Jul 2026· Frontiers in Virology· 0 citations· 208 references
TL;DR
This review systematically summarizes the evolution, technical framework and optimization strategies of RNA virus reverse genetics, with three prototype viruses covering all major RNA genome types: SARS-CoV-2 (+ssRNA), non-segmented negative-sense Newcastle disease virus (NDV), and segmented negative-sense influenza A virus.
Abstract
Viral reverse genetics enables the rescue of infectious virions from cloned cDNA and serves as a core technique for mapping viral genotype–phenotype relationships, dissecting RNA viral life cycles, and developing antiviral countermeasures. This review systematically summarizes the evolution, technical framework and optimization strategies of RNA virus reverse genetics, with three prototype viruses covering all major RNA genome types: SARS-CoV-2 (+ssRNA), non-segmented negative-sense Newcastle disease virus (NDV), and segmented negative-sense influenza A virus. Core modules including infectious clone construction, diverse promoter systems, hammerhead/HDV ribozymes, and solutions for large unstable genomes such as BAC and ISA are elaborated. We summarize its irreplaceable applications in vaccine development, pathogenesis research, virus-host interaction analysis and high-throughput antiviral screening. Current bottlenecks include low rescue efficiency, cDNA genetic instability and biosafety hazards. We also introduce non-infectious surrogate platforms and establish a three-tier antiviral screening pipeline. Future advances will integrate CRISPR/Cas editing, standardized modular tools, biosafety engineering, AI and big data. Distinct from previous reviews focusing on single viral genera, this work conducts cross-type horizontal comparisons and summarizes universal technical obstacles and tailored optimizations, offering comprehensive references for basic virology, accelerated vaccine innovation and precise antiviral design.
Reverse genetics systems are crucial for facilitating the precise manipulation of viruses across a wide spectrum of translational and fundamental research pipelines. Here, we compared Circular polymerase extension reaction (CPER), Gibson assembly, and infectious subgenomic amplicons (ISA) for bacteria-free recovery of a positive sense RNA virus. Through optimisation of CPER, we demonstrated accelerated virus recovery and enhanced viral yields. We further investigated strategies to improve rescue efficiency across diverse positive-sense RNA virus families through incorporation of alternative promoters and non-coding elements. To evaluate the performance of the Aedes aegypti polyubiquitin promoter (AePUb) in tandem with a hammerhead ribozyme (HH Rbz) and a polymerase pause site for virus recovery in insect cells, we constructed a new fluorescent reporter genome using a 20 kb insect-specific mesonivirus. In vitro recovery by CPER of the mesonivirus was achievable in 1 day when using AePUb with HH Rbz, in comparison to a four-day recovery when using the minimal OpIE2-CA promoter. These elements were additionally assessed for rescue of the orthoflaviviruses, Binjari virus (BinJV) and dengue virus 2 (DENV-2), in insect cells (using AePUb); or in mammalian cells (using the CMV promoter) and for launch of DENV2 and SARS-CoV-2. Both BinJV and DENV-2 demonstrated improved rescue with the AePUb promoter and HH Rbz. However, the addition of the HH Rbz and the polymerase pause site to the CMV linker fragment showed no substantial differences to the standard CMV promoter systems for both DENV-2 and SARS-CoV-2, highlighting the context-specific benefits of their implementation. In summary, we demonstrated that a potent constitutive promoter system and a hammerhead ribozyme enhance the efficiency of positive-sense RNA virus rescue using CPER. Importance Reverse genetics systems are often limited by plasmid instability and variable efficiency of promoters across diverse cell lines. Extensive comparative approaches have yielded improvements across a variety of systems, however, there has been a paucity of publications that empirically compare novel advancements to established approaches. Here, we formalised and compared a series of reverse genetics advancements in the form of bacteria-free assembly methods, host promoters, pause sites, and ribozymes. These streamlined approaches expedite the existing methodologies and provide fundamental improvements to the field of synthetic virology. The advancements herein may support applications requiring efficient recovery of low fitness mutants and diverse mutational libraries and barcoded virus populations.
James R. Potter, Helen Mostafavi, A. Amarilla et al.· bioRxiv· 0 citations
This review discusses selected RNA viruses, focusing on the structure of their RNA polymerases and interactions with host factors during the different stages of the viral lifecycle, as well as the traditional antivirals targeting these structures and pathways.
Hana Krnjić, Adna Hrapović, Aiša Galijatović et al.· Viruses· 0 citations
This review comprehensively evaluates the rational design of classical animal herpesvirus vectors, including pseudorabies virus, herpesvirus of turkeys, and feline herpesvirus type 1, providing perspectives on how continuous biotechnological innovations will empower herpesvirus vectors to serve as formidable prophylactic tools against emerging and re-emerging infectious diseases.
Jia-Hui Guo, Chen Mei, Xin-Yao Sun et al.· Frontiers in Microbiology· 0 citations
A modular, helper-virus-free and high-efficiency rescue platform based on an orthogonal transcription system utilizing orthogonal promoters and engineered RNA polymerases fused to an mRNA capping enzyme is developed and provides a proof-of-concept methodological framework for the rapid development of vaccine candidates.
Zihan Ma, Weijun Wang, Qiuli Lou et al.· Synthetic and Systems Biotec...· 0 citations
RNA vaccines—comprising linear mRNA, self-amplifying RNA, and circular RNA (circRNA)—constitute a core next-generation platform for the prevention and control of viral diseases; among these, circRNA vaccines possess notable structural stability, yet their technical bottlenecks and application prospects in veterinary medicine have not been systematically reviewed. This review synthesizes current research on circRNA vaccine design, circularization strategies, translation mechanisms, delivery systems, and immunological outcomes, and compares their antiviral performance with that of linear mRNA vaccines. Owing to their covalently closed circular conformation, circRNA vaccines exhibit enhanced resistance to nucleases and superior thermal stability, enabling sustained transfection activity at ambient temperatures without reliance on strict cold chains; through cap-independent translation driven by internal ribosome entry sites or N6-methyladenosine modifications, and in conjunction with optimized circularization protocols and lipid nanoparticle carriers, circRNA vaccines elicit substantially higher antiviral IgG titers and durable antigen-specific T-cell memory relative to linear mRNA vaccines. These vaccines have been deployed against COVID-19, monkeypox, influenza, and livestock viral diseases, demonstrating strong adaptability to viral variants and compatibility with mucosal or needle-free administration routes. CircRNA vaccines are well suited for both emergency outbreak response and routine immunization programs; nevertheless, challenges persist, including low circularization efficiency for long sequences, elevated manufacturing costs, and inadequate quality control standards. Addressing these issues through improved production workflows and delivery technologies adapted to resource-limited settings will be critical to establishing circRNA vaccines as a pillar of livestock disease management and as a strategic reserve for emerging zoonotic threats.
Dong-Jie Cai, Xing-Ling Li, Ruo-Xu Wang et al.· Vaccines· 0 citations
Ambiviruses are fungal-infecting circular RNA viruses that uniquely combine viroid-like and viral features, yet the function of their conserved ORF-B protein and their effects on hosts remain unknown, hindered by the lack of a reverse genetics system. Here, we constructed the first infectious cDNA clone of an ambivirus, Fusarium graminearum ambivirus 1 (FgAV1), using a head-to-tail dimer placed downstream of a fungal promoter. FgAV1 was horizontally transmitted via hyphal anastomosis to virus-free Fusarium graminearum strains. Notably, a reverse-oriented dimer construct was also infectious and transmissible. Targeted mutagenesis revealed that both ORF-A- and ORF-B-encoded proteins and the presence of embedded ribozymes are indispensable for ambivirus replication. Our results further demonstrate that FgAV1 infection triggers a fungal RNAi response, extending the antiviral role of host sRNAs to circular RNA viruses. Furthermore, FgAV1 infection suppressed fungal growth and significantly reduced the virulence of F. graminearum on wheat. These findings provide novel insights into ambivirus replication and their potential in fungal pathogen biocontrol.
Yi Guo, M. Forgia, N. Serale et al.· Science Advances· 0 citations
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