Genetic stability of Rift Valley fever virus MP-12 vaccine during serial passages in culture cells

Rift Valley fever is a mosquito-borne zoonotic disease endemic to Africa, which affects both ruminants and humans. Rift Valley fever causes serious damage to the livestock industry and is also a threat to public health. The Rift Valley fever virus has a segmented negative-stranded RNA genome consisting of Large (L)-segment, Medium (M)-segment, and Small (S)-segment. The live-attenuated MP-12 vaccine is immunogenic in livestock and humans, and is conditionally licensed for veterinary use in the US. The MP-12 strain encodes 23 mutations (nine amino acid substitutions) and is attenuated through a combination of mutations in the L-segment, M-segment, and S-segment. Among them, the M-U795C, M-A3564G, and L-G3104A mutations contribute to viral attenuation through the L-segment and M-segment. The M-U795C, M-A3564G, L-U533C, and L-G3750A mutations are also independently responsible for temperature-sensitive phenotype. We hypothesized that a serial passage of the MP-12 vaccine in culture cells causes reversions of the MP-12 genome. The MP-12 vaccine and recombinant rMP12-Delta NSs16/198 were serially passaged 25 times. Droplet digital polymerase chain reaction analysis revealed that the reversion occurred at L-G3750A during passages of MP-12 in Vero or MRC-5 cells. The reversion also occurred at M-A3564G and L-U533C of rMP12-Delta NSs16/198 in Vero cells. Reversion mutations were not found in MP-12 or the variant, rMP12-TOSNSs, in the brains of mice with encephalitis. This study characterized genetic stability of the MP-12 vaccine and the potential risk of reversion mutation at the L-G3750A temperature-sensitive mutation after excessive viral passages in culture cells.