Oncolytic viruses, which selectively infect and kill cancer cells, have emerged as a promising cancer treatment (Fountzilas et al., 2017, Sinkovics and Horvath, 2008). They can also synergize with immunotherapy drugs to enhance their efficacy and reduce side effects (Lawler et al., 2017, Russell et al., 2012). The oncolytic virus M1 (OVM), initially isolated from mosquitoes in Hainan Island, China, classified as a Getah virus, a member of the genus Alphavirus (Li et al., 1992). It is known to be transmitted between horses and pigs(Fukunaga et al., 2000, Wen et al., 2007). Alphaviruses are a group of enveloped, single-stranded positive-sense RNA viruses with a diameter of 60–70 nm (Lefkowitz et al., 2018), and possess an enveloped architecture comprising an icosahedral nucleocapsid core and a lipid envelope studded with 80 glycoprotein spikes arranged in T = 4 icosahedral symmetry. Each spike is a trimer of E1–E2 heterodimers, with E2 primarily mediating receptor binding and E1 facilitating membrane fusion during viral entry (Jose et al., 2009, Mukhopadhyay et al., 2006, Sjoberg et al., 2011). Alphavirus, a genus in the family Togaviridae, includes several prominent human pathogens—Semliki Forest virus (SFV), Ross River virus (RRV), and Chikungunya virus (CHIKV) etc. (Lefkowitz et al., 2018, Payne, 2017). These viruses are arboviruses transmitted by arthropods, particularly mosquitoes, which can infect humans and lead to various diseases such as fever, encephalitis, and arthritis, rash, etc., posing a significant threat to human health (Suhrbier et al., 2012, Zaid et al., 2021).

However, OVM is non-pathogenic in humans (Fukunaga et al., 2000, Wen et al., 2007). Previous studies have characterized OVM’s intrinsic oncolytic properties and established it as a novel effective oncolytic virus (Lin et al., 2014). The molecular mechanisms behind OVM's ability to induce cancer cell death have been elucidated (Lin et al., 2014). Its life cycle and the characteristic structures it induces in the infected cells have been described previously (Dan et al., 2021). Like other alphaviruses, such as SFV and CHIKV, which can cause vacuolization in host cells (Grimley et al., 1968, Kaariainen et al., 1975), OVM can cause vacuolization in cancer cells during infection, leading to the formation of various cytopathic vesicles (CPVs). Previously reported ultrastructural features of CPVs have greatly facilitated their identification (Kujala et al., 2001a)(Griffiths et al., 1983, Grimley et al., 1968; Soonsawad et al., 2010).Three major types of CPVs have been historically defined based on morphological characteristics (Dan et al., 2021). CPV-I features ∼ 50 nm regular, bulb-shaped invaginations (spherules) on the inner surface of the limiting membrane. These spherules, which often contain a central electron-dense spot, represent the established sites of viral RNA replication (Acheson and Tamm, 1967, Friedman et al., 1972, Grimley et al., 1968). CPV-II, in contrast, is filled with mature virions which is marked by an inner electron-dense nucleocapsid core surrounded by an outer light halo with diameter of ∼ 60–70 nm and has viral nucleocapsids, with diameter of ∼ 30–40 nm, associated with its outer membrane (Grimley et al., 1968, Kaariainen et al., 1975). Based on previous 2D observations, CPV-II is further subclassified into CPV-II-1 and CPV-II-2 according to the presence or absence of associated tubular structures(Dan et al., 2021). The third type, CPV-0 is a large, irregular vesicle containing only mature virions (Dan et al., 2021). Previous observations of these CPVs were limited to two-dimensional views of a specific area, obtained through transmission electron microscopy (TEM) of ultra-thin sections. A more comprehensive understanding of the OVM life cycle within cancer cells is needed to fully describe the three-dimensional morphology of these vesicles and their interrelationships.

In this study, we utilized serial slice imaging by Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) to reconstruct three-dimensional volumes of virus-related structures in OVM-infected Hs578T cells. This approach allowed us to explore the spatial–temporal relationships among these vesicles within the entire cell and capture evidence of new transport pathways for progeny viruses and the release of mature virions through CPV.