In December 2019, a coronavirus disease (named as COVID-19 later) was reported in Wuhan 39 city, which rapidly spread throughout China and then to many other countries in the world ( showed two distinct clinical phenotypes from 4 DPI. One mouse (G-D5-M3) was found dead 4 104 DPI, and two lost weight 4 DPI and were humanely euthanized 5 DPI. The remaining mouse 105 began to lose weight 6 DPI and was humanely euthanized 7 DPI. In total, these four mice 106 showed significant body weight decreases from days 4 to 6 and generated noticeable respiratory 107 distress and neurological symptoms before death or humane euthanisation. All other mice 108 survived to the endpoint without evident symptoms (Table S1 and Figure 1B) . 109 110 White blood cells (WBC) analysis indicated that the levels of most neutrophils and monocytes 111 began to increase in the peripheral blood at day 1, peaked at day 3, and decreased 5 and 7 DPI in 112 the infected group, compared with those of control mice ( Figure S1 ). The infected mice 113 demonstrated a reduced ratio of lymphocytes 1 DPI. However, lymphocytes ratio recovered, 114 beginning on 3 DPI. All the dying mice (dead or lost >20% body weigh) showed significantly 115 decreased lymphocytes numbers, which is one of the key clinical hallmarks of COVID-19 116 patients . Five mice showed significantly higher levels of creatine kinase 117 compared with those of the control group, which may be linked to muscle fatigue, soreness, or 118 myocarditis (Table S2 ). The deceased mice showed higher aspartate transaminase and creatine 119 kinase levels than the control group. These data suggested that SARS-CoV-2-infected mice 120 generate symptoms that partly resembled human infection symptoms (Chen et To better understand viral replication in hACE2 mice, viral RNA was quantified by RT-qPCR in 153 different tissues from mice euthanized at 1, 3, 5, and 7 DPI (Figures 3A-D) . The viral nucleic 154 acid was detected in all lungs from 1 DPI, and slowly increased until 5 DPI, and then decreased 155 beginning 7 DPI ( Figure 3A ). We also found low viral RNA copies in the eyes and hearts in 156 eight and two infected mice, respectively ( Figures 3B and 3C) . No viral RNA was detected in 157 other organs. Viral antigens were detected in bronchial epithelial cells and alveolar cells of the 158 lungs using immunohistological assays with cross-reactive antibody that has been used for 159 SARS-CoV-2 detection previously (Zhou et al., 2020) ( Figure 3E) . Notably, four of the animals 160 demonstrated SARS-CoV-2 neuroinvasion of the brain, but only in the deceased mice ( Figure  161 3D). However, the exact details and mechanisms regulating SARS-CoV-2 neuroinvasion remain 162 unclear, as this phenomenon has been previously described with this mouse model (Menachery 163 et al., 2016) . We then tried to re-isolate SARS-CoV-2 from infected animals. We inoculated 164
Vero E6 cells with infected mouse (G-D7-M1) lung and brain homogenate supernatant and 165 observed a noticeable cytopathic effect (CPE) in the infected cells ( Figures S4B-C) compared 166 with the control ( Figure S4A) . The re-isolated virus from lung and brain of G-D7-M1 was 167 confirmed by indirect imunofluorescence assays (IFA) and genome sequencing ( Figures S4D-F , 168 Table S3 ). The lung isolates indicated two mutations, C14554T (nsp13, L372F) and C23525T 169 no.EPI_ISL_402124). The mutation at nsp13 was unique compared with published viral 171 sequences identified by online BLAST. The mutation in the spike gene was located at the end of 172 the S1 region of the spike gene. This mutation was identified using BLAST in SARS-CoV-2 173 genome sequence isolated from a patient from the United States (Genbank no. MT163720). The 174 brain isolates exhibited only one mutation, C23525T (H644Y), at the S1 region of the spike 175 gene. The viral titres in the infected lung ranged from 9.4×10 3 to 9.1×10 5 TCID 50 per gram of 176 tissue, as determined by serial dilution of VeroE6 cell lines ( Figure S5) . Conclusively, SARS-177
CoV-2 mainly replicated in mouse lung, although it may also target the brain, heart, and eyes of 178 with the infection group. Upon infection, three mice died 6 DPI. All the deceased mice were 188 male, with total mortality of 50% (3/6). In general, there were two infection outcomes, those that 189 lost more than 20% body weight from 4 or 5 DPI and died eventually, or those that lost less than 190 10% of body weight and survived (Figures 4B and 4C) Three mice, one male and two females who survived the infection, were observed for 21 days. 194
These mice generated antibodies against SARS-CoV-2 that could neutralise 100 TCID 50 viruses 195 at dilutions of 1:10 to 1:40 (Table S4) We also demonstrated that the lung is the primary target of this virus, although the eye, heart, 248 and brain could also be infected. In human cases, it has been suspected that SARS-CoV or 249 SARS-CoV-2 could also attack the eye, heart and brain, however, only limited data are available 250 as control (P), severely infected mice (Q) show hyaline membrane (R, green arrow) and hyaline 337 thrombus (R, black arrow). Images were collected using a Pannoramic MIDI system. Scale bar: 338 500 µm (A, B, D, E, F, J, K, L, P, Q) and 100 µm (C, G, H, I, M, N, O, R) . See also Figure S2 . surviving mice 21 DPI and three previously mock-infected mice were infected with 7*10 5 358 TCID 50 SARS-CoV-2 at 21 DPI, respectively. Body weights (A) and survival outcomes (B) were 359 monitored for up to 5 days. Mice were euthanised 5 DPI, and the viral loads were measured 360 using RT-qPCR (C). Naïve-infected mouse lung showed peri-bronchial and peri-vascular 361 infiltration and oedema with hyaline membrane formation (D). In contrast, previously challenged 362 mice presented mild peri-bronchial and peri-vascular infiltration (E). Viral antigen was widely 363 present in the naïve-infected mouse lung (F) and brain (H) but barely observed in previously 364 challenged mouse lung (G) and brain (I). Error bars indicate the standard error. ND: not 365 detected. Scale bar: 500 µm (D, E), 100 µm (F, G) and 200 µm (H, I). See also Table S3 . Infected mouse lung and brain tissues were homogenised with DMEM, and the 386 supernatant was used to infect Vero E6 cells. CPE was observed in cells infected by the