Development of a Simian RNA polymerase I promoter-driven reverse genetics system for the rescue of recombinant rift valley fever virus from vero cells

Rift Valley fever (RVF), which has been designated a priority disease by the World Health Organization (WHO), is one of the most pathogenic zoonotic diseases endemic to Africa and the Arabian Peninsula. Human vaccine preparation requires the use of appropriate cell substrates to support the efficient production of a seed vaccine with minimum concerns of tumorigenicity, oncogenicity, or adventitious agents. Vero cells, which were derived from the African green monkey kidney, represent one of the few mammalian cell lines that are used for vaccine manufacturing. This study demonstrates the rescue of RVF virus (RVFV) MP-12 infectious clones in Vero cells using plasmids encoding the Macaca mulatta RNA polymerase I promoter. Although Vero cells demonstrated an;20% transfection efficiency, only 0.5% of transfected cells showed the replication of viral genomic RNA, supported by the coexpression of RVFV N and L helper proteins. RVFV infectious clones were detectable in the culture supernatants at approximately 4 to 9 days posttransfection, reaching maximum titers during the following 5 days. The reamplification of rescued recombinant MP-12 (rMP-12) in Vero cells led to an increase in the genetic subpopulations, affecting the viral phenotype via amino acid substitutions in the NSs gene, whereas rMP-12 reamplified in human diploid MRC-5 cells did not increase viral subpopulations with NSs gene mutations. The strategy in which RVFV infectious clones are rescued in Vero cells and then subsequently amplified in MRC-5 cells will support the vaccine seed lot systems of live-attenuated recombinant RVFV vaccines for human use. IMPORTANCE RVF is a mosquito-transmitted, viral, zoonotic disease endemic to Africa and the Arabian Peninsula, and its spread outside the area of endemicity will potentially cause devastating economic damage and serious public health problems. Different from classical live-attenuated vaccines, live-attenuated recombinant vaccines allow rational improvement of vaccine production efficiency, protective efficacy, and vaccine safety via genetic engineering. This study demonstrated the generation of infectious Rift Valley fever (RVF) virus from cloned cDNA using Vero cells, which are one of a few mammalian cell lines used for vaccine manufacturing. Subsequent reamplification of virus clones in Vero cells unexpectedly increased viral subpopulations encoding unfavorable mutations, whereas viral reamplification in human diploid MRC-5 cells could minimize the emergence of such mutants. Rescue of recombinant RVFV from Vero cells and reamplification in MRC-5 cells will support the vaccine seed lot systems of live-attenuated recombinant RVFV vaccines for human use.