Soft X-ray cryotomography will be applied in ALBA MISTRAL to gain detailed insight into the components and architecture of the viral entry process and the replication organelles or viral factories (VFs) of African swine fever virus (ASFV).ASFV is a member of the Asfarviridae family, which is part of the Nucleocytoplasmic Large DNA Viruses (NCLDV) group and is closely related to Poxvirus. The mechanism of ASFV internalization in the host cell and the fusion membrane events that occur during early stages of infection are not well understood. The aim of this study is to elucidate the role of viral components, organelles, membranes, lipids, and other structures in the entry and viral replication site process. We will focus specially on the role of endosomes and microtubules as well as and how mitochondria are affected during the infection. This cellular complex is important for endosomal membrane fusion and subsequent release into the cytoplasm. Furthermore, this study aims to describe the remodeling of cellular compartments and cellular modifications induced at the viral factories. To achieve this, we will analyze the ASFV viral replication sites using Soft X-ray tomography of cryopreserved samples (Cryo-SXT), a synchrotron-based imaging technique, in two relevant cell types for ASFV: Vero cells and PK15 cells (Porcine kidney cells). Previously, we identified the recruitment of endosomal membranes to early African Swine Fever Virus (ASFV) factories, which combined with cholesterol are required for a productive infection. Additionally, we found that certain cellular proteins related to cholesterol transport are important for viral entry and the formation of viral factories.