Optimizing Yeast Homologous Recombination for Splicing Large Coronavirus Genome Fragments DOI Open Access
Guoqing Xiong, Xuan Huang, Ao Hu

et al.

International Journal of Molecular Sciences, Journal Year: 2024, Volume and Issue: 25(24), P. 13742 - 13742

Published: Dec. 23, 2024

Reverse genetics is a useful tool for studying viruses and developing vaccines coronaviruses. However, constructing manipulating the coronavirus genome in Escherichia coli can be time-consuming challenging due to its large size instability. Homologous recombination, genetic manipulation mechanism found organisms, essential DNA repair, gene engineering. In yeast, particularly Saccharomyces cerevisiae, homologous recombination technology commonly used expression plasmids editing. this study, we successfully split spliced 30 kb viral fragment using yeast recombination. By optimizing program parameters, such as arm lengths fragment-to-vector ratios, achieved splicing efficiency of up 97.9%. The optimal parameters selected were 60 bp sequence vector ratio 1:2:2:2:2:2 fragments.

Language: Английский

Advances in the development of infectious clones of human coronaviruses and related applications DOI Creative Commons
Guangya Liu, Rayner Kay Jin Tan, Yiyi Wu

et al.

Biosafety and Health, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 1, 2025

Language: Английский

Citations

1
An optimized high-throughput SARS-CoV-2 dual reporter trans-complementation system for antiviral screening in vitro and in vivo DOI Creative Commons

Yingjian Li,

Xue Tan,

Jikai Deng

et al.

Virologica Sinica, Journal Year: 2024, Volume and Issue: 39(3), P. 447 - 458

Published: March 26, 2024

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still epidemic around the world. manipulation of SARS-CoV-2 restricted to biosafety level 3 laboratories (BSL-3). In this study, we developed a ΔN-GFP-HiBiT replicon delivery particles (RDPs) encoding dual reporter gene, GFP-HiBiT, capable producing both GFP signal and luciferase activities. Through optimal selection GFP-HiBiT demonstrated superior stability convenience for antiviral evaluation. Additionally, established RDP infection mouse model by delivering N gene into K18-hACE2 KI through lentivirus. This supports replication can be utilized in vivo evaluations. summary, system serves as valuable tool efficient screening studying function SARS-CoV-2. Importantly, manipulated BSL-2 laboratories, decreasing threshold experimental requirements.

Language: Английский

Citations

7
Developing Next-Generation Live Attenuated Vaccines for Porcine Epidemic Diarrhea Using Reverse Genetic Techniques DOI Creative Commons

Ruisong Yu,

Shijuan Dong,

Bingqing Chen

et al.

Vaccines, Journal Year: 2024, Volume and Issue: 12(5), P. 557 - 557

Published: May 19, 2024

Porcine epidemic diarrhea virus (PEDV) is the etiology of porcine (PED), a highly contagious digestive disease in pigs and especially neonatal piglets, which mortality rate up to 100% will be induced. Immunizing pregnant sows remains most promising effective strategy for protecting their offspring from PEDV. Although half century has passed since its first report Europe several prophylactic vaccines (inactivated or live attenuated) have been developed, PED still poses significant economic concern swine industry worldwide. Hence, there an urgent need novel clinical practice, attenuated (LAVs) that can induce strong protective lactogenic immune response sows. Reverse genetic techniques provide robust tool virological research function viral proteins generation rationally designed vaccines. In this review, after systematically summarizing progress on virulence-related proteins, we reviewed reverse genetics PEDV application development LAVs. Then, probed into potential methods generating safe, effective, genetically stable LAV candidates, aiming new ideas rational design

Language: Английский

Citations

5
Efficiency of reverse genetics methods for rescuing severe acute respiratory syndrome coronavirus 2 DOI Creative Commons
Chang‐Joo Park, Tae-Hun Kim,

Seung‐Min Yoo

et al.

The Journal of Microbiology, Journal Year: 2025, Volume and Issue: 63(2), P. e2411023 - e2411023

Published: Feb. 27, 2025

Language: Английский

Citations

0
Isolation, pathogenicity, and an infectious cDNA clone of the prevalent G2c variant of porcine epidemic diarrhea virus in China DOI
Qi Yang,

Y. Zhang,

Long-Fei Chen

et al.

Microbial Pathogenesis, Journal Year: 2025, Volume and Issue: unknown, P. 107637 - 107637

Published: April 1, 2025

Language: Английский

Citations

0
Development of viral infectious clones and their applications based on yeast and bacterial artificial chromosome platforms DOI Creative Commons
Yiyi Wu,

Shangqing Gao,

G. Liu

et al.

Molecular Biomedicine, Journal Year: 2025, Volume and Issue: 6(1)

Published: April 29, 2025

Abstract Infectious Clones represent a foundational technique in the field of reverse genetics, allowing for construction and manipulation full-length viral genomes. The main methods currently used constructing infectious clones include Transformation-associated recombination (TAR), which is based on Yeast Artificial Chromosome (YAC) Bacterial (BAC). YAC BAC systems are powerful tools that enable large DNA fragments, making them well-suited These have been successfully applied to construct wide range viruses, including coronaviruses, herpesviruses, flaviviruses baculoviruses. rescued recombinant viruses from these widely various research areas, such as vaccine development, antiviral drug screening, pathogenesis virulence studies, gene therapy vector design. However, different possess unique biological characteristics, challenge remains how rapidly obtain future research. In summary, this review introduced development applications clones, with focus YAC, combined YAC-BAC technologies. We emphasize importance platforms areas aim provide deeper insights can advance platform broaden its application horizons.

Language: Английский

Citations

0
Plasmid-Based Reverse Genetics System Enabling One-Step Generation of Genotype 3 Hepatitis E Virus DOI Creative Commons
Tominari Kobayashi, Takashi Nishiyama, Kentaro Yamada

et al.

Viruses, Journal Year: 2025, Volume and Issue: 17(5), P. 669 - 669

Published: May 3, 2025

Hepatitis E virus (HEV) is a positive-sense, single-stranded RNA that poses significant public health risk, yet its study 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 susceptible to degradation. In this study, we developed single-step, plasmid-based HEV expression system enabled direct intracellular transcription full-length under cytomegalovirus immediate-early (CMV-IE) promoter. The viral was flanked hammerhead (HH) hepatitis delta (HDV) ribozymes ensure precise self-cleavage generation authentic 5′ 3′ termini. This successfully supported replication, protein expression, progeny virion production at levels comparable those obtained using vitro-transcribed, capped RNA. Additionally, genetic marker introduced into plasmid construct stably retained virions, demonstrating feasibility targeted modifications. However, plasmid-derived exhibited delayed replication kinetics, likely due absence an immediate cap. Attempts enhance capping efficiency through co-expression vaccinia enzyme failed improve suggesting alternative strategies, such as optimizing promoter design for may be required. simplifies pathogenesis provides versatile platform engineering genome.

Language: Английский

Citations

0
Key Amino Acids in RNA Polymerase and Helicase Proteins Regulate RNA Synthesis Efficiency in Porcine Reproductive and Respiratory Syndrome Virus DOI Creative Commons
Hui Li, Riteng Zhang, Honglin Xie

et al.

Journal of Biological Chemistry, Journal Year: 2025, Volume and Issue: unknown, P. 110247 - 110247

Published: May 1, 2025

Porcine reproductive and respiratory syndrome virus (PRRSV) exhibits rapid evolution due to its high mutation rate frequent recombination, posing significant challenges for disease control. In this study, we investigated the molecular mechanisms underlying strain-specific variations in PRRSV replication phenotypes. Using reverse genetics biology approaches, established a non-infectious replicon model that simulates genomic subgenomic (sg) mRNA transcription at cellular level. This enabled evaluation of regulatory effects viral non-structural proteins (nsps) transcription-regulating sequences (TRSs) on transcription, revealing crucial roles nsp9 nsp12 RNA synthesis. Furthermore, developed system (sg-Rep-PRRSV) driven by minimal replication-transcription complex (mini-RTC) investigate impact specific mutations replicase-associated synthesis efficiency. Our findings demonstrated mini-RTC components derived from XM-2020 exhibited significantly higher transcriptional driving efficiency compared those GD strain (p < 0.01). Site-directed mutagenesis analysis identified critical amino acid residues contributing differential between strains: E141N, N416H, S591A nsp9, S51D, L57T, K349E nsp10. These adaptive likely modulate catalytic conformations RNA-dependent polymerase (RdRp) helicase, ultimately distinct phenotypes observed among strains. provide an insight into adaptation, which have implications mitigating future PRRS outbreak risks maintaining sustainable development swine industry.

Language: Английский

Citations

0
Functional dissection of the spike glycoprotein S1 subunit and identification of cellular cofactors for regulation of swine acute diarrhea syndrome coronavirus entry DOI
Yong‐Le Yang, Bin Wang, Wentao Li

et al.

Journal of Virology, Journal Year: 2024, Volume and Issue: 98(4)

Published: March 19, 2024

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel porcine enteric coronavirus, and the broad interspecies infection of SADS-CoV poses potential threat to human health. This study provides experimental evidence dissect roles distinct domains within spike S1 subunit in cellular entry. Specifically, we expressed its subdomains,

Language: Английский

Citations

3
Highs and Lows in the Calicivirus Reverse Genetics DOI Open Access
Ángel L. Álvarez, Aroa Arboleya, Fábio A. Abade dos Santos

et al.

Published: March 26, 2024

In Virology, the term reverse genetics refers to a set of methodologies in which changes are introduced into viral genome and their effects on generation infectious progeny phenotypic features assessed. Reverse emerged thanks advances recombinant DNA technology made isolation, cloning, modification genes through mutagenesis possible. Most virus studies depend our capacity rescue an wild-type from cell cultures transfected with “infectious clone”. This clone generally consists circular plasmid containing functional copy full-length genome, under control appropriate RNA polymerase promoter. For most viruses, systems very straightforward, since genomes relatively easy handle modify also (with few notable exceptions) per se. is not true for whose need be transcribed cDNA before any -such as restriction enzyme digestion, gene knock-out or knock-in, site directed mutagenesis, ligation, etc.- can done. Establishing members Caliciviridae has proven exceptionally challenging, due low number this family that propagate cultures. Despite successful Feline calicivirus (FCV) more than 20 years ago, methods routine procedures easily extrapolated other caliciviruses. Reports have been far between, review, we discuss main pitfalls, failures, delays behind several clones.

Language: Английский

Citations

1