A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses

Amarilla, Alberto A.; Sng, Julian D. J.; Parry, Rhys; Deerain, Joshua M.; Potter, James R.; Setoh, Yin Xiang; Rawle, Daniel J.; Le, Thuy T.; Modhiran, Naphak; Wang, Xiaohui; Peng, Nias Y. G.; Torres, Francisco J.; Pyke, Alyssa T.; Harrison, Jessica J.; Freney, Morgan E.; Liang, Bojian; McMillan, Christopher L. D.; Cheung, Stacey T. M.; Guevara, Darwin J. Da Costa; Hardy, Joshua M.; Bettington, Mark; Muller, David A.; Coulibaly, F.; Moore, Frederick; Hall, Roy A.; Young, Paul R.; Mackenzie, Jason M.; Hobson‐Peters, Jody; Suhrbier, Andreas; Watterson, Daniel; Khromykh, Alexander A.

doi:10.1038/s41467-021-23779-5

Cited by 98 publications

(163 citation statements)

References 67 publications

(81 reference statements)

Supporting

Mentioning

150

Contrasting

Order By: Relevance

“…Leukocyte infiltration and bronchiolar membrane sloughing have previously been described as histopathological features of SARS-CoV-2 infection in K18-hACE2 [ 9 , 57 ] and Ad5-hACE2 mouse lungs [ 18 , 19 , 53 ]. These features were examined in H&E stained lung sections from hACE2-lentivirus transduced mice and compared with mock transduced mice, with both groups receiving an intrapulmonary inoculum of SARS-CoV-2.…”

Section: Resultsmentioning

confidence: 99%

“…Bronchiole occlusion and oedema, smooth muscle hyperplasia and alveolar thickening (measured by white space) [ 9 , 57 ] were also analyzed. Although the former approached significance, the remaining features did not show significant differences ( S8A–S8D Fig ), with viral inoculation even in the absence of active replication able to induce lung pathology.…”

Section: Resultsmentioning

confidence: 99%

“…Lungs were fixed in 10% formalin, embedded in paraffin, and sections stained with H&E (Sigma Aldrich). Lung histopathology was scored blinded by examining whole lung sections and evaluating infiltration, bronchiole membrane sloughing, occluded bronchioles, bronchiolar oedema, and smooth muscle hyperplasia as criteria for disease [ 57 ]. Areas with infiltrate lesions greater than approximately 1500 μm were counted and measured using Aperio ImageScope software (Leica Biosystems, Mt Waverley, Australia) (v10).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

ACE2-lentiviral transduction enables mouse SARS-CoV-2 infection and mapping of receptor interactions

Rawle

Dumenil

et al. 2021

PLoS Pathog

Self Cite

View full text Add to dashboard Cite

SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intrapulmonary hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lung epithelium. RNA-Seq and histological analyses illustrated that this model involved an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mouse lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

ACE2-lentiviral transduction enables mouse SARS-CoV-2 infection and mapping of receptor interactions

Rawle

Dumenil

et al. 2021

PLoS Pathog

Self Cite

View full text Add to dashboard Cite

show abstract

“…Several different methods were already used to generate recombinant SARS-CoV-2 viruses, including in vitro ligation [ 3 ], homologous recombination in yeast [ 4 ], and circular polymerase extension reaction [ 5 , 6 ], as well as plasmid- and bacterial artificial chromosome (BAC)-based approaches [ 7 , 8 , 9 ].…”

Section: Introductionmentioning

confidence: 99%

Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination

Herrmann

Jungnickl

Cordsmeier

et al. 2021

IJMS

View full text Add to dashboard Cite

The ongoing pandemic coronavirus (CoV) disease 2019 (COVID-19) by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has already caused substantial morbidity, mortality, and economic devastation. Reverse genetic approaches to generate recombinant viruses are a powerful tool to characterize and understand newly emerging viruses. To contribute to the global efforts for countermeasures to control the spread of SARS-CoV-2, we developed a passage-free SARS-CoV-2 clone based on a bacterial artificial chromosome (BAC). Moreover, using a Lambda-based Red recombination, we successfully generated different reporter and marker viruses, which replicated similar to a clinical isolate in a cell culture. Moreover, we designed a full-length reporter virus encoding an additional artificial open reading frame with wild-type-like replication features. The virus-encoded reporters were successfully applied to ease antiviral testing in cell culture models. Furthermore, we designed a new marker virus encoding 3xFLAG-tagged nucleocapsid that allows the detection of incoming viral particles and, in combination with bio-orthogonal labeling for the visualization of viral RNA synthesis via click chemistry, the spatiotemporal tracking of viral replication on the single-cell level. In summary, by applying BAC-based Red recombination, we developed a powerful, reliable, and convenient platform that will facilitate studies answering numerous questions concerning the biology of SARS-CoV-2.

show abstract

“…The drug (at 4 times the indicated final concentration) was diluted in twofold serial dilutions in RPMI 1640 supplemented with 2% FCS in a 96 well round bottom plate, and 50 µl was then transferred to cells using a multichannel pipette. For antiviral screening assay, SARS-CoV-2 (hCoV-19/Australia/ QLD02/2020 [37], kindly provided by Queensland Health Forensic and Scientific Services, Queensland Department of Health, Brisbane, Australia) was diluted in RPMI 1640 supplemented with 2% FCS to a final concentration of 2 × 10 3 CCID 50 /ml and 50 µl was added per well using a multichannel pipette for a final MOI ~ 0.01. For cytomorbidity or cytotoxicity assay, 50 µl RPMI 1640 supplemented with 2% FCS (instead of virus) was then added per well to give a final volume of 200 µl at the desired drug concentration.…”

mentioning

confidence: 99%

Simple rapid in vitro screening method for SARS-CoV-2 anti-virals that identifies potential cytomorbidity-associated false positives

Yan

Rawle

et al. 2021

Virol J

Self Cite

View full text Add to dashboard Cite

Background The international SARS-CoV-2 pandemic has resulted in an urgent need to identify new anti-viral drugs for treatment of COVID-19. The initial step to identifying potential candidates usually involves in vitro screening that includes standard cytotoxicity controls. Under-appreciated is that viable, but stressed or otherwise compromised cells, can also have a reduced capacity to replicate virus. A refinement proposed herein for in vitro drug screening thus includes a simple growth assay to identify drug concentrations that cause cellular stress or “cytomorbidity”, as distinct from cytotoxicity or loss of viability. Methods A simple rapid bioassay is presented for antiviral drug screening using Vero E6 cells and inhibition of SARS-CoV-2 induced cytopathic effects (CPE) measured using crystal violet staining. We use high cell density for cytotoxicity assays, and low cell density for cytomorbidity assays. Results The assay clearly illustrated the anti-viral activity of remdesivir, a drug known to inhibit SARS-CoV-2 replication. In contrast, nitazoxanide, oleuropein, cyclosporine A and ribavirin all showed no ability to inhibit SARS-CoV-2 CPE. Hydroxychloroquine, cyclohexamide, didemnin B, γ-mangostin and linoleic acid were all able to inhibit viral CPE at concentrations that did not induce cytotoxicity. However, these drugs inhibited CPE at concentrations that induced cytomorbidity, indicating non-specific anti-viral activity. Conclusions We describe the methodology for a simple in vitro drug screening assay that identifies potential anti-viral drugs via their ability to inhibit SARS-CoV-2-induced CPE. The additional growth assay illustrated how several drugs display anti-viral activity at concentrations that induce cytomorbidity. For instance, hydroxychloroquine showed anti-viral activity at concentrations that slow cell growth, arguing that its purported in vitro anti-viral activity arises from non-specific impairment of cellular activities. The cytomorbidity assay can therefore rapidly exclude potential false positives.

show abstract

A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses

Cited by 98 publications

References 67 publications

ACE2-lentiviral transduction enables mouse SARS-CoV-2 infection and mapping of receptor interactions

ACE2-lentiviral transduction enables mouse SARS-CoV-2 infection and mapping of receptor interactions

Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination

Simple rapid in vitro screening method for SARS-CoV-2 anti-virals that identifies potential cytomorbidity-associated false positives

Contact Info

Product

Resources

About