Molecular mechanisms of oncolytic properties of Newcastle disease virus (NDV) in human cancer cells

Newcastle disease virus (NDV) is a single-stranded, non-segmented, negative-sense enveloped RNA virus, which belongs to the Orthoavulavirus genus of Paramyxoviridae family. It is a well-known, economically important poultry pathogen worldwide. Over the last five decades, NDV is known to have oncolytic properties based on its tumourselective replication sparing the healthy cells and immunostimulation affirming potential candidate for oncovirotherapy. In this PhD project, the efficacy of naturally occurring and genetically modified GFP expressing non-pathogenic NDV strains investigated in various human cancer cell lines as an oncolytic virus. This study has demonstrated that the heterogeneous colorectal cancer cell line Caco-2 was the most susceptible to the cytotoxicity induced by avirulent NDV strains amongst the panel of diverse cell lines tested. Despite the high levels of cell cytotoxicity induced by NDV in Caco-2 cells, a small population of surviving cells was isolated from NDV superinfected Caco-2 cells 12 days post infection and named VR Caco-2 cells (virus-resistant Caco-2 cells). The persistent infection with avirulent strains of NDV was demonstrated in VR Caco-2 cells for the first time. An integrated approach of cellular, transcriptomic, and proteomic data was used to study differences between persistently NDV-infected VR Caco-2 cells,and acutely NDVinfected and uninfected Caco-2 cells. This study has shown that persistent NDVinfection resulted in slower cell proliferation of VR Caco-2 cells than parental Caco-2 cells, and production of recoverable and replicating virus at lower titres. VR Caco-2 cells have demonstrated resistance to NDV re-infection. In contrast, VR Caco-2 cells were susceptible to other viruses such as avian influenza virus (H9N2) and vesicular stomatitis virus (VSV). Persistent NDV infection in VR Caco-2 cells resulted in reduced NDV-induced cytotoxicity, although VR Caco-2 cells were still sensitive to the increased cell cytotoxicity induced by the VSV infection. This would suggest that a combination of oncolytic viruses could be used to overcome the effects of NDV persistent infection. Potential mechanisms of persistence of NDV in Caco-2 cells were explored. Both Caco2 and VR Caco-2 cells demonstrated the lack of efficient induction of IFN-β and ISG15 mRNAs, which could be a potential reason for the establishment of NDV persistent infection. RNA-seq data analysis demonstrated extensive regulation of thousands of genes and hundreds of cellular pathways in VR Caco-2 cells while maintaining 50-times lower viral load compared to acute NDV-infection (in Caco-2 cells). Fluctuation in SQSTM1/p62, a multifunctional and multidomain signalling adapter protein required in selective autophagy, was also demonstrated suggesting the induction of autophagy in NDV-infected Caco-2 cells. The results generated in this PhD project strongly suggest that NDV not only establishes persistent infection but that too reduces NDV-induced cytotoxicity while regulating thousands of genes in Caco-2 cells. It also suggests the need for detailed investigation of persistent NDV-infection in vitro and in vivo models of Caco-2 cells before and after developing the NDV as an oncolytic therapeutic vaccine for cancer.