Comparison of viral propagation and drug response among SARS-CoV-2 VOCs using replicons capable of recapitulating virion assembly and release DOI Creative Commons

Lingqian Tian,

Qiuhong Liu, Rongjuan Pei

et al.

Virologica Sinica, Journal Year: 2022, Volume and Issue: 37(5), P. 695 - 703

Published: July 1, 2022

Several variants of concern (VOCs) have emerged since the WIV04 strain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first isolated in January 2020. Due to mutations spike (S) protein, these VOCs evolved enhance viral infectivity and immune evasion. However, whether other proteins lead altered propagation drug resistance remains obscure. The replicon is a noninfectious surrogate capable recapitulating certain steps life cycle. Although several SARS-CoV-2 replicons been developed, none them were derived from emerging could only recapitulate genome replication subgenomic RNA (sgRNA) transcription. In this study, two (the Beta Delta variants) prepared by removing S gene their genomes, while structural genes remained untouched. These not sgRNA transcription but also support assembly release viral-like particles, as manifested electron microscopic assays. Thus, S-deletion virtually all post-entry cycle provides versatile tool for measuring intracellular screening novel antiviral drugs, including inhibitors virion release. Through quantification released into supernatant, we demonstrate that response remdesivir yet substantially changed during evolution VOCs.

Language: Английский

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
Ultrarapid and ultrasensitive detection of SARS‐CoV‐2 antibodies in COVID‐19 patients via a 3D‐printed nanomaterial‐based biosensing platform DOI Creative Commons
Md. Azahar Ali, Fei Zhang,

Chunshan Hu

et al.

Journal of Medical Virology, Journal Year: 2022, Volume and Issue: 94(12), P. 5808 - 5826

Published: Aug. 19, 2022

Abstract Rapid detection of antibodies during infection and after vaccination is critical for the control infectious outbreaks, understanding immune response, evaluating vaccine efficacy. In this manuscript, we evaluate a simple ultrarapid test SARS‐CoV‐2 in COVID‐19 patients, which gives quantitative results (i.e., antibody concentration) 10–12 s using previously reported nanomaterial‐based three‐dimensional (3D)‐printed biosensing platform. This platform consists micropillar array electrode fabricated via 3D printing aerosolized gold nanoparticles coated with nanoflakes graphene specific antigens, including spike S1, S1 receptor‐binding domain (RBD) nucleocapsid (N). The sensor works on principle electrochemical transduction, where change impedance realized by interactions between viral proteins attached to surface antibodies. three sensors were used samples from 17 patients 3 without COVID‐19. Unlike other serological tests, quantitatively detected at concentration as low picomole within human plasma samples. We found that studied had higher concentrations (RBD S1) than N protein. These demonstrate enormous potential rapid patients' immunity, disease epidemiology efficacy, facilitating prevention epidemics.

Language: Английский

Citations

20
Reverse genetics systems for SARS-CoV-2: Development and applications DOI Creative Commons

Hou-Li Cai,

Yao‐Wei Huang

Virologica Sinica, Journal Year: 2023, Volume and Issue: 38(6), P. 837 - 850

Published: Oct. 11, 2023

The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused serious harm to human health and struck a blow global economic development. Research on SARS-CoV-2 has greatly benefited from the use reverse genetics systems, which have been established artificially manipulate viral genome, generating recombinant reporter infectious viruses or biosafety level (BSL-2)-adapted non-infectious replicons with desired modifications. These tools instrumental in studying molecular biological characteristics virus, investigating antiviral therapeutics, facilitating development attenuated vaccine candidates. Here, we review construction strategies, development, applications systems for SARS-CoV-2, may be applied other CoVs as well.

Language: Английский

Citations

13
Construction and characterization of two SARS‐CoV‐2 minigenome replicon systems DOI
Hu Zhang,

D Fischer,

Masahiro Shuda

et al.

Journal of Medical Virology, Journal Year: 2022, Volume and Issue: 94(6), P. 2438 - 2452

Published: Feb. 9, 2022

The ongoing COVID-19 pandemic severely impacts global public health and economies. To facilitate research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virology antiviral discovery, a noninfectious viral replicon system operating under biosafety level containment is warranted. We report herein the construction characterization of two SARS-CoV-2 minigenome systems. First, we constructed IVT-CoV2-Rep complementary DNA template to generate messenger RNA (mRNA) with nanoluciferase (NLuc) reporter via in vitro transcription (IVT). mRNA transfection assay demonstrated rapid transient replication variety cell lines, which could be completely abolished by known inhibitors. Our data also suggest that phenotype not due host innate responses. In addition, have developed DNA-launched BAC-CoV2-Rep, supports in-cell as initial template. BAC-CoV2-Rep exhibited much stronger longer signal compared version. found portion NLuc was derived from spliced resistant treatment, especially during early phase after transfection. summary, established systems are suitable for basic research, hold promise stable line development further optimization.

Language: Английский

Citations

17
One-pot Golden Gate Assembly of an avian infectious bronchitis virus reverse-genetics system DOI Creative Commons
Katharina Bilotti, Sarah Keep, Andrew P. Sikkema

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Nov. 22, 2023

Abstract Avian infectious bronchitis is an acute respiratory disease of poultry particular concern for global food security. Investigation Infectious Bronchitis Virus (IBV), the causative agent avian bronchitis, via reverse genetics enables deeper understanding virus biology and a rapid response to emerging variants. Classic methods IBV can be time consuming, rely on recombination introduction mutations, and, depending system, subject genome instability unreliable success rates. In this study, we have applied data-optimized Golden Gate Assembly design create rapidly executable, flexible, faithful system IBV. The was divided into 12 fragments at high-fidelity fusion site breakpoints. All were synthetically produced propagated in E. coli plasmids, amenable standard molecular techniques DNA manipulation. assembly carried out single reaction, with products used directly subsequent viral rescue steps. We demonstrate use generation point mutants gene replacements. This Assembly-based will enable variants IBV, particularly important vaccine development controlling spread within populations.

Language: Английский

Citations

9
Optimization of a DNA‐launched SARS‐CoV‐2 replicon with RNA splicing inhibitor Isoginkgetin DOI
Hu Zhang, Haitao Guo

Journal of Medical Virology, Journal Year: 2024, Volume and Issue: 96(3)

Published: March 1, 2024

Abstract We have previously developed a bacterial artificial chromosome (BAC)‐vectored SARS‐CoV‐2 replicon, namely BAC‐CoV2‐Rep, which, upon transfection into host cells, serves as transcription template for replicon mRNA to initiate replication and produce nanoluciferase (Nluc) reporter from the subgenomic viral mRNA. However, an inherent issue of such DNA‐launched system is that nascent full‐length transcript undergoes process by RNA splicing machinery, which reduces generates spliced species expressing NLuc independent replication. To mitigate this problem, we employed Isoginkgetin, universal eukaryotic inhibitor, treat cells transfected with BAC‐CoV2‐Rep. Isoginkgetin effectively increased level transcripts while concurrently reducing Nluc signal derived mRNA, making more correlated replication, evidenced treatment known inhibitors including Remdesivir, GC376, EIDD‐1931. Thus, our study emphasizes confounding factor systems, can be mitigated treatment.

Language: Английский

Citations

2
Development of a Cell Culture Model for Inducible SARS-CoV-2 Replication DOI Creative Commons

Xiaoyan Wang,

Yuanfei Zhu, Qiong Wu

et al.

Viruses, Journal Year: 2024, Volume and Issue: 16(5), P. 708 - 708

Published: April 29, 2024

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces direct cytopathic effects, complicating the establishment of low-cytotoxicity cell culture models for studying its replication. We initially developed a DNA vector-based replicon system utilizing CMV promoter to generate recombinant viral genome bearing reporter genes. However, this frequently resulted in drug resistance and cytotoxicity, impeding model establishment. Herein, we present novel with SARS-CoV-2 replication induced by Cre/LoxP-mediated recombination. An engineered transcription unit was subcloned into bacterial artificial chromosome (BAC) vector. To enhance biosafety, spike protein gene deleted, nucleocapsid replaced gene. exogenous sequence inserted within NSP1 as modulatory cassette that is removable after recombination subsequent RNA splicing. Using PiggyBac transposon strategy, integrated host chromatin, yielding stable line capable inducing The exhibited sensitivity potential antivirals forsythoside A verteporfin. innovative inducible introduced further explore pathogenesis virus facilitate screening assessment anti-SARS-CoV-2 therapeutics.

Language: Английский

Citations

2
Indiscriminate activities of different henipavirus polymerase complex proteins allow for efficient minigenome replication in hybrid systems DOI Creative Commons
X. D. Li, Yanling Yang, Carolina B. López

et al.

Journal of Virology, Journal Year: 2024, Volume and Issue: 98(6)

Published: May 23, 2024

ABSTRACT The henipaviruses, including Nipah virus (NiV) and Hendra (HeV), are biosafety level 4 (BSL-4) zoonotic pathogens that cause severe neurological respiratory disease in humans. To study the replication machinery of these viruses, we developed robust minigenome systems can be safely used BSL-2 conditions. nucleocapsid (N), phosphoprotein (P), large protein (L) henipaviruses critical elements their thus essential support components systems. Here, tested effects diverse combinations proteins on capacity NiV HeV minigenomes by exchanging helper plasmids coding for among two viruses. We demonstrate all one or more heterologous were capable replicating both minigenomes. Sequence alignment showed identities 92% N protein, 67% P, 87% L. Notably, variations amino acid residues not concentrated N-P P-L interacting regions implying dissimilarities composition polymerase complex may impact interactions. observed indiscriminate activity is different from related which genomes only when whole belongs to same virus. This newly promiscuous property henipavirus likely attributed conserved interaction could potentially harnessed develop universal anti-henipavirus antivirals. IMPORTANCE Given severity induced viruses humans continuous emergence new as well henipa-like it necessary conduct a comprehensive investigation biology with host. development antiviral agents studied allow experiments performed under 2 (HeV) provide convenient alternative studying replication. Using systems, any combination three effectively initiate viral minigenomes, suggests effective targets interventions.

Language: Английский

Citations

2
One-pot Golden Gate Assembly of an avian infectious bronchitis virus reverse genetics system DOI Creative Commons
Katharina Bilotti, Sarah Keep, Andrew P. Sikkema

et al.

PLoS ONE, Journal Year: 2024, Volume and Issue: 19(7), P. e0307655 - e0307655

Published: July 25, 2024

Avian infectious bronchitis is an acute respiratory disease of poultry particular concern for global food security. Investigation virus (IBV), the causative agent avian bronchitis, via reverse genetics enables deeper understanding biology and a rapid response to emerging variants. Classic methods IBV can be time consuming, rely on recombination introduction mutations, and, depending system, subject genome instability unreliable success rates. In this study, we have applied data-optimized Golden Gate Assembly design create rapidly executable, flexible, faithful system IBV. The was divided into 12 fragments at high-fidelity fusion site breakpoints. All were synthetically produced propagated in E . coli plasmids, amenable standard molecular techniques DNA manipulation. assembly carried out single reaction, with products used directly subsequent viral rescue steps. We demonstrate use generation point mutants gene replacements. This Assembly-based will enable variants IBV, particularly important vaccine development controlling spread within populations.

Language: Английский

Citations

2
Development and validation of Mayaro virus with luciferase reporter genes as a tool for antiviral assays DOI Creative Commons
Mikaela dos Santos Marinho, Yanan Zhang, Natasha Marques Cassani

et al.

Heliyon, Journal Year: 2024, Volume and Issue: 10(13), P. e33885 - e33885

Published: July 1, 2024

Arboviruses are etiological agents in an extensive group of emerging diseases with great clinical relevance Brazil, due to the wide distribution their vectors and favorable environmental conditions. Among them, Mayaro virus (MAYV) has drawn attention since its emergence as etiologic agent fever, a highly debilitating disease. To study viral replication identify new drug candidates, traditional antiviral assays based on antigens and/or plaque have been demonstrating low throughput, making it difficult carry out larger-scale assays. Therefore, we developed characterized two DNA-launched infectious clones reporter viruses MAYV strain BeAr 20290 containing genes

Language: Английский

Citations

1