盐霉素体外对猪流行性腹泻病毒的抑制效果

doi:10.11843/j.issn.0366-6964.2024.04.028

摘要/Abstract

摘要： 猪流行性腹泻(porcine epidemic diarrhea，PED)是由猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV)引起的一种以水样腹泻、呕吐和脱水为主要特征的猪肠道传染病，给全球养猪业造成巨大的经济损失，但目前仍无有效的疫苗和治疗药物。盐霉素(salinomycin，SLM)是一种常用的抗生素，具有不易产生耐药性、排泄迅速、残留量极低的优点。为明确SLM对PEDV的抑制作用，首先通过TCID₅₀ (tissue culture infective dose)检测PEDV在Vero细胞上的增殖规律，进一步经CCK-8试验和细胞病变效应(cytopathic effect，CPE)测定SLM的半数细胞毒性浓度(half-cytotoxic concentration，CC₅₀)和其对PEDV的半数抑制浓度(median inhibition concentration，IC₅₀)，最后建立SLM、PEDV和Vero细胞的作用模型，利用RT-qPCR、免疫印迹(Western blot)和间接免疫荧光(indirect immunofluorescent assay，IFA)技术检测SLM对PEDV复制周期的影响。结果表明PEDV感染Vero细胞后24 h病毒滴度最高(10^7.7·0.1 mL^-1)；SLM对Vero细胞的CC₅₀为7.698 μmol·L^-1，对PEDV的IC₅₀为1.617 μmol·L^-1；随着SLM浓度的增加，PEDV滴度、N蛋白和N基因mRNA的表达量逐渐下降；SLM抑制PEDV的复制阶段，对病毒的吸附、入侵和释放阶段无明显影响。该研究结果可以为进一步挖掘PEDV抑制药物提供新思路。

关键词: PEDV CV777毒株, Vero细胞, 盐霉素, 复制周期

Abstract: Porcine epidemic diarrhea (PED) is a porcine intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV), characterized by watery diarrhea, vomiting and dehydration. It has caused huge economic losses to the global pig industry, but there are still no effective vaccines and treatments. Salinomycin (SLM) is a commonly used antibiotic with the advantages of low resistance, rapid excretion and very low residue. To explore the inhibitory effect of SLM on PEDV, firstly, the proliferation pattern of PEDV on Vero cells was detected by TCID₅₀, and then the CC₅₀ and IC₅₀ of SLM were measured by CCK-8 test and cytopathic effect (CPE). Finally, the co-culture models of SLM, PEDV and Vero cell were established. The effects of SLM on the replication cycle of PEDV were determined by RT-qPCR, Western blot and indirect immunofluorescent assay. The results showed that the highest virus titer (10^7.7·0.1 mL^-1) was observed in Vero cells at 24 hours after PEDV infection. The CC₅₀ of SLM on Vero cells was 7.698 μmol·L^-1, and the IC₅₀ for PEDV was 1.617 μmol·L^-1. The study showed that viral titer, N protein and N gene mRNA expression of PEDV gradually decreased with the increase of SLM concentration. SLM mainly inhibited the replication phase of PEDV, and had no significant effect on the adsorption, invasion and release phases of the virus. The research results could provide new ideas for further exploring PEDV inhibitory drugs.

Key words: PEDV CV777, Vero cell, salinomycin, replication cycle

中图分类号:

R978.7

马亚娟, 苏恺, 林依丹, 王亚文, 张亚楠, 袁洪兴, 袁晨, 宋勤叶. 盐霉素体外对猪流行性腹泻病毒的抑制效果[J]. 畜牧兽医学报, 2024, 55(4): 1661-1671.

MA Yajuan, SU Kai, LIN Yidan, WANG Yawen, ZHANG Yanan, YUAN Hongxing, YUAN Chen, SONG Qinye. The Inhibitory Effect of Salinomycin on Porcine Epidemic Diarrhea Virus in vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1661-1671.

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