摘要Objective This study aimed to identify internal ribosome entry sites (IRESs) in the open reading frame (ORF) of the Coxsackievirus B3 (CVB3) genome. Methods The sequences of P1, P2, or P3 of the CVB3 genome or the truncated sequences from each antithymocyte globulin (ATG) to the end of the P1, P2, or P3 gene were inserted into the pEGFP-N1 vector. After transfection, possible IRES-dependent green fluorescent protein (GFP)-fused proteins were detected by anti-GFP western blotting. The sequences of possible IRESs were inserted into specific Fluc/Rluc bicistronic vectors, in which the potential IRESs were determined according to the Fluc/Rluc activity ratio. Expression of Fluc and Rluc mRNA of the bicistronic vector was detected by RT-qPCR. Results After transfection of full length or truncated sequences of the P1, P2, or P3 plasmids, six GFP-fused protein bands in P1, six bands in P2 and nine bands in P3 were detected through western blotting. Two IRESs in VP2 (1461–1646 nt) and VP1 (2784–2983 nt) of P1; one IRES in 2C (4119–4564 nt) of P2;and two IRESs in 3C (5634–5834 nt) and 3D (6870–7087 nt) of P3 were identified according to Fluc/Rluc activity ratio. The cryptic promoter was also excluded by RT-qPCR. Conclusion Five IRESs are present in the CVB3 coding region.