The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice
Authors: Chunxia Ma
Chuan Qin
Weimin Zhou
Li Guo
Feifei Qi
Xiaojuan Zhang
Guizhen Wu
Qiangming Sun
Wei Zhen
Na Zhu
Xiaozhong Peng
Wenling Wang
Jun Han
Yanfeng Xu
Baoying Huang
Mengli Yang
Conghui Wang
Wenbo Xu
Yajin Qu
Zhiqi Song
Yan Xiao
Linlin Bao
Haisheng Yu
Qi Jin
Ying Wang
Linna Zhao
Huijuan Wang
Shunyi Wang
Fei Ye
Mingya Liu
Li Zhao
Hongqi Liu
Shuran Gong
Qiang Wei
Xinming Wang
Guanpeng Wang
Hong Gao
Fanli Zhu
Fengdi Li
Wei Deng
Wenjie Tan
Qi Lv
Peipei Liu
Pin Yu
Jing Xue
Lingmei Yan
Lan Chen
Jiangning Liu
Lili Ren
Journal: Nature (2020)
Discipline: Multidisciplinary
Keywords: SARS-CoV-2; COVID-19; ACE2; transgenic mice; pathogenicity
Affiliations: Beijing Key Laboratory for Animal Models of Emerging and Remerging Infectious Diseases, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing, China., NHC Key Laboratory of Human Disease Comparative Medicine, Comparative Medicine Center, Peking Union Medical College, Beijing, China., MHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China., Institute of Pathogen Biology, Chinese Academy of Medical Sciences, Beijing, China., Institute of Medical Biology, Chinese Academy of Medical Sciences, Beijing, China., MHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China. ., Beijing Key Laboratory for Animal Models of Emerging and Remerging Infectious Diseases, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing, China. ., NHC Key Laboratory of Human Disease Comparative Medicine, Comparative Medicine Center, Peking Union Medical College, Beijing, China. .

Abstract:  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), which has become a public health emergency of international concern1. Angiotensin-converting enzyme 2 (ACE2) is the cell-entry receptor for severe acute respiratory syndrome coronavirus (SARS-CoV)2. Here we infected transgenic mice that express human ACE2 (hereafter, hACE2 mice) with SARS-CoV-2 and studied the pathogenicity of the virus. We observed weight loss as well as virus replication in the lungs of hACE2 mice infected with SARS-CoV-2. The typical histopathology was interstitial pneumonia with infiltration of considerable numbers of macrophages and lymphocytes into the alveolar interstitium, and the accumulation of macrophages in alveolar cavities. We observed viral antigens in bronchial epithelial cells, macrophages and alveolar epithelia. These phenomena were not found in wild-type mice infected with SARS-CoV-2. Notably, we have confirmed the pathogenicity of SARS-CoV-2 in hACE2 mice. This mouse model of SARS-CoV-2 infection will be valuable for evaluating antiviral therapeutic agents and vaccines, as well as understanding the pathogenesis of COVID-19. 

Retracted: No

PubPeer comments:
 Figure 2A. * Blue boxes: In the H E 100x row, the WT-HB-01 and ACE2-Mock panels appear to show an overlap.  , 
 https: twitter.com houndcl status 1260461431381733376 Source code: https: github.com houndcl nodup I can confirm #1 s finding based on the following evidence. 1. Intensity and edges are highly similar (R2 0.9)   2. A  symmetric focal spike can be found when we explore the regions around the optimal merge. This is a strong indication of duplicate because when you overlay two images taken from the same object, you will find two images become less and less blurred and **suddenly** overlap perfectly.  , 
 The data points painted on top of bar graphs curiously follow the same pattern: Fig 1c  Fig 1b  Also there s a wonderful anagram of data points in Fig 1e:  And yet another instance of the authors favorite pattern of data points in Extended Fig 5  In this case the data points were painted on wrong: their mean is higher than the bar. , 
 Hi, Elisabeth, Thank you very much for your comments on our online publication, suggesting that there was an overlapped histopathological picture in two controls (Figure 2A), and possible errors in statistics analyses (Figure 1). The current online version of publication is u201cuneditedu201d, indeed there was a misused histopathological picture in a control, we had already noticed it, contacted the Nature editor on May 12, and submitted the correct image for a correction. Specifically, one of the control image (WT-HB-01) was misused for another control group (ACE2-Mock) (Figure 2A). Fig 1 are generated by Graphpad Prism 8.0, all original experiment results and records were sent to the Nature, and all the data were support the major conclusions of this manuscript. We thank you for the feedbacks and comments, apologize for the misused image and possible misunderstanding for scientific community. , 
 #4 Dear Dr. Chuan Qin, Could you confirm that the mice in the study were indeed 6 to 11 months old? The body weight increase for the control groups is highly inconsistent with that old mice. I would expect to see no change in their weight, and definitely not a consistent 5 % increase. What were the mice s starting weights?  , 
 Thank you for the comments on our online paper. The mice used in present experiment were conserved as seeds (which was the only available mice then) in the isolator for long time, the body weights of hACE2 mice were 27-33 g, and the wild-type mice were 33-38 g. The attending veterinarian noticed the body weight of mice in this experiment were relatively lower. Given the fact that a series of sampling, including blood collections and viral challenging need to be performed from these experimental animals, thus protein-enriched feed was used in experiment according to the suggestions of the veterinarian, which might lead to the increase (1-2 g) in body weight during the observation period. , 
 #5 I intuitively agree with you, so I went to find some information. In the Reporting Summary , the information about animals is:  Male and female transgenic hACE2 mice (specific pathogen-free, 6-11-month-old) were obtained from the Institute of Laboratory Animal Science, Peking Union Medical College, China. Transgenic mice were generated by microinjection of the cytomegalovirus promoter driving the human ACE2 coding sequence into Institute of Cancer Research (ICR) mice, the presence of human ACE2 was confirmed in the mice used by PCR. I don t know if the mice used by them is similar to K18-hACE2 provided by The Jackson Laboratory(JAX). On JAX website , K18-hACE2 is described as a line may be useful in studies related to the study of 2019 novel coronavirus (SARS-CoV-2) pathogenesis and the disease outbreak COVID-19. In Breeding Considerations, JAX suggest investigators to breed hemizygous mice to wildtype (non-carrier) mice from the colony, or to C57BL 6J inbred mice (Stock No. 000664). It is reasonable to speculate that the physiological characteristics of these two mice are very similar. And I found the Body Weight information for aged C57BL 6J (000664) on their website( https: www.jax.org jax-mice-and-services strain-data-sheet-pages body-weight-chart-aged-b6#). It can be seen that the weight of mice aged 24-44 week(6-11-month-old) gains slowly.   I tried to estimate the growth rate of JAX data using the mean. The red number means estimated. B3 is =B2+(B4-B2) 2, and so on. It can be seen that on every 14 days, the weight gain of both male and female mice is less than 5%. Of course, I admit that such estimates are hasty.  I doubt whether the author presented the results of the younger mice? As they mentioned in the review report : (Referee #1)4.Why were 6-11 month old mice used? Did this reflect availability? Author response: Thank you for your valuable comment. We have performed the experiments using 4-5 months old mice after the manuscript was submitted. We have observed the similar clinical manifestation and pathogenic changes. We compared the preliminary data between 611-month mice and 4-5-month mice and found no significant disparity. Herein, 6-11-month mice were used in our experiment. , 
 #6 Dear Prof. Qin, Considering that you are building animal models, I respectfully think that this information should appear in some form in the text or supplementary materials, because there are few studies discussing the impact of host nutritional status on COVID-19 disease. , 
 #8 Dear Professor The mice used in present study was different from the K-18 hACE2 mice of JAX. Generally, the weight gain of mice in 6-11 months old was as slowly as 1-3%. Our seed mice are used for conservation in long time, and they are fed with maintain feed. The weight of our adult mice is lower than that of JAX. We also compared the younger mice of 4-6 m in the later period, but the paper only included the data of 6-11 months mice. Your question about feed is meaningful and worth thinking about. I hope we can pay attention to it and perform it together in the future. , 
 Dear Prof Parashorea Tomentella, The mice used in present study was different from the K-18 hACE2 mice of JAX. Generally, the weight gain of mice in 6-11 months old was as slowly as 1-3%. Our seed mice are used for conservation in long time, and they are fed with maintain feed. The weight of our adult mice is lower than that of JAX. We also compared the younger mice of 4-6 m in the later period, but the paper only included the data of 6-11 months mice. Your question about feed is meaningful and worth thinking about. I hope we can pay attention to it and perform it together in the future. , 
 #10 Thank you for your reply. ,