Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus

Abstract

Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans, and a high rate of abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is attenuated in the M- and L-segments, but the S-segment retains the virulent phenotype. MP-12 was manufactured as an Investigational New Drug vaccine by using MRC-5 cells and encodes a functional NSs gene, the major virulence factor of RVFV which 1) induces a shutoff of the host transcription, 2) inhibits interferon (IFN)-β promoter activation, and 3) promotes the degradation of dsRNA-dependent protein kinase (PKR). MP-12 lacks a marker for differentiation of infected from vaccinated animals (DIVA). Although MP-12 lacking NSs works for DIVA, it does not replicate efficiently in type-I IFN-competent MRC-5 cells, while the use of type-I IFN-incompetent cells may negatively affect its genetic stability. To generate modified MP-12 vaccine candidates encoding a DIVA marker, while still replicating efficiently in MRC-5 cells, we generated recombinant MP-12 encoding Punta Toro virus Adames strain NSs (rMP12-PTNSs) or Sandfly fever Sicilian virus NSs (rMP12-SFSNSs) in place of MP-12 NSs. We have demonstrated that those recombinant MP-12 viruses inhibit IFN-β mRNA synthesis, yet do not promote the degradation of PKR. The rMP12-PTNSs, but not rMP12-SFSNSs, replicated more efficiently than recombinant MP-12 lacking NSs in MRC-5 cells. Mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs induced neutralizing antibodies at a level equivalent to those vaccinated with MP-12, and were efficiently protected from wild-type RVFV challenge. The rMP12-PTNSs and rMP12-SFSNSs did not induce antibodies cross-reactive to anti-RVFV NSs antibody and are therefore applicable to DIVA. Thus, rMP12-PTNSs is highly efficacious, replicates efficiently in MRC-5 cells, and encodes a DIVA marker, all of which are important for vaccine development for Rift Valley fever.

Hijacking of RIG-I Signaling Proteins into Virus-Induced Cytoplasmic Structures Correlates with the Inhibition of Type I Interferon Responses

“…In addition, RVFV NSs-mediated posttranslational downregulation of the p62 subunit of TFIIH has recently been described (34). Interestingly, this mechanism was not shared by sand fly fever Sicilian phlebovirus, another member of the genus Phlebovirus (68).…”

The two faces of Rift Valley fever virus virulence factor NSs: The development of a vaccine and the elucidation of pathogenesis

“…So far, it has been shown by in vitro analyses that both PTV and SFSV NSs have the ability to inhibit induction of the IFN-b gene, yet neither degrades PKR. 25 Despite their similar characteristics as an IFN antagonist, there was a significant difference in survival in mice infected with rZH501-PTVNSs and mice infected with rZH501-SFSNSs. 13 The basis for such a discrepancy is unclear, although it may be due to as yet undescribed differences in the function of PTV and SFSV NSs.…”

“…21,22 The research group led by Tetsuro Ikegami has had a longtime interest in developing an effective RVF vaccine with a DIVA marker by genetically manipulating the NSs gene. [23][24][25] They have previously generated a recombinant MP-12 lacking a functional NSs by the deletion of amino acid residues 16 to 198 [rMP-12-DNSs 16/198 (also referred as rMP-12-C13type)] and used the mouse model to demonstrate that rMP-12-DNSs 16/198 vaccination fails to produce detectable viremia but does induce detectable IFN-a in the sera. 23 Conversely, MP-12 vaccination induces viremia in 30% of mice without any detectable induction of IFN-a.…”

“…13 The interesting idea that Ikegami's group next put forward was not to completely disrupt NSs, but "to replace the RVFV NSs with a functional NSs derived from other phleboviruses." 24,25 RVFV belongs to the sandfly fever phlebovirus group, which also includes the Naples serocomplex (e.g., Toscana virus [TOSV]), Punta Toro serocomplex (e.g., Punta Toro virus (PTV) Adames strain [PTA]), Sicilian serocomplex (e.g., Sandfly fever Sicilian virus [SFSV]), Icoaraci serocomplex (e.g., Icoaraci virus), and Frijoles serocomplex (e.g., Frijoles virus). 27 The fact that the NSs proteins of TOSV, PTV, and SFSV all have the ability to inhibit the expression of the IFN-b gene 24,25,28,29 implies that RVFV possessing a functional NSs derived from other phleboviruses can replicate in vivo, providing effective immunity to vaccinated animals.…”

“…Such a clear conclusion could not be obtained using an attenuated RVFV encoding other phlebovirus NSs proteins since there was no clear difference in survival in mice infected with MP-12 and mice infected with rMP-12 encoding TOSNSs, 24 PTVNSs, or SFSNSs. 25 This is probably because MP-12 is highly attenuated by several mechanisms, masking the function of NSs as a virulence factor. Moreover, the approach proposed by Nishiyama et al 13 may also be a useful tool to compare the function of NSs proteins between phleboviruses in vivo.…”