Reverse genetics system for Tick-borne encephalitis virus using Circular Polymerase Extension Reaction

IF 2.6 4区医学 Q3 INFECTIOUS DISEASES

Infection Genetics and Evolution Pub Date : 2025-06-01 DOI:10.1016/j.meegid.2025.105776

Saki Mitsunaga , Tomokazu Tamura , Samuel Nyampong , Takasuke Fukuhara , Hiroshi Shimoda , Daisuke Hayasaka

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引用次数: 0

Abstract

Tick-borne encephalitis virus (TBEV) belongs to the Family Flaviviridae, Genus Orthoflavivirus, and causes severe neurological diseases in humans. The reverse genetics system is a basic tool for viral research; however, cloning the genome of orthoflavivirus using bacterial plasmids is difficult because of their toxicity to Escherichia coli. Polymerase chain reaction-based Circular Polymerase Extension Reaction (CPER), an E. coli-free reverse genetics system, has been recently applied to several RNA viruses. However, there have been no reports on recombinant TBEV produced using CPER. In this study, we attempted to produce recombinant TBEV Oshima, Sofjin and Hypr strains by CPER. Genome sequencing and plaque-forming assays were performed to determine the properties of rescued TBEVs. In addition, infectivity and pathogenicity of rescued TBEVs was also monitored in C57BL/6 mice. Rescued TBEVs of Oshima, Sofjin and Hypr caused apparent cytopathic effect and efficient propagations in BHK cells, and showed intrinsic virulence in mice. The rescued TBEVs showed several nucleotide and amino acid substitutions compared to the original viral sequences. These results showed that infectious TBEVs from the Oshima, Sofjin, and Hypr strains were produced using CPER. We propose that future applications of this method will contribute to related research on TBEV.

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利用循环聚合酶延伸反应的蜱传脑炎病毒反向遗传系统

蜱传脑炎病毒（TBEV）属于黄病毒科正黄病毒属，可引起人类严重的神经系统疾病。逆向遗传系统是病毒研究的基本工具；然而，利用细菌质粒克隆正黄病毒基因组是困难的，因为它们对大肠杆菌有毒性。基于聚合酶链反应的环状聚合酶延伸反应（CPER）是一种无大肠杆菌的反向遗传系统，近年来已应用于几种RNA病毒。然而，目前还没有使用CPER生产重组TBEV的报道。在本研究中，我们尝试用CPER法制备重组TBEV Oshima、Sofjin和Hypr菌株。通过基因组测序和斑块形成分析来确定获救的tbev的特性。此外，在C57BL/6小鼠中监测了获救的tbev的传染性和致病性。Oshima、Sofjin和Hypr三种被拯救的tbev在小鼠BHK细胞中产生明显的细胞病变作用和高效繁殖，并表现出内在的毒力。与原始病毒序列相比，获救的tbev显示出几个核苷酸和氨基酸的取代。结果表明，利用CPER技术可从Oshima、Sofjin和Hypr菌株中获得传染性tbev。我们认为，该方法的进一步应用将有助于TBEV的相关研究。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Infection Genetics and Evolution 医学-传染病学

CiteScore

8.40

自引率

0.00%

发文量

215

审稿时长

82 days

期刊介绍： (aka Journal of Molecular Epidemiology and Evolutionary Genetics of Infectious Diseases -- MEEGID) Infectious diseases constitute one of the main challenges to medical science in the coming century. The impressive development of molecular megatechnologies and of bioinformatics have greatly increased our knowledge of the evolution, transmission and pathogenicity of infectious diseases. Research has shown that host susceptibility to many infectious diseases has a genetic basis. Furthermore, much is now known on the molecular epidemiology, evolution and virulence of pathogenic agents, as well as their resistance to drugs, vaccines, and antibiotics. Equally, research on the genetics of disease vectors has greatly improved our understanding of their systematics, has increased our capacity to identify target populations for control or intervention, and has provided detailed information on the mechanisms of insecticide resistance. However, the genetics and evolutionary biology of hosts, pathogens and vectors have tended to develop as three separate fields of research. This artificial compartmentalisation is of concern due to our growing appreciation of the strong co-evolutionary interactions among hosts, pathogens and vectors. Infection, Genetics and Evolution and its companion congress [MEEGID](http://www.meegidconference.com/) (for Molecular Epidemiology and Evolutionary Genetics of Infectious Diseases) are the main forum acting for the cross-fertilization between evolutionary science and biomedical research on infectious diseases. Infection, Genetics and Evolution is the only journal that welcomes articles dealing with the genetics and evolutionary biology of hosts, pathogens and vectors, and coevolution processes among them in relation to infection and disease manifestation. All infectious models enter the scope of the journal, including pathogens of humans, animals and plants, either parasites, fungi, bacteria, viruses or prions. The journal welcomes articles dealing with genetics, population genetics, genomics, postgenomics, gene expression, evolutionary biology, population dynamics, mathematical modeling and bioinformatics. We also provide many author benefits, such as free PDFs, a liberal copyright policy, special discounts on Elsevier publications and much more. Please click here for more information on our author services .