Development of a Simian RNA Polymerase I Promoter-Driven Reverse Genetics for the Rescue of Recombinant Rift Valley Fever Virus from Vero Cells.

Rift Valley fever (RVF), which has been designated as 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 efficient production of 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 demonstrated the rescue of RVFV MP-12 infectious clones in Vero cells using plasmids encoding the Macaca mulatta RNA polymerase I promoter. Although Vero cells demonstrated an approximately 20% transfection efficiency, only 0.5% of transfected cells showed the replication of viral genomic RNA, supported by the co-expression of RVFV N and L helper proteins. RVFV Infectious clones were detectable in the culture supernatants approximately 4 to 9 days posttransfection reaching maximum titers during the following 5 days. The re-amplification 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 the rMP-12 re-amplified in human diploid MRC-5 cells did not increase viral sub-populations 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 of the endemic area will potentially cause devastating economic damages 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 the 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 re-amplification of virus clones in Vero cells unexpectedly increased viral subpopulations encoding unfavorable mutations, whereas viral re-amplification in human diploid MRC-5 cells could minimize the emergence of such mutants. Rescue of recombinant RVFV from Vero cells and re-amplification in MRC-5 cells will support the vaccine seed lot systems of live-attenuated recombinant RVFV vaccines for human use.