鸭干扰素诱导的跨膜蛋白抑制基因3型鸭甲型肝炎病毒的增殖

doi:10.11843/j.issn.0366-6964.2022.03.015

摘要/Abstract

摘要： 旨在研究鸭干扰素诱导的跨膜蛋白(duck interferon-induced transmembrane proteins,duIFITMs)和相关细胞因子在鸭甲型肝炎病毒3型(duck hepatitis A virus genotype 3,DHAV-3)感染早期的变化规律,以及duIFITMs对DHAV-3的抑制作用,本研究对DHAV-3感染早期雏鸭肝中mRNA进行高通量测序分析,通过Real-time PCR验证duIFITMs及其相关细胞因子的变化;分别用pEGFP-duIFITM1和duIFITM1-siRNA构建duIFITM1过表达和敲减的鸭胚肝原代细胞(duck embryo liver cell,DELC)模型,通过该模型评价了duIFITM1对DHAV-3增殖的影响。结果显示:转录组测序分析及验证结果均表明,DHAV-3感染雏鸭后24和36 h duIFITM1显著上调(P<0.01),IFITM5无明显变化。进一步用DHAV-3感染duIFITM1过表达和敲减的DELC细胞,与对照组和敲减组相比在过表达细胞中,在48、60 h DHAV-3拷贝数和病毒滴度均明显下降(P<0.01)。此外,经转录组测序分析共筛选出211个与抗DHAV-3免疫反应相关分子和74条显著富集的信号通路。经Real-time PCR检测,在攻毒后12和24 h肝中RIG-I和MDA5的表达水平与对照组差异不显著,但在36 h上调到对照组的4.23倍(P<0.01)和3.61倍(P<0.05),这与IFN-α/IFN-β早期表达滞后的趋势也恰好一致。同时,感染早期IRF1和IRF3的表达水平也显著上调。本研究首次探明duIFITM1对DHAV-3具有抑制作用,为以此开发新型抗病毒药物奠定了基础。对抗病毒感染早期的多个关键免疫分子表达变化规律的系统研究分析,可为DHAV-3感染与免疫的分子机制研究提供重要参考。

关键词: 干扰素诱导跨膜蛋白, 鸭甲型肝炎病毒, 转录组学, 细胞因子, 过表达, RNA干扰

Abstract: In order to investigate the changes of expression levels of duck interferon-induced transmembrane proteins (duIFITMs) and related cytokines in the early stage of duck hepatitis A virus genotype 3 (DHAV-3) infection, and to evaluate the inhibitory effect of duIFITMs against proliferation of DHAV-3, the high-throughput sequencing and real-time PCR were used to evaluate the expression levels of duIFITMs and their related cytokines in the liver of ducklings at the early stage by the infection of DHAV-3. The cell models of DELC with duIFITM1 over-expression or knockdown were constructed by transfection of pEGFP-duIFITM1 and duIFITM1-siRNA, respectively. Then, the effect of duIFITM1 on DHAV-3 proliferation was evaluated. The results showed that duIFITM1 was significantly up-regulated at 24 and 36 h after DHAV-3 infection in ducklings (P<0.01), but duIFITM5 had no change in the early stage of DHAV-3 infection. Then dulIFITM1 over-expressing and knockdown DELC were infected with DHAV-3. Compared with control group and siRNA interference group, the number of DHAV-3 copies and virus titers decreased in the duIFITM1 overexpression group significantly at both 48 and 60 h (P<0.01). Furthermore, a total of 211 molecules and 74 enriched signaling pathways, which related to immune response against DHAV-3 were founded by transcriptome analyses. The results of real-time PCR detection confirmed that the levels of mRNA of RIG-I and MDA5 had no changes in livers at 12 and 24 h after inoculated by DHAV-3, but it increased to 4.23 times (P<0.01) and 3.61 times (P<0.05) in 36 h compared with the control group, these expression changes of RLRs coincided with that of IFN-α/IFN-β in early stage of DHAV-3 infection. Besides, the expression levels of IRF1 and IRF3 were upregulated significantly in early stage. It was the first time to be demonstrated that the duIFITM1 have an inhibitory effect against DHAV-3 proliferation, which could provide theoretical foundations for the development of new anti-DHAV drugs. Systematic study and analysis of the expression levels of a variety of key immune molecules in the early stage of anti-viral infection might be an important reference for the further exploration of the molecular mechanisms of DHAV-3 infection and immunity.

Key words: interferon-inducible transmembrane protein, duck hepatitis A virus, transcriptome, cytokine, overexpression, RNA interference

中图分类号:

S852.659.6

王一丹, 陈弟诗, 向华, 张焕容, 任玉鹏. 鸭干扰素诱导的跨膜蛋白抑制基因3型鸭甲型肝炎病毒的增殖[J]. 畜牧兽医学报, 2022, 53(3): 822-833.

WANG Yidan, CHEN Dishi, XIANG Hua, ZHANG Huanrong, REN Yupeng. Inhibitory Effect of Duck Interferon-induced Transmembrane Proteins against Proliferation of DHAV-3[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 822-833.

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