Characterization of Two Heterogeneous Lethal Mouse-Adapted SARS-CoV-2 Variants Recapitulating Representative Aspects of Human COVID-19

Abstract

New emerging severe acute respiratory syndrome 2 (SARS-CoV-2) has caused a worldwide pandemic. Several animal models of coronavirus disease 2019 (COVID-19) have been developed and applied to antiviral research. In this study, two lethal mouse-adapted SARS-CoV-2 variants (BMA8 and C57MA14) with different virulence were generated from different hosts, which are characterized by high viral replication titers in the upper and lower respiratory tract, pulmonary pathology, cytokine storm, cellular tropism, lymphopenia, and neutrophilia. Two variants exhibit host genetics-related and age-dependent morbidity and mortality in mice, exquisitely reflecting the clinical manifestation of asymptomatic, moderate, and severe COVID-19 patients. Notably, both variants equally weaken the neutralization capacity of the serum derived from COVID-19 convalescent, but the C57MA14 variant showed a much higher virulence than the BMA8 variant in vitro. Q489H substitution in the receptor-binding domain (RBD) of BMA8 and C57MA14 variants results in the receptors of SARS-CoV-2 switching from human angiotensin-converting enzyme 2 (hACE2) to murine angiotensin-converting enzyme 2 (mACE2). Additionally, A22D and A36V mutation in E protein were first reported in our study, which potentially contributed to the virulence difference between the two variants. Of note, the protective efficacy of the novel bacterium-like particle (BLP) vaccine candidate was validated using the BMA8- or C57MA14-infected aged mouse model. The BMA8 variant- and C57MA14 variant-infected models provide a relatively inexpensive and accessible evaluation platform for assessing the efficacy of vaccines and novel therapeutic approaches. This will promote further research in the transmissibility and pathogenicity mechanisms of SARS-CoV-2.

Keywords: BLP vaccine; COVID-19; SARS-CoV-2; mouse model; mutation; pathogenesis.

Figure 1

Two mouse-adapted strains of severe acute respiratory syndrome 2 (SARS-CoV-2) from outbred mice carry unique amino acid substitution. Schematic diagram of SARS-CoV-2 genome and all the adaptive mutations of amino acid identified in BMA8 (A) and C57MA14 (B) compared with the SARS-CoV-2 Wuhan01. “nsp”, nonstructural protein; “S”, Spike Protein; “ORF”, Open Reading Frame; “E”, Envelope Protein; “M”, Membrane Protein; “N”, Nucleocapsid Protein. (C) Table of mutations present in plaque purified mouse-adapted SARS-CoV-2 BMA8 and C57MA14. Red font showed the same mutations in both mouse-adapted variants. Blue font showed the mutations only occurring in SARS-CoV-2 BMA8. Green font showed the mutation only occurring in SARS-CoV-2 C57MA14. The proportions of all the amino acid mutations in both mouse-adapted strains were calculated in each passage (D–G). One-step growth curves of SARS-CoV-2 Wuhan01, BMA8, and C57MA14 were measured in Vero E6 cells. n = 3 technical replicates for each group, representative of 2 independent experiments (H). Neutralization capabilities of the serum samples from 32 SARS-CoV-2-infected individuals were measured with BMA8, C57MA14, and SARS-CoV-2 Wuhan01 (I). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 2

Mouse-adapted strains severe acute respiratory syndrome 2 (SARS-CoV-2) BMA8 and C57MA14 cause severe disease in aged BALB/c and C57BL/6N mice. Nine-month-old female BALB/c mice or C57BL/6N mice were mock infected (n = 13) or infected with 50 LD₅₀ SARS-CoV-2 BMA8 or C57MA14 (n = 13), respectively. The survival rate, weight change, body temperature, and clinical scores of BALB/c mice were monitored daily after SARS-CoV-2 BMA8 infection (A–D) (n = 8) or those parameters of C57BL/6N mice were monitored daily after SARS-CoV-2 C57MA14 infection (J–M) (n = 8). The hematological values of BALB/c mice were analyzed, including lymphocyte percentage (LYM%), neutrophil percentage (Neu%), monocyte percentage (Mon%), platelet (PLT) count, and white blood cell (WBC) count, at 3 dpi after SARS-CoV-2 BMA8 infection (E–I) (n = 5), or those measurement indicators of C57BL/6N mice were also observed daily after SARS-CoV-2 C57MA14 (N-R) (n = 5). Data are presented as mean ± SEM. (*P < 0.05, ****P < 0.0001).

Figure 3

Tissue distribution of the viral RNAs and the pathological lung lesions in passaged mice infected with the mouse-adapted severe acute respiratory syndrome 2 (SARS-CoV-2) BMA8 and C57MA14. To screen virus replication, groups of aged BALB/c mice (n = 10) or aged C57BL/6N mice (n = 10) were infected with 50 LD₅₀ of SARS-CoV-2 BMA8 or C57MA14, and infected mice were sacrificed at 3 dpi and 5 dpi (n = 4 or 5), respectively. The blood and internal organs were harvested to analysis the viral RNA loads by qRT-PCR and TCID₅₀ (A–D). Data are presented as mean ± SEM (n = 5). The gross pathology and histopathology of lungs from 9-month-old female BALB/c mice intranasally inoculated with 50 LD₅₀ of SARS-CoV-2 BMA8 or PBS (Mock) at 3 dpi (E). The thickened and narrowed alveolar cavity, the congested capillaries in the alveolar wall (black arrow), the infiltration of neutrophils (yellow arrow), and perivascular edema and a small amount of inflammatory cell infiltration (yellow arrow) were observed in lung tissues at 3 dpi after viral infection in BALB/c mice (E). Lung tissue changes of infected C57BL/6N mice are characterized by exudation in airway (green arrow), thickened alveolar wall (black arrow), alveolar space stenosis, macrophage infiltration (yellow arrow), inflammatory cell infiltration (yellow arrow), and perivascular edema and exudation of protein-like substance in alveolar sac (blue arrow). Similarly, the gross pathology and histopathology of lungs from 9-month-old female C57BL/6N mice intranasally inoculated with 50 LD₅₀ of SARS-CoV-2 C57MA14 or PBS (Mock) at 3 dpi (F). Photographs of gross pathological lungs shown in left panels. Local lesions are indicated by arrows. Hematoxylin and eosin stain (H&E) shown in middle panels. Right panels show immunohistochemistry (IHC) labeling against SARS-CoV-2. Scale bar, 100 µm.

Figure 4

Cytokine and chemokine production in serum of aged mice infected with the mouse-adapted severe acute respiratory syndrome 2 (SARS-CoV-2) BMA8 or C57MA14. Groups of aged BALB/c mice (n = 10) (A) or aged C57BL/6N mice (n = 10) (B) were infected with 50 LD₅₀ SARS-CoV-2 BMA8 or C57MA14, and the sera were harvested at 3 dpi to detect the cytokine and chemokine profiles in serum, respectively. Groups of aged BALB/c mice (n = 10) (A) and aged C57BL/6N mice (n = 10) (B) were intranasally inoculated with the same volume of PBS as control. Data are presented as mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5

Characterization of age-related and host genetics-dependent mortality and morbidity of severe acute respiratory syndrome 2 (SARS-CoV-2) BMA8 and C57MA14 in mice. To characterize the pathogenesis of SARS-CoV-2 BMA8 associated with age and host genetics, groups of 6-week-old female C57BL/6N mice, 6-week-old female BALB/c mice, and 9-month-old female C57BL/6N mice were infected intranasally with 50 LD₅₀ of SARS-CoV-2 BMA8 in a volume of 50 µl. The survival rate, weight change, and body temperature in each group (n = 8) were monitored daily after infection (A–C). At 3, 5, and 7 dpi, five mice of each group were euthanized, and turbinates and lungs were sampled for virus RNA loads by qRT-PCR (D, E). Similarly, to assess the pathogenesis of SARS-CoV-2 C57MA14, groups of 23 6-week-old female C57BL/6N mice, 6-week-old female BALB/c mice, and 9-month-old female BALB/c mice were infected intranasally with 50 LD₅₀ of SARS-CoV-2 C57MA14 in a volume of 50 µl. The survival rate, weight change, body temperature, and clinical score in each group (n = 8) were monitored daily after infection (F–H). At 3, 5, and 7 dpi, five mice of each group were euthanized, and turbinates and lungs were sampled for viral RNA load test by qRT-PCR (I, J). Data are presented as mean ± SEM. (*P < 0.05, **P < 0.01,****P < 0.0001).

Figure 6

Q498H substitution increased the binding affinity between receptor-binding domain (RBD) and murine angiotensin-converting enzyme 2 (mACE2). (A) Multiplex immunofluorescence staining of mouse lung section, severe acute respiratory syndrome 2 (SARS-CoV-2) (red), mACE2 (green), nuclei (blue). (B) Homology modeling of mouse ACE2 (purple) in complex with SARS-CoV-2 variant RBD (green) with Q498 (left) or H498 (right). (C, D) Contribution of each amino acid to the binding free energy in complex of RBD containing Q498 or H498 binding with mACE2. (E, F) ELISA for detecting the binding ability between SARS-CoV-2 RBD and ACE2. (G, H) Survival rate and weight change of BALB/c^-ACE2 mice (n = 3) and C57BL/6J^-ACE2 mice (n = 3), while normal BALB/c mice (n = 4) and C57BL/6J mice (n = 4) were as control.

Figure 7

Protection efficacy of the severe acute respiratory syndrome 2 (SARS-CoV-2) bacterium-like particle (BLP) vaccine candidate against BMA8 and C57MA14 infection in aged mice. The vaccination schedule, viral challenge, and characterization of immunologic responses in aged BALB/c and C57BL/6N mice (A). Groups of 9-month-old female BALB/c (n = 10) or C57BL/6N mice (n = 10) were immunized with three doses of 10 µg/dose BLP combined with Freund’s incomplete Freund’s adjuvant (IFA) in 3-week intervals. Empty Gram-positive enhancer matrix (GEM) vector or PBS with same adjuvant was given as negative controls. Serum was harvested at 2 weeks after the last immunization and at 1 week after SARS-CoV-2 challenge to detect the viral neutralizing antibody (B, H). Mice were challenged with 50 LD50 of SARS-CoV-2 BMA8 or C57MA14 via i.n. route at 21 days after the third immunization. The survival rate (C, I), weight change (D, J), and body temperature (E, K) in each group were monitored daily after BMA8 or C57MA14 challenge. At 3 dpi, three mice in each group were euthanized, and turbinates (F, L) and lungs (G, M) were sampled for viral RNA loads by qRT-PCR. Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001.