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

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 ({Delta}ORF) viruses producing smaller plaques than those of the wild-type (rSARS-CoV-2/WT). However, growth kinetics of {Delta}ORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice with the {Delta}ORF rSARS-CoV-2 identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while {Delta}ORF7a, {Delta}ORF7b and {Delta}ORF8 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. IMPORTANCEDespite 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.

Generation and Characterization of recombinant SARS-CoV-2 expressing reporter genes

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. ImportanceSevere 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.

Rescue of SARS-CoV-2 from a single bacterial artificial chromosome

An infectious coronavirus disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, Severe Acute Respiratory Syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant (r)SARS-CoV-2 was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, the BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the SARS-CoV-2 natural isolate. Likewise, rSARS-CoV-2 showed similar levels of replication to that of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays similar features in vivo to that of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.

Lethality of SARS-CoV-2 infection in K18 human angiotensin converting enzyme 2 transgenic mice

ABSTRACTVaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease currently lacks a validated small animal model. 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 4. 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.

SARS-CoV-2 infection leads to acute infection with dynamic cellular and inflammatory flux in the lung that varies across nonhuman primate species

There are no known cures or vaccines for COVID-19, the defining pandemic of this era. Animal models are essential to fast track new interventions and nonhuman primate (NHP) models of other infectious diseases have proven extremely valuable. Here we compare SARS-CoV-2 infection in three species of experimentally infected NHPs (rhesus macaques, baboons, and marmosets). During the first 3 days, macaques developed clinical signatures of viral infection and systemic inflammation, coupled with early evidence of viral replication and mild-to-moderate interstitial and alveolar pneumonitis, as well as extra-pulmonary pathologies. Cone-beam CT scans showed evidence of moderate pneumonia, which progressed over 3 days. Longitudinal studies showed that while both young and old macaques developed early signs of COVID-19, both groups recovered within a two-week period. Recovery was characterized by low-levels of viral persistence in the lung, suggesting mechanisms by which individuals with compromised immune systems may be susceptible to prolonged and progressive COVID-19. The lung compartment contained a complex early inflammatory milieu with an influx of innate and adaptive immune cells, particularly interstitial macrophages, neutrophils and plasmacytoid dendritic cells, and a prominent Type I-interferon response. While macaques developed moderate disease, baboons exhibited prolonged shedding of virus and extensive pathology following infection; and marmosets demonstrated a milder form of infection. These results showcase in critical detail, the robust early cellular immune responses to SARS-CoV-2 infection, which are not sterilizing and likely impact development of antibody responses. Thus, various NHP genera recapitulate heterogeneous progression of COVID-19. Rhesus macaques and baboons develop different, quantifiable disease attributes making them immediately available essential models to test new vaccines and therapies.