Rift valley fever virus lacking the NSs and NSm genes is highly attenuated, confers protective immunity from virulent virus challenge, and allows for differential identification of infected and vaccinated animals

Abstract

Rift Valley fever (RVF) virus is a mosquito-borne human and veterinary pathogen associated with large outbreaks of severe disease throughout Africa and more recently the Arabian peninsula. Infection of livestock can result in sweeping "abortion storms" and high mortality among young animals. Human infection results in self-limiting febrile disease that in approximately 1 to 2% of patients progresses to more serious complications including hepatitis, encephalitis, and retinitis or a hemorrhagic syndrome with high fatality. The virus S segment-encoded NSs and the M segment-encoded NSm proteins are important virulence factors. The development of safe, effective vaccines and tools to screen and evaluate antiviral compounds is critical for future control strategies. Here, we report the successful reverse genetics generation of multiple recombinant enhanced green fluorescent protein-tagged RVF viruses containing either the full-length, complete virus genome or precise deletions of the NSs gene alone or the NSs/NSm genes in combination, thus creating attenuating deletions on multiple virus genome segments. These viruses were highly attenuated, with no detectable viremia or clinical illness observed with high challenge dosages (1.0 x 10(4) PFU) in the rat lethal disease model. A single-dose immunization regimen induced robust anti-RVF virus immunoglobulin G antibodies (titer, approximately 1:6,400) by day 26 postvaccination. All vaccinated animals that were subsequently challenged with a high dose of virulent RVF virus survived infection and could be serologically differentiated from naïve, experimentally infected animals by the lack of NSs antibodies. These rationally designed marker RVF vaccine viruses will be useful tools for in vitro screening of therapeutic compounds and will provide a basis for further development of RVF virus marker vaccines for use in endemic regions or following the natural or intentional introduction of the virus into previously unaffected areas.

FIG. 1.

(A) A schematic depiction of the rRVF cDNA plasmid constructions utilized for the generation of rRVF virus stocks. (B) All rRVF viruses were passaged serially 15 times postrescue to confirm the stability of eGFP insert. Results of direct live cell UV imaging 24 h postinfection of Vero E6 cells with each rRVF virus after 15 passages. Note the strong expression of the eGFP signal in all cases, with the filamentous intranuclear accumulation of eGFP in the rRVF-NSs-(Ala)₃GFP and rRVF-NSs(Ala)₁₀GFP viruses compared with the diffuse cytoplasmic distribution of eGFP in the rZH501-ΔNSs:GFP or rZH501-ΔNSs:GFP-ΔNSm viruses. (C) Results of Vero E6 cell infection (multiplicity of infection, 1) with the rRVF-NSs-(Ala)₃GFP and rRVF-NSs(Ala)₁₀GFP viruses 24 h postinfection. Cells were fixed and stained with monoclonal antibodies specific for eGFP (green channel) and RVF virus NSs (red channel) and counterstained with DAPI (blue channel) to confirm intranuclear colocalization of eGFP and NSs (merged channel).

FIG. 2.

Results of anti-RVF virus total IgG adjusted Sum_OD enzyme-linked immunosorbent assay of all vaccinated (40) and sham-inoculated (5) control animals on day 26 postimmunization. Note the lack of anti-RVF virus total IgG among sham-inoculated controls compared with that of vaccinated animal groups. A positive/negative cutoff value was established as the mean ± three standard deviations of the sham-inoculated Sum_OD values (open circles-dashed line). Significant differences between the vaccinated and the control groups (P value of <0.05) are indicated by a “§” symbol. No significant differences were found among vaccinated groups, regardless of the dose or the vaccine used. Mean Sum_OD values are shown as a heavy black line; error bars indicate the mean ± 2 standard deviations.

FIG. 3.

(A) Representative results of indirect fluorescence testing of serum collected from Wistar-Furth rats surviving challenge with RVF virus to demonstrate the presence of anti-NP (left panel) and anti-NSs (middle panel) antibody to RVF virus NP and NSs proteins expressed separately in Vero E6 cells. To confirm the intranuclear accumulation of anti-NSs antibody, cells were counterstained with DAPI (right panel). Note the strong staining of both the NP and the NSs proteins among the wild-type-infected animals. (B) Representative results of vaccinated Wistar-Furth rats (day 26 postvaccination) illustrating robust anti-NP antibody production and the concomitant lack of detectable anti-NSs antibody. (C) Representative results of the negative control sham-inoculated rats (day 26 postvaccination) illustrating the lack of detectable anti-NP and anti-NSs antibody. (D) Representative results from naturally infected convalescent livestock (goat) sera obtained during the RVF virus outbreak in Saudi Arabia in 2000. Note the presence of both the anti-NP and the anti-NSs antibodies.