Transient acquisition of cross-species infectivity during the evolution of SARS-CoV-2

Abstract

Since the coronavirus disease 2019 (COVID-19) pandemic began, its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread worldwide. During the global transmission of SARS-CoV-2, mutations in the viral genome have gradually accumulated and have led to the emergence of variants. These emerging variants, including 501Y.V1, 501Y.V2 and 501Y.V3 (also called the alpha, beta and gamma variants, respectively), rapidly became the predominant epidemic strains and subsequently spread worldwide. All three of these SARS-CoV-2 variants contain specific amino acid mutations in the S protein and share an amino acid mutation, N501Y, in the receptor binding domain (RBD) of the S protein (Fig. S1). The RBD specifically binds to the receptor angiotensin-converting enzyme 2 (ACE2) on human cells and mediates host cell entry of SARS-CoV-2. Interestingly, the N501Y mutation was first documented during in vivo passaging of SARS-CoV-2 in mice (Fig. S1), and the resulting mouse-adapted strains MASCp6 and MASCp36 are fully capable of infecting standard laboratory mice [1,2], unlike isolates of the original SARS-CoV-2 strain. Most importantly, we and others have demonstrated that theN501Ymutation significantly enhances the binding affinity of the SARSCoV-2 RBD for mouse ACE2 [2,3], thus contributing to the acquired infectivity and pathogenicity phenotype in mice. However, whether naturally occurring SARS-CoV-2 variants (501Y.V1, 501Y.V2 and 501Y.V3) that contain this unique N501Y mutation have acquired the capability to infectmice remains to be determined. Herein, we adopt a contemporary 501Y.V2 variant, GDPCC, isolated from an imported case in a patient from South Africa to assay infectivity in mice. The SARS-CoV-2 clinical strain IME-BJ05 (wild-type, WT) isolated in the early stage of the COVID-19 pandemic was used as the control strain.Groups of ninemonth-old female BALB/cmice were intranasally challenged with the 501Y.V2 variant orWTstrain at a doseof 1.2×104 pfu. Remarkably, all 501Y.V2-infected mice began to show ruffled fur, hunched posture and reduced activity on day 3 post infection, and significant weight loss was seen in 501Y.V2-infected mice on days 4–6 post infection (Fig. 1A). The 501Y.V2-infected mice finally recovered, and no deaths occurred during the observation period. However, none of the animals challenged with WT virus developed obvious weight loss or clinical symptoms, as expected. To characterize viral replication dynamics in mice, 501Y.V2or mockinfected animals were sacrificed, and the major tissues and serum were collected. SARS-CoV-2 subgenomic RNA (sgRNA) quantitation showed that the highest abundance of viral RNA was detected in lung tissues from 501Y.V2-infected mice, with an obvious increasing trend during the first two days post infection (Fig. 1B). Viral sgRNA remained detectable in the trachea until day 8 post infection (Fig. 1B). However, no detectable sgRNA was present in other tissues or serum. An in situ hybridization (ISH) assay with the RNAScope approach showed that viral RNA was located mainly in cells along the airway and at the alveolar walls (Fig. 1C). Immunostaining of lung sections showed that SARS-CoV-2 N protein was expressed mainly in bronchiolar epithelial cells and alveolar cells, consistent with the ISH results (Fig. 1D). More importantly, gross necropsy showed visible lung injury, characterized by lung enlargement and local perihilar consolidation, upon 501Y.V2 challenge (Fig. 1E, left panel). Microscopic observation of lung sections from 501Y.V2-infected mice also showed that lung injury occurred mainly in the perihilar region (Fig. 1E, middle panel) and was characterized by large quantities of desquamating necrotic epithelial cells in bronchioles (yellow arrow), scattered hemorrhage (blue arrow) and inflammatory cell infiltration within fused alveolar walls (white arrow)