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Recombinant SARS-CoV-2 housing dual detectors for COVID-19 surveillance

In a recent article on the bioRxiv* Preprint servers, researchers at the Texas Biomedical Research Institute, Georgia State University, and the University of Alabama at Birmingham have developed a bireporter-expressing recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) coronavirus to infect a SARS-CoV -2 to track infection.

Study: Surveillance of SARS-CoV-2 infection with a double-reporter-expressing virus.  Photo Credit: ktsdesign/ShutterstockStudy: Surveillance of SARS-CoV-2 infection with a double-reporter-expressing virus. Photo Credit: ktsdesign/Shutterstock

background

The 2019 coronavirus disease (COVID-19) pandemic is caused by the highly contagious virus known as coronavirus 2 (SARS-CoV-2) with the severe acute respiratory syndrome. The SARS-CoV-2 pandemic is still wreaking havoc on medical facilities worldwide, despite the availability of vaccines and antivirals for COVID-19. Therefore, robust techniques to screen for the presence of SARS-CoV-2 in animal models or infected cells are a must to study the basic biology of COVID-19 and the impact of antiviral therapies.

Recombinant viruses that express reporters and are replication competent have historically proven to be an excellent way to study topics such as SARS-CoV-2 infection, pathogenesis, replication and transmission. Despite the establishment and efficient synthesis of rSARS-CoV-2 carrying fluorescent or luciferase reporter genes, the findings from their application are overarching in vitro Experiments or live animals have been limited to the properties of the fluorescent or luciferase reporter genes.

About the study

In the present research, the authors have for the first time engineered a replication-competent rSARS-CoV-2 expressing both luciferase (Nluc) and fluorescent (mCherry) reporter genes (rSARS-CoV-2/mCherry-Nluc) to bypass the Disadvantages of using a single reporter gene.

The team developed the first rSARS-CoV-2 expressing both the luciferase-Nluc and fluorescence mCherry reporter gene, ie rSARS-CoV-2/mCherry-Nluc, using their previously disclosed, on the bacterial artificial chromosome (BAC) based reverse genetics and the innovative 2A method. Expression of the two reporter genes was confirmed using luciferase activity with a microplate reader (Nluc) and fluorescence microscopy (mCherry). Researchers used Western blotting to validate reporter expression.

Researchers created a bireporter-based microneutralization experiment to detect and classify antivirals and neutralizing antibodies (NAbs). This assay was based on the advantages of using a rSARS-CoV-2 expressing both fluorescent and bioluminescent proteins over those expressing either fluorescent or luciferase reporter genes. They infected keratin 18 human angiotensin-converting enzyme 2 (K18-hACE2) transgenic mice and Syrian golden hamsters with rSARS-CoV-2/mCherry-Nluc to see whether expression of mCherry in conjunction with Nluc reduced SARS-CoV -2 replication altered in vivo.

Generation of a bireporter rSARS-CoV-2 expressing mCherry and Nluc (rSARS-CoV-2/mCherry-Nluc).  A) Schematic representation of the rSARS-CoV-2/mCherry-Nluc viral genome: SARS-CoV-2 structural, nonstructural, and accessory open reading frame proteins (ORF) are indicated in white boxes.  mCherry (red), Nluc (blue) and the autoproteolytic sequence of PTV-1 2A (black) were inserted in front of the viral N protein.  B) mCherry expression and immunofluorescence assays: Vero E6 cells (1.2 x 106 cells/well, 6-well format, triplicate) were mock infected or infected (MOI 0.01) with rSARS-CoV-2 WT , rSARS-CoV-2/ mCherry, rSARS-CoV-2/Nluc or rSARS-CoV-2/mCherry-Nluc.  Cells were fixed in 10% neutral buffered formalin 24 hpi before direct visualization of mCherry expression under a fluorescence microscope or viral N-protein using a specific 1C7C7 MAb.  Cell nuclei were loaded with DAPI.  Representative images are shown.  Scale bar = 100 µm.  Magnification = X20.  C) Western blots: Vero E6 cells (1.2 x 106 cells/well, 6-well format, triplicate) were mock infected or infected (MOI 0.01).  -CoV-2/Nluc or rSARS-CoV-2/mCherry-Nluc.  At 24 hpi, cells were collected and protein expression in cell lysates assessed by Western blot using specific antibodies against the SARS-CoV-2 N protein or the mCherry and Nluc reporter proteins.  Tubulin was included as a loading control.  The molecular mass of proteins is given in kilodaltons (kDa) on the left.  D) Deep sequence analysis of reporter-expressing rSARS-CoV-2: The non-reference cell frequencies of rSARS-CoV-2/mCherry (top), rSARS-CoV-2/Nluc (middle), and rSARS-CoV-2/mCherry-Nluc ( below) was calculated by comparing the short reads to the respective SARS-CoV-2 WA-1 reference virus genome (MN985325.1).  Non-reference alleles present in less than 10% of the reads are not shown (dashed line) and the frequency of non-reference alleles greater than 10% is shown.

Generation of a bireporter rSARS-CoV-2 expressing mCherry and Nluc (rSARS-CoV-2/mCherry-Nluc). A) Schematic representation of the rSARS-CoV-2/mCherry-Nluc viral genome: SARS-CoV-2 structural, nonstructural, and accessory open reading frame proteins (ORF) are indicated in white boxes. mCherry (red), Nluc (blue) and the autoproteolytic sequence of PTV-1 2A (black) were inserted in front of the viral N protein. B) mCherry expression and immunofluorescence assays: Vero E6 cells (1.2 x 106 cells/well, 6-well format, triplicate) were mock infected or infected (MOI 0.01) with rSARS-CoV-2 WT , rSARS-CoV-2/ mCherry, rSARS-CoV-2/Nluc or rSARS-CoV-2/mCherry-Nluc. Cells were fixed in 10% neutral buffered formalin 24 hpi before direct visualization of mCherry expression under a fluorescence microscope or viral N-protein using a specific 1C7C7 MAb. Cell nuclei were loaded with DAPI. Representative images are shown. Scale bar = 100 µm. Magnification = X20. C) Western blots: Vero E6 cells (1.2 x 106 cells/well, 6-well format, triplicate) were mock infected or infected (MOI 0.01). -CoV-2/Nluc or rSARS-CoV-2/mCherry-Nluc. At 24 hpi, cells were collected and protein expression in cell lysates assessed by Western blot using specific antibodies against the SARS-CoV-2 N protein or the mCherry and Nluc reporter proteins. Tubulin was included as a loading control. The molecular mass of proteins is given in kilodaltons (kDa) on the left. D) Deep sequence analysis of reporter-expressing rSARS-CoV-2: The non-reference cell frequencies of rSARS-CoV-2/mCherry (top), rSARS-CoV-2/Nluc (middle), and rSARS-CoV-2/mCherry-Nluc ( below) was calculated by comparing the short reads to the respective SARS-CoV-2 WA-1 reference virus genome (MN985325.1). Non-reference alleles present in less than 10% of the reads are not shown (dashed line) and the frequency of non-reference alleles greater than 10% is shown.

Results

Study results showed that rSARS-CoV-2/mCherry-Nluc had identical viral fitness in cultured cells as rSARS-CoV-2 expressing single reporter luciferase (rSARS-CoV-2/Nluc) and fluorescence (rSARS-CoV- 2/mCherry) ) genes or wild-type (WT) rSARS-CoV-2, which was an rSARS-CoV-2 without reporter genes. rSARS-CoV-2/mCherry-Nluc also showed similar expression levels for the two reporter genes.

Similarly, the new rSARS-CoV-2/mCherry-Nluc plaque phenotype was similar in size to the plaque phenotypes of rSARS-CoV-2/Nluc, rSARS-CoV-2/mCherry, or rSARS-CoV-2/ WT comparable. However, only rSARS-CoV-2/mCherry-Nluc showed measurable expression levels of both reporter genes. The hypothesis that reporter genes are a reliable surrogate for studying viral infection was further supported by the finding that Nluc or mCherry reporter expression levels were consistent with the level of viral replication.

The authors discovered that the half-maximal effective concentration (EC50) of antiviral agents and the 50% neutralizing titer (NT50) of NAbs obtained in bireporter-based microneutralization experiments with either fluorescence or luciferase signal were identical to those obtained with rSARS -CoV-2 /WT, rSARS-CoV-2/Nluc, or rSARS-CoV-2/mCherry reporter genes and those documented in previous literature.

in vivo, rSARS-CoV-2/mCherry-Nluc, shows similar pathogenicity in K18-hACE2 transgenic mice compared to rSARS-CoV-2 with single reporter genes or WT rSARS-CoV-2. Identical findings have been reported in the Syrian golden hamster model of transmission and infection of SARS-CoV-2. In addition, rSARS-CoV-2/mCherry-Nluc in golden hamsters allows for the assessment of COVID-19 and SARS-CoV-2 transmission in vivo imaging systems (IVIS).

Taken together, the current study shows that SARS-CoV-2 could be analyzed in vivo and in vitro using the currently established new bireporter-expressing rSARS-CoV-2, i.e. rSARS-CoV-2/mCherry-Nluc.

Conclusions

Overall, in the present study, the team created an rSARS-CoV-2 expressing the luciferase (Nluc) and fluorescence (mCherry) genes, namely rSARS-CoV-2/mCherry-Nluc. They showed that rSARS-CoV-2/mCherry-Nluc could be used to research SARS-CoV-2 biology in vivo and or in vitroB. Characterization and identification of NAbs and/or antivirals. The scientists map SARS-CoV-2 infection and transmission via IVIS using rodent models.

rSARS-CoV-2/mCherry-Nluc was suitable for various experimental applications not currently available by using rSARS-CoV-2 expressing a single luciferase or fluorescent reporter gene. According to the investigators, this SARS-CoV-2/mCherry-Nluc virus was the first replication-competent rSARS-CoV-2 to consistently express two reporter genes.

The viability of producing rSARS-CoV-2, which expresses a combination of two reporter genes, illustrates the flexibility of the viral genome to express extensive open reading frames (ORFs) from the SARS-CoV-2 nucleocapsid (N) protein locus express. Additionally, the ability to express foreign genes without having to remove a viral protein (such as ORF7a) and the robust thresholds of reporter gene expression acquired with the 2A autoproteolytic approach make rSARS704 CoV-2/mCherry-Nluc an excellent one Choice for studying viral infections and transmission and pathogenesis, such as. B. newly discovered SARS-CoV-2 variants of concern (VOCs).

*Important NOTE

bioRxiv publishes preliminary scientific reports that have not been peer-reviewed and therefore should not be considered conclusive, guide clinical practice/health behavior or treated as established information.

Magazine reference:

  • Monitoring SARS-CoV-2 infection with a double-reporter-expressing virus; Kevin Chiem, Jun-Gyu Park, Desarey Morales Vasquez, Richard K Plemper, Jordi B Torrelles, James Kobie, Mark R Walter, Chengjin Ye, Luis Martinez-Sobrido. bioRxiv Preprint 2022, DOI: https://doi.org/10.1101/2022.06.23.497376, https://www.biorxiv.org/content/10.1101/2022.06.23.497376v1

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