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

KEYWORDS:

• differentiate infected from vaccinated animals (DIVA)
• interferon antagonism
• live-attenuated vaccine
• nonstructural protein NSs
• Rift Valley fever

This article is referred to by:

Attenuation of pathogenic Rift Valley fever virus strain through the chimeric S-segment encoding sandfly fever phlebovirus NSs or a dominant-negative PKR

In the summer of 1930, an excessive rainfall in Kenya, followed by an increase in the mosquito population, ultimately led to a large number of abortions in ewe flocks.¹ This was to become the first recorded outbreak of Rift Valley fever (RVF), which is endemic to sub-Saharan Africa² and threatens to spread even further, regionally and internationally.^3,4 RVF is a mosquito-borne zoonotic disease caused by Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus of the family Bunyaviridae.⁵ RVFV primarily affects livestock, such as cattle, sheep, and goats, although it also causes illness in humans.⁶ Since the disease causes a high rate of abortion in pregnant animals and high lethality in newborn animals,⁷ outbreaks of RVF result in serious economic losses. Moreover, while most human cases of RVF result in a self-limiting febrile illness, some patients develop a more severe disease that can include fulminant hepatitis, encephalitis, retinitis, blindness, or a lethal hemorrhagic syndrome.^5,8 Due to its potential impact on both agriculture and public health, RVFV is classified as a Category A Priority pathogen and an overlap select agent by the CDC and USDA.⁹ The fact that livestock are highly susceptible to RVFV−and therefore act as a source for human RVF disease−makes veterinary vaccination a critical factor in preventing RVF outbreaks.^7,10,11

In the United States, a live-attenuated vaccine for RVF has been licensed for veterinary purposes, but its use is limited to emergency situations only.¹¹ One of the most substantial barriers preventing the routine use of this vaccine is the lack of a marker to differentiate infected from vaccinated animals (a concept known as DIVA).^10,12 The lack of a DIVA marker is a serious disadvantage for the use of RVF vaccines in non-endemic areas since it makes it impossible to serologically distinguish animals naturally infected with RVFV from the vaccinated population, hampering disease control efforts. Thus, the establishment of an RVF vaccine with a DIVA marker is much needed. The study entitled “Attenuation of pathogenic Rift Valley fever virus strain through the chimeric S-segment encoding sandfly fever phlebovirus NSs or a dominant-negative PKR” published by Nishiyama et al.¹³ in Virulence focuses on this issue and proposes an interesting approach, which will not only be useful for vaccine development but also for in vivo functional analysis of sandfly fever phlebovirus virulence factors.

Like other phleboviruses, RVFV has a tripartite, negative-sense RNA genome composed of so-called large (L), medium (M), and small (S) segments.⁴ The L-segment encodes the RNA-dependent RNA polymerase, known as L; the M-segment encodes the envelope glycoproteins Gn and Gc, a 78-k_D protein, and the nonstructural protein NSm; and the S-segment encodes the nucleoprotein, known as N. In addition, the S-segment also encodes the nonstructural protein NSs in an ambisense manner. The current live-attenuated RVF vaccine MP-12, which was generated from wild-type RVFV strain ZH548 by serial passaging and plaque purification in human cells in the presence of a mutagen,¹⁴ is largely attenuated by point mutations in the M- and L-segments.¹⁵ Although the MP-12 vaccine is known to be safe and immunogenic, it lacks a DIVA marker.

Since the establishment of a reverse genetics system for RVFV,¹⁶ it has become possible to genetically manipulate the virus and, therefore, implement various strategies for developing an RVF DIVA vaccine. The DIVA strategy can usually be described as the use of a vaccine carrying at least one less antigenic protein than the corresponding wild-type virus.¹⁷ In the case of RVFV, NSs is an attractive target for deletion, since it is not only a major virulence factor but also dispensable for viral replication.^5,18 NSs suppresses host general transcription,¹⁹ inhibits transcription from the interferon β (IFN-β) promoter,²⁰ and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR).^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.^23-25 They have previously generated a recombinant MP-12 lacking a functional NSs by the deletion of amino acid residues 16 to 198 [rMP-12-ΔNSs 16/198 (also referred as rMP-12-C13type)] and used the mouse model to demonstrate that rMP-12-ΔNSs 16/198 vaccination fails to produce detectable viremia but does induce detectable IFN-α in the sera.²³ Conversely, MP-12 vaccination induces viremia in 30% of mice without any detectable induction of IFN-α.²³ Moreover, it was found that 80% of rMP-12-ΔNSs 16/198-vaccinated mice are protected from challenge with wild-type RVFV.²³ Although these results demonstrate the ability to generate effective DIVA vaccines with increased safety, it was also shown that the immunogenicity of rMP-12-ΔNSs 16/198 is not as high as the MP-12 vaccine in sheep.²⁶ A likely explanation for this result is that complete disruption of NSs obviates the virus's ability to counteract the host IFN response and reduces viral replication in vivo.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-β 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. Moreover, the proposed approach would satisfy the DIVA concept since anti-RVFV NSs antibodies detected in animals would indicate RVFV infection but not vaccination.

While this strategy is very attractive, it was important to address the safety of this approach. To examine whether NSs genes derived from other sandfly fever phlebovirus contribute to the attenuation of RVFV, Nishiyama et al.¹³ generated chimeric recombinant RVFV based on the pathogenic RVFV strain ZH501 (rZH501), and they evaluated attenuation by using a mouse model. Several of the chimeras, including rZH501 encoding TOSV NSs (rZH501-TOSNSs), PTA NSs (rZH501-PTANSs), PTV Balliet strain NSs (rZH501-PTBNSs), or SFSV NSs (rZH501-SFSNSs), were inoculated into mice, and the survival rate was observed. Compared to the wild-type rZH501, all mice infected with the chimeric viruses showed delayed onset of disease, although the mice infected with rZH501-TOSNSs, rZH501-PTANSs, or rZH501-PTBNSs eventually died. Importantly, whereas all mice infected with wild-type rZH501 succumbed by day 7 post-infection, 50% of mice infected with rZH501-SFSNSs survived for 21 days. These results indicate that the exogenous NSs contributes to the attenuation of RVFV, and they also suggest the applicability of SFSV NSs as a DIVA marker in the next generation of MP-12 vaccine.

Another important aspect of the approach used by Nishiyama et al.¹³ is that it demonstrated that phlebovirus NSs proteins function as virulence factors in vivo. It was shown in the same study that 95% of mice infected with rZH501 lacking a functional NSs survived.¹³ On the other hand, all mice infected with rZH501-TOSNSs, rZH501-PTANSs, or rZH501-PTBNSs, and 50% of mice infected with rZH501-SFSNSs succumbed to infection, as described earlier. These results suggest that, although the insertion of TOSV, PTV, or SFSV NSs contributes to the attenuation of RVFV, each NSs itself still contributes to virus virulence. 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. 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-β 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.

In addition to MP-12 possessing exogenous NSs, MP-12 encoding a dominant-negative PKR is also an attractive RVF vaccine platform. Dominant-negative PKR inhibits the antiviral activity of wild-type PKR and therefore functionally replaces NSs, itself a PKR inhibitor, in the virus rMP-12-ΔNSs 16/198. Nishiyama et al.¹³ demonstrated the significant attenuation of rZH501 encoding a PKR gene in place of NSs, suggesting the applicability of PKR as a DIVA marker in the MP-12 vaccine. Moreover, rZH501 lacking both NSs and NSm has been tested in pregnant ewes and demonstrated to be efficacious and safe for veterinary use.^12,30 Those viruses are also promising live-attenuated RVF vaccine candidates possessing DIVA markers. Further research, including functional analysis of various phlebovirus NSs in vivo pathogenesis, will provide valuable information for better understanding of the role of NSs as a virulence factor as well as development of novel RVF vaccines, which will definitely become a powerful tool for preventing and mitigating RVF outbreaks.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

The authors (S.Y. and H.E.) are supported by the Division of Intramural Research, NIAID, NIH. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the NIAID and NIH.