{"title":"表达增强型绿色荧光蛋白的重组塞内卡病毒 A 的开发与表征。","authors":"Weihong Huang, Yongjie Chen, Ting Xu, Ting Xiong, Yadi Lv, Dingxiang Liu, Ruiai Chen","doi":"10.3389/fmicb.2024.1443696","DOIUrl":null,"url":null,"abstract":"<p><strong>Introduction: </strong>Senecavirus A (SVA), belonging to the genus <i>Senecavirus</i> in the family <i>Picornaviridae</i>, is an emerging pathogen causing vesicular disease in pigs. The main clinical manifestations of SVA infection include high mortality in neonatal piglets, skin ulceration, and vesicular lesions. So far, there is no commercially available vaccines or drugs against SVA. Construction of SVA infectious clones carrying reporter genes will help understand the characteristics of SVA and promote vaccine development.</p><p><strong>Methods: </strong>In this study, we established a reverse genetics system for a local SVA isolate and used it to rescue a recombinant SVA, rSVA-eGFP, expressing the enhanced green fluorescent protein (eGFP) by inserting eGFP, GSG linker and the P2A sequence between 2A and 2B genes.</p><p><strong>Results: </strong>We found that rSVA-eGFP exhibited a high replication efficiency comparable to the parental virus, was able to express the eGFP reporter efficiently and stable in maintaining the reporter gene up to six rounds of serial passages in BHK-21 cells. In mice, rSVA-eGFP also showed similar replication kinetics and pathogenicity to the parental virus, both causing mild lung lesions. In addition, a high-throughput viral neutralization assay was developed using eGFP as a surrogate readout in a fluorescence-based direct titration (FBT) assay based on rSVA-eGFP, facilitating rapid and accurate determination of the neutralizing antibody (nAb) titers.</p><p><strong>Discussion: </strong>The successful establishment of an SVA reverse genetics system and the rescue of rSVA-eGFP would create a powerful tool for future studies of SVA replication mechanisms and pathogenicity as well as for antiviral development.</p>","PeriodicalId":12466,"journal":{"name":"Frontiers in Microbiology","volume":null,"pages":null},"PeriodicalIF":4.0000,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11464439/pdf/","citationCount":"0","resultStr":"{\"title\":\"Development and characterization of a recombinant Senecavirus A expressing enhanced green fluorescent protein.\",\"authors\":\"Weihong Huang, Yongjie Chen, Ting Xu, Ting Xiong, Yadi Lv, Dingxiang Liu, Ruiai Chen\",\"doi\":\"10.3389/fmicb.2024.1443696\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Introduction: </strong>Senecavirus A (SVA), belonging to the genus <i>Senecavirus</i> in the family <i>Picornaviridae</i>, is an emerging pathogen causing vesicular disease in pigs. The main clinical manifestations of SVA infection include high mortality in neonatal piglets, skin ulceration, and vesicular lesions. So far, there is no commercially available vaccines or drugs against SVA. Construction of SVA infectious clones carrying reporter genes will help understand the characteristics of SVA and promote vaccine development.</p><p><strong>Methods: </strong>In this study, we established a reverse genetics system for a local SVA isolate and used it to rescue a recombinant SVA, rSVA-eGFP, expressing the enhanced green fluorescent protein (eGFP) by inserting eGFP, GSG linker and the P2A sequence between 2A and 2B genes.</p><p><strong>Results: </strong>We found that rSVA-eGFP exhibited a high replication efficiency comparable to the parental virus, was able to express the eGFP reporter efficiently and stable in maintaining the reporter gene up to six rounds of serial passages in BHK-21 cells. 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引用次数: 0
摘要
导言:猪细小病毒 A(SVA)属于细小病毒科猪细小病毒属,是一种新出现的导致猪水泡病的病原体。SVA 感染的主要临床表现包括新生仔猪死亡率高、皮肤溃疡和水泡病。迄今为止,市场上还没有针对 SVA 的疫苗或药物。构建携带报告基因的 SVA 感染克隆将有助于了解 SVA 的特征并促进疫苗的开发:本研究中,我们建立了本地 SVA 分离物的反向遗传学系统,并利用该系统通过插入 eGFP、GSG 连接子以及 2A 和 2B 基因之间的 P2A 序列,挽救了表达增强型绿色荧光蛋白(eGFP)的重组 SVA rSVA-eGFP:结果:我们发现,rSVA-eGFP具有与亲本病毒相当的高复制效率,能高效表达eGFP报告基因,并能在BHK-21细胞中稳定地维持报告基因达6轮系列传代。在小鼠体内,rSVA-eGFP 也表现出与亲本病毒相似的复制动力学和致病性,都会引起轻微的肺部病变。此外,在基于 rSVA-eGFP 的荧光直接滴定(FBT)检测中,使用 eGFP 作为替代读数,开发了一种高通量病毒中和检测方法,有助于快速准确地测定中和抗体(nAb)滴度:讨论:成功建立 SVA 反向遗传学系统并挽救 rSVA-eGFP 将为未来研究 SVA 复制机制和致病性以及开发抗病毒药物提供强有力的工具。
Development and characterization of a recombinant Senecavirus A expressing enhanced green fluorescent protein.
Introduction: Senecavirus A (SVA), belonging to the genus Senecavirus in the family Picornaviridae, is an emerging pathogen causing vesicular disease in pigs. The main clinical manifestations of SVA infection include high mortality in neonatal piglets, skin ulceration, and vesicular lesions. So far, there is no commercially available vaccines or drugs against SVA. Construction of SVA infectious clones carrying reporter genes will help understand the characteristics of SVA and promote vaccine development.
Methods: In this study, we established a reverse genetics system for a local SVA isolate and used it to rescue a recombinant SVA, rSVA-eGFP, expressing the enhanced green fluorescent protein (eGFP) by inserting eGFP, GSG linker and the P2A sequence between 2A and 2B genes.
Results: We found that rSVA-eGFP exhibited a high replication efficiency comparable to the parental virus, was able to express the eGFP reporter efficiently and stable in maintaining the reporter gene up to six rounds of serial passages in BHK-21 cells. In mice, rSVA-eGFP also showed similar replication kinetics and pathogenicity to the parental virus, both causing mild lung lesions. In addition, a high-throughput viral neutralization assay was developed using eGFP as a surrogate readout in a fluorescence-based direct titration (FBT) assay based on rSVA-eGFP, facilitating rapid and accurate determination of the neutralizing antibody (nAb) titers.
Discussion: The successful establishment of an SVA reverse genetics system and the rescue of rSVA-eGFP would create a powerful tool for future studies of SVA replication mechanisms and pathogenicity as well as for antiviral development.
期刊介绍:
Frontiers in Microbiology is a leading journal in its field, publishing rigorously peer-reviewed research across the entire spectrum of microbiology. Field Chief Editor Martin G. Klotz at Washington State University is supported by an outstanding Editorial Board of international researchers. This multidisciplinary open-access journal is at the forefront of disseminating and communicating scientific knowledge and impactful discoveries to researchers, academics, clinicians and the public worldwide.