title: Using reverse genetics to manipulate the NSs gene of the Rift Valley fever virus MP-12 strain to improve vaccine safety and efficacy.
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 ruminants, 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 strain as well as wild-type RVFV strains, 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 RNA, 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 mRNA and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level. 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), 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 response. 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.
authors.affiliation: Department of Pathology, University of Texas Medical Branch, USA.
authors.forename: Birte
authors.initials: B
authors.lastname: Kalveram
authors.forename: Olga
authors.initials: O
authors.lastname: Lihoradova
authors.forename: Sabarish V
authors.initials: SV
authors.lastname: Indran
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Ikegami
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D006224:Cricetinae; D012296:Rift Valley fever virus / Q000235:genetics* / Q000276:immunology*; D014613:Vaccines, Attenuated / Q000235:genetics / Q000276:immunology; D014709:Vero Cells; D014765:Viral Vaccines / Q000235:genetics* / Q000276:immunology*
chemical_list: D014613:Vaccines, Attenuated; D014765:Viral Vaccines
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