**1. Introduction**

Observations that naturally occurring viral infections could cause spontaneous tumor regressions led to the search for viruses that could selectively lyse tumor cells with limited pathogenicity in humans [1]. In the 1950s, it was discovered that Newcastle disease virus (NDV), a highly virulent pathogen to over 240 species of birds, has oncolytic properties [2,3]. A decade later, NDV was injected intraperitoneally in mice with Ehrlich ascites, leading to tumor cell lysis and durable immunity upon tumor re-challenge [4,5]. Around the same time, NDV was tested clinically in a patient with acute myelogenous leukemia, who experienced transient anti-leukemic effect and clinical improvement with limited side effects [6].

NDV is an avian paramyxovirus type I virus belonging to the *Avulavirus* genus. NDV has a spherical morphology, formed by a lipid bilayer which surrounds the RNA genome. The genome consists of a 15,186-nucleotide negative single-strand RNA encoding six different genes: nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), haemagglutinin-neuraminidase (HN), and RNA-dependent RNA polymerase (L). NP, P, and L proteins form a ribonucleotide protein complex that embeds the genomic RNA. The lipid envelope surrounds the ribonucleotide protein complex [7–9]. NDV infection is initiated by binding of the viral surface HN and F glycoproteins to sialic acid-containing host cell surface proteins [10,11]. This triggers a conformational change in the F protein, which results in fusion of the viral envelope and the cell plasma membrane. Viral particles are internalized by endocytosis, and adjacent cells with attached particles may form syncytia due to the fusogenic F protein [8,11,12]. After viral entry, the M protein dissociates from the ribonucleotide protein complex in the cytoplasm, and the P and L proteins form a polymerase complex that initiates transcription of the viral RNA [10,13].

There are three main pathotypes of NDV, classified by the severity of disease caused in birds: lentogenic (avirulent), mesogenic (intermediate), and velogenic (highly virulent) [8]. Virulence is primarily determined by sequence variation in the F gene, which affects F protein cleavage efficiency [14,15]. Lentogenic viruses possess a monobasic F cleavage site and exhibit reduced capacity for multicycle replication and lysis. The mesogenic and velogenic NDV types possess a polybasic F cleavage site and have superior capacity for multicycle replication, syncytia formation, and tumor cell lysis. In birds, mesogenic strains cause mild respiratory and gastrointestinal disease, while velogenic strains cause severe respiratory and gastrointestinal disease as well as neurotoxicity [14–16]. In preclinical studies, the most commonly used strains are the mesogenic strains MTH-68/H, PV701, 73T, Italien, Beaudette C, and AF2240, and the lentogenic strains HUJ, Ulster, LaSota, Hitchner B1, and V40-UPM. Among these strains, the lentogenic NDV LaSota strain is a proven and safe vaccine vector that is commonly used as a live attenuated vaccine in the poultry industry [17]. Due to capacity for multicycle replication, mesogenic and velogenic exhibit superior capacity for direct virus-mediated lysis. It is incorrect, however, to classify the lentogenic NDV strains as completely nonlytic. In a number of studies using lentogenic NDV strains lacking the polybasic F cleavage site, the viruses still demonstrate capacity to infect and lyse cancer cells at multiplicity of infection as low as 0.001 [18].

Oncolytic properties of NDV derive primarily from deficient type I IFN signaling pathways and less sensitive type I IFN receptor-mediated signaling in tumor cells [19–21]. Mutations in genes related to the type I IFN pathway and the downstream Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway are associated with NDV susceptibility and cytotoxicity [19,22,23]. Tumor cell susceptibility to NDV infection may also be based on the presence of sialic acid-containing cell surface proteins. It was proposed that the combination of altered type I IFN-related gene expression and sialic acid content could act as a clinical biomarker for determining susceptible tumor types [24]. Finally, defects in apoptotic pathways such as the Fas-FasL interaction or overexpression of antiapoptotic genes such as Livin and BcL-xL, which are documented in many tumor types, may increase susceptibility to NDV allowing for viral persistence, increased replication, and spread to surrounding cells [25–27].

NDV has been shown to cause cell death by apoptosis, necrosis, or autophagy mechanisms [26,28–30]. Viral HN protein can directly trigger the release of type I IFN and upregulates tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) [31]. In human peripheral blood mononuclear cells (PBMCs), TRAIL signaling in turn upregulates apoptotic genes (FasL, Bax, caspase-8, caspase-9, and caspase-3) [32]. HN gene expression alone has been reported to induce apoptosis in human breast

cancer MCF-7 cells [33]. NDV can also induce apoptosis through interferon-independent mechanisms such as the intrinsic mitochondrial death pathway [34]. Finally, the formation of syncytia by some NDV strains (termed "fusogenic" strains) ultimately leads syncytium disintegration either through necrosis or apoptosis [35].
