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.

<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>

Wir haben nichts zu offenbaren.

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 ...

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Universidad de Cartagena UDEC

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JoVE Journal

Immunology and Infection

17.3K Aufrufe.  University of Texas Medical Branch. 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.

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

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Paramyxoviruses für Tumor-gezielte Immunmodulation: Gestaltung und Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Forschung

Krebsforschung

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. 
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. 
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

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 ...

Immunologie und Infektion

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

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, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

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, ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

Rettung von rekombinanten Newcastle Disease Virus aus cDNA

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA viruses. By integrating a full RNA genome error-prone PCR and reverse genetics, random genome-wide substitution mutagenesis can be induced. We have developed a method using this technique to synthesize diverse libraries to identify viral mutants with phenotypes of interest. This method, called full-length mutant RNA synthesis (FL-MRS), offers the following advantages: (i) the ability to create a large library via a highly efficient one-step error-prone PCR; (ii) the ability to create groups of libraries with varying levels of genetic diversity by manipulating the fidelity of DNA polymerase; (iii) the creation of a full-length PCR product that can directly serve as a template for mutant RNA synthesis; and (iv) the ability to create RNA that can be delivered into host cells as a non-selected input pool to screen for viral mutants of the desired phenotype. We have found, using a reverse genetics approach, that FL-MRS is a reliable tool to study viral-directed evolution at all stages in the life cycle of the hepatitis C virus, JFH1 isolate. This technique appears to be an invaluable tool to employ directed evolution to understand adaptation, replication, and the role of viral genes in pathogenesis and antiviral resistance in positive-sense RNA viruses.

The protocol describes a method for introducing controllable genetic diversity in the hepatitis C virus genome by combining full-length mutant RNA synthesis using error-prone PCR and reverse genetics. The method provides a model for phenotype selection and can be used for 10 kb long positive-sense RNA virus genomes.

Reverse Genetik zur Entwicklung von RNA-Virusvarianten mit positivem Sinn

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA ...

Genetik

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Eine einfache und effiziente Methode zu konstruieren Mutant Vacciniavirusvektoren

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Generierung von rekombinanten aviären Herpesvirus Vektoren mit CRISPR/Cas9 gen bearbeiten

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

Erzeugung, Verstärkung und Titration von rekombinanten Respiratory Syncytial Viren

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian and avian host species, including humans. With the recent advent of rotavirus reverse genetics systems, it has become possible to use directed mutagenesis to explore rotavirus biology, modify and optimize existing rotavirus vaccines, and develop rotavirus multitarget vaccine vectors. In this report, we describe a simplified reverse genetics system that allows the efficient and reliable recovery of recombinant rotaviruses. The system is based on co-transfection of T7 transcription vectors expressing full-length rotavirus (+)RNAs and a CMV vector encoding an RNA capping enzyme into BHK cells constitutively producing T7 RNA polymerase (BHK-T7). Recombinant rotaviruses are amplified by overseeding the transfected BHK-T7 cells with MA104 cells, a monkey kidney cell line that is highly permissive for virus growth. In this report, we also describe an approach for generating recombinant rotaviruses that express a separate fluorescent reporter protein through the introduction of a 2A translational stop-restart element into genome segment 7 (NSP3). This approach avoids deleting or modifying any of the viral open reading frames, thus allowing the production of recombinant rotaviruses that retain fully functional viral proteins while expressing a fluorescent protein.

Generation of recombinant rotaviruses from plasmid DNA provides an essential tool for the study of rotavirus replication and pathogenesis, and the development of rotavirus expression vectors and vaccines. Herein, we describe a simplified reverse genetics approach for generating recombinant rotaviruses, including strains expressing fluorescent reporter proteins.

Vereinfachte Reverse Genetics-Methode zur Wiederherstellung rekombinanter Rotaviren, die Reporterproteine ausdrücken

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian ...

Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACV&#8217;s easily manipulated genome and broad host range as an outstanding platform to generate recombinant viruses with a variety of therapeutic applications. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host range selection method coupled with visual identification of recombinant viruses. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. The cassette, including the gene of interest and the mCherry-E3L fusion is flanked by sequences derived from the VACV genome. Between the gene of interest and mCherry-E3L is a smaller region that is identical to the first ~150 nucleotides of the 3&#8217; arm, to promote homologous recombination and loss of the mCherry-E3L gene after selection. We demonstrate that this method permits efficient, seamless generation of rVACV in a variety of cell types without requiring drug selection or extensive screening for mutant viruses.

This is a method to generate &#8220;scarless&#8221; recombinant vaccinia viruses using host-range selection and visual identification of recombinant viruses.