Development and characterization of mouse-adapted recombinant SARS-CoV-2 expressing reporter genes

SARS-CoV-2 is the causative agent of COVID-19. The ancestral SARS-CoV-2 Washington-1 (WA1) strain does not infect standard laboratory mouse strains, necessitating the use of mouse-adapted (MA) viruses. A MA SARS-CoV-2, SARS-CoV-2-N501Y MA30 (hereafter MA30), has been developed to allow infection of wild-type (WT) mice. However, SARS-CoV-2 MA30 cannot be tracked in vitro, ex vivo, or in vivo. To address this problem, we generated recombinant (r)SARS-CoV-2 MA30 expressing fluorescent (mCherry) and nanoluciferase (Nluc) reporter genes, alone or in combination, that enable tracking viral infections in WT C57BL/6 and BALB/c mice. Insertion of the reporter genes resulted in minor viral attenuation in vitro, with ~0.5-1.0 log lower titers than rSARS-CoV-2 MA30 WT in A549 hACE2 cells, while maintaining similar plaque morphology and replication kinetics in Vero AT cells. In vivo, reporter-expressing rSARS-CoV-2 MA30 caused transient weight loss, contrasting with lethal rSARS-CoV-2 MA30 WT infection. Bioluminescence imaging of rSARS-CoV-2 MA30 Nluc in C57BL/6 and BALB/c mice revealed peak pulmonary replication at 2 days post-infection, with resolution by day 4, and correlated with tissue viral loads. Our results demonstrate the feasibility of using rSARS-CoV-2 MA30 expressing reporter genes to track viral infection in vitro, ex vivo, and in vivo without a need for secondary approaches to monitor viral infection required for rSARS-CoV-2 MA30 WT. Our system is highly suitable to evaluate prophylactic vaccines and therapeutic antibodies or antiviral approaches in WT or transgenic C57BL/6 and BALB/c mice without the shortcomings of K18-hACE2 mice and with the added advantage of non-invasive monitoring of treatment efficacy.

Mouse-adapted (MA) SARS-CoV-2 that infect wild-type (WT) mice are critical tools for preclinical studies. While the previously described SARS-CoV-2-N501Y MA30 enables infection of WT mice, it does not allow non-invasive tracking of viral infections. Recombinant viruses expressing reporter genes enable real-time monitoring of infection dynamics, opening an avenue to study viral tropism and easily evaluate prophylactic and therapeutic approaches. They furthermore support longitudinal studies, which reduces the number of research animals required. Here, we show that a recombinant (r)SARS-CoV-2 expressing fluorescent (mCherry) and nanoluciferase (Nluc) reporter genes, alone or in combination, can be used to track viral infections in vitro, ex vivo, and in vivo without the need for secondary approaches that are required to detect SARS-CoV-2 MA30 in WT mice. These reporter-expressing rSARS-CoV-2 MA30 may accelerate vaccine development and antiviral drug discovery in WT or transgenic mice bypassing the need for hACE2 overexpression in K18-hACE2 transgenic mice.