Infection of wild-type mice by SARS-CoV-2 B.1.351 variant indicates a possible novel cross-species transmission route

Abstract

COVID-19 is identified as a zoonotic disease caused by SARS-CoV-2, which also can cross-transmit to many animals but not mice. Genetic modifications of SARS-CoV-2 or mice enable the mice susceptible to viral infection. Although neither is the natural situation, they are currently utilized to establish mouse infection models. Here we report a direct contact transmission of SARS-CoV-2 variant B.1.351 in wild-type mice. The SARS-CoV-2 (B.1.351) replicated efficiently and induced significant pathological changes in lungs and tracheas, accompanied by elevated proinflammatory cytokines in the lungs and sera. Mechanistically, the receptor-binding domain (RBD) of SARS-CoV-2 (B.1.351) spike protein turned to a high binding affinity to mouse angiotensin-converting enzyme 2 (mACE2), allowing the mice highly susceptible to SARS-CoV-2 (B.1.351) infection. Our work suggests that SARS-CoV-2 (B.1.351) expands the host range and therefore increases its transmission route without adapted mutation. As the wild house mice live with human populations quite closely, this possible transmission route could be potentially risky. In addition, because SARS-CoV-2 (B.1.351) is one of the major epidemic strains and the mACE2 in laboratory-used mice is naturally expressed and regulated, the SARS-CoV-2 (B.1.351)/mice could be a much convenient animal model system to study COVID-19 pathogenesis and evaluate antiviral inhibitors and vaccines.

Fig. 1

Infectivity of pseudotyped SARS-CoV-2 variants to mACE2-expressing cells. a Schematics of the Spike proteins of different SARS-CoV-2 variants which included D614 (Wuhan-Hu-1) virus, G614 (SYSU-IHV), B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.429 (Epsilon), B.1.525 (Eta), B.1.617.1 (Kappa), B.1.617.2 (Delta), and C.37 (Lambda). The mutation sites and types were indicated next to each backbone and written in red. b HEK293T-hACE2 and HEK293T-mACE2 cells were incubated with different pseudotyped SARS-CoV-2 viruses, followed by detecting the expression of luciferase at 48 h post infection. The fold changes of luciferase expression were normalized to viral titers followed by normalizing to the mock group and indicated as normalized infectivity (n = 3 for each group). The pseudotyped viruses were quantified and normalized with western blot with HIV-1 p24 protein antibody. c HEK293T cells were linearly transfected with different amounts (50, 100, and 200 ng in 24-well plates) of hACE2- or mACE2-expressing plasmids, followed by infecting with different pseudotyped SARS-CoV-2 viruses which included B.1.1.7, B.1.351, P.1, and B.1.617.1. The luciferase expression levels were quantified and indicated normalized infectivity. The expression levels of different amounts of hACE2- or mACE2-expressing plasmids transfected HEK293T cells were verified by western blot with antibodies against hACE2 and mACE2. GAPDH proteins were immunoblotted as the internal control (n = 3 for each group). d The pseudotyped B.1.1.7, B.1.351, P.1, and B.1.617.1 viruses (the initial copies of all the viruses were 2 × 10⁵ copies per μl) were twofold serially diluted and infected HEK293T-mACE2 cells. The normalized infectivity for each variant in each dilution was calculated as in c (n = 3 for each group). The pseudotyped viruses were quantified with western blot with HIV-1 p24 protein antibody. Data in b–d represented as mean ± SEM in triplicate. p Values were calculated by two-way ANOVA with Dunnett’s multiple comparisons test. ns = p ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 2

Binding affinities of RBD mutants to hACE2 and mACE2. a–f The binding affinities of different RBD or S1 mutants to hACE2 and mACE2 were measured by surface plasmon resonance (SPR) assays with a Biacore^TM T100 instrument. The K_a, K_d, and K_D values were measured and calculated by the software BIAevaluation. The K_D value shown was a mean of three independent experiments. These different RBD or S1 mutants included original RBD (a), S1 (D614G) (b), S1 (B.1.1.7) (c), RBD (K417N, E484K, N501Y) (d), RBD (K417T, E484K, N501Y) (e), and RBD (L452R, E484Q) (f)

Fig. 3

Infectivity and pathogenesis of B.1.351 in wild-type mice. a The hACE2 transgenic mice, BALB/c mice, and C57BL/6 mice were intranasally challenged with 5 × 10⁵ FFU of B.1.351 virus. Four C57BL/6 mice were assigned to the uninfected group. Four mice were assigned for each time point in each group. The virus-challenged and uninfected mice were euthanized at 0, 1, 2, 3, 5, and 8 days post infection (d.p.i.). The lungs of each mouse were harvested and homogenized and proceeded to total RNA extraction and RT-qPCR for determining viral RNA copies, which were plotted as log10 copies per ml (n = 4). b The tracheas of B.1.351-infected hACE2 mice, BALB/c mice, and C57BL/6 mice in each time point were homogenized and proceeded to RNA extraction, followed by RT-qPCR to determine viral copies that were plotted as log10 copies per ml (n = 4). c The hACE2 transgenic mice, BALB/c mice, and C57BL/6 mice were virus-challenged and euthanized as in a. Lungs and tracheas that were harvested on Day 5 were fixed with 4% paraformaldehyde buffer, followed by staining with hematoxylin and eosin (HE). The scale bar in each figure represented 100 μm. Each picture was a representation of four mice. The HE results of lung tissues were 200-fold amplified and shown below the original images. Arrows indicated pathological lung damages. d Lungs and tracheas that were harvested on Day 2 were fixed with 4% paraformaldehyde buffer. Then lungs and tracheas of B.1.351-infected and uninfected mice proceeded to immunohistochemical (IHC) assays with antibodies against SARS-CoV-2 Nucleoprotein (N) proteins. The scale bar in each figure represented 1 mm. Each picture was a representation from four mice. e The hearts, livers, spleens, kidneys, intestines, lymph nodes, and brains of B.1.351-infected hACE2 mice, BALB/c mice, and C57BL/6 mice in each time point were homogenized and proceeded to RNA extraction, followed by RT-qPCR to determine viral copies that were plotted as log10 copies per ml (n = 4). The dotted line indicated the limit of detection (L.O.D.) which was 100 copies per ml. Data in a, b, e are represented as mean ± SEM in quadruplicate. p Values were calculated by two-way ANOVA with Dunnett’s multiple comparisons test. Viral load on Days 1, 2, 3, 5, and 8 was compared with viral load on Day 0 for all the mice groups. ns = p ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

Inflammatory responses in B.1.351-infected mice. a–c The hACE2 transgenic mice, BALB/c mice, and C57BL/6 mice were challenged with B.1.351 variant as in Fig. 2. The lungs of each mice in each time point were harvested and homogenized. Total RNAs from lungs were extracted and proceeded to RT-qPCR for quantifying the mRNA expression of each cytokine gene, which included Il1b, Il5, Il6, Il9, Il10, Il12a/Il12p70, Il17a, Ifng, Ccl4, Ccl5, Tnfa, Gcsf, Cxcl1, Gmcsf, Il1a, and Il3. The RT-qPCR results were normalized to mouse GAPDH. The relative expression of each gene was calculated as 2^−ΔΔCt method (n = 4). d The cytokines in sera were also quantified by corresponding ELISA kits, including IL-6, IL-10, IL-1α, IL-1β, and CCL5 (n = 4). Data represented as mean ± SEM in quadruplicate. p Values were calculated by two-way ANOVA with Dunnett’s multiple comparisons test. ns = p ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5

Contact transmission of B.1.351 in wild-type mice. a Schematic of experimental design and sample collection of close-contact transmission assay. Twelve C57BL/6 mice were inoculated with 5 × 10⁶ FFU of B.1.351 virus on Day 0 and assigned to two cages (n = 6 for each cage). On Day 1 post inoculation, six naive mice were assigned into each cage. On Day 7, three inoculated mice and three contact mice from both cages were euthanized to detect viral RNA copies. On Day 14, three inoculated mice and three contact mice from both cages were euthanized for serological detection. As a control, B.1.351-infected hACE2 mice were co-housed with naive hACE2 mice (6:6 ratio, 2 cages). Viral and serological detections were conducted as above. The body weight changes were monitored every 2 days. b, c The viral RNA copies of lung and trachea samples from B.1.351-infected and close-contact mice were quantified and represented as log10 copies per ml (n = 6). d, e Reactivity of the serum samples from B.1.351-infected and close-contact mice with SARS-CoV-2 S antigens. Both anti-S IgM and anti-S IgG of these sera were detected. The absorbance at 450 nm represented the relative amount of antibodies (n = 6). f The body weight changes of both B.1.351-inoculated and close-contact mice. The body weight of each mouse was monitored every 2 days. Data in b–f are represented as mean ± SEM in sextuplicate. p Values in b–e were calculated by one-way ANOVA with Tukey’s multiple comparison test, which compared the mean of each group with the mean of every other group. p Values in f were calculated by two-way ANOVA with Dunnett’s multiple comparisons test, which compared the mean of each group with the mean of the first group. ns = p ≥ 0.05, *p < 0.05, ***p < 0.001, ****p < 0.0001