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.
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Method Article
Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy
In This Article
Summary
Abstract
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.
Protocol
1. Recovery of recombinant MP-12 encoding NSs mutation(s) from plasmid DNAs2
- 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 μg/ml) (Invitrogen, Cat#15140122), and 600 μg/ml of hygromycin B (Cellgro, Cat#30-240-CR).
* The efficiency of viral recovery is higher in 6-cm dishes than in 35-mm dishes. BHK/T7-9 cells with low passage level support higher rates of recovery. Alternatively, other BHK cell lines that....
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Discussion
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 .......
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Disclosures
We have nothing to disclose.
Acknowledgements
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.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Minimum Essential Medium (MEM)-alpha | Invitrogen | 32561037 | |
| Dulbecco’s modified minimum essential medium | Invitrogen | 11965092 | |
| Modified Eagle Medium (MEM 2x) | Invitrogen | 11935046 | |
| Penicillin-Streptomycin | Invitrogen | 15140122 | |
| Hygromycin B | Cellgro | 30-240-CR | |
| Tryptose phosphate broth | MP Biomedicals | 1682149 | |
| Noble agar | VWR international | 101170-362 | |
| TransIT-LT1 | Mirus Bio LLC | MIR2300 | |
| Opti-MEM | Invitrogen | 31985070 | |
| Aerosol tight lid | Eppendorf | C-2223-25 | |
| 0.33% neutral red solution | Sigma-Aldrich | N2889-100ML | |
| C57/WT MEF cells | Invitrogen | mef-c57wt | |
| Blasticidin S | Invitrogen | Ant-bl-1 | |
| Zeocin | Invitrogen | ant-zn-1 | |
| QUANTI-Blue | Invitrogen | rep-qb1 | |
| BHK/T7-9 cells15 | Gifu university, Japan | ||
| Vero E6 cells | ATCC | CRL-1586 |
References
- Bird, B. H., Ksiazek, T. G., Nichol, S. T., Maclachlan, N. J. Rift Valley fever virus. J. Am. Vet. Med. Assoc. 234, 883-893 (2009).
- Ikegami, T., Won, S., Peters, C. J., Makino, S. Rescue of infectious rift valley fever virus entirely from cDNA, an....
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