Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). SP40 peptide was previously identified to inhibit EV-A71 strains from genotypes A, B and C. However, the stability and antiviral activity of SP40 peptide in human serum are yet to be established. To address this, we evaluated the stability and anti-EV-A71 activity of SP40 peptide after incubation in 25 % human serum. Reverse-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC/MS) were utilized to evaluate serum stability and cleavage patterns of SP40 peptide after incubation in human serum. Cell protection assay was used to evaluate the anti-EV-A71 activity of SP40 peptide after incubation in human serum and to identify the minimal active sequence of SP40 peptide that retained antiviral activity. The results showed that the SP40 peptide was stable in human serum with 56 % of the full-length SP40 peptide being detected after 48 h incubation in human serum. The SP40 peptide was mainly cleaved by exopeptidases and no endoprotease recognition sites were identified within the SP40 peptide. Cell protection assays revealed that the SP40 peptide retained substantial activity after 24 and 48 h incubation in human serum. Furthermore, the data revealed that three amino acids at the N-terminus and one amino acid at the C-terminus of the SP40 peptide were dispensable for its antiviral activity. Importantly, the four truncated peptides displayed better potency than the full-length SP40 peptide. Overall, this study provided insights into the stability and activity of SP40 peptide in human serum and will facilitate the development of SP40 peptide as an anti-EV-A71 agent.

肠道病毒 A71 (EV-A71) 是手足口病 (HFMD) 的主要病原体之一。SP40 肽先前被鉴定为抑制基因型 A、B 和 C 的 EV-A71 菌株。然而，SP40 肽在人血清中的稳定性和抗病毒活性尚未确定。为了解决这个问题，我们评估了 SP40 肽在 25% 人血清中孵育后的稳定性和抗 EV-A71 活性。反相高效液相色谱 (RP-HPLC) 和液相色谱-质谱 (LC/MS) 用于评估 SP40 肽在人血清中孵育后的血清稳定性和切割模式。细胞保护试验用于评估 SP40 肽在人血清中孵育后的抗 EV-A71 活性，并确定保留抗病毒活性的 SP40 肽的最小活性序列。结果表明，SP40 肽在人血清中是稳定的，在人血清中孵育 48 小时后检测到 56% 的全长 SP40 肽。SP40 肽主要被外肽酶切割，在 SP40 肽内未鉴定出内切蛋白酶识别位点。细胞保护测定表明，SP40 肽在人血清中孵育 24 和 48 小时后仍保持显着活性。此外，数据显示，SP40 肽的 N 端的三个氨基酸和 C 端的一个氨基酸对其抗病毒活性是可有可无的。重要的是，四种截短肽显示出比全长 SP40 肽更好的效力。总体，