिकी सिस्टम उल्टा T7 प्रमोटर 2,4,5 या 3 माउस या मानव 4 प्रमोटर नीति मैं का उपयोग करके कई समूहों द्वारा विकसित किया गया है . इस पांडुलिपि में, हम BHK/T7-9 15 कि stably व्यक्त T7 शाही सेना पोलीम?...

<table border="1"><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियां (वैकल्पिक) </td></tr><tr><td> न्यूनतम आवश्यक (सदस्य) मध्यम अल्फा </td><td> Invitrogen </td><td> 32561037 </td><td></td></tr><tr><td> Dulbecco संशोधित न्यूनतम आवश्यक मध्यम </td><td> Invitrogen </td><td> 11965092 </td><td></td></tr><tr><td> संशोधित ईगल मध्यम (सदस्य 2x) </td><td> Invitrogen </td><td> 11935046 </td><td></td></tr><tr><td> पेनिसिलिन - स्ट्रेप्टोमाइसिन </td><td> Invitrogen </td><td> 15140122 </td><td></td></tr><tr><td> Hygromycin बी </td><td> Cellgro </td><td> 30-240-सीआर </td><td></td></tr><tr><td> Tryptose फॉस्फेट शोरबा </td><td> सांसद Biomedicals </td><td> 1682149 </td><td></td></tr><tr><td> नोबल अगर </td><td> VWR </td><td> 101170-362 </td><td></td></tr><tr><td> ट्रांजिट-LT1 </td><td> Mirus </td><td> MIR2300</td><td></td></tr><tr><td> Opti-सदस्य </td><td> Invitrogen </td><td> 31985070 </td><td></td></tr><tr><td> एयरोसोल तंग ढक्कन </td><td> Eppendorf </td><td> सी २२२३-25 </td><td></td></tr><tr><td> 0.33% तटस्थ लाल समाधान </td><td> सिग्मा Aldrich </td><td> N2889-100ml </td><td></td></tr><tr><td> C57/WT MEF कोशिकाओं </td><td> InvivoGen </td><td> MEF - c57wt </td><td></td></tr><tr><td> Blasticidin एस </td><td> InvivoGen </td><td> चींटी - बीएल-1 </td><td></td></tr><tr><td> Zeocin </td><td> InvivoGen </td><td> चींटी-Zn-1 </td><td></td></tr><tr><td> Quanti-ब्लू </td><td> InvivoGen </td><td> प्रतिनिधि qb1 </td><td></td></tr><tr><td> BHK/T7-9 15 कोशिकाओं </td><td> Gifu विश्वविद्यालय, जापान </td><td></td><td></td></tr><tr><td> वेरो E6 कोशिकाओं </td><td> ATCC </td><td> CRL - 1586 </td><td></td></tr></table>

using reverse genetics to manipulate the nss gene of the rift valley fever virus mp-12 strain to improve vaccine safety and efficacy

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants1, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus in the family Bunyaviridae and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3 as well as wild-type RVFV strains 4-6, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8 and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10 IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.

Watch this Scientific Journal Video about Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy at JoVE.com

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

रिवर्स आनुवंशिकी प्रणाली दरार घाटी बुखार वायरस सांसद 12 टीका तनाव के लिए बढ़ क्षीणन और प्रतिरक्षाजनकता साथ अतिरिक्त सांसद 12 म्यूटेंट बनाने के लिए एक उपयोगी उपकरण है. हम प्रोटोकॉल का वर्णन करने के लिए पैदा करते हैं और एनएसएस उत्परिवर्ती उपभेदों विशेषताएँ.

रिवर्स आनुवंशिकी का उपयोग करने के लिए दरार घाटी बुखार वायरस सांसद 12 वैक्सीन सुरक्षा और प्रभावकारिता में सुधार तनाव के एनएसएस जीन हेरफेर

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal ...

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In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

वेस्ट नील वायरस उत्परिवर्ती तनाव संक्रमण को Myd88 की मध्यस्थता सेलुलर प्रतिरक्षा प्रतिक्रिया के इन विट्रो विश्लेषण में

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

जीन या ड्रग थेरेपी में उपयोग के लिए लक्ष्य निर्धारण एचआईवी -1 उत्पादन प्रभावकारिता और आरएनए की विषाक्तता का मूल्यांकन

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

बचने इंफ्लूएंजा वायरस मोनोक्लोनल एंटीबॉडी के भागने के वेरिएंट की पीढ़ी

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

ओवेरेक्स्पेशन एंड हेरिंग ऑफ ह्यूमन<em&gt; सीआईएस</em&gt; -प्रेनिलट्रांसफ़्रेज़ इन<em&gt; एस्चेरिचिया कोली</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Eukaryotic जीन मूल की जांच करने के लिए वंशावली विश्लेषण का उपयोग

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

Assembly and Purification of Prototype Foamy Virus Intasomes

विधानसभा और प्रोटोटाइप फोम वायरस Intasomes का शुद्धिकरण

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

चूहों में मेजबान-रोगज़नक़ प्रतिक्रियाओं और वैक्सीन प्रभावकारिता का मूल्यांकन

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...