Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (7): 1407-1416.doi: 10.3864/j.issn.0578-1752.2024.07.015

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

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. 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 Online:2024-04-01 Published:2024-04-09
  • Contact: YANG DeCheng, BIAN Chuanzhou

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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

Table 1

Primers information"

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

Fig. 1

Sequence alignment (A) and RNA structure analysis (B) of core IRES sequences of different SVA strains"

Fig. 2

Viral infection assays of different SVA strains in PK-15 cells"

Fig. 3

Indirect immunofluorescence assays of different SVA strains in pk-5 cells"

Fig. 4

Western blot assays of different SVA strains VP2 protein expression during infection"

Fig. 5

Identification of SVA 5’UTR region of different passages recombinant virus and parental virus A: PCR amplifications of 5’UTR region of different passages SVA; B: Sequencing analysis of 5’UTR region of different passages SVA"

Fig. 6

Growth kinetics of the recombinant virus and parental virus in different cells"

[1]
GUO B Q, PIÑEYRO P E, RADEMACHER C J, ZHENG Y, LI G W, YUAN J, HOANG H, GAUGER P C, MADSON D M, SCHWARTZ K J, CANNING P E, ARRUDA B L, COOPER V L, BAUM D H, LINHARES D C, MAIN R G, YOON K J. Novel Senecavirus A in swine with vesicular disease, United States, July 2015. Emerging Infectious Diseases, 2016, 22(7): 1325-1327.

doi: 10.3201/eid2207.151758 pmid: 27314645
[2]
CANNING P, CANON A, BATES J L, GERARDY K, LINHARES D C L, PIÑEYRO P E, SCHWARTZ K J, YOON K J, RADEMACHER C J, HOLTKAMP D, KARRIKER L. Neonatal mortality, vesicular lesions and lameness associated with senecavirus A in a U.S. sow farm. Transboundary and Emerging Diseases, 2016, 63(4): 373-378.

doi: 10.1111/tbed.12516 pmid: 27213868
[3]
ZHANG X L, ZHU Z X, YANG F, CAO W J, TIAN H, ZHANG K S, ZHENG H X, LIU X T. Review of Seneca valley virus: A call for increased surveillance and research. Frontiers in Microbiology, 2018, 9: 940.

doi: 10.3389/fmicb.2018.00940 pmid: 29867849
[4]
WU H G, LI C, JI Y C, MOU C X, CHEN Z H, ZHAO J W. The evolution and global spatiotemporal dynamics of senecavirus A. Microbiology Spectrum, 2022, 10(6): e0209022.
[5]
陶倩, 曹飞, 彭珂楠, 朱玲, 徐志文. 猪塞内卡病毒病原学研究进展. 病毒学报, 2022, 38(2): 505-512.
TAO Q, CAO F, PENG K N, ZHU L, XU Z W. Research progress on etiology of porcine Seneca virus. Chinese Journal of Virology, 2022, 38(2): 505-512. (in Chinese)
[6]
WILLCOCKS M M, LOCKER N, GOMWALK Z, ROYALL E, BAKHSHESH M, BELSHAM G J, IDAMAKANTI N, BURROUGHS K D, REDDY P S, HALLENBECK P L, ROBERTS L O. Structural features of the Seneca Valley virus internal ribosome entry site (IRES) element: A picornavirus with a pestivirus-like IRES. Journal of Virology, 2011, 85(9): 4452-4461.

doi: 10.1128/JVI.01107-10 pmid: 21325406
[7]
HALES L M, KNOWLES N J, REDDY P S, XU L, HAY C, HALLENBECK P L. Complete genome sequence analysis of Seneca Valley virus-001, a novel oncolytic picornavirus. The Journal of General Virology, 2008, 89(Pt 5): 1265-1275.

doi: 10.1099/vir.0.83570-0
[8]
LIU F X, WANG Q, WANG N, SHAN H. Impacts of single nucleotide deletions from the 3' end of Senecavirus A 5' untranslated region on activity of viral IRES and on rescue of recombinant virus. Virology, 2021, 563: 126-133.

doi: 10.1016/j.virol.2021.09.002 pmid: 34530232
[9]
LEE K M, CHEN C J, SHIH S R. Regulation mechanisms of viral IRES-driven translation. Trends in Microbiology, 2017, 25(7): 546-561.

doi: 10.1016/j.tim.2017.01.010
[10]
MARTINEZ-SALAS E, FRANCISCO-VELILLA R, FERNANDEZ- CHAMORRO J, EMBAREK A M. Insights into structural and mechanistic features of viral IRES elements. Frontiers in Microbiology, 2018, 8: 2629.

doi: 10.3389/fmicb.2017.02629
[11]
FRASER C S, DOUDNA J A. Structural and mechanistic insights into hepatitis C viral translation initiation. Nature Reviews Microbiology, 2007, 5: 29-38.

doi: 10.1038/nrmicro1558 pmid: 17128284
[12]
WANG M M, CHEN L L, PAN S N, MOU C X, SHI K C, CHEN Z H. Molecular evolution and characterization of novel Seneca Valley virus (SVV) strains in South China. Infection, Genetics and Evolution: Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases, 2019, 69: 1-7.
[13]
WANG Z, ZHANG X Z, YAN R Q, YANG P P, WU Y Y, YANG D D, BIAN C Z, ZHAO J. Emergence of a novel recombinant Seneca Valley virus in Central China, 2018. Emerging Microbes & Infections, 2018, 7(1): 180.
[14]
ZHANG X Z, LU J Z, DENG T W, ZHAO P D, PENG Z F, CHEN L L, QIAN M W, GUO Y W, QIAO H X, SONG Y Z, XIA Y X, BIAN C Z, WANG Z. Development of an improved dual-promoter-based reverse genetics system for emerging Senecavirus A. Journal of Virological Methods, 2020, 286: 113973.

doi: 10.1016/j.jviromet.2020.113973
[15]
SIEVERS F, HIGGINS D G. Clustal Omega for making accurate alignments of many protein sequences. Protein Science: a Publication of the Protein Society, 2018, 27(1): 135-145.
[16]
张晓战, 杨磊, 邓同炜, 赵攀登, 彭志锋, 陈露露, 郭懿文, 夏艳勋, 乔宏兴, 边传周, 王增. 表达绿色荧光蛋白重组猪塞内卡病毒的构建及初步应用. 畜牧兽医学报, 2021, 52(10): 2978-2985.
ZHANG X Z, YANG L, DENG T W, ZHAO P D, PENG Z F, CHEN L L, GUO Y W, XIA Y X, QIAO H X, BIAN C Z, WANG Z. Development and application of recombinant senecavirus A expressing the green fluorescent protein. Acta Veterinaria et Zootechnica Sinica, 2021, 52(10): 2978-2985. (in Chinese)
[17]
ROBERTS L, WIEDEN H J. Viruses,IRESs, and a universal translation initiation mechanism. Biotechnology & Genetic Engineering Reviews, 2018, 34(1): 60-75.
[18]
朱元源, 韩焘, 邹兴启, 范学政, 徐璐, 王琴, 赵启祖. 猪瘟病毒石门重组标记毒株的构建与拯救. 中国农业科学, 2013, 46(1): 187-194. doi: 10.3864/j.issn.0578-1752.2013.01.022.
ZHU Y Y, HAN T, ZOU X Q, FAN X Z, XU L, WANG Q, ZHAO Q Z. Construction and rescue of recombinant classical swine fever virus with Shimen structure protein and flag marker. Scientia Agricultura Sinica, 2013, 46(1): 187-194. doi: 10.3864/j.issn.0578-1752.2013.01.022. (in Chinese)
[19]
ARHAB Y, BULAKHOV A G, PESTOVA T V, HELLEN C U T. Dissemination of internal ribosomal entry sites (IRES) between viruses by horizontal gene transfer. Viruses, 2020, 12(6): 612.
[20]
樊帅, 钟函, 杨中元, 何文瑞, 万博, 魏战勇, 韩世充, 张改平. 非洲猪瘟病毒MGF110-5 L-6L蛋白诱导宿主细胞翻译阻滞和应激颗粒形成的作用机制. 中国农业科学, 2023, 56(7): 1401-1416. doi: 10.3864/j.issn.0578-1752.2023.07.016.
FAN S, ZHONG H, YANG Z Y, HE W R, WAN B, WEI Z Y, HAN S C, ZHANG G P. African swine fever virus MGF110-5L-6L induces host cell translation arrest and stress granule formation by activating the PERK/PKR-eIF2α pathway. Scientia Agricultura Sinica, 2023, 56(7): 1401-1416. doi: 10.3864/j.issn.0578-1752.2023.07.016. (in Chinese)
[21]
傅美贤, 龙健儿. 病毒IRES的结构及IRES介导的蛋白质翻译研究进展. 生命科学, 2021, 33(4): 407-418.
FU M X, LONG J E. Research progress on the structure of viral IRES and IRES-mediated protein translation. Chinese Bulletin of Life Sciences, 2021, 33(4): 407-418. (in Chinese)
[22]
AVANZINO B C, JUE H, MILLER C M, CHEUNG E, FUCHS G, FRASER C S. Molecular mechanism of poliovirus Sabin vaccine strain attenuation. The Journal of Biological Chemistry, 2018, 293(40): 15471-15482.

doi: 10.1074/jbc.RA118.004913
[23]
GUEST S, PILIPENKO E, SHARMA K, CHUMAKOV K, ROOS R P. Molecular mechanisms of attenuation of the Sabin strain of poliovirus type 3. Journal of Virology, 2004, 78(20): 11097-11107.

doi: 10.1128/JVI.78.20.11097-11107.2004 pmid: 15452230
[24]
SUN C, YANG D C, GAO R Y, LIANG T, WANG H W, ZHOU G H, YU L. Modification of the internal ribosome entry site element impairs the growth of foot-and-mouth disease virus in porcine-derived cells. The Journal of General Virology, 2016, 97(4): 901-911.

doi: 10.1099/jgv.0.000406
[25]
DI LIBERTO G, ROQUE-AFONSO A M, KARA R, DUCOULOMBIER D, FALLOT G, SAMUEL D, FERAY C. Clinical and therapeutic implications of hepatitis C virus compartmentalization. Gastroenterology, 2006, 131(1): 76-84.

pmid: 16831592
[26]
罗玉子, 孙元, 王涛, 仇华吉. 非洲猪瘟: 我国养猪业的重大威胁. 中国农业科学, 2018, 51(21): 4177-4187. doi: 10.3864/j.issn.0578-1752.2018.21.016.
LUO Y Z, SUN Y, WANG T, QIU H J. African swine fever: a major threat to the Chinese swine industry. Scientia Agricultura Sinica, 2018, 51(21): 4177-4187. doi: 10.3864/j.issn.0578-1752.2018.21.016. (in Chinese)
[27]
LIU F X, WANG Q Q, HUANG Y L, WANG N, SHAN H. A 5-year review of senecavirus A in China since its emergence in 2015. Frontiers in Veterinary Science, 2020, 7: 567792.

doi: 10.3389/fvets.2020.567792
[28]
ZHANG H W, CHEN P, HAO G X, LIU W Q, CHEN H C, QIAN P, LI X M. Comparison of the pathogenicity of two different branches of senecavirus a strain in China. Pathogens, 2020, 9(1): 39.
[29]
GAO H, CHEN Y J, XU X Q, XU Z Y, XU S J, XING J B, LIU J, ZHA Y F, SUN Y K, ZHANG G H. Comprehensive phylogeographic and phylodynamic analyses of global Senecavirus A. Frontiers in Microbiology, 2022, 13: 980862.

doi: 10.3389/fmicb.2022.980862
[30]
ZHAO K, ZHANG S X, LIU X N, GUO X R, GUO Z M, ZHANG X Z, YUAN W Z. The game between host antiviral innate immunity and immune evasion strategies of senecavirus A-A cell biological perspective. Frontiers in Immunology, 2022, 13: 1107173.

doi: 10.3389/fimmu.2022.1107173
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