Plasmid-Based Reverse Genetics System Enabling One-Step Generation of Genotype 3 Hepatitis E Virus
Hepatitis E virus (HEV) is a positive-sense, single-stranded RNA virus that poses a significant public health risk, yet its study is hindered by the complexity of conventional RNA-based reverse genetics systems. These systems require multiple steps, including genome cloning, in vitro transcription,...
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2025-05-01
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| author | Tominari Kobayashi Takashi Nishiyama Kentaro Yamada Kazumoto Murata Hiroaki Okamoto |
| author_facet | Tominari Kobayashi Takashi Nishiyama Kentaro Yamada Kazumoto Murata Hiroaki Okamoto |
| author_sort | Tominari Kobayashi |
| collection | DOAJ |
| description | Hepatitis E virus (HEV) is a positive-sense, single-stranded RNA virus that poses a significant public health risk, yet its study is hindered by the complexity of conventional RNA-based reverse genetics systems. These systems require multiple steps, including genome cloning, in vitro transcription, and capping, making them labor-intensive and susceptible to RNA degradation. In this study, we developed a single-step, plasmid-based HEV expression system that enabled direct intracellular transcription of the full-length HEV genome under a cytomegalovirus immediate-early (CMV-IE) promoter. The viral genome was flanked by hammerhead (HH) and hepatitis delta virus (HDV) ribozymes to ensure precise self-cleavage and the generation of authentic 5′ and 3′ termini. This system successfully supported HEV genome replication, viral protein expression, and progeny virion production at levels comparable to those obtained using in vitro-transcribed, capped HEV RNA. Additionally, a genetic marker introduced into the plasmid construct was stably retained in progeny virions, demonstrating the feasibility of targeted genetic modifications. However, plasmid-derived HEV exhibited delayed replication kinetics, likely due to the absence of an immediate 5′ cap. Attempts to enhance capping efficiency through co-expression of the vaccinia virus capping enzyme failed to improve HEV replication, suggesting that alternative strategies, such as optimizing the promoter design for capping, may be required. This plasmid-based HEV reverse genetics system simplifies the study of HEV replication and pathogenesis and provides a versatile platform for the genetic engineering of the HEV genome. |
| format | Article |
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| institution | DOAJ |
| issn | 1999-4915 |
| language | English |
| publishDate | 2025-05-01 |
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| spelling | doaj-art-e9c8e9e97ccd4f898741b61e7cd0f3bc2025-08-20T03:12:11ZengMDPI AGViruses1999-49152025-05-0117566910.3390/v17050669Plasmid-Based Reverse Genetics System Enabling One-Step Generation of Genotype 3 Hepatitis E VirusTominari Kobayashi0Takashi Nishiyama1Kentaro Yamada2Kazumoto Murata3Hiroaki Okamoto4Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-Shi 329-0498, Tochigi, JapanDivision of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-Shi 329-0498, Tochigi, JapanDivision of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-Shi 329-0498, Tochigi, JapanDivision of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-Shi 329-0498, Tochigi, JapanDivision of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-Shi 329-0498, Tochigi, JapanHepatitis E virus (HEV) is a positive-sense, single-stranded RNA virus that poses a significant public health risk, yet its study is hindered by the complexity of conventional RNA-based reverse genetics systems. These systems require multiple steps, including genome cloning, in vitro transcription, and capping, making them labor-intensive and susceptible to RNA degradation. In this study, we developed a single-step, plasmid-based HEV expression system that enabled direct intracellular transcription of the full-length HEV genome under a cytomegalovirus immediate-early (CMV-IE) promoter. The viral genome was flanked by hammerhead (HH) and hepatitis delta virus (HDV) ribozymes to ensure precise self-cleavage and the generation of authentic 5′ and 3′ termini. This system successfully supported HEV genome replication, viral protein expression, and progeny virion production at levels comparable to those obtained using in vitro-transcribed, capped HEV RNA. Additionally, a genetic marker introduced into the plasmid construct was stably retained in progeny virions, demonstrating the feasibility of targeted genetic modifications. However, plasmid-derived HEV exhibited delayed replication kinetics, likely due to the absence of an immediate 5′ cap. Attempts to enhance capping efficiency through co-expression of the vaccinia virus capping enzyme failed to improve HEV replication, suggesting that alternative strategies, such as optimizing the promoter design for capping, may be required. This plasmid-based HEV reverse genetics system simplifies the study of HEV replication and pathogenesis and provides a versatile platform for the genetic engineering of the HEV genome.https://www.mdpi.com/1999-4915/17/5/669hepatitis E virusplasmid-based reverse genetics systemmammalian expression promoterribozymevaccinia virus capping enzyme |
| spellingShingle | Tominari Kobayashi Takashi Nishiyama Kentaro Yamada Kazumoto Murata Hiroaki Okamoto Plasmid-Based Reverse Genetics System Enabling One-Step Generation of Genotype 3 Hepatitis E Virus Viruses hepatitis E virus plasmid-based reverse genetics system mammalian expression promoter ribozyme vaccinia virus capping enzyme |
| title | Plasmid-Based Reverse Genetics System Enabling One-Step Generation of Genotype 3 Hepatitis E Virus |
| title_full | Plasmid-Based Reverse Genetics System Enabling One-Step Generation of Genotype 3 Hepatitis E Virus |
| title_fullStr | Plasmid-Based Reverse Genetics System Enabling One-Step Generation of Genotype 3 Hepatitis E Virus |
| title_full_unstemmed | Plasmid-Based Reverse Genetics System Enabling One-Step Generation of Genotype 3 Hepatitis E Virus |
| title_short | Plasmid-Based Reverse Genetics System Enabling One-Step Generation of Genotype 3 Hepatitis E Virus |
| title_sort | plasmid based reverse genetics system enabling one step generation of genotype 3 hepatitis e virus |
| topic | hepatitis E virus plasmid-based reverse genetics system mammalian expression promoter ribozyme vaccinia virus capping enzyme |
| url | https://www.mdpi.com/1999-4915/17/5/669 |
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