Mouse-adapted SARS-CoV-2 replicates efficiently in the upper and lower respiratory tract of BALB/c and C57BL/6J mice

As of June, 2020, more than ten million cases of COVID-19 have been reported worldwide. The causative pathogen of the disease is a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (World Health Organization, 2020). Animal infection models are important to characterize the infection, pathogenesis, and immunology of SARS-CoV-2, as well as for the development of medications and vaccines against COVID-19. Mice are particularly attractive animal models for their identical genetic background, reliable reproducibility, well characterized biology, and the huge availability of research reagents and knockout animals. Models in inbreed mice such as BALB/c and C57BL/6J (C57), which are widely used in research, are highly desired. The ideal model should mimic the infection of SARS-CoV-2 in humans, in whom the virus efficiently replicates in both the upper and lower respiratory tracts. Several SARS-CoV-2 mouse infection models, including human angiotensin-converting enzyme 2 (ACE2) transgenic mouse models (Bao et al., 2020; Jiang et al., 2020), a BALB/c mouse-adapted virus model (Gu et al., 2020), a reverse genetically modified SARS-CoV-2 infection model (Dinnon III et al., 2020), and recombinant adenovirusmediated transient expression of human ACE2 mouse models (Hassan et al., 2020; Sun et al., 2020), have been reported that viruses efficiently replicated in the lung. However, none of these reported models showed significant and stable infection in the upper respiratory tract. SARS-CoV-2 infection of the upper respiratory tract in humans is highly associated with the initial infection, the shedding of the offspring virus, and the transmission capability of the disease. Prevention of virus replication in the upper and lower respiratory tract is, therefore, highly desirable and an important aspect of the development of antiviral medications and vaccines against COVID-19. The mouse-adapted virus infected mouse model established here resembling the infection of SARS-CoV-2 in humans will be helpful to achieve the goals. Firstly, we found SARS-CoV-2/HRB26/human/2020/CHN (HRB26) is able to establish infection in the respiratory tract of BALB/c mice (Fig. S1). 4–6-week-old female BALB/c mice were intranasally (i.n.) infected with HRB26 at a dose of 10 plaque forming unit (PFU). On day 3 post inoculation (p.i.), the nasal turbinates and lungs were respectively collected and homogenized for viral RNA detection by qPCR and virus titration in Vero E6 cells. HRB26 only infected the nasal turbinates of 2 of the 3 inoculated mice and the lung of 1 of the 3 inoculated mice (Fig. S1). We serially passaged HRB26 in 4–6-week-old female BALB/c mice. A mixture of nasal turbinate and lung homogenate from the mouse of each passage with the highest viral RNA copies was used to inoculate three mice via intranasal inoculation. The viral RNA loads increased by passages in the nasal turbinates (Fig. S1A) and lungs (Fig. S1B). The infectious titres in the nasal turbinates and lungs at passage 14 (P14) were 10 PFU/g and 10 PFU/g on day 3 p.i., respectively (Fig. S1C and S1D). The virus of P14 was propagated in Vero E6 cells and the resultant mouse-adapted virus was designated as HRB26M (10 PFU/mL). The 50% mouse infectious dose (MID50) of HRB26M in 4–6-week-old female BALB/c mice was 1.4 PFU (Fig. S2). In the mice infected i.n. with HRB26M, the viral RNA was detected in the nasal turbinates on day 3, 5, and 7 p.i., and the infectious virus was detected on day 3 and 5 p.i. (Fig. 1A and 1B). The viral RNA was also detected in the heart, liver, kidney and spleen on day 3 p.i., but not on day 5 and day 7 p.i., respectively (Fig. S3A and S3B). Mild pathological changes were observed in the respiratory tract of 4–6-week-old BALB/c mice infected i.n. with HRB26M (Fig. 1G–I). Viral antigens were detected in the epithelium of the nasal respiratory mucosa (Fig. 1J), the epithelial cells of the bronchiole (Fig. 1K) and the alveolar septa cells (Fig. 1L) on day 3 p.i. These results demonstrate that SARS-CoV-2 successfully adapted and efficiently infected the upper and lower respiratory tract of young BALB/c mice. SARS-CoV-2 infection causes more serious disease and higher mortality in people older than 65 years (Guan et al., 2020). We then assessed the infectivity and pathogenicity of HRB26M in 8–9-month-old (aging adult) male BALB/c mice. Aging adult mice inoculated i.n. with 10 PFU of HRB26M


Figure S2 50% mouse infectious dose (MID50) of mouse-adapted virus in young female
BALB/c mice. Groups of three 4-6-week-old female BALB/c mice were inoculated i.n. with 10-fold serially diluted HRB26M. On day 3 p.i., three mice in each group were euthanized and the viral RNA copies in the nasal turbinates (A) and lungs (B) were detected by qPCR.
The horizontal dashed lines indicate the limit of detection.

Figure S3 Replication of mouse-adapted SARS-CoV-2 virus in the organs of young and
aging adult male BALB/c and C57 mice. Groups of nine 4-6-week-old female BALB/c (A, B), 4-6-week-old female C57 (C, D) or 8-9-month-old male BALB/c mice (E, F) were inoculated i.n. with 10 4.4 PFU of HRB26M in a volume of 50 μL. On days 3, 5, and 7 p.i., three mice in each group were euthanized. The viral RNA copies (A, C, E) and infectious titres (B, D, F) in the hearts, brains, kidneys, small intestines, spleens, and livers were detected by qPCR and virus titration. The horizontal dashed lines indicate the limit of detection.
Body weights were monitored daily for 7 days and are presented as a percentage of the weight on the day of inoculation (day 0).

Biosafety and Facility
All experiments with infectious SARS-CoV-2 were performed in the biosafety level 4 and animal biosafety level 4 facilities in the Harbin Veterinary Research Institute (HVRI) of the Chinese Academy of Agricultural Sciences (CAAS), which is approved for such use by the Ministry of Agriculture and Rural Affairs of China.

EPI_ISL_459909) was isolated from a patient in Vero E6 cells. Viral stocks were prepared in
Vero E6 cells with DMEM containing 5% FBS. Mouse-adapted SARS-CoV-2/HRB26/human/2020/CHN (HRB26M, GISAID access no. EPI_ISL_459910) was obtained by serially passaging the HRB26 virus in 4-6-week-old female mice until passage 14 and was propagated in Vero E6 cells. Infectious virus titers were determined by using a plaque forming unit (PFU) assay in Vero E6 cells.

qPCR and sequencing of viral genomes
Viral genomic RNA of SARS-CoV-2 was extracted by using a QIAamp vRNA Minikit

Mouse study
The mice for this study, 4-6-week-old female and 8-9-month-old male BALB/c, and 4-6-week-old female C57BL/6J (C57) were obtained from Beijing Charles River Labs (Beijing, China). Mice were lightly anesthetized with CO2 and intranasally (i.n.) inoculated with 50 μL dilutions of SARS-CoV-2. Body weights and clinical symptoms were monitored daily. On days 3, 5, or 7 post-inoculation (p.i.), animals were euthanized and their organs, including nasal turbinates, lungs, hearts, brains, kidneys, small intestines, spleens, and livers, were collected for viral RNA detection by qPCR, virus titration by use of a PFU assay, and histopathological study.
To determine the 50% mouse infectious dose (MID50) of HRB26M virus, groups of three young female mice were inoculated i.n. with 10-fold serially diluted HRB26M virus. On day 3 p.i., three mice in each group were euthanized and their nasal turbinates and lungs were collected for virus detection; 50% mouse infectious dose (MID50) at day 3 p.i. were calculated by the method of Reed and Muench (Reed and Muench, 1938).

Histopathologic and immunohistochemical studies
Animal tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, and cut into 4-µm sections. The sections were stained with hematoxylin-eosin for histopathologic observation. The sections used for immunohistochemistry were dewaxed in xylene and hydrated through a series of descending concentrations of alcohol to water. For viral antigen retrieval, sections were immersed in citric acid/sodium citrate solution, treated with 3% hydrogen peroxide, and blocked with 8% skim milk. Primary rabbit anti-SARS-CoV-2 nucleoprotein monoclonal antibody (1:500; Frdbio, Wuhan, China) and HRP-conjugated antirabbit IgG (whole molecule) secondary antibody (1:600, Sigma-Aldrich) were used.
Immunostaining was visualized with DAB and counterstained with hematoxylin.

Evaluation of antiviral activity in mice
Groups of six 4-6-week-old female mice were treated intramuscularly (i.m.) with a loading dose of 50 mg/kg(high dose)or 10 (low dose) mg/kg remdesivir, followed by a daily maintenance dose of 25 mg/kg(high dose)or 5 mg/kg (low dose). Alternatively, mice were treated i.n. alone or a combination of i.n. and i.m. with a loading dose of 50 mg/kg remdesivir, followed by a daily maintenance dose of 25 mg/kg. As a control, mice were administered vehicle solution (12% sulfobutylether-β-cyclodextrin, pH 3.5) daily. One hour after administration of the loading dose of remdesivir or vehicle solution, each mouse was inoculated i.n. with10 3.6 PFU of HRB26M in 50 μL. Three mice from each group were euthanized on days 3 and 5 p.i.. The nasal turbinates and lungs were collected for virus detection by qPCR and PFU assay.