Uncorrected Proof Int J Cancer Manag. 2022 September; 15(9):e122781. Published online 2022 September 19. doi: 10.5812/ijcm-122781. Research Article Isolation and Characterization of a Discrete Genetically Homogeneous Viral Subpopulation of Mumps Virus RS-12 Strain with Superior Oncolytic Potency Compared to Its Progenitor Virus Behnam Alirezaie 1, * , Abolhasan Foroughi 1 and Ashraf Mohammadi 1 1 Department of Human Viral Vaccines, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization, Karaj, Iran * Corresponding author: Department of Human Viral Vaccines, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization, Karaj, Iran. Email: b.alirezaie@rvsri.ac.ir Received 2022 January 25; Revised 2022 September 10; Accepted 2022 September 12. Abstract Background: Oncolytic virotherapy can serve as a novel therapeutic strategy in oncology. In this study, we aimed to evaluate the oncolytic activity of the mumps virus RS-12 strain after its adaptation to cancer cells via serial passaging. Methods: To adapt the RS-12 strain-based vaccine to cancer cells, it was passaged eight times in the HT1080 cell line and was isolated via two terminal endpoint dilutions. The genetic homogeneity of isolated cancer cell-adapted RS-12 variant was confirmed by direct sequencing of regions, encompassing four known heterogeneous genomic positions. The in vitro cytotoxic effects of viruses was assessed in two different cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptosis-inducing effects of the cancer cell-adapted variant and its parental virus on cancer cells were quantified by flow cytometry. Results: According to the chromatograms, the RS-12 strain vaccine seed exhibited two peaks at the genomic nucleotide positions 1591, 2417, 3774, and 12977. On the contrary, cancer cell-adapted RS-12, isolated by terminal endpoint serial dilutions, contained no vi- ral subpopulations in these positions. A significant improvement was observed in the oncolytic potency of our cancer cell-adapted variant compared to its parental virus vaccine seed in vitro. Besides, the variant efficiently induced apoptosis in the human fibrosar- coma and adenocarcinoma cell lines. Conclusions: Considering the increased oncolytic potency and apoptosis-inducing capacity of this variant in cancer cells, it can be a promising option for future experiments. Keywords: Apoptosis, Cytotoxicity, Oncolytic Virotherapy, Vaccines 1. Background Malignancies are currently the leading cause of mor- tality worldwide, imposing a significant burden on human health and the global economy. Several therapeutic ap- proaches, such as surgery, chemotherapy, and radiother- apy are traditionally used for cancer treatment, and these treatments have been found to be effective for some types of malignancies. Despite the success of these treatments, cancer treatment still remains a major clinical challenge and there is a great need for finding novel methods of treat- ment for different cancers. Many research efforts have been made to find effective anti-cancer treatments based on immunotherapeutic ap- proaches, such as virotherapy. Today, some viruses from different viral families are receiving increasing attention as oncolytic therapeutic agents (1). In a previous study, the wild-type Urabe strain of mumps virus (MuV) was investi- gated clinically for its oncolytic activity (2). Since the dis- covery of the oncolytic activity of MuV in the mid-1970s, dif- ferent MuV strains (natural/chimeric and wild/attenuated) have been found to be effective against a variety of cancers (3-11). According to previous studies, a combination of MuV and measles virus (MeV) vaccine strains can exert synergis- tic anti-tumor effects on cancerous cells (12, 13). MuV is an enveloped virus having a linear negative- sense RNA genome, and belongs to the family paramyx- oviridae, and the genus Orthorubulavirus. Until now, sev- eral MuV vaccine strains have been developed in different countries (14). The RS-12 strain-based vaccine was devel- oped in Iran from a wild MuV, which was originally iso- lated from a throat-wash specimen of a patient with typical symptoms of mumps, including fever and bilateral paroti- tis, without any other complications (15). The virus was isolated in green monkey kidney cells (GMKCs) and under- went several passages at a low temperature in normal hu- Copyright © 2022, Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.