|
|
|
| Construction and Biological Characteristics Analysis of Recombinant Senecavirus A with 5' UTR Deletion |
| ZHANG Yan1, YANG Fan2,3, CAO Wei-Jun2,3, DU Xiao-Li4, ZHENG Hai-Xue1,2,3,*, BAO Shi-Jun1,* |
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China;
2 Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences/College of Veterinary Medicine, Lanzhou University/State Key Laboratory for Animal Disease Control and Prevention/National Foot-and-Mouth Disease Reference Laboratory, Lanzhou 730000, China;
3 Gansu Province Research Center for Basic Disciplines of Pathogen Biology, Lanzhou 730046, China;
4 College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730030, China |
|
|
|
|
Abstract Senecavirus A (SVA) is a small RNA virus belonging to the genus Senecavirus that causes vesicular disease in pigs (Sus scrofa), which is clinically difficult to distinguish from Foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. In this study, a mutant SVA strain with a 13-nucleotide (nt) deletion in the 5' untranslated region (5' UTR) was successfully identified. To investigate the impact of this deletion on SVA's biological characteristics, this study employed reverse genetics technology, using the full-length cDNA infectious clone of the SVA CH/FJ/2017 strain as a template, and a targeted deletion in the genomic 5'UTR sequence was introduced.The recombinant plasmid was then transfected into BHK-21 cells, successfully rescuing a 5'UTR-deleted Seneca virus strain designated rSVA-5UTRM. The recombinant virus was characterized by indirect immunofluorescence assay (IFA) and Western blot, and its genetic stability was analyzed through serial passages in BHK-21 cells. Subsequently, plaque assays and viral growth curves were performed to compare the replication characteristics between the recombinant virus and parental strain in BHK-21 cells. The results demonstrated that the recombinant virus exhibited specific reactivity with SVA antibodies and SVA VP2 antibodies, confirming the successful rescue of the recombinant strain. Genetic stability analysis was performed by serially passaging the recombinant virus in BHK-21 cells for up to 20 generations, indicating excellent genetic stability of the recombinant strain. Virus biological characterization revealed that the recombinant strain exhibited similar replication properties to the parental SVA CH/FJ/2017 strain in BHK-21 cells and maintained a high viral titer. These findings suggested that the 13-nt deletion in the 5' UTR did not affect viral replication in BHK-21 cells. This study successfully rescued a recombinant SVA strain with a 5' UTR deletion, investigated the functions of the deleted region, and provided new scientific data for elucidating the non-coding functions of SVA.
|
|
Received: 19 December 2024
|
|
|
|
Corresponding Authors:
* zhenghaixue@caas.cn; bsjdy@126.com
|
|
|
|
[1] Barton D J, O'Donnell B J, Flanegan J B.2001. 5' cloverleaf in Poliovirus RNA is a cis-acting replication element required for negative-strand synthesis[J]. EMBO Journal, 20(6): 1439-1448.
[2] Ben M H, Olivia M, Joshua D, et al.2016. Senecavirus A in pigs, United States, 2015[J]. Emerging Infectious Diseases, 22(7): 1323-1325.
[3] Bouin A, Vu M N, Al-Hakeem A, et al.2023. Enterovirus-cardiomyocyte interactions: Impact of terminally deleted genomic RNAs on viral and host functions[J]. Journal of Virology, 97(1): 1-16.
[4] Brierley I, Pennell S, Gilbert J C.2007. Viral RNA pseudoknots: Versatile motifs in gene expression and replication[J]. Nature Reviews Microbiology, 5(8): 598-610.
[5] Bullman S, Kearney K, O' Mahony M, et al.2014. Identification and genetic characterization of a novel Picornavirus from chickens[J]. Journal of General Virology, 95(5): 1094-1103.
[6] Corbett V, Hallenbeck P, Rychahou P, et al.2022. Evolving role of Seneca valley virus and its biomarker TEM8/ANTXR1 in cancer therapeutics[J]. Frontiers in Molecular Biosciences, 9: 930207.
[7] Flather D, Semler B L.2015. Picornaviruses and nuclear functions: Targeting a cellular compartment distinct from the replication site of a positive-strand RNA virus[J]. Frontiers in Microbiology, 6: 594
[8] Glenet M, Heng L, Callon D, et al.2020. Structures and functions of viral 5' non-coding genomic RNA domain-I in group-B enterovirus[J]. Viruses, 12(9): 919.
[9] Joshi L R, Fernandes M H V, Clement T, et al.2016. Pathogenesis of Senecavirus A infection in finishing pigs[J]. Journal of General Virology, 97(12): 3267-3279.
[10] Lian K, Yang F, Zhu Z, et al.2015. Recovery of infectious type Asia1 foot-and-mouth disease virus from suckling mice directly inoculated with an RNA polymeraseⅠ/Ⅱ-driven unidirectional transcription plasmid[J]. Virus Research, 208: 73-81.
[11] Liu F X, Wang Q, Meng H L, et al.2022. Experimental evidence for occurrence of putative copy-choice recombination between two Senecavirus A genomes[J]. Veterinary Microbiology, 271: 109487.
[12] Liu F X, Wang Q, Wang N, et al.2021. 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[J]. Virology, 563: 126-133.
[13] Liu Z G, Zhao X M, Mao H, et al.2013. Intravenous injection of oncolytic Picornavirus SVV-001 prolongs animal survival in a panel of primary tumor-based orthotopic xenograft mouse models of pediatric glioma[J]. Neuro-Oncology, 15(9): 1173-1185.
[14] Lyons T, Murray K E, Roberts A W, et al.2001. Poliovirus 5'-terminal cloverleaf RNA is required in for VPg uridylylation and the initiation of negative-strand RNA synthesis[J]. Journal of Virology, 75(22): 10696-10708.
[15] Martínez-Salas E, Francisco-Velilla R, Fernandez-Chamorro J, et al.2015. Picornavirus IRES elements: RNA structure and host protein interactions[J]. Virus Research, 206: 62-73.
[16] Meng H L, Wang Q, Liu M L, et al.2022. The 5'-end motif of Senecavirus A cDNA clone is genetically modified in 36 different ways for uncovering profiles of virus recovery[J]. Frontiers in Microbiology, 13: 957849.
[17] Montiel N, Buckley A, Guo B Q, et al.2016. Vesicular disease in 9-week-old pigs experimentally infected with Senecavirus A[J]. Emerging Infectious Diseases, 22(7): 1246-1248.
[18] Nagashima S, Sasaki J, Taniguchi K.2005. The 5'-terminal region of the Aichi virus genome encodes acting replication elements required for positive-and negative-strand RNA synthesis[J]. Journal of Virology, 79(11): 6918-6931.
[19] Pascal, S M, Garimella R, Warden M S,et al., 2020. Structural biology of the enterovirus replication-linked 5'-cloverleaf RNA and associated virus proteins[J]. Microbiology and Molecular Biology Reviews, 84(2): e00062-19.
[20] Paul A V, Wimmer E.2015. Initiation of protein-primed Picornavirus RNA synthesis[J]. Virus Research, 206: 12-26.
[21] Segalés J, Barcellos D, Alfieri A, et al.2016. Senecavirus A[J]. Veterinary Pathology, 54(1): 11-21.
[22] Sasaki J, Kusuhara Y, Maeno Y, et al.2001. Construction of an infectious cDNA clone of Aichi virus (a new member of the family picornaviridae) and mutational analysis of a stem-loop structure at the 5' end of the genome[J]. Journal of Virology, 75(17): 8021-8030.
[23] Vannucci F A, Linhares D C, Barcellos D E, et al.2015. Identification and complete genome of Seneca valley virus in vesicular fluid and sera of pigs affected with idiopathic vesicular disease, Brazil[J]. Transboundary and Emerging Diseases, 62(6): 589-593.
[24] Willcocks M M, Locker N, Gomwalk Z, et al.2011. Structural features of the Seneca valley virus internal ribosome entry site (IRES) element: A Picornavirus with a pestivirus-like IRES[J]. Journal of Virology, 85(9): 4452-4461.
[25] Xu W H, Hole K, Goolia M, et al.2017. Genome wide analysis of the evolution of Senecavirus A from swine clinical material and assembly yard environmental samples[J]. PLOS ONE, 12(5): e0176964.
[26] Zhu S, Guo X, Keyes L R, et al.2015. Recombinant Encephalomyocarditis viruses elicit neutralizing antibodies against PRRSV and CSFV in mice[J]. PLOS ONE, 10(6): e0129729.
[27] Zhu Z, Yang F, Chen P, et al.2017. Emergence of novel Seneca valley virus strains in China, 2017[J]. Transboundary and Emerging Diseases, 64: 1024-1029. |
| [1] |
YAN Hui-Xuan, YAO Ting-Ting, CAO Yi-Fan, LI Xin-Yi, CIREN Luo-Bu, SUOLANG Qu-Ji, NIMA Cang-Jue, DANZENG Luo-Sang, SILANG Wang-Mu, LEI Chu-Zhao, BASANG Zhu-Zha, CHEN Ning-Bo. Study on Paternal Genetic Diversity of Cattle (Bos taurus) in Qinghai-Tibet Plateau[J]. 农业生物技术学报, 2025, 33(8): 1755-1766. |
|
|
|
|
