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Dr. Luis Martinez-Sobrido
Texas Biomedical Research Institute, San Antonio, TX 78245, USA

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0 Innate Immunity
0 Vaccines
0 Virology
0 antivirals
0 Zika virus

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Influenza viruses
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Zika virus
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Article
Published: 26 August 2021
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Recombinant viruses expressing reporter genes allow visualization and quantification of viral infections and can be used as valid surrogates to identify the presence of the virus in infected cells and animal models. However, one of the limitations of recombinant viruses expressing reporter genes is the use of either fluorescent or luciferase proteins that are used alternatively for different purposes. Vaccinia virus (VV) is widely used as a viral vector, including recombinant (r)VV singly expressing either fluorescent or luciferase reporter genes that are useful for specific purposes. In this report, we engineered two novel rVV stably expressing both fluorescent (Scarlet or GFP) and luciferase (Nluc) reporter genes from different loci in the viral genome. In vitro, these bi-reporter expressing rVV have similar growth kinetics and plaque phenotype than those of the parental WR VV isolate. In vivo, rVV Nluc/Scarlet and rVV Nluc/GFP effectively infected mice and were easily detected using in vivo imaging systems (IVIS) and ex vivo in the lungs from infected mice. We used these bi-reporter expressing rVV to assess viral pathogenesis, infiltration of immune cells in the lungs, and to directly identify the different subsets of cells infected by VV in the absence of antibody staining. Collectively, these rVV expressing two reporter genes open the feasibility to study the biology of viral infections in vitro and in vivo, including host-pathogen interactions and dynamics or tropism of viral infections. Moreover, they represent an excellent approach for the discovery of new prophylactics and/or therapeutics for the treatment of poxvirus infections. IMPORTANCE Despite the eradication of variola virus (VARV), the causative agent of smallpox, poxviruses still represent an important threat to human health due to their possible use as bioterrorism agents and the emergence of zoonotic poxvirus diseases. Recombinant vaccinia viruses (rVV) expressing easily traceable fluorescent or luciferase reporter genes have significantly contributed to the progress of poxvirus research. However, rVV expressing one marker gene have several constraints for in vitro and in vivo studies, since both fluorescent and luciferase proteins impose certain limitations for specific applications. To overcome these limitations, we generated optimized rVV stably expressing both fluorescent (Scarlet or GFP) and luciferase (Nluc) reporter genes to easily track viral infection in vitro and in vivo. This new generation of double reporter-expressing rVV represent an excellent option to study viral infection dynamics in cultured cells and validated animal models of infection, and for the discovery of new poxvirus antiviral treatments.

ACS Style

Kevin Chiem; Maria Lorenzo; Javier Rangel-Moreno; Maria De La Luz Garcia-Hernandez; Jun-Gyu Park; Aitor Nogales; Rafael Blasco; Luis Martínez-Sobrido. Bi-reporter vaccinia virus for tracking viral infections in vitro and in vivo. 2021, 1 .

AMA Style

Kevin Chiem, Maria Lorenzo, Javier Rangel-Moreno, Maria De La Luz Garcia-Hernandez, Jun-Gyu Park, Aitor Nogales, Rafael Blasco, Luis Martínez-Sobrido. Bi-reporter vaccinia virus for tracking viral infections in vitro and in vivo. . 2021; ():1.

Chicago/Turabian Style

Kevin Chiem; Maria Lorenzo; Javier Rangel-Moreno; Maria De La Luz Garcia-Hernandez; Jun-Gyu Park; Aitor Nogales; Rafael Blasco; Luis Martínez-Sobrido. 2021. "Bi-reporter vaccinia virus for tracking viral infections in vitro and in vivo." , no. : 1.

Review
Published: 20 August 2021 in Viruses
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Equine influenza virus (EIV) is a constantly evolving viral pathogen that is responsible for yearly outbreaks of respiratory disease in horses termed equine influenza (EI). There is currently no evidence of circulation of the original H7N7 strain of EIV worldwide; however, the EIV H3N8 strain, which was first isolated in the early 1960s, remains a major threat to most of the world’s horse populations. It can also infect dogs. The ability of EIV to constantly accumulate mutations in its antibody-binding sites enables it to evade host protective immunity, making it a successful viral pathogen. Clinical and virological protection against EIV is achieved by stimulation of strong cellular and humoral immunity in vaccinated horses. However, despite EI vaccine updates over the years, EIV remains relevant, because the protective effects of vaccines decay and permit subclinical infections that facilitate transmission into susceptible populations. In this review, we describe how the evolution of EIV drives repeated EI outbreaks even in horse populations with supposedly high vaccination coverage. Next, we discuss the approaches employed to develop efficacious EI vaccines for commercial use and the existing system for recommendations on updating vaccines based on available clinical and virological data to improve protective immunity in vaccinated horse populations. Understanding how EIV biology can be better harnessed to improve EI vaccines is central to controlling EI.

ACS Style

Fatai S. Oladunni; Saheed Oluwasina Oseni; Luis Martinez-Sobrido; Thomas M. Chambers. Equine Influenza Virus and Vaccines. Viruses 2021, 13, 1657 .

AMA Style

Fatai S. Oladunni, Saheed Oluwasina Oseni, Luis Martinez-Sobrido, Thomas M. Chambers. Equine Influenza Virus and Vaccines. Viruses. 2021; 13 (8):1657.

Chicago/Turabian Style

Fatai S. Oladunni; Saheed Oluwasina Oseni; Luis Martinez-Sobrido; Thomas M. Chambers. 2021. "Equine Influenza Virus and Vaccines." Viruses 13, no. 8: 1657.

Article
Published: 10 August 2021 in Journal of Virology
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Despite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and the contribution of viral proteins to disease outcome remain elusive.

ACS Style

Jesus A. Silvas; Desarey Morales Vasquez; Jun-Gyu Park; Kevin Chiem; Anna Allué-Guardia; Andreu Garcia-Vilanova; Roy Neal Platt; Lisa Miorin; Thomas Kehrer; Anastasija Cupic; Ana S. Gonzalez-Reiche; Harm van Bakel; Adolfo García-Sastre; Tim Anderson; Jordi B. Torrelles; Chengjin Ye; Luis Martinez-Sobrido. Contribution of SARS-CoV-2 Accessory Proteins to Viral Pathogenicity in K18 Human ACE2 Transgenic Mice. Journal of Virology 2021, 95, JVI0040221 .

AMA Style

Jesus A. Silvas, Desarey Morales Vasquez, Jun-Gyu Park, Kevin Chiem, Anna Allué-Guardia, Andreu Garcia-Vilanova, Roy Neal Platt, Lisa Miorin, Thomas Kehrer, Anastasija Cupic, Ana S. Gonzalez-Reiche, Harm van Bakel, Adolfo García-Sastre, Tim Anderson, Jordi B. Torrelles, Chengjin Ye, Luis Martinez-Sobrido. Contribution of SARS-CoV-2 Accessory Proteins to Viral Pathogenicity in K18 Human ACE2 Transgenic Mice. Journal of Virology. 2021; 95 (17):JVI0040221.

Chicago/Turabian Style

Jesus A. Silvas; Desarey Morales Vasquez; Jun-Gyu Park; Kevin Chiem; Anna Allué-Guardia; Andreu Garcia-Vilanova; Roy Neal Platt; Lisa Miorin; Thomas Kehrer; Anastasija Cupic; Ana S. Gonzalez-Reiche; Harm van Bakel; Adolfo García-Sastre; Tim Anderson; Jordi B. Torrelles; Chengjin Ye; Luis Martinez-Sobrido. 2021. "Contribution of SARS-CoV-2 Accessory Proteins to Viral Pathogenicity in K18 Human ACE2 Transgenic Mice." Journal of Virology 95, no. 17: JVI0040221.

Article
Published: 05 July 2021 in bioRxiv
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Drug development for specific antiviral agents against coronavirus disease 2019 (COVID-19) is still an unmet medical need as the pandemic continues to spread globally. Although huge efforts for drug repurposing and compound screens have put forth, only few compounds remain in late stage clinical trials. New approaches and assays are needed to accelerate COVID-19 drug discovery and development. Here we report a time-resolved fluorescence resonance energy transfer-based assay that detects the severe acute respiratory syndrome coronavirus 2 (SARS-CoV‑2) nucleocapsid protein (NP) produced in infected cells. It uses two specific anti-NP monoclonal antibodies (MAbs) conjugated to donor and acceptor fluorophores that produces a robust ratiometric signal for high throughput screening of large compound collections. Using this assay, we measured a half maximal inhibitory concentration (IC50) for Remdesivir of 9.3 μM against infection with SARS-CoV-2 USA/WA1/2020 (WA-1). The assay also detected SARS-CoV-2 South African (Beta, β), Brazilian/Japanese variant P.1 (Gamma, γ), and Californian (Epsilon, ε), variants of concern or interest (VoC). Therefore, this homogeneous SARS-CoV-2 NP detection assay can be used for accelerating lead compound discovery for drug development and for evaluating drug efficacy against emerging SARS-CoV-2 VoC.

ACS Style

Kirill Gorshkov; Desarey Morales Vasquez; Kevin Chiem; Chengjin Ye; Bruce Nguyen Tran; Juan Carlos de la Torre; Thomas Moran; Catherine Z. Chen; Luis Martinez-Sobrido; Wei Zheng. A SARS-CoV-2 nucleocapsid protein TR-FRET assay amenable to high-throughput screening. bioRxiv 2021, 1 .

AMA Style

Kirill Gorshkov, Desarey Morales Vasquez, Kevin Chiem, Chengjin Ye, Bruce Nguyen Tran, Juan Carlos de la Torre, Thomas Moran, Catherine Z. Chen, Luis Martinez-Sobrido, Wei Zheng. A SARS-CoV-2 nucleocapsid protein TR-FRET assay amenable to high-throughput screening. bioRxiv. 2021; ():1.

Chicago/Turabian Style

Kirill Gorshkov; Desarey Morales Vasquez; Kevin Chiem; Chengjin Ye; Bruce Nguyen Tran; Juan Carlos de la Torre; Thomas Moran; Catherine Z. Chen; Luis Martinez-Sobrido; Wei Zheng. 2021. "A SARS-CoV-2 nucleocapsid protein TR-FRET assay amenable to high-throughput screening." bioRxiv , no. : 1.

Journal article
Published: 30 June 2021 in Viruses
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Influenza B viruses (IBV) circulate annually, with young children, the elderly and immunocompromised individuals being at high risk. Yearly vaccinations are recommended to protect against seasonally influenza viruses, including IBV. Live attenuated influenza vaccines (LAIV) provide the unique opportunity for direct exposure to the antigenically variable surface glycoproteins as well as the more conserved internal components. Ideally, LAIV Master Donor Viruses (MDV) should accurately reflect seasonal influenza strains. Unfortunately, the continuous evolution of IBV have led to significant changes in conserved epitopes compared to the IBV MDV based on B/Ann Arbor/1/1966 strain. Here, we propose a recent influenza B/Brisbane/60/2008 as an efficacious MDV alternative, as its internal viral proteins more accurately reflect those of circulating IBV strains. We introduced the mutations responsible for the temperature sensitive (ts), cold adapted (ca) and attenuated (att) phenotype of B/Ann Arbor/1/1966 MDV LAIV into B/Brisbane/60/2008 to generate a new MDV LAIV. In vitro and in vivo analysis demonstrated that the mutations responsible of the ts, ca, and att phenotype of B/Ann Arbor/1/1966 MDV LAIV were able to infer the same phenotype to B/Brisbane/60/2008, demonstrating its potential as a new MDV for the development of LAIV to protect against contemporary IBV strains.

ACS Style

Chantelle White; Kevin Chiem; Daniel Perez; Jefferson Santos; Stivalis Cardenas Garcia; Aitor Nogales; Luis Martínez-Sobrido. A New Master Donor Virus for the Development of Live-Attenuated Influenza B Virus Vaccines. Viruses 2021, 13, 1278 .

AMA Style

Chantelle White, Kevin Chiem, Daniel Perez, Jefferson Santos, Stivalis Cardenas Garcia, Aitor Nogales, Luis Martínez-Sobrido. A New Master Donor Virus for the Development of Live-Attenuated Influenza B Virus Vaccines. Viruses. 2021; 13 (7):1278.

Chicago/Turabian Style

Chantelle White; Kevin Chiem; Daniel Perez; Jefferson Santos; Stivalis Cardenas Garcia; Aitor Nogales; Luis Martínez-Sobrido. 2021. "A New Master Donor Virus for the Development of Live-Attenuated Influenza B Virus Vaccines." Viruses 13, no. 7: 1278.

Preprint content
Published: 28 June 2021
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and has been responsible for the still ongoing coronavirus disease 2019 (COVID-19) pandemic. Prophylactic vaccines have been authorized by the United States (US) Food and Drug Administration (FDA) for the prevention of COVID-19. Identification of SARS-CoV-2 neutralizing antibodies (NAbs) is important to assess vaccine protection efficacy, including their ability to protect against emerging SARS-CoV-2 variants of concern (VoC). Here we report the generation and use of a recombinant (r)SARS-CoV-2 USA/WA1/2020 (WA-1) strain expressing Venus and a rSARS-CoV-2 expressing mCherry and containing mutations K417N, E484K, and N501Y found in the receptor binding domain (RBD) of the spike (S) glycoprotein of the South African (SA) B.1.351 (beta, β) VoC, in bifluorescent-based assays to rapidly and accurately identify human monoclonal antibodies (hMAbs) able to neutralize both viral infections in vitro and in vivo. Importantly, our bifluorescent-based system accurately recapitulated findings observed using individual viruses. Moreover, fluorescent-expressing rSARS-CoV-2 and the parental wild-type (WT) rSARS-CoV-2 WA-1 had similar viral fitness in vitro, as well as similar virulence and pathogenicity in vivo in the K18 human angiotensin converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 infection. We demonstrate that these new fluorescent-expressing rSARS-CoV-2 can be used in vitro and in vivo to easily identify hMAbs that simultaneously neutralize different SARS-CoV-2 strains, including VoC, for the rapid assessment of vaccine efficacy or the identification of prophylactic and/or therapeutic broadly NAbs for the treatment of SARS-CoV-2 infection.

ACS Style

Kevin Chiem; Desarey Morales Vasquez; Jesus A. Silvas; Jun-Gyu Park; Michael S. Piepenbrink; Julien Sourimant; Michelle J. Lin; Alexander L. Greninger; Richard K. Plemper; Jordi B. Torrelles; Mark R. Walter; Juan C. de la Torre; James K. Kobie; Luis Martinez-Sobrido. A bifluorescent-based assay for the identification of neutralizing antibodies against SARS-CoV-2 variants of concern in vitro and in vivo. 2021, 1 .

AMA Style

Kevin Chiem, Desarey Morales Vasquez, Jesus A. Silvas, Jun-Gyu Park, Michael S. Piepenbrink, Julien Sourimant, Michelle J. Lin, Alexander L. Greninger, Richard K. Plemper, Jordi B. Torrelles, Mark R. Walter, Juan C. de la Torre, James K. Kobie, Luis Martinez-Sobrido. A bifluorescent-based assay for the identification of neutralizing antibodies against SARS-CoV-2 variants of concern in vitro and in vivo. . 2021; ():1.

Chicago/Turabian Style

Kevin Chiem; Desarey Morales Vasquez; Jesus A. Silvas; Jun-Gyu Park; Michael S. Piepenbrink; Julien Sourimant; Michelle J. Lin; Alexander L. Greninger; Richard K. Plemper; Jordi B. Torrelles; Mark R. Walter; Juan C. de la Torre; James K. Kobie; Luis Martinez-Sobrido. 2021. "A bifluorescent-based assay for the identification of neutralizing antibodies against SARS-CoV-2 variants of concern in vitro and in vivo." , no. : 1.

Preprint content
Published: 04 June 2021
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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.

ACS Style

Chengjin Ye; Kevin Chiem; Jun-Gyu Park; Jesus A. Silvas; Desarey Morales Vasquez; Jordi B. Torrelles; James J. Kobie; Mark R. Walter; Juan Carlos de la Torre; Luis Martinez-Sobrido. Visualization of SARS-CoV-2 infection dynamic. 2021, 1 .

AMA Style

Chengjin Ye, Kevin Chiem, Jun-Gyu Park, Jesus A. Silvas, Desarey Morales Vasquez, Jordi B. Torrelles, James J. Kobie, Mark R. Walter, Juan Carlos de la Torre, Luis Martinez-Sobrido. Visualization of SARS-CoV-2 infection dynamic. . 2021; ():1.

Chicago/Turabian Style

Chengjin Ye; Kevin Chiem; Jun-Gyu Park; Jesus A. Silvas; Desarey Morales Vasquez; Jordi B. Torrelles; James J. Kobie; Mark R. Walter; Juan Carlos de la Torre; Luis Martinez-Sobrido. 2021. "Visualization of SARS-CoV-2 infection dynamic." , no. : 1.

Journal article
Published: 02 June 2021 in Nature Communications
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The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2′-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2′-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2′-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.

ACS Style

Thiruselvam Viswanathan; Anurag Misra; Siu-Hong Chan; Shan Qi; Nan Dai; Shailee Arya; Luis Martinez-Sobrido; Yogesh K. Gupta. A metal ion orients SARS-CoV-2 mRNA to ensure accurate 2′-O methylation of its first nucleotide. Nature Communications 2021, 12, 1 -7.

AMA Style

Thiruselvam Viswanathan, Anurag Misra, Siu-Hong Chan, Shan Qi, Nan Dai, Shailee Arya, Luis Martinez-Sobrido, Yogesh K. Gupta. A metal ion orients SARS-CoV-2 mRNA to ensure accurate 2′-O methylation of its first nucleotide. Nature Communications. 2021; 12 (1):1-7.

Chicago/Turabian Style

Thiruselvam Viswanathan; Anurag Misra; Siu-Hong Chan; Shan Qi; Nan Dai; Shailee Arya; Luis Martinez-Sobrido; Yogesh K. Gupta. 2021. "A metal ion orients SARS-CoV-2 mRNA to ensure accurate 2′-O methylation of its first nucleotide." Nature Communications 12, no. 1: 1-7.

Journal article
Published: 20 May 2021 in Microorganisms
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The influenza A virus (IAV) PA-X protein is a virulence factor that selectively degrades host mRNAs leading to protein shutoff. This function modulates host inflammation, antiviral responses, cell apoptosis, and pathogenesis. In this work we describe a novel approach based on the use of bacteria and plasmid encoding of the PA-X gene under the control of the bacteriophage T7 promoter to identify amino acid residues important for A/Brevig Mission/1/1918 H1N1 PA-X’s shutoff activity. Using this system, we have identified PA-X mutants encoding single or double amino acid changes, which diminish its host shutoff activity, as well as its ability to counteract interferon responses upon viral infection. This novel bacteria-based approach could be used for the identification of viral proteins that inhibit host gene expression as well as the amino acid residues responsible for inhibition of host gene expression.

ACS Style

Kevin Chiem; Luis Martinez-Sobrido; Aitor Nogales; Marta DeDiego. Amino Acid Residues Involved in Inhibition of Host Gene Expression by Influenza A/Brevig Mission/1/1918 PA-X. Microorganisms 2021, 9, 1109 .

AMA Style

Kevin Chiem, Luis Martinez-Sobrido, Aitor Nogales, Marta DeDiego. Amino Acid Residues Involved in Inhibition of Host Gene Expression by Influenza A/Brevig Mission/1/1918 PA-X. Microorganisms. 2021; 9 (5):1109.

Chicago/Turabian Style

Kevin Chiem; Luis Martinez-Sobrido; Aitor Nogales; Marta DeDiego. 2021. "Amino Acid Residues Involved in Inhibition of Host Gene Expression by Influenza A/Brevig Mission/1/1918 PA-X." Microorganisms 9, no. 5: 1109.

Preprint content
Published: 05 May 2021
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SARS-CoV-2 variants of concern (VoCs) are impacting responses to the COVID-19 pandemic. Here we present a comparison of the SARS-CoV-2 USA-WA1/2020 (WA-1) strain with B.1.1.7 and B.1.351 VoCs and identify significant differences in viral propagation in vitro and pathogenicity in vivo using K18-hACE2 transgenic mice. Passive immunization with plasma from an early pandemic SARS-CoV-2 patient resulted in significant differences in the outcome of VoC-infected mice. WA-1-infected mice were protected by plasma, B.1.1.7-infected mice were partially protected, and B.1.351-infected mice were not protected. Serological correlates of disease were different between VoC-infected mice, with B.1.351 triggering significantly altered cytokine profiles than other strains. In this study, we defined infectivity and immune responses triggered by VoCs and observed that early 2020 SARS-CoV-2 human immune plasma was insufficient to protect against challenge with B.1.1.7 and B.1.351 in the mouse model.

ACS Style

Alexander M. Horspool; Chengjin Ye; Ting Y. Wong; Brynnan P. Russ; Katherine S. Lee; Michael T. Winters; Justin R. Bevere; Theodore Kieffer; Ivan Martinez; Julien Sourimant; Alexander L. Greninger; Richard K. Plemper; James Denvir; Holly A. Cyphert; Jordi B Torrelles; Luis Martinez-Sobrido; Fredrick Heath Damron. SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern induce lethal disease in K18-hACE2 transgenic mice despite convalescent plasma therapy. 2021, 1 .

AMA Style

Alexander M. Horspool, Chengjin Ye, Ting Y. Wong, Brynnan P. Russ, Katherine S. Lee, Michael T. Winters, Justin R. Bevere, Theodore Kieffer, Ivan Martinez, Julien Sourimant, Alexander L. Greninger, Richard K. Plemper, James Denvir, Holly A. Cyphert, Jordi B Torrelles, Luis Martinez-Sobrido, Fredrick Heath Damron. SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern induce lethal disease in K18-hACE2 transgenic mice despite convalescent plasma therapy. . 2021; ():1.

Chicago/Turabian Style

Alexander M. Horspool; Chengjin Ye; Ting Y. Wong; Brynnan P. Russ; Katherine S. Lee; Michael T. Winters; Justin R. Bevere; Theodore Kieffer; Ivan Martinez; Julien Sourimant; Alexander L. Greninger; Richard K. Plemper; James Denvir; Holly A. Cyphert; Jordi B Torrelles; Luis Martinez-Sobrido; Fredrick Heath Damron. 2021. "SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern induce lethal disease in K18-hACE2 transgenic mice despite convalescent plasma therapy." , no. : 1.

Journal article
Published: 17 April 2021 in Viruses
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The influenza A virus (IAV) is able to infect multiple mammalian and avian species, and in humans IAV is responsible for annual seasonal epidemics and occasional pandemics of respiratory disease with significant health and economic impacts. Studying IAV involves laborious secondary methodologies to identify infected cells. Therefore, to circumvent this requirement, in recent years, multiple replication-competent infectious IAV expressing traceable reporter genes have been developed. These IAVs have been very useful for in vitro and/or in vivo studies of viral replication, identification of neutralizing antibodies or antivirals, and in studies to evaluate vaccine efficacy, among others. In this report, we describe, for the first time, the generation and characterization of two replication-competent influenza A/Puerto Rico/8/1934 H1N1 (PR8) viruses where the viral non-structural protein 1 (NS1) was substituted by the monomeric (m)Cherry fluorescent or the NanoLuc luciferase (Nluc) proteins. The ΔNS1 mCherry was able to replicate in cultured cells and in Signal Transducer and Activator of Transcription 1 (STAT1) deficient mice, although at a lower extent than a wild-type (WT) PR8 virus expressing the same mCherry fluorescent protein (WT mCherry). Notably, expression of either reporter gene (mCherry or Nluc) was detected in infected cells by fluorescent microscopy or luciferase plate readers, respectively. ΔNS1 IAV expressing reporter genes provide a novel approach to better understand the biology and pathogenesis of IAV, and represent an excellent tool to develop new therapeutic approaches against IAV infections.

ACS Style

Aitor Nogales; Michael Schotsaert; Raveen Rathnasinghe; Marta DeDiego; Adolfo García-Sastre; Luis Martinez-Sobrido. Replication-Competent ΔNS1 Influenza A Viruses Expressing Reporter Genes. Viruses 2021, 13, 698 .

AMA Style

Aitor Nogales, Michael Schotsaert, Raveen Rathnasinghe, Marta DeDiego, Adolfo García-Sastre, Luis Martinez-Sobrido. Replication-Competent ΔNS1 Influenza A Viruses Expressing Reporter Genes. Viruses. 2021; 13 (4):698.

Chicago/Turabian Style

Aitor Nogales; Michael Schotsaert; Raveen Rathnasinghe; Marta DeDiego; Adolfo García-Sastre; Luis Martinez-Sobrido. 2021. "Replication-Competent ΔNS1 Influenza A Viruses Expressing Reporter Genes." Viruses 13, no. 4: 698.

Preprint content
Published: 12 March 2021
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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 (ΔORF) viruses producing smaller plaques than those of the wild-type (rSARS-CoV-2/WT). However, growth kinetics of ΔORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice with the ΔORF rSARS-CoV-2 identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while ΔORF7a, ΔORF7b and ΔORF8 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. IMPORTANCE Despite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and contribution of viral proteins to disease outcome remains elusive. Our study aims to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins in viral pathogenesis and disease outcome, and develop a synergistic platform combining our robust reverse genetics system to generate recombinant (r)SARS-CoV-2 with a validated rodent model of infection and disease. We demonstrated that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as the foundation to generate attenuated forms of the virus to develop live-attenuated vaccines for the treatment of SARS-CoV-2.

ACS Style

Jesus Silvas; Desarey Morales-Vasquez; Jun-Gyu Park; Kevin Chiem; Jordi B. Torrelles; Roy Neal Platt; Tim Anderson; Chengjin Ye; Luis Martinez-Sobrido. Contribution of SARS-CoV-2 accessory proteins to viral pathogenicity in K18 hACE2 transgenic mice. 2021, 1 .

AMA Style

Jesus Silvas, Desarey Morales-Vasquez, Jun-Gyu Park, Kevin Chiem, Jordi B. Torrelles, Roy Neal Platt, Tim Anderson, Chengjin Ye, Luis Martinez-Sobrido. Contribution of SARS-CoV-2 accessory proteins to viral pathogenicity in K18 hACE2 transgenic mice. . 2021; ():1.

Chicago/Turabian Style

Jesus Silvas; Desarey Morales-Vasquez; Jun-Gyu Park; Kevin Chiem; Jordi B. Torrelles; Roy Neal Platt; Tim Anderson; Chengjin Ye; Luis Martinez-Sobrido. 2021. "Contribution of SARS-CoV-2 accessory proteins to viral pathogenicity in K18 hACE2 transgenic mice." , no. : 1.

Journal article
Published: 10 March 2021 in Journal of Virology
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has significantly impacted the human health and economic status worldwide. There is an urgent need to identify effective prophylactics and therapeutics for the treatment of SARS-CoV-2 infection and associated COVID-19.

ACS Style

Kevin Chiem; Desarey Morales Vasquez; Jun-Gyu Park; Roy Neal Platt; Tim Anderson; Mark R. Walter; James J. Kobie; Chengjin Ye; Luis Martinez-Sobrido. Generation and Characterization of Recombinant SARS-CoV-2 Expressing Reporter Genes. Journal of Virology 2021, 95, 1 .

AMA Style

Kevin Chiem, Desarey Morales Vasquez, Jun-Gyu Park, Roy Neal Platt, Tim Anderson, Mark R. Walter, James J. Kobie, Chengjin Ye, Luis Martinez-Sobrido. Generation and Characterization of Recombinant SARS-CoV-2 Expressing Reporter Genes. Journal of Virology. 2021; 95 (7):1.

Chicago/Turabian Style

Kevin Chiem; Desarey Morales Vasquez; Jun-Gyu Park; Roy Neal Platt; Tim Anderson; Mark R. Walter; James J. Kobie; Chengjin Ye; Luis Martinez-Sobrido. 2021. "Generation and Characterization of Recombinant SARS-CoV-2 Expressing Reporter Genes." Journal of Virology 95, no. 7: 1.

Journal article
Published: 24 February 2021 in Cell Reports Medicine
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Summary SARS-CoV-2 infection results in viral burden in the respiratory tract, enabling transmission and leading to substantial lung pathology. The 1212C2 fully human monoclonal antibody was derived from an IgM memory B cell of a COVID-19 patient, has high affinity for the Spike protein receptor binding domain, neutralizes SARS-CoV-2, and exhibits in vivo prophylactic and therapeutic activity in hamsters when delivered intraperitoneally, reducing upper and lower respiratory viral burden and lung pathology. Inhalation of nebulized 1212C2 at levels as low as 0.6 mg/kg, corresponding to 0.03 mg/kg lung-deposited dose, reduced the viral burden below the detection limit and mitigated lung pathology. The therapeutic efficacy of an exceedingly low dose of inhaled 1212C2 supports the rationale for local lung delivery for dose-sparing benefits, as compared to the conventional parenteral route of administration. These results suggest that the clinical development of 1212C2 formulated and delivered via inhalation for the treatment of SARS-CoV-2 infection should be considered.

ACS Style

Michael S. Piepenbrink; Jun-Gyu Park; Fatai S. Oladunni; Ashlesha Deshpande; Madhubanti Basu; Sanghita Sarkar; Andreas Loos; Jennifer Woo; Phillip Lovalenti; Derek Sloan; Chengjin Ye; Kevin Chiem; Christopher W. Bates; Reuben E. Burch; Nathaniel B. Erdmann; Paul A. Goepfert; Vu L. Truong; Mark R. Walter; Luis Martinez-Sobrido; James J. Kobie. Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters. Cell Reports Medicine 2021, 2, 100218 .

AMA Style

Michael S. Piepenbrink, Jun-Gyu Park, Fatai S. Oladunni, Ashlesha Deshpande, Madhubanti Basu, Sanghita Sarkar, Andreas Loos, Jennifer Woo, Phillip Lovalenti, Derek Sloan, Chengjin Ye, Kevin Chiem, Christopher W. Bates, Reuben E. Burch, Nathaniel B. Erdmann, Paul A. Goepfert, Vu L. Truong, Mark R. Walter, Luis Martinez-Sobrido, James J. Kobie. Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters. Cell Reports Medicine. 2021; 2 (3):100218.

Chicago/Turabian Style

Michael S. Piepenbrink; Jun-Gyu Park; Fatai S. Oladunni; Ashlesha Deshpande; Madhubanti Basu; Sanghita Sarkar; Andreas Loos; Jennifer Woo; Phillip Lovalenti; Derek Sloan; Chengjin Ye; Kevin Chiem; Christopher W. Bates; Reuben E. Burch; Nathaniel B. Erdmann; Paul A. Goepfert; Vu L. Truong; Mark R. Walter; Luis Martinez-Sobrido; James J. Kobie. 2021. "Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters." Cell Reports Medicine 2, no. 3: 100218.

Journal article
Published: 26 January 2021 in Journal of Virological Methods
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The use of monoclonal neutralizing antibodies (mNAbs) is being actively pursued as a viable intervention for the treatment of Severe Acute Respiratory Syndrome CoV-2 (SARS-CoV-2) infection and associated coronavirus disease 2019 (COVID-19). While highly potent mNAbs have great therapeutic potential, the ability of the virus to mutate and escape recognition and neutralization of mNAbs represents a potential problem in their use for the therapeutic management of SARS-CoV-2. Studies investigating natural or mNAb-induced antigenic variability in the receptor binding domain (RBD) of SARS-CoV-2 Spike (S) glycoprotein, and their effects on viral fitness are still rudimentary. In this manuscript we described experimental approaches for the selection, identification, and characterization of SARS-CoV-2 monoclonal antibody resistant mutants (MARMs) in cultured cells. The ability to study SARS-CoV-2 antigenic drift under selective immune pressure by mNAbs is important for the optimal implementation of mNAbs for the therapeutic management of COVID-19. This will help to identify essential amino acid residues in the viral S glycoprotein required for mNAb-mediated inhibition of viral infection, to predict potential natural drift variants that could emerge upon implementation of therapeutic mNAbs, as well as vaccine prophylactic treatments for SARS-CoV-2 infection. Additionally, it will also enable the assessment of MARM viral fitness and its potential to induce severe infection and associated COVID-19 disease.

ACS Style

Fatai S. Oladunni; Jun-Gyu Park; Kevin Chiem; Chengjin Ye; Michael Pipenbrink; Mark R. Walter; James Kobie; Luis Martinez-Sobrido. Selection, identification, and characterization of SARS-CoV-2 monoclonal antibody resistant mutants. Journal of Virological Methods 2021, 290, 114084 -114084.

AMA Style

Fatai S. Oladunni, Jun-Gyu Park, Kevin Chiem, Chengjin Ye, Michael Pipenbrink, Mark R. Walter, James Kobie, Luis Martinez-Sobrido. Selection, identification, and characterization of SARS-CoV-2 monoclonal antibody resistant mutants. Journal of Virological Methods. 2021; 290 ():114084-114084.

Chicago/Turabian Style

Fatai S. Oladunni; Jun-Gyu Park; Kevin Chiem; Chengjin Ye; Michael Pipenbrink; Mark R. Walter; James Kobie; Luis Martinez-Sobrido. 2021. "Selection, identification, and characterization of SARS-CoV-2 monoclonal antibody resistant mutants." Journal of Virological Methods 290, no. : 114084-114084.

Journal article
Published: 30 November 2020 in Nature Communications
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Vaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease would benefit from validated small animal models. Here, we show that transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2 transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2 transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 6. K18 hACE2 transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.

ACS Style

Fatai S. Oladunni; Jun-Gyu Park; Paula A. Pino; Olga Gonzalez; Anwari Akhter; Anna Allué-Guardia; Angélica Olmo-Fontánez; Shalini Gautam; Andreu Garcia-Vilanova; Chengjin Ye; Kevin Chiem; Colwyn Headley; Varun Dwivedi; Laura M. Parodi; Kendra J. Alfson; Hilary M. Staples; Alyssa Schami; Juan I. Garcia; Alison Whigham; Roy Neal Platt Ii; Michal Gazi; Jesse Martinez; Colin Chuba; Stephanie Earley; Oscar H. Rodriguez; Stephanie Davis Mdaki; Katrina N. Kavelish; Renee Escalona; Cory R. A. Hallam; Corbett Christie; Jean L. Patterson; Tim J. C. Anderson; Ricardo Carrion Jr; Edward J. Dick Jr; Shannan Hall-Ursone; Larry S. Schlesinger; Xavier Alvarez; Deepak Kaushal; Luis D. Giavedoni; Joanne Turner; Luis Martinez-Sobrido; Jordi B. Torrelles. Lethality of SARS-CoV-2 infection in K18 human angiotensin-converting enzyme 2 transgenic mice. Nature Communications 2020, 11, 1 -17.

AMA Style

Fatai S. Oladunni, Jun-Gyu Park, Paula A. Pino, Olga Gonzalez, Anwari Akhter, Anna Allué-Guardia, Angélica Olmo-Fontánez, Shalini Gautam, Andreu Garcia-Vilanova, Chengjin Ye, Kevin Chiem, Colwyn Headley, Varun Dwivedi, Laura M. Parodi, Kendra J. Alfson, Hilary M. Staples, Alyssa Schami, Juan I. Garcia, Alison Whigham, Roy Neal Platt Ii, Michal Gazi, Jesse Martinez, Colin Chuba, Stephanie Earley, Oscar H. Rodriguez, Stephanie Davis Mdaki, Katrina N. Kavelish, Renee Escalona, Cory R. A. Hallam, Corbett Christie, Jean L. Patterson, Tim J. C. Anderson, Ricardo Carrion Jr, Edward J. Dick Jr, Shannan Hall-Ursone, Larry S. Schlesinger, Xavier Alvarez, Deepak Kaushal, Luis D. Giavedoni, Joanne Turner, Luis Martinez-Sobrido, Jordi B. Torrelles. Lethality of SARS-CoV-2 infection in K18 human angiotensin-converting enzyme 2 transgenic mice. Nature Communications. 2020; 11 (1):1-17.

Chicago/Turabian Style

Fatai S. Oladunni; Jun-Gyu Park; Paula A. Pino; Olga Gonzalez; Anwari Akhter; Anna Allué-Guardia; Angélica Olmo-Fontánez; Shalini Gautam; Andreu Garcia-Vilanova; Chengjin Ye; Kevin Chiem; Colwyn Headley; Varun Dwivedi; Laura M. Parodi; Kendra J. Alfson; Hilary M. Staples; Alyssa Schami; Juan I. Garcia; Alison Whigham; Roy Neal Platt Ii; Michal Gazi; Jesse Martinez; Colin Chuba; Stephanie Earley; Oscar H. Rodriguez; Stephanie Davis Mdaki; Katrina N. Kavelish; Renee Escalona; Cory R. A. Hallam; Corbett Christie; Jean L. Patterson; Tim J. C. Anderson; Ricardo Carrion Jr; Edward J. Dick Jr; Shannan Hall-Ursone; Larry S. Schlesinger; Xavier Alvarez; Deepak Kaushal; Luis D. Giavedoni; Joanne Turner; Luis Martinez-Sobrido; Jordi B. Torrelles. 2020. "Lethality of SARS-CoV-2 infection in K18 human angiotensin-converting enzyme 2 transgenic mice." Nature Communications 11, no. 1: 1-17.

Preprint content
Published: 18 November 2020
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The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible of coronavirus disease 2019 (COVID-19), has devastated public health services and economies worldwide. Despite global efforts to contain the COVID-19 pandemic, SARS-CoV-2 is now found in over 200 countries and has caused an upward death toll of over 1 million human lives as of November 2020. To date, only one Food and Drug Administration (FDA)-approved therapeutic drug (Remdesivir) and a monoclonal antibody, MAb (Bamlanivimab), but no vaccines, are available for the treatment of SARS-CoV-2. As with other viruses, studying SARS-CoV-2 requires the use of secondary approaches to detect the presence of the virus in infected cells. To overcome this limitation, we have generated replication-competent recombinant (r)SARS-CoV-2 expressing fluorescent (Venus or mCherry) or bioluminescent (Nluc) reporter genes. Vero E6 cells infected with reporter-expressing rSARS-CoV-2 can be easily detected via fluorescence or luciferase expression and display a good correlation between reporter gene expression and viral replication. Moreover, rSARS-CoV-2 expressing reporter genes have comparable plaque sizes and growth kinetics to those of wild-type virus, rSARS-CoV-2/WT. We used these reporter-expressing rSARS-CoV-2 to demonstrate their feasibility to identify neutralizing antibodies (NAbs) or antiviral drugs. Our results demonstrate that reporter-expressing rSARS-CoV-2 represent an excellent option to identify therapeutics for the treatment of SARS-CoV-2, where reporter gene expression can be used as valid surrogates to track viral infection. Moreover, the ability to manipulate the viral genome opens the feasibility of generating viruses expressing foreign genes for their use as vaccines for the treatment of SARS-CoV-2 infection. Importance Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has significantly impacted the human health and economic status worldwide. There is an urgent need to identify effective prophylactics and therapeutics for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. The use of fluorescent- or luciferase-expressing reporter expressing viruses has significantly advanced viral research. Here, we generated recombinant (r)SARS-CoV-2 expressing fluorescent (Venus and mCherry) or luciferase (Nluc) reporter genes and demonstrate that they represent an excellent option to track viral infections in vitro. Importantly, reporter-expressing rSARS-CoV-2 display similar growth kinetics and plaque phenotype that their wild-type counterpart (rSARS-CoV-2/WT), demonstrating their feasibility to identify drugs and/or neutralizing antibodies (NAbs) for the therapeutic treatment of SARS-CoV-2. Henceforth, these reporter-expressing rSARS-CoV-2 can be used to interrogate large libraries of compounds and/or monoclonal antibodies (MAb), in high-throughput screening settings, to identify those with therapeutic potential against SARS-CoV-2.

ACS Style

Kevin Chiem; Desarey Morales Vasquez; Jun-Gyu Park; Roy Neal Platt; Tim Anderson; Mark R. Walter; James J. Kobie; Chengjin Ye; Luis Martinez-Sobrido. Generation and Characterization of recombinant SARS-CoV-2 expressing reporter genes. 2020, 1 .

AMA Style

Kevin Chiem, Desarey Morales Vasquez, Jun-Gyu Park, Roy Neal Platt, Tim Anderson, Mark R. Walter, James J. Kobie, Chengjin Ye, Luis Martinez-Sobrido. Generation and Characterization of recombinant SARS-CoV-2 expressing reporter genes. . 2020; ():1.

Chicago/Turabian Style

Kevin Chiem; Desarey Morales Vasquez; Jun-Gyu Park; Roy Neal Platt; Tim Anderson; Mark R. Walter; James J. Kobie; Chengjin Ye; Luis Martinez-Sobrido. 2020. "Generation and Characterization of recombinant SARS-CoV-2 expressing reporter genes." , no. : 1.

Journal article
Published: 27 October 2020 in mBio
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The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate in vitro and in vivo . This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19.

ACS Style

Chengjin Ye; Kevin Chiem; Jun-Gyu Park; Fatai Oladunni; Roy Nelson Platt; Tim Anderson; Fernando Almazan; Juan Carlos de la Torre; Luis Martinez-Sobrido. Rescue of SARS-CoV-2 from a Single Bacterial Artificial Chromosome. mBio 2020, 11, 1 .

AMA Style

Chengjin Ye, Kevin Chiem, Jun-Gyu Park, Fatai Oladunni, Roy Nelson Platt, Tim Anderson, Fernando Almazan, Juan Carlos de la Torre, Luis Martinez-Sobrido. Rescue of SARS-CoV-2 from a Single Bacterial Artificial Chromosome. mBio. 2020; 11 (5):1.

Chicago/Turabian Style

Chengjin Ye; Kevin Chiem; Jun-Gyu Park; Fatai Oladunni; Roy Nelson Platt; Tim Anderson; Fernando Almazan; Juan Carlos de la Torre; Luis Martinez-Sobrido. 2020. "Rescue of SARS-CoV-2 from a Single Bacterial Artificial Chromosome." mBio 11, no. 5: 1.

Journal article
Published: 14 October 2020 in Journal of Virological Methods
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Towards the end of 2019, a novel coronavirus (CoV) named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), genetically similar to severe acute respiratory syndrome coronavirus (SARS-CoV), emerged in Wuhan, Hubei province of China, and has been responsible for coronavirus disease 2019 (COVID-19) in humans. Since its first report, SARS-CoV-2 has resulted in a global pandemic, with over 10 million human infections and over 560,000 deaths reported worldwide at the end of June 2020. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines and/or antivirals licensed against SARS-CoV-2. The high economical and health impacts of SARS-CoV-2 has placed global pressure on the scientific community to identify effective prophylactic and therapeutic treatments for SARS-CoV-2 infection and associated COVID-19 disease. While some compounds have been already reported to reduce SARS-CoV-2 infection and a handful of monoclonal antibodies (mAbs) have been described that neutralize SARS-CoV-2, there is an urgent need for the development and standardization of assays which can be used in high through-put screening (HTS) settings to identify new antivirals and/or neutralizing mAbs against SARS-CoV-2. Here, we described a rapid, accurate, and highly reproducible plaque reduction microneutralization (PRMNT) assay that can be quickly adapted for the identification and characterization of both neutralizing mAbs and antivirals against SARS-CoV-2. Importantly, our MNA is compatible with HTS settings to interrogate large and/or complex libraries of mAbs and/or antivirals to identify those with neutralizing and/or antiviral activity, respectively, against SARS-CoV-2.

ACS Style

Jun-Gyu Park; Fatai S. Oladunni; Kevin Chiem; Chengjin Ye; Michael Pipenbrink; Thomas Moran; Mark R. Walter; James Kobie; Luis Martinez-Sobrido. Rapid in vitro assays for screening neutralizing antibodies and antivirals against SARS-CoV-2. Journal of Virological Methods 2020, 287, 113995 -113995.

AMA Style

Jun-Gyu Park, Fatai S. Oladunni, Kevin Chiem, Chengjin Ye, Michael Pipenbrink, Thomas Moran, Mark R. Walter, James Kobie, Luis Martinez-Sobrido. Rapid in vitro assays for screening neutralizing antibodies and antivirals against SARS-CoV-2. Journal of Virological Methods. 2020; 287 ():113995-113995.

Chicago/Turabian Style

Jun-Gyu Park; Fatai S. Oladunni; Kevin Chiem; Chengjin Ye; Michael Pipenbrink; Thomas Moran; Mark R. Walter; James Kobie; Luis Martinez-Sobrido. 2020. "Rapid in vitro assays for screening neutralizing antibodies and antivirals against SARS-CoV-2." Journal of Virological Methods 287, no. : 113995-113995.

Protocol
Published: 13 October 2020 in Current Protocols in Microbiology
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SARS‐CoV‐2, the causative agent of COVID‐19, has been responsible for a million deaths worldwide as of September 2020. At the time of this writing, there are no available US FDA−approved therapeutics for the treatment of SARS‐CoV‐2 infection. Here, we describe a detailed protocol to generate recombinant (r)SARS‐CoV‐2 using reverse‐genetics approaches based on the use of a bacterial artificial chromosome (BAC). This method will allow the production of mutant rSARS‐CoV‐2—which is necessary for understanding the function of viral proteins, viral pathogenesis and/or transmission, and interactions at the virus‐host interface—and attenuated SARS‐CoV‐2 to facilitate the discovery of effective countermeasures to control the ongoing SARS‐CoV‐2 pandemic. © 2020 Wiley Periodicals LLC. Basic Protocol: Generation of recombinant SARS‐CoV‐2 using a bacterial artificial chromosome Support Protocol: Validation and characterization of rSARS‐CoV‐2

ACS Style

Kevin Chiem; Chengjin Ye; Luis Martinez‐Sobrido. Generation of Recombinant SARS‐CoV‐2 Using a Bacterial Artificial Chromosome. Current Protocols in Microbiology 2020, 59, e126 .

AMA Style

Kevin Chiem, Chengjin Ye, Luis Martinez‐Sobrido. Generation of Recombinant SARS‐CoV‐2 Using a Bacterial Artificial Chromosome. Current Protocols in Microbiology. 2020; 59 (1):e126.

Chicago/Turabian Style

Kevin Chiem; Chengjin Ye; Luis Martinez‐Sobrido. 2020. "Generation of Recombinant SARS‐CoV‐2 Using a Bacterial Artificial Chromosome." Current Protocols in Microbiology 59, no. 1: e126.