Generation and Characterization of Recombinant SARS-CoV-2 Expressing Reporter Genes

Chiem, Kevin; Vasquez, Desarey Morales; Park, Jun-Gyu; Platt, Roy Neal; Anderson, Tim; Walter, Mark R.; Kobie, James J.; Ye, Chengjin; Martínez-Sobrido, Luis

doi:10.1128/jvi.02209-20

Cited by 47 publications

(106 citation statements)

References 65 publications

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“…media at 37°C. At the indicated times after infection, viral titers in tissue culture supernatants were determined by plaque assay and immunostaining using the SARS-CoV N protein cross-reactive monoclonal antibody 1C7C7, as previously described (39) .…”

Section: Virus Growth Kineticsmentioning

confidence: 99%

Contribution of SARS-CoV-2 accessory proteins to viral pathogenicity in K18 hACE2 transgenic mice

Silvas

Vasquez

Park

et al. 2021

Preprint

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Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. To date, it is estimated that over 113 million individuals have been infected with SARS-CoV-2 and over 2.5 million human deaths have been recorded worldwide. Currently, three vaccines have been approved by the Food and Drug Administration for emergency use only. However much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse genetics system approach to successfully engineer recombinant (r)SARS-CoV-2, where we individually removed viral 3a, 6, 7a, 7b, and 8 ORF proteins, and characterized these recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF deficient (DORF) viruses producing smaller plaques than those of the wild-type (rSARS-CoV-2/WT). However, growth kinetics of DORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice with the DORF rSARS-CoV-2 identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while DORF7a, DORF7b and DORF8 rSARS-CoV-2 induced comparable pathology to rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse genetics system to generate rSARS-CoV-2 and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live-attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19.

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Section: Virus Growth Kineticsmentioning

confidence: 99%

Contribution of SARS-CoV-2 accessory proteins to viral pathogenicity in K18 hACE2 transgenic mice

Silvas

Vasquez

Park

et al. 2021

Preprint

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“…We observed different adaptation rate in different strain that positively co-relates with the size of the subdominant population in these strains, which indicates that adaptation dynamics are likely dependent on the presence and size of subdominant populations in virus swarm. Furthermore, absence of S1/S2 mutation in BAC derived SARS-CoV-2 clones after five passages in Vero E6 cells (17) suggest that the rapid emergence of mutation that we observed is likely due to the presence of minor variants in clinical isolates. These observations suggest that SARS-CoV-2 maintains a considerable diversity in the quasispecies, facilitating natural selection and a rapid virus adaptation to changing environment and conditions.…”

Section: Discussionmentioning

confidence: 69%

“…However, this stability does not hinder the ability of the virus to adapt quickly. Furthermore, high diversity in the clinical SARS-CoV-2 isolates as compared to BAC derived clones suggest that these minor variants play an important role in overall virus fitness and its adaptation potential (17).…”

Section: Discussionmentioning

confidence: 99%

“…Patient one sequence had 17410 positions with >0.1% and 2163 with >1% mutation as compared to consensus genotype, while SARS-CoV-2 genome from patient two had 6920 positions with >0.1% mutation frequency and 1161 positions with >1% allele frequency. On the other hand, BAC derived SARS-CoV-2 clone has been shown to possess relatively low diversity and lower number of genome position with >1% mutation frequency (17). These data suggested that SARS-CoV-2 virus quasispecies diversity despite stable consensus genotype.…”

Section: Sars-cov-2 Rapidly Adapted Upon Culture On Vero E6 Cellsmentioning

confidence: 97%

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Rapid SARS-CoV-2 Adaptation to Available Cellular Proteases

Chaudhry

Eschke

Hoffmann

et al. 2020

Preprint

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The pandemic spread of SARS-CoV-2 and the resulting global healthcare emergency warrants a better understanding of its biology.The potential of SARS-CoV-2 evolution to create novel dangerous variants remain underexplored. Thus, we passaged SARS-CoV-2 in defined conditions and determined its genomic adaptation dynamics. We demonstrate the presence of remarkably stable SARS-CoV-2 quasispecies. We further show that the quasispecies nature of the virus population ensured rapid adaptation of the spike PRRARS motif upon passaging in Vero cells. On the other hand, SARS-CoV-2 replication in TMPRSS2 expressing cells led to a reverse mutation at the same site. We observed the emergence of novel mutations in envelope protein upon virus culture in Calu-3 and Caco-2 cells. Finally, we show that the heparan sulfate-binding motif (PRRARS) of the SARS-CoV-2 S protein acted as a determinant of negative growth selection. Overall, our research has far-reaching implications for development of antiviral strategies, suggesting viral quasispecies may facilitate rapid emergence of escape mutants under selection pressure, such as the treatment with antivirals against SARS-CoV-2.

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“…Laboratory studies of SARS-CoV-2 allow the use of additional methods to check for the presence of the virus in infected cells. The ability to generate reporter-expressing recombinant viruses represents a powerful tool to address important questions about the pathogenesis of viral infection as well as for the easy and rapid identification of candidate antiviral drug [ 59 ].…”

Section: Recombinant Sars-cov-2 Expressing Reporter Genesmentioning

confidence: 99%

Studying SARS-CoV-2 with Fluorescence Microscopy

Putlyaeva

Lukyanov

2021

IJMS

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The COVID-19 pandemic caused by SARS-CoV-2 coronavirus deeply affected the world community. It gave a strong impetus to the development of not only approaches to diagnostics and therapy, but also fundamental research of the molecular biology of this virus. Fluorescence microscopy is a powerful technology enabling detailed investigation of virus–cell interactions in fixed and live samples with high specificity. While spatial resolution of conventional fluorescence microscopy is not sufficient to resolve all virus-related structures, super-resolution fluorescence microscopy can solve this problem. In this paper, we review the use of fluorescence microscopy to study SARS-CoV-2 and related viruses. The prospects for the application of the recently developed advanced methods of fluorescence labeling and microscopy—which in our opinion can provide important information about the molecular biology of SARS-CoV-2—are discussed.

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Generation and Characterization of Recombinant SARS-CoV-2 Expressing Reporter Genes

Cited by 47 publications

References 65 publications

Contribution of SARS-CoV-2 accessory proteins to viral pathogenicity in K18 hACE2 transgenic mice

Contribution of SARS-CoV-2 accessory proteins to viral pathogenicity in K18 hACE2 transgenic mice

Rapid SARS-CoV-2 Adaptation to Available Cellular Proteases

Studying SARS-CoV-2 with Fluorescence Microscopy

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