Optimization of an infectious subgenomic amplicons reverse genetics protocol for the rescue of synthetic coronaviruses

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods Pub Date : 2025-04-04 DOI:10.1016/j.jviromet.2025.115152

Ilaria Puglia , Marialuigia Caporale , Giovanni Di Teodoro , Massimo Spedicato , Francesca Profeta , Maurilia Marcacci , Chiara Di Pancrazio , Fabrizia Valleriani , Emanuela Rossi , Heidi Auerswald , Alessio Lorusso

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引用次数: 0

Abstract

Reverse genetics (rg) systems are indispensable tools for investigating the pathogenesis of RNA viruses, facilitating vaccine design, and advancing antiviral therapeutic strategies. In this study, we optimized the Infectious Subgenomic Amplicons (ISA) method for generating synthetic r-wt SARS-CoV-2 Wuhan-Hu-1. This system was validated by demonstrating the successful rescue of infectious viral particles from overlapping DNA fragments and their propagation in vitro. Sequencing confirmed 100 % identity of the recovered virus with the Wuhan-Hu-1 reference genome. Importantly, in vivo experiments using K18-hACE2 mice revealed that the r-wt SARS-CoV-2 Wuhan-Hu-1 strain caused clinical symptoms, weight loss, and mortality comparable to those induced by a virulent SARS-CoV-2 field variant. This ISA rg method offers a rapid and reproducible approach to generating synthetic coronaviruses, with potential applications in pathogenesis studies, antiviral testing, and vaccine development.

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来源期刊

Journal of virological methods 医学-病毒学

CiteScore

5.80

自引率

0.00%

发文量

209

审稿时长

41 days

期刊介绍： The Journal of Virological Methods focuses on original, high quality research papers that describe novel and comprehensively tested methods which enhance human, animal, plant, bacterial or environmental virology and prions research and discovery. The methods may include, but not limited to, the study of: Viral components and morphology- Virus isolation, propagation and development of viral vectors- Viral pathogenesis, oncogenesis, vaccines and antivirals- Virus replication, host-pathogen interactions and responses- Virus transmission, prevention, control and treatment- Viral metagenomics and virome- Virus ecology, adaption and evolution- Applied virology such as nanotechnology- Viral diagnosis with novelty and comprehensive evaluation. We seek articles, systematic reviews, meta-analyses and laboratory protocols that include comprehensive technical details with statistical confirmations that provide validations against current best practice, international standards or quality assurance programs and which advance knowledge in virology leading to improved medical, veterinary or agricultural practices and management.