Chloroquine phosphate targets the MAPK-ERK pathway to inhibit ASFV SY-1 replication in vitro

African swine fever (ASF) is a highly fatal disease caused by the African swine fever virus (ASFV). It infects domestic pigs and wild boars, causing significant economic losses worldwide. However, effective vaccines against this virus remain not be commercialized because of its large genome and high mutation frequency. Thus, antiviral therapies need to be developed urgently. Chloroquine phosphate (CQP) has been demonstrated in previous studies to exert inhibitory effects against a variety of viruses, but its inhibitory effect against the SY-1 strain of ASFV remains unclear. Therefore, we selected CQP as the research subject to investigate its anti-ASFV function. In this study, we confirmed that CQP has a significant inhibitory effect on the ASFV SY-1 strain by RT-qPCR, Western Blot, and HAD50. Transcriptome sequencing and KEGG pathway enrichment analysis showed that CQP treatment significantly affected multiple signaling pathways, including the cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, tumour necrosis factor (TNF) signaling pathway and IL-17 signaling pathway. Western Blot results further indicated that CQP can inhibit ERK phosphorylation. Treatment with the MAPK agonist C16-PAF reversed the inhibitory effect of CQP, verifying the key role of this pathway in the anti-viral mechanism of CQP. In sum, the results of this study indicate that CQP effectively inhibits ASFV replication by suppressing the MAPK-ERK signaling pathway. This study provides a theoretical basis and technical support for the development of anti-viral strategies targeting ASFV.