牛病毒性腹泻病毒诱导铁死亡对其复制水平的影响

doi:10.11843/j.issn.0366-6964.2024.12.034

摘要/Abstract

摘要：

本研究旨在探究牛病毒性腹泻病毒(BVDV)感染对牛肾细胞(Madin-Darby Bovine Kidney cells, MDBK)铁死亡的调控作用及其对病毒复制的影响。本研究通过激光共聚焦、Western blot、qPCR和电镜技术等技术检测BVDV感染MDBK细胞铁死亡发生与病毒复制水平，另外，使用铁死亡抑制剂Fer-1研究铁死亡对病毒复制和细胞炎性因子表达的影响。结果表明：BVDV(MOI=5)感染细胞较正常对照组细胞死亡率显著增加(P < 0.05)，感染细胞内Fe²⁺和脂质过氧化水平较正常细胞显著升高(P < 0.05)，透射电镜检测可见线粒体表面积变小、嵴减少以及膜密度增加等铁死亡发生特征；将铁死亡抑制剂Fer-1预处理MDBK细胞后，可显著抑制BVDV感染病毒复制水平(P < 0.05)；此外，BVDV感染诱导铁死亡对MDBK细胞中IL-1β、IL-18以及IFN-β等炎性因子呈正调控效应。以上结果提示，BVDV感染可引起宿主细胞铁死亡，且铁死亡发生可显著促进病毒复制水平以及宿主细胞炎性因子表达。

关键词: 牛病毒性腹泻病毒(BVDV), 铁死亡, 病毒复制, 炎性因子

Abstract:

This study aimed to investigate the regulatory effect of bovine viral diarrhoea virus(BVDV) infection on ferroptosis in Madin-Darby Bovine Kidney(MDBK) cells and its effect on viral replication. Technologies such as confocal microscopy, Western blot, qPCR and electron microscopy were used to detect ferroptosis and the level of viral replication in BVDV-infected cells. Furthermore, the viral replication level and inflammatory cytokines expression in BVDV-infected cells pretreated with Fer-1, a commonly used ferroptosis inhibitor, were also detected. The results showed that, compared with the normal control group, the mortality rate of cells infected with BVDV (MOI=5) was significantly increased (P < 0.05), which was accompanied with increased levels of Fe²⁺ and lipid peroxidation (P < 0.05). Transmission electron microscopy (TEM) analysis revealed that BVDV-infected and Erastin-treated cells displayed shrunk mitochondria with fewer cristae, increased mitochondrial membrane density and decreased mitochondrial mean areas compared with those of mock-infected cells, which was a typical morphological feature of ferroptosis. Fer-1 treatment significantly inhibited the levels of viral replication in BVDV-infected cells (P < 0.05). In addition, ferroptosis induced by BVDV infection had positive regulatory effects on inflammatory cytokines expression, including IL-1β, IL-18 and IFN-β. The results suggested that BVDV infection can induce ferroptosis in host cells, and the induction of ferroptosis enhanced the level of viral replication and the expression of inflammatory cytokines.

Key words: bovine viral diarrhoea virus (BVDV), ferroptosis, virus replication, inflammatory cytokines

中图分类号:

S852.653

张梓璇, 张颖, 李志军, 杨婧玲, 蒋子豪, 黄华敏, 齐雪峰. 牛病毒性腹泻病毒诱导铁死亡对其复制水平的影响[J]. 畜牧兽医学报, 2024, 55(12): 5716-5724.

ZHANG Zixuan, ZHANG Ying, LI Zhijun, YANG Jingling, JIANG Zihao, HUANG Huamin, QI Xuefeng. The Effects of Bovine Viral Diarrhoea Virus (BVDV)-induced Ferroptosis on Virus Replication[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(12): 5716-5724.

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基因名称 Genes name	引物 Primer	序列 Sequence	登录号 Accession No.	长度/bp Length
BVDV N^pro	F	CAACGCTAAAACTGCCACATAA	JX065783	124
BVDV N^pro	R	CCCTGGTTTTAAATAGATTCCACTC	JX065783	124
IFN-β^[10]	F	CCTGTGCCTGATTTCATCATGA	NM_174350	97
IFN-β^[10]	R	GCAAGCTGTAGCTCCTGGAAAG	NM_174350	97
IL-18^[11]	F	GAAAATGATGAAGACCTGGAATCA	NM_174091	84
IL-18^[11]	R	AACTTGGTCATTCAATTTCGTATGA	NM_174091	84
IL-1β^[11]	F	GCCTTCAATAACTGTGGAACCAAT	NM_174093	80
IL-1β^[11]	R	GTATATTTCAGGCTTGGTGAAAGGA	NM_174093	80
GAPDH^[10]	F	GGCAAGTTCAACGGCACAG	NM_001034034	248
GAPDH^[10]	R	TTCACGCCCATCACAAACA	NM_001034034	248

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