Fig.2 Detection of reversion mutants of the MP-12 vaccine during viral passages in Vero cells.
在MRC-5细胞，MP-12也MP-795,M-3564,L-533和L-3104 也没有出现回复突变超过0.2%,除了L-3750（图3）。
Fig. 3 Detection of reversion mutants of the MP-12 vaccine during viral passages in MRC-5 cells.
为评估MP-12疫苗株和变异株rMP12-TOSVSs在体内病毒复制时的回复突变的发生，研究者提取了两者引起脑炎的老鼠的脑组织的RNA，用微滴式数字PCR仪分析，没有一个脑标本在上述五个变异位点有回复突变（图4）。
Fig. 4 Detection of reversion mutations in MP-12 and rMP12-TOSNSs in brains of mice
综上，这项研究显示了MP-12疫苗在Vero和MRC-5细胞上有限的复制周期中显著的稳定性，尽管L-V172A, L-M1244I和M-R1182G 的回复突变在病毒连续传代时被确证。对MP-12疫苗株进一步的安全性分析，着重点应在于体内和体外实验产生的突变基因型的相关毒力。
Title:Genetic stability of Rift Valley fever virus MP-12 vaccine during serial passages in culture cells
Abtract: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, Msegment, 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 characterizedgenetic 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.
doi:10.1038/s41541-017-0021-9.