This work was funded by Grant Number 5 U54 AI057156-07 through Western Regional Center of Excellence (WRCE), 1 R01 AI08764301-A1 from National Institute of Allergy and Infectious Diseases, and an internal funding from Sealy Center for Vaccine Development at the University of Texas Medical Branch.

1. Recovery of recombinant MP-12 encoding NSs mutation(s) from plasmid DNAs2
<ol>
<li>Spread baby hamster kidney (BHK)/T7-9 cells15, which stably express T7 RNA polymerase, into 6-cm dishes in Minimum Essential Medium (MEM)-alpha (Invitrogen, Cat# 32561037) containing 10% fetal bovine serum (FBS), Penicillin-Streptomycin (Penicillin:100 U/ml, Streptomycin: 100 &mu;g/ml) (Invitrogen, Cat#15140122), and 600 &mu;g/ml of hygromycin B (Cellgro, Cat#30-240-CR). 
* The efficiency of viral recovery i...

<ol>
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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. 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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. 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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>

We have nothing to disclose.

Reverse genetics systems for RVFV have been developed by several groups by utilizing T7 promoter2,4,5 or mouse3 or human4 pol-I promoter. In this manuscript, we describe a protocol to generate recombinant RVFV MP-12 strains by using BHK/T7-9 cells15 that stably express T7 RNA polymerase. The efficiency of viral recovery varied depending on the condition of BHK/T7-9 cells, the amount of plasmids, the number of transfected cells and so on. We always amplify the P0 virus in ...

<table border="1">
<tr>
<td>Name of the reagent</td>
<td>Company</td>
<td>Catalogue number</td>
<td>Comments (optional)</td>
</tr>
<tr>
<td>Minimum Essential Medium (MEM)-alpha </td>
<td>Invitrogen</td>
<td>32561037 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Dulbecco&rsquo;s modified minimum essential medium </td>
<td>Invitrogen</td>
<td>11965092 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Modified Eagle Medium (MEM 2x) </td>
<td>Invitrogen</td>
<td>11935046 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Penicillin-Streptomycin </td>
<td>Invitrogen</td>
<td>15140122 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Hygromycin B </td>
<td>Cellgro</td>
<td>30-240-CR </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Tryptose phosphate broth </td>
<td>MP biomedicals</td>
<td>1682149 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Noble agar </td>
<td>VWR </td>
<td>101170-362 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>TransIT-LT1 </td>
<td>Mirus</td>
<td>MIR2300 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Opti-MEM </td>
<td>Invitrogen</td>
<td>31985070 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Aerosol tight lid </td>
<td>Eppendorf </td>
<td>C-2223-25 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>0.33% neutral red solution </td>
<td>Sigma Aldrich </td>
<td>N2889-100ML </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>C57/WT MEF cells</td>
<td>InvivoGen</td>
<td>mef-c57wt</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Blasticidin S</td>
<td>InvivoGen</td>
<td>Ant-bl-1</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Zeocin</td>
<td>InvivoGen</td>
<td>ant-zn-1</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>QUANTI-Blue</td>
<td>InvivoGen</td>
<td>rep-qb1</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>BHK/T7-9 cells15 </td>
<td>Gifu university, Japan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Vero E6 cells</td>
<td>ATCC</td>
<td>CRL-1586</td>
<td>&nbsp;</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

The reverse genetics system for the Rift Valley fever virus MP-12 vaccine strain is a useful tool for creating additional MP-12 mutants with increased attenuation and immunogenicity. We describe the protocol to generate and characterize NSs mutant strains.

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

using-reverse-genetics-to-manipulate-nss-gene-rift-valley-fever-virus

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JoVE Journal

Immunology and Infection

17.2K visualizações.  University of Texas Medical Branch. O sistema de genética reversa para o vírus da febre Rift Vale cepa vacinal MP-12 é uma ferramenta útil para a criação de adicionais MP-12 mutantes com atenuação aumentada e imunogenicidade. Nós descrevemos o protocolo para gerar e caracterizar NSS cepas mutantes.