蓝舌病病毒感染对绵羊微血管内皮细胞中Ⅰ型干扰素应答的影响

doi:10.11843/j.issn.0366-6964.2024.12.026

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (12): 5631-5640.doi: 10.11843/j.issn.0366-6964.2024.12.026

蓝舌病病毒感染对绵羊微血管内皮细胞中Ⅰ型干扰素应答的影响

罗世美¹(), 陈韵伊¹(), 李其沙¹, 周艳梅¹, 胡雪柔¹, 李铭悦¹, 魏子昱¹, 李芳², 马鲜平¹^,*(), 易华山¹^,*()

1. 西南大学动物医学院, 重庆 402460
2. 重庆市大足区动物疫病预防控制中心, 重庆 402360

收稿日期:2024-01-23 出版日期:2024-12-23 发布日期:2024-12-27
通讯作者: 马鲜平,易华山 E-mail:1918239281@qq.com;2249029810@qq.com;124520096@qq.com;dyxyihuashan@swu.edu.cn
作者简介:罗世美(1998-), 女, 云南昭通人, 硕士生, 主要从事兽医临床感染与免疫研究, E-mail: 1918239281@qq.com
陈韵伊(2000-), 女, 云南丽江人, 硕士生, 主要从事兽医临床感染与免疫研究, E-mail: 2249029810@qq.com
第一联系人：
罗世美和陈韵伊为同等贡献作者
基金资助:
重庆市自然科学面上项目基金(CSTB2022NSCQ-MSX0419);重庆市基础研究与前沿探索专项项目基金(cstc2018jcyjAX0615)

Effect of Bluetongue Virus Infection on Type Ⅰ Interferon Responses in Sheep Lung Microvascular Endothelial Cells

LUO Shimei¹(), CHEN Yunyi¹(), LI Qisha¹, ZHOU Yanmei¹, HU Xuerou¹, LI Mingyue¹, WEI Ziyu¹, LI Fang², MA Xianping¹^,*(), YI Huashan¹^,*()

1. College of Veterinary Medicine, Southwest University, Chongqing 402460, China
2. Dazu Center for Animal Disease Prevention and Control, Chongqing 402360, China

Received:2024-01-23 Online:2024-12-23 Published:2024-12-27
Contact: MA Xianping, YI Huashan E-mail:1918239281@qq.com;2249029810@qq.com;124520096@qq.com;dyxyihuashan@swu.edu.cn

摘要/Abstract

摘要：

蓝舌病病毒(bluetongue virus，BTV)是一种严重危害绵羊等反刍动物的虫媒病毒，而绵羊微血管内皮细胞(sheep lung microvascular endothelial cells，SLMECs)构成肺半选择性屏障，为研究BTV感染与SLMECs干扰素抗病毒免疫应答的分子机制。本研究通过BTV噬斑试验以感染复数(MOI)为1的BTV诱导18、24、36 h的SLMECs，通过实时荧光定量PCR分析干扰素相关通路基因mRNA表达特征，以及Western blot分析MDA5、TRAF3、RIG-I和TBK1蛋白表达。结果表明，在诱导36 h时，干扰素信号通路基因mRNA表达的变化最明显，RIG-I、MDA5、IKKε、IFN-β、TBK1和TRAF6基因mRNA上调表达，差异极显著；而IFN-α、VISA、USP18基因mRNA下调表达，差异极显著；在蛋白水平上MDA5、TBK1、RIG-I和TRAF3蛋白都上调表达。本研究发现BTV可感染SLMECs诱导Ⅰ型干扰素免疫应答，为进一步分析IFN-I信号通路的激活在抗BTV感染天然免疫应答中的作用机制研究奠定基础。

关键词: 蓝舌病病毒, Ⅰ型干扰素, 绵羊肺微血管内皮细胞

Abstract:

Bluetongue virus (BTV) is an arbovirus that seriously harms ruminants such as Sheep, while sheep lung microvascular endothelial cells (SLMECs) constitute a semi-selective barrier in order to study the molecular mechanism of interferon antiviral immune response in BTV infection and SLMECs. In this study, BTV plaque assay was used to induce SLMECs at 18 h, 24 h and 36 h with multiple infection (MOI) 1. The mRNA expression characteristics of interferon-related pathway genes were analyzed by real-time fluorescence quantitative PCR, and the protein expressions of MDA5, TRAF3, RIG-I and TBK1 were analyzed by Western blot. The results showed that the changes of interferon signaling pathway gene expression were most obvious, and the mRNA expressions of RIG-I, MDA5, IKKε, IFN-β, TBK1 and TRAF6 genes were up-regulated, and the differences were extremely significant (P < 0.01) at 36 h of BTV induction, while the mRNA expressions of IFN-α, VISA and USP18 genes were significantly downregulated (P < 0.01). At the protein level, the protein expressions of MDA5, TBK1, RIG-I and TRAF3 were mainly upregulated. This study found that BTV infection can induce an IFN immune response of SLMECs, which lays a foundation for further analysis of the mechanism of IFN-Ⅰ signaling pathway activation in the innate immune response against BTV infection.

Key words: bluetongue virus, type Ⅰ interferon, sheep lung microvascular endothelial cells

中图分类号:

S852.659.4

罗世美, 陈韵伊, 李其沙, 周艳梅, 胡雪柔, 李铭悦, 魏子昱, 李芳, 马鲜平, 易华山. 蓝舌病病毒感染对绵羊微血管内皮细胞中Ⅰ型干扰素应答的影响[J]. 畜牧兽医学报, 2024, 55(12): 5631-5640.

LUO Shimei, CHEN Yunyi, LI Qisha, ZHOU Yanmei, HU Xuerou, LI Mingyue, WEI Ziyu, LI Fang, MA Xianping, YI Huashan. Effect of Bluetongue Virus Infection on Type Ⅰ Interferon Responses in Sheep Lung Microvascular Endothelial Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(12): 5631-5640.

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目的基因 Target genes	引物序列(5′→3′) Primer sequences(5′→3′)	用途 Usage
β-Actin	F: CCACTGTCGAGTCGCGTCC	qRT-PCR
	R: ATTCCCACCATCACACCCTGG
RIG-Ⅰ	F: CTGACTGCCTCGGTTGGTGTTG	qRT-PCR
	R: CTTGCTCCAGTTCCTCCAGATTGTG
MDA5	F: AGCTTCACCTGGTGTTGGAG	qRT-PCR
	R: TGGCAAACTTCTTGCATGGC
VISA	F: AGCCATCCCAAGGAATCCAG	qRT-PCR
	R: GAGACAGATGGAGACACAGGC
TBK1	F: TGGAAGCGGCAGAGTTAGGTGAA	qRT-PCR
	R: TTCGGATGAGTGCCTTCTTGATGTG
IKKε	F: TGGGAATTCATGCAGAGCACAGCCAATT	qRT-PCR
	R: TGTGGTACCTCAGACATCAGGAGGTGCT
TRAF3	F: CTGCAGGTTTCCTTGTTGCAG	qRT-PCR
	R: TGCTTGGCTGTCTATCACTCG
TRAF6	F: GAGATCCATGACCAGAACTGTCCTT	qRT-PCR
	R: GGCGTGCCAAGTGATTCCTCT
USP18	F: ATCACGAATGAGCAAGGCGT	qRT-PCR
	R: CAACCAGGCCATGAGGGTAG
IFN-α	F: CTCATACACCAGGTCACGCT	qRT-PCR
	R: AGTGTAAAGGTGCACATGACG
IFN-β	F: TAGGCGACACTGTTCGTGTT	qRT-PCR
	R：CCATTCAATTGCCACAGGAGC
VP7	F: ATGGACACTATCGCTGCAAG	RT-PCR
	R：CTACACATAGGCGGCGCGT

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蓝舌病病毒感染对绵羊微血管内皮细胞中Ⅰ型干扰素应答的影响

Effect of Bluetongue Virus Infection on Type Ⅰ Interferon Responses in Sheep Lung Microvascular Endothelial Cells

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