IRES核心区12-bp非连续插入突变对猪塞内卡病毒复制和细胞嗜性的影响

doi:10.3864/j.issn.0578-1752.2024.07.015

中国农业科学 ›› 2024, Vol. 57 ›› Issue (7): 1407-1416.doi: 10.3864/j.issn.0578-1752.2024.07.015

• 畜牧·兽医 • 上一篇

IRES核心区12-bp非连续插入突变对猪塞内卡病毒复制和细胞嗜性的影响

张晓战¹(), 董轩志¹, 吕楠楠¹, 刘懿雯¹, 马新甜¹, 王林青², 夏艳勋¹, 蒋增海¹, 郭运泽¹, 赵攀登¹, 宋予震¹, 杨德成³(), 边传周¹()

¹ 河南牧业经济学院动物医药学院，郑州 450046
² 郑州师范学院/分子生物学郑州市重点实验室, 郑州 450044
³ 中国农业科学院哈尔滨兽医研究所/动物疫病防控全国重点实验室，哈尔滨 150069

收稿日期:2023-09-12 接受日期:2023-12-31 出版日期:2024-04-01 发布日期:2024-04-09
通信作者:
边传周，Tel：0371-86176197；E-mail：chuanzhou-bian@126.com。
杨德成，Tel：0451-51051774；E-mail：yangdecheng@caas.cn
联系方式: 张晓战，Tel：0371-86176275；E-mail：zhangxz@hnuahe.edu.cn。
基金资助:
国家自然科学基金(32002264); 河南省高校科技创新人才支持计划(24HASTIT061); 河南省科技攻关项目(232102110109); 河南省自然科学基金(222300420586); 河南牧业经济学院兽医重点学科基金(XJXK202202)

Effect of 12 Nucleotides Natural Insertion within the Internal Ribosome Entry Site Core Region on the Replication and Cellular Tropism of Porcine Senecavirus A

ZHANG XiaoZhan¹(), DONG XuanZhi¹, LÜ NanNan¹, LIU YiWen¹, MA XinTian¹, WANG LinQing², XIA YanXun¹, JIANG ZengHai¹, GUO YunZe¹, ZHAO PanDeng¹, SONG YuZhen¹, YANG DeCheng³(), BIAN Chuanzhou¹()

¹ College of Veterinary Medicine, Henan University of Animal Husbandry and Economy, Zhengzhou 450046,
² Zhengzhou Key Laboratory of Molecular Biology, Zhengzhou Normal University, Zhengzhou 450044,
³ Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences/State Key Laboratory for Animal Disease Control and Prevention, Harbin 150069

Received:2023-09-12 Accepted:2023-12-31 Published:2024-04-01 Online:2024-04-09

摘要/Abstract

摘要：

【背景】塞内卡病毒（senecavirus A, SVA）是新近暴发的一种引起猪特发性水疱病及仔猪死亡的小核糖核酸病毒。该病毒基因组5′非编码区（untranslated region, UTR）中的内部核糖体进入位点（IRES）元件在病毒复制过程中发挥重要作用。2017年我国出现IRES核心区Domain II 12个碱基非连续插入的自然突变SVA毒株，在病毒复制和致病性方面存在明显改变。【目的】探讨IRES Domain II区域发生的突变对SVA的复制及细胞嗜性的影响，为进一步了解SVA致病机制奠定基础。【方法】以实验室前期构建的含HeN-1/2018株全基因组感染性克隆pHeN-1/2018为基础，通过定点突变的方式，逐步将其IRES Domain II核心区域308—317 nt的9个碱基（ACTCAAGCG）替换为GD04/2017株基因组308—328 nt的21个碱基（CACGCCTGCCGATAGACGATT），构建重组载体pHeN-1/2018-i12并进行了病毒拯救。随后，利用病毒基因组克隆测序、间接免疫荧光试验和Western blot试验对所拯救病毒进行鉴定，同时验证了IRES核心区12个碱基插入突变对SVA病毒体外复制能力和细胞嗜性的影响。【结果】将构建完成的重组载体pHeN-1/2018-i12转染细胞，盲传至P2代，获得致使感染细胞病变明显、病变时间稳定的IRES突变病毒rHeN-1/2018-i12。病毒连续传代后测序结果表明rHeN-1/2018-i12遗传稳定，P5代病毒基因组序列未发生碱基突变，P10代病毒IRES区域没有出现突变现象。用低代次的突变病毒rHeN-1/2018-i12体外感染本体动物猪源细胞系PK-15和IBRS-2，及仓鼠源细胞系BHK-21，进行细胞感染试验，结果表明rHeN-1/2018-i12与亲本毒株rHeN-1/2018在PK-15、IBRS-2和BHK-21中均能导致明显的细胞病变，表现出相似的生长曲线趋势，表明IRES核心区Domain II 12个碱基突变不能够改变对上述细胞的嗜性；但SVA突变病毒和亲本株在不同细胞中的病变时间及病毒滴度上存在较大的差异，rHeN-1/2018-i12株在细胞中生长能力较其亲本株rHeN-1/2018差，诱使细胞病变的时间较晚，在感染24 hpi，两者病毒滴度相差可达10倍。【结论】本研究基于SVA反向遗传操作系统构建并拯救SVA IRES突变毒株，确定了IRES突变对SVA生物学特性的影响，有助于我们更好地了解SVA致病机制，拓宽我们对病毒IV型IRES功能的认识。

关键词: 塞内卡病毒, 内部核糖体进入位点, 反向遗传操作系统, 病毒复制, 细胞嗜性

Abstract:

【Background】Senecavirus A (SVA) is a newly emerged picornavirus causing swine idiopathic vesicular disease and epidemic transient neonatal losses. The internal ribosome entry site (IRES) located in 5’ untranslated region (UTR) of SVA genome plays a critical role in virus replication. In 2017, a natural mutant SVA strain, with 12 nucleotides discontinuously inserted into the IRES core region Domain II, was identified in China, and its replication capacity and pathogenicity changed significantly. 【Objective】The aim of this study was to investigate the effects of IRES Domain II mutations on SVA replication and cell tropism, and to lay a foundation for further understanding the pathogenesis of SVA. 【Method】An IRES mutant plasmid of pHeN-1/2018-i12 based on the background of pHeN-1/2018 were constructed, and the DNA-launched infectious clone of HeN-1/2018, with 9 nucleotides in IRES region of HeN-1/2018 genome (308-317 nt, ACTCAAGCG), were gradually replaced by the 21 nucleotides in GD04/2017 genome (308-328 nt, CACGCCTGCCGATAGACGATT) through multiple site-directed mutagenesis. The recombinant virus rHeN-1/2018-i12 was rescued and then identified by viral nucleotide genome examination, indirect immunofluorescence assay and Western blot assay, which was further examined the effect of 12 nucleotides natural insertion within the IRES core region Domain II on the replication and cellular tropism of SVA. 【Result】The pHeN-1/2018-i12 was then directly transfected into PK-15 cells and the recombinant virus rHeN-1/2018-i12 caused stable cytopathic effect was harvested after twice blind passages. Furthermore, the cellular tropism and growth kinetic of rHeN-1/2018-i12 was further investigated via virus infection assays. The viral genome of the IRES mutant virus rHeN-1/2018-i12 in the fifth and tenth passage were sequenced, and results showed that the IRES mutations passed on to the progeny viruses stably, with no nucleotide mutation in viral genome at fifth passage, and no nucleotide mutation in viral 5’-UTR region at tenth passage. Moreover, the growth characteristics of low passage recombinant virus rHeN-1/2018-i12 were further investigated in porcine cell lines PK-15 and IBRS-2, and hamster cell line BHK-21. The results showed that the recombinant virus rHeN-1/2018-i12 shared similar cellular tropism and growth dynamics with parental virus rHeN-1/2018, and all the two viruses could cause obvious CPE in PK-15 cells, IBRS-2 cells and BHK-21 cells, which indicated the mutation of 12 nucleotides insertion in the IRES core region Domain II had no significant difference in cellular tropism. Importantly, the virus-induced CPE time of rHeN-1/2018-i12 was later than that of rHeN-1/2018, and the viral titer of rHeN-1/2018-i12 was also lower than that of rHeN-1/2018 at the same time point post infection, especially at the 24 hpi, the difference of virus titer between the two viruses can be up to 10 times. 【Conclusion】The IRES mutant virus rHeN-1/2018-i12 was constructed and rescued, and further confirmed the influence of IRES mutation on SVA viral biological characteristics in this study, which provided an insight of the pathogenesis of SVA, and broadened our understanding of the function of viral type IV IRES.

Key words: sencavirus A, internal ribosome entry site, reverse genetics system, virus replication, cellular tropism

张晓战, 董轩志, 吕楠楠, 刘懿雯, 马新甜, 王林青, 夏艳勋, 蒋增海, 郭运泽, 赵攀登, 宋予震, 杨德成, 边传周. IRES核心区12-bp非连续插入突变对猪塞内卡病毒复制和细胞嗜性的影响[J]. 中国农业科学, 2024, 57(7): 1407-1416.

ZHANG XiaoZhan, DONG XuanZhi, LÜ NanNan, LIU YiWen, MA XinTian, WANG LinQing, XIA YanXun, JIANG ZengHai, GUO YunZe, ZHAO PanDeng, SONG YuZhen, YANG DeCheng, BIAN Chuanzhou. Effect of 12 Nucleotides Natural Insertion within the Internal Ribosome Entry Site Core Region on the Replication and Cellular Tropism of Porcine Senecavirus A[J]. Scientia Agricultura Sinica, 2024, 57(7): 1407-1416.

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名称 Name	引物序列 Primer sequences（5′→3′）
IRESmut-1 F	CAAGATTGCACGCCTCAAGCGCGGAATGCGCTG
IRESmut-1 R	TCCGCGCTTGAGGCGTGCAATCTTGTGGTTATC
IRESmut-2 F	GATTGCACGCCTGCCGAAGCGCGGAATGCGCTGTAAC
IRESmut-2 R	CATTCCGCGCTTCGGCAGGCGTGCAATCTTGTGG
IRESmut-3 F	CACGCCTGCCGATAGACGCGGAATGCGCTGTAACCAC
IRESmut-3 R	GCGCATTCCGCGTCTATCGGCAGGCGTGCAATCTTG
IRESmut-4 F	GCCGATAGACGATTCGGAATGCGCTGTAACCAC
IRESmut-4 R	GTTACAGCGCATTCCGAATCGTCTATCGGCAGG
SVA-UTR F	CTGGCTTATCGAAATTAATAC
SVA-UTR R	GTTCTGCATATTTGTATGTGC

IRES核心区12-bp非连续插入突变对猪塞内卡病毒复制和细胞嗜性的影响

Effect of 12 Nucleotides Natural Insertion within the Internal Ribosome Entry Site Core Region on the Replication and Cellular Tropism of Porcine Senecavirus A

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