Abstract
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-ΔNSs16/198 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-ΔNSs16/198 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.
Highlights
Rift Valley fever virus (RVFV) is a high consequence zoonotic pathogen that is classified as a Category A priority pathogen by the National Institute of Health (NIH), and an overlap select agent by the US Department of Health and Human Service and the US
The deletion of the RVFV S-segment is known to occur during serial passages in BHK-21 cells (0.1 MOI),[25] and high MOI passage of virus may lead to the generation of defective interfering (DI) particles.[26]
In MRC-5 cells, the MP-12 vaccine demonstrated no increases in genetic subpopulations encoding reversions in M-795, M-3564, L-533, or L-3104 exceeding 0.2% (Fig. 4); as shown by the bars labeled in red in Fig. 4e, a reversion at L-3750 occurred at passage 15 (31% of population in extracted from culture supernatants of rMP12-ΔNSs16/198 (Exp-1); 29%, Exp-2) or passage 10 (11%, Exp-3), and the reversion population became dominant at passage 25 (87–89%)
Summary
Rift Valley fever virus (RVFV) is a high consequence zoonotic pathogen that is classified as a Category A priority pathogen by the National Institute of Health (NIH), and an overlap select agent by the US Department of Health and Human Service and the US. In the US, a formalininactivated RVF vaccine (TSI-GSD-200) and the live-attenuated MP-
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