Modulation of Fe3+ homeostasis by African Swine Fever virus facilitates its efficient replication in Ornithodoros lahorensis and pigs

African swine fever virus (ASFV) can be transmitted through direct contact and, in specific ecological settings, by Ornithodoros spp . soft ticks. However, the cellular mechanisms that support ASFV replication in both porcine host and tick vector remain poorly understood. Iron is essential for cellular metabolism and viral replication, but excessive labile iron can induce oxidative stress and ferroptosis-like morphological changes in mitochondria. Whether and how ASFV exploits intracellular Fe 3+ homeostasis to sustain its replication has not been fully defined. In this study, proteomic analysis of ASFV-infected PAM (pig) and OME/CTVM24 (derived from Ornithodoros moubata ) cells revealed a marked reduction in ferritin/FTH1 abundance, suggesting that ASFV infection alters intracellular iron storage and availability. Further analyses showed that ASFV dynamically regulated labile Fe 3+ levels during infection. At the early stage, ASFV promoted NCOA4-associated ferritin turnover, increased labile Fe 3+ release, enhanced mitochondria-endoplasmic reticulum contact, and induced ferroptosis-like changes in mitochondrial morphology. At later stages, ASFV restrained excessive iron-associated damage, thereby maintaining an intracellular environment compatible with continued viral replication. Pharmacological depletion of labile iron with deferoxamine mesylate markedly suppressed ASFV replication in both porcine and tick-derived cells, indicating that controlled Fe 3+ availability is required for efficient viral propagation. Mechanistically, the ASFV-encoded protein pA151R interacted with NCOA4 and promoted autophagic degradation of FTH1 in a manner dependent on its intact LC3-interacting region motif. pA151R also impaired mitochondrial outer membrane integrity and disrupted MDA5-MAVS/VISA signaling. Deletion of A151R impaired the ability of ASFV to modulate Fe 3+ homeostasis and significantly reduced viral replication. ASFV infection increased serum iron levels in pigs and caused abnormal iron redistribution and depletion in splenic tissues in vivo . Similar iron depletion and transient ferroptosis-like morphological changes in mitochondria were observed in the salivary glands of ASFV-infected O. lahorensis . Collectively, these findings demonstrate that ASFV exploits Fe 3+ homeostasis through a pA151R-NCOA4-FTH1-dependent mechanism to support replication in host and vector cells. This study provides new insights into ASFV metabolic regulation and identifies iron homeostasis and pA151R-mediated ferritinophagy as potential targets for strategies aimed at limiting ASFV replication and tick-associated transmission.