Rapid Accumulation of Virulent Rift Valley Fever Virus in Mice from an Attenuated Virus Carrying a Single Nucleotide Substitution in the M RNA

Abstract

BackgroundRift Valley fever virus (RVFV), a member of the genus Phlebovirus within the family Bunyaviridae, is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, while in livestock it causes fever and high abortion rates.Methodology/Principal FindingsSequence analysis showed that a wild-type RVFV ZH501 preparation consisted of two major viral subpopulations, with a single nucleotide heterogeneity at nucleotide 847 of M segment (M847); one had a G residue at M847 encoding glycine in a major viral envelope Gn protein, while the other carried A residue encoding glutamic acid at the corresponding site. Two ZH501-derived viruses, rZH501-M847-G and rZH501-M847-A, carried identical genomic sequences, except that the former and the latter had G and A, respectively, at M847 were recovered by using a reverse genetics system. Intraperitoneal inoculation of rZH501-M847-A into mice caused a rapid and efficient viral accumulation in the sera, livers, spleens, kidneys and brains, and killed most of the mice within 8 days, whereas rZH501-M847-G caused low viremia titers, did not replicate as efficiently as did rZH501-M847-A in these organs, and had attenuated virulence to mice. Remarkably, as early as 2 days postinfection with rZH501-M847-G, the viruses carrying A at M847 emerged and became the major virus population thereafter, while replicating viruses retained the input A residue at M847 in rZH501-M847-A-infected mice.Conclusions/SignificanceThese data demonstrated that the single nucleotide substitution in the Gn protein substantially affected the RVFV mouse virulence and that a virus population carrying the virulent viral genotype quickly emerged and became the major viral population within a few days in mice that were inoculated with the attenuated virus.

The Dominant-Negative Inhibition of Double-Stranded RNA-Dependent Protein Kinase PKR Increases the Efficacy of Rift Valley Fever Virus MP-12 Vaccine

“…Lyophilized MP-12 vaccine was reconstituted and passaged once either in MRC-5 cells or in Vero E6 cells. RVFV strain ZH501 stock was generated after one Vero E6 cell passage of an original ZH501 reference collection vial (serial number JM1137) at the University of Texas Medical Branch (UTMB) (49).…”

Rift Valley Fever Virus MP-12 Vaccine Is Fully Attenuated by a Combination of Partial Attenuations in the S, M, and L Segments

“…BHK/T7-9 cells that express T7 RNA polymerase (62) were maintained in alpha minimum essential medium containing 10% FBS, penicillin-streptomycin, and 600 g/ml of hygromycin. Rescue and work with infectious recombinant RVFV strain ZH501 (rZH501) or recombinant strain MP-12 (rMP-12) were performed at The University of Texas Medical Branch at Galveston (UTMB) Robert E. Shope biosafety level 4 (BSL-4) laboratory as described previously (63). Rescued viruses were amplified once in VeroE6 cells after recovery in BHK/T7-9 cells and used for subsequent experiments.…”

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

“…Recombinant RVFV were rescued in BHK/T7-9 cells by reverse genetics as described previously. 44 Rescued RVFV were amplified once in VeroE6 cells, and used for subsequent experiments. Virus titration of RVFV was performed by plaque assay using VeroE6 cells as described previously.…”