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

Diese Arbeit wurde durch Grant Number 5 U54 AI057156-07 durch Western Regional Center of Excellence (WRCE), 1 R01 AI08764301-A1 von National Institute of Allergy and Infectious Diseases, und eine interne Finanzierung von Sealy Center for Vaccine Development an der Universität finanziert Texas Medical Branch.

1. Rückgewinnung von rekombinanten MP-12-Codierung NSs Mutation (en) von Plasmid-DNA 2 <ol><li> Spread-Baby-Hamster-Nierenzellen (BHK) / T7-9-Zellen 15, die stabil exprimieren, T7-RNA-Polymerase, in 6-cm-Schalen in Minimum Essential Medium (MEM)-alpha (Invitrogen, Kat. Nr. 32561037) mit 10% fötalem Rinderserum (FBS ), Penicillin-Streptomycin (Penicillin: 100 U / ml, Streptomycin: 100 pg / ml) (Invitrogen, Kat. Nr. 15140122) und 600 pg / ml Hygromycin B (CellGro, Kat. Nr. 30 bis 240-CR). ...

<ol>
	<li>Bird, B. H., Ksiazek, T. G., Nichol, S. T., Maclachlan, N. J. <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.">Rift Valley fever virus.</a> J. Am. Vet. Med. Assoc. 234, 883-893 (2009).</li><li>Ikegami, T., Won, S., Peters, C. J., Makino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+infectious+rift+valley+fever+virus+entirely+from+cDNA,+analysis+of+virus+lacking+the+NSs+gene,+and+expression+of+a+foreign+gene.">Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene.</a> J. Virol. 80, 2933-2940 (2006).</li><li>Billecocq, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+polymerase+I-mediated+expression+of+viral+RNA+for+the+rescue+of+infectious+virulent+and+avirulent+Rift+Valley+fever+viruses.">RNA polymerase I-mediated expression of viral RNA for the rescue of infectious virulent and avirulent Rift Valley fever viruses.</a> Virology. 378, 377-384 (2008).</li><li>Habjan, M., Penski, N., Spiegel, M., Weber, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T7+RNA+polymerase-dependent+and+-independent+systems+for+cDNA-based+rescue+of+Rift+Valley+fever+virus.">T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus.</a> J. Gen. Virol. 89, 2157-2166 (2008).</li><li>Gerrard, S. R., 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=The+NSm+proteins+of+Rift+Valley+fever+virus+are+dispensable+for+maturation,+replication+and+infection.">The NSm proteins of Rift Valley fever virus are dispensable for maturation, replication and infection.</a> Virology. 359, 459-465 (2007).</li><li>Billecocq, A. <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+blocks+interferon+production+by+inhibiting+host+gene+transcription.">NSs protein of Rift Valley fever virus blocks interferon production by inhibiting host gene transcription.</a> J. Virol. 78, 9798-9806 (2004).</li><li>May, N. L. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TFIIH+transcription+factor,+a+target+for+the+Rift+Valley+hemorrhagic+fever+virus.">TFIIH transcription factor, a target for the Rift Valley hemorrhagic fever virus.</a> Cell. 116, 541-550 (2004).</li><li>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=Rift+Valley+fever+virus+NSs+protein+promotes+post-transcriptional+downregulation+of+protein+kinase+PKR+and+inhibits+eIF2alpha+phosphorylation.">Rift Valley fever virus NSs protein promotes post-transcriptional downregulation of protein kinase PKR and inhibits eIF2alpha phosphorylation.</a> PLoS Pathog. 5, e1000287-e1000287 (2009).</li><li>Habjan, M. <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+induces+the+specific+degradation+of+the+double-stranded+RNA-dependent+protein+kinase.">NSs protein of Rift valley fever virus induces the specific degradation of the double-stranded RNA-dependent protein kinase.</a> J. Virol. 83, 4365-4375 (2009).</li><li>Garcia-Sastre, A., Biron, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+1+interferons+and+the+virus-host+relationship:+a+lesson+in+detente.">Type 1 interferons and the virus-host relationship: a lesson in detente.</a> Science. 312, 879-882 (2006).</li><li>Bon, A. L. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+i+interferons+potently+enhance+humoral+immunity+and+can+promote+isotype+switching+by+stimulating+dendritic+cells+in+vivo.">Type i interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo.</a> Immunity. 14, 461-470 (2001).</li><li>Le Bon, A., Tough, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Links+between+innate+and+adaptive+immunity+via+type+I+interferon.">Links between innate and adaptive immunity via type I interferon.</a> Curr. Opin. Immunol. 14, 432-436 (2002).</li><li>Tough, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+interferon+as+a+link+between+innate+and+adaptive+immunity+through+dendritic+cell+stimulation.">Type I interferon as a link between innate and adaptive immunity through dendritic cell stimulation.</a> Leuk. Lymphoma. 45, 257-264 (2004).</li><li>Ito, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+recovery+of+rabies+virus+from+cloned+cDNA+using+a+vaccinia+virus-free+reverse+genetics+system.">Improved recovery of rabies virus from cloned cDNA using a vaccinia virus-free reverse genetics system.</a> Microbiol. Immunol. 47, 613-617 (2003).</li><li>Terasaki, K., Murakami, S., Lokugamage, K. G., Makino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+tripartite+RNA+genome+packaging+in+Rift+Valley+fever+virus.">Mechanism of tripartite RNA genome packaging in Rift Valley fever virus.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 804-809 (2010).</li><li>Buchholz, U. J., Finke, S., Conzelmann, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+bovine+respiratory+syncytial+virus+(BRSV)+from+cDNA:+BRSV+NS2+is+not+essential+for+virus+replication+in+tissue+culture,+and+the+human+RSV+leader+region+acts+as+a+functional+BRSV+genome+promoter.">Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter.</a> J. Virol. 73, 251-259 (1999).</li><li>Diaz, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homozygous+deletion+of+the+alpha-+and+beta+1-interferon+genes+in+human+leukemia+and+derived+cell+lines.">Homozygous deletion of the alpha- and beta 1-interferon genes in human leukemia and derived cell lines.</a> Proc. Natl. Acad. Sci. U.S.A. 85, 5259-5263 (1988).</li><li>Mosca, J. D., Pitha, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+and+posttranscriptional+regulation+of+exogenous+human+beta+interferon+gene+in+simian+cells+defective+in+interferon+synthesis.">Transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis.</a> Mol. Cell. Biol. 6, 2279-2283 (1986).</li><li>Constantinescu, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+signaling+specificity+of+the+IFNAR+chain+of+the+type+I+interferon+receptor+complex.">Expression and signaling specificity of the IFNAR chain of the type I interferon receptor complex.</a> Proc. Natl. Acad. Sci. U.S.A. 92, 10487-10491 (1995).</li><li>Kumar, K. 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. 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:+use+of+recombinant+vaccinia+viruses+to+study+Phlebovirus+gene+expression.">Rift Valley fever virus M segment: use of recombinant vaccinia viruses to study Phlebovirus gene expression.</a> J. 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. Virol. 85, 6234-6243 (2011).</li><li>Taniguchi, T., Ogasawara, K., Takaoka, A., Tanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRF+family+of+transcription+factors+as+regulators+of+host+defense.">IRF family of transcription factors as regulators of host defense.</a> Annu. Rev. Immunol. 19, 623-655 (2001).</li><li>Marie, I., Durbin, J. E., Levy, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+viral+induction+of+distinct+interferon-alpha+genes+by+positive+feedback+through+interferon+regulatory+factor-7.">Differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7.</a> EMBO J. 17, 6660-6669 (1998).</li><li>Ikegami, T., Won, S., Peters, C. J., Makino, 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+NSs+mRNA+is+transcribed+from+an+incoming+anti-viral-sense+S+RNA+segment.">Rift Valley fever virus NSs mRNA is transcribed from an incoming anti-viral-sense S RNA segment.</a> J. Virol. 79, 12106-12111 (2005).</li><li>Mims, C. A. <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+in+mice.+I.+General+features+of+the+infection.">Rift Valley Fever virus in mice. I. General features of the infection.</a> Br. J. Exp. 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>

Die reverse Genetik für RVFV wurden von mehreren Gruppen wurden durch den Einsatz T7-Promotor 2,4,5 oder Maus 3 oder menschliche 4 pol-I-Promotor entwickelt. In diesem Manuskript, beschreiben wir ein Protokoll zur rekombinanten RVFV MP-12-Stämme mit BHK/T7-9 Zellen 15, die stabil exprimieren, T7-RNA-Polymerase zu erzeugen. Die Effizienz der viralen Erholung variiert je nach Zustand des BHK/T7-9 Zellen, die Menge von Plasmiden, die Anzahl der transfizierten Zellen und so weit...

<table border="1"><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> Minimum Essential Medium (MEM)-alpha </td><td> Invitrogen </td><td> 32561037 </td><td></td></tr><tr><td> Dulbecco modifiziertem Minimum Essential Medium </td><td> Invitrogen </td><td> 11965092 </td><td></td></tr><tr><td> Modified Eagle Medium (MEM 2x) </td><td> Invitrogen </td><td> 11935046 </td><td></td></tr><tr><td> Penicillin-Streptomycin </td><td> Invitrogen </td><td> 15140122 </td><td></td></tr><tr><td> Hygromycin B </td><td> CellGro </td><td> 30 bis 240-CR </td><td></td></tr><tr><td> Tryptosephosphat Brühe </td><td> MP Biomedicals </td><td> 1682149 </td><td></td></tr><tr><td> Edle Agar </td><td> VWR </td><td> 101170-362 </td><td></td></tr><tr><td> Transit-LT1 </td><td> Mirus </td><td> MIR2300</td><td></td></tr><tr><td> Opti-MEM </td><td> Invitrogen </td><td> 31985070 </td><td></td></tr><tr><td> Aerosol dichten Deckel </td><td> Eppendorf </td><td> C-2223 bis 25 </td><td></td></tr><tr><td> 0,33% Neutralrot-Lösung </td><td> Sigma Aldrich </td><td> N2889-100ML </td><td></td></tr><tr><td> C57/WT MEF-Zellen </td><td> InvivoGen </td><td> MEF-c57wt </td><td></td></tr><tr><td> Blasticidin S </td><td> InvivoGen </td><td> Ant-bl-1 </td><td></td></tr><tr><td> Zeocin </td><td> InvivoGen </td><td> ant-Zn-1 </td><td></td></tr><tr><td> Quanti-Blue </td><td> InvivoGen </td><td> rep-QB1 </td><td></td></tr><tr><td> BHK/T7-9 Zellen 15 </td><td> Gifu University, Japan </td><td></td><td></td></tr><tr><td> Vero E6-Zellen </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-Fieber-Virus (RVFV), die hämorrhagisches Fieber, neurologischen Erkrankungen oder Erblindung bei Menschen, und eine hohe Rate Abtreibung und fetale Missbildungen bei Wiederkäuern 1 bewirkt hat als HHS / USDA eingestuft überschneiden wählen Agent und eine Risikogruppe 3 Erreger. Es gehört zur Gattung Phlebovirus in der Familie Bunyaviridae und ist einer der bösartigsten Mitglieder dieser Familie. Mehrere reversen Genetik-Systeme für den RVFV MP-12 Impfstamm 2,3 sowie Wildtyp-RVFV Stämme 4-6, einschließlich ZH548 und ZH501, seit 2006 entwickelt worden. Der MP-12-Stamm (das ist ein Risiko, Gruppe 2 Erreger und einem Nicht-wählen-Agent) ist stark gedämpft durch mehrere Mutationen im M-und L-Segmente, aber trägt noch virulent S-Segment RNA 3, die eine funktionelle Virulenz kodiert Faktor, NSS. Die rMP12-C13type (C13type) tragen 69% in-frame-Deletion von NSS ORF fehlt allen bekannten NSs Funktionen, während es als effic repliziertient ebenso wie MP-12 in VeroE6 Zellen ohne Typ-I-IFN. NSs induziert eine Abschaltung von Host-Transkription einschließlich Interferon (IFN)-beta mRNA 7,8 und fördert Abbau von doppelsträngigen RNA-abhängige Proteinkinase (PKR) in der post-translationalen Ebene. 9,10 IFN-beta ist transkriptionell hochreguliert durch Interferon regulatory factor 3 (IRF-3), NF-kB und Aktivator-Protein-1 (AP-1), und die Bindung von IFN-beta zu IFN-alpha/beta Rezeptor (IFNAR) stimuliert die Transkription von IFN-alpha Gene oder anderen Interferon-stimulierten Genen (ISGs) 11, welcher Host antivirale Aktivitäten induziert, während Host Transkription Unterdrückung einschließlich IFN-beta-Gen von NSS verhindert das Gen upregulations dieser ISGs in Reaktion auf die virale Replikation, obwohl IRF-3, NF-kB und Aktivator Protein-1 (AP-1) kann durch RVFV7 aktiviert werden. . So ist NSs ein hervorragendes Ziel weiter dämpfen MP-12 und zum Host angeborenen Immunantwort durch die Abschaffung der IFN-beta Unterdrückung Funktion zu verbessern. HierWir beschreiben ein Protokoll zur Erzeugung eines rekombinanten MP-12-Codierung mutierte NSS und liefern ein Beispiel für ein Screening-Verfahren auf NSS-Mutanten fehlt die Funktion, IFN-beta-mRNA-Synthese zu unterdrücken identifizieren. Zusätzlich zu ihrer wesentlichen Rolle in der angeborenen Immunität, ist der Typ-I IFN für die Reifung von dendritischen Zellen und die Induktion einer adaptiven Immunantwort 12-14 wichtig. So induzieren NSs Mutanten-Typ-I IFN sind weiter abgeschwächt, aber gleichzeitig sind effizienter zu stimulieren Host Immunreaktionen als Wildtyp-MP-12, die sie zu idealen Kandidaten für die Impfung Ansätze macht.

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

Die reverse Genetik für die Rift Valley-Fieber-Virus MP-12 Impfstamm ist ein nützliches Werkzeug für die Schaffung zusätzlicher MP-12-Mutanten mit erhöhter Dämpfung und Immunogenität. Wir beschreiben das Protokoll zu erzeugen und zu charakterisieren NSs Mutanten.

Mit Reverse-Genetics der NSS Gene des Rift Valley Fever Virus MP-12-Stamm zu Vaccine Safety und Wirksamkeit zu verbessern Manipulieren

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

Research

JoVE Journal

Immunology and Infection

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Reverse-Genetics vermittelte Wiedergewinnung eines infektiösen Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

Forschung

Immunologie und Infektion

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Mit Klick-Chemie, um die Wirkung von Viral Infection auf Host-Zell-RNA Synthese Messen

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

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

In-vitro-Analyse Myd88-vermittelte zelluläre Immunantwort gegen das West-Nil-Virus-Infektion Mutantenstamm

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

Bewertung der Wirksamkeit und Toxizität von RNAs Targeting HIV-1-Produktion für den Einsatz in Gene oder Arzneimitteltherapie

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

Generation von Escape Varianten des Influenza-Virus Monoclonal Antikörper neutralisieren

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

Überexpression und Reinigung des Menschen<em&gt; Cis</em&gt; -prenyltransferase in<em&gt; Escherichia coli</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

Mit der phylogenetischen Analyse zu eukaryotischen Gens Herkunft untersuchen

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

Montage und Reinigung der Prototyp schaumig Virus Intasomes

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

Bewertung der Wirt-Pathogen-Antworten und Wirksamkeit des Impfstoffes bei Mäusen

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