Visualization of SARS-CoV-2 infection dynamic
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Abstract
Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing rSARS-CoV-2 have jeopardized their use to monitor the dynamics of SARS-CoV-2 infection in vitro or in vivo . Here, we report an alternative strategy where reporter genes were placed upstream of the viral nucleocapsid gene followed by a 2A cleavage peptide. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and K18 hACE2 transgenic mice. Importantly, real-time viral infection was readily tracked using a non-invasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo . Notably, these reporter-expressing rSARS-CoV-2 retained wild-type virus like pathogenicity in vivo , supporting their use to investigate viral infection, dissemination, pathogenesis and therapeutic interventions for the treatment of SARS-CoV-2 in vivo .
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SciScore for 10.1101/2021.06.03.446942: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Biosafety (IBC) and Animal Care and Use (IACUC) committees.
Euthanasia Agents: For the body weight and survival studies, five-week-old female K18 hACE2 transgenic mice were infected intranasally with 105 PFU/animal following gaseous sedation in an isoflurane chamber.Sex as a biological variable Five-week-old female K18 hACE2 transgenic mice were purchased from The Jackson Laboratory and maintained in the animal facility at Texas Biomed under specific pathogen-free conditions. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Immunofluorescence assay (IFA): Vero … SciScore for 10.1101/2021.06.03.446942: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Biosafety (IBC) and Animal Care and Use (IACUC) committees.
Euthanasia Agents: For the body weight and survival studies, five-week-old female K18 hACE2 transgenic mice were infected intranasally with 105 PFU/animal following gaseous sedation in an isoflurane chamber.Sex as a biological variable Five-week-old female K18 hACE2 transgenic mice were purchased from The Jackson Laboratory and maintained in the animal facility at Texas Biomed under specific pathogen-free conditions. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Immunofluorescence assay (IFA): Vero E6 cells (106 cells/well, Vero E6suggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:To overcome this limitation, we established a rapid method based on a non-invasive measurement of Nluc expression. By using this in vivo imaging system, NAbs could be easily identified as early as 1 dpi and in a relative high throughput method. This was further supported by Nluc activity, viral titration, body weight changes and survival rate (Fig. 7), indicating our strategy provides a rapid in vivo screening method to identify NAbs against SARS-CoV-2. In the present work, we have documented a new strategy to generate replication-competent reporter rSARS-CoV-2 expressing higher levels of reporter gene than those previously described by substituting the viral ORF7a protein with the report gene. This novel strategy does not eliminate any viral gene and these new reporter-expressing rSARS-CoV-2 were genetically stable and replicated as efficiently as rSARS-CoV-2/WT both in vitro and in vivo, with comparable pathogenicity in K18 hACE2 transgenic mice. Notably, the robust levels of reporter gene expression of these new reporter-expressing rSARS-CoV-2 represent an excellent option to study viral pathogenesis, tissue tropism, and replication kinetics of SARS-CoV-2, including recently identified VoC.
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 40, 43, 44 and 38. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- No funding statement was detected.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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