SARS-CoV-2 z-bo 2020-12-09 292

　　Results: Humanized hACE2 mice were established using CRISPR/Cas9

　　 Previous studies have shown that transgenic mice expressing human ACE2 are highly susceptible to SARS-CoV infection. Here, we aim to create humanized ACE2 mice using CRISPR/Cas9 knock-in technology. The complete cDNA of hACE2 was inserted into exon 2 of mAce2 gene located at the GRC m38.p6 site of X chromosome, the first encoding exon. This disrupted the mAce2 gene and stopped its expression. Insert the tdTomato gene downstream of hAce2, the internal ribosome entry site (IRES). Allow hACE2 and tdTomato co-expression. The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and poly(A) sequences have been added to enhance mRNA stability and translation efficiency. This targeting strategy allows the intrinsic expression of hACE2 under the control of the mAce2 promoter. The targeting construct was injected into 370 fertilized eggs of C57BL/6 mice along with subgenomic RNA (sgRNA) and Cas9 mRNA. PCR screening confirmed that 10 of the 46 offspring (21.74%) were successfully inserted, then the three successfully inserted mice were backcrossed with C57BL/6 mice, and 37 F1 positive offspring were screened. The targeting results of 12 F1 positive mice were further confirmed by sequencing and Southern blotting. As expected, the WPRE internal probe showed no random insertion of animals. Importantly, western blotting results show that hACE2 is strongly expressed in the lung and kidney, but weakly in the spleen and liver, and shows a similar expression pattern of mACE2 protein in wild-type (WT) animals. More importantly, the mAce2 gene of homozygous hACE2 mice was not detected by PCR, and the mAce2 gene expression of heterozygous mice was significantly reduced, while the lung, small intestine, spleen and kidney of hACE2 heterozygous mice and homozygous mice Significant expression of hACE2 gene was detected, but not in WT mice. Using co-expression design, the overall expression pattern of hACE2 mice was observed by bioluminescence imaging. In addition, immunofluorescence staining of the lungs of homozygous mice showed that hACE2 and tdTomato were mainly expressed in airway CC10 + Clara cells and a small amount of surfactant protein C-positive alveolar type II cells. Therefore, we established a homozygous mouse model that stably expresses hACE2 under the control of the mACE2 promoter, so we called it hACE2-KI/NIFDC mouse (abbreviated as hACE2 mouse).

　　HACE2 mice are highly susceptible to SARS-CoV-2 via the nasal route: To further understand the susceptibility of hACE2 mice to SARS-CoV-2 infection, young (4.5 weeks old) and old (30 weeks old) hACE2 mice were respectively infected with SARS-CoV-2 at 4×105 pfu through the nose, and Set wild-type (WT) C57BL/6 mice as controls. The clinical symptoms and weight changes of all animals were monitored every day, and they were sacrificed on the 6th day after infection (dpi). The vaccinated animals showed no obvious clinical symptoms. Only the elderly hACE2 mice lost 10% of their body weight at 3dpi and then recovered. A large amount of viral RNA replication was found in the lung, trachea and brain tissues of young and old hACE2 mice, but no viral RNA was found in the spleen, kidney, liver, intestine and serum. In particular, the plaque method was used to successfully detect infectious SARS-CoV-2 from lung specimens. As expected, no viral RNA was detected in any tissue or serum tested in WT C57BL/6 mice. Interestingly, we also detected high levels of viral RNA (2.9×105 copies/g) in the feces of elderly hACE2 mice. In order to confirm the main target cells of SARS-CoV-2 in the model, we further stained the lungs of infected hACE2 mice with different cell markers, including Clara cells (labeled with Clara cell 10 kDa protein/CC10) , Ciliated cells (labeled with β-IV-tubulin), type 1 alveolar cells (AT1, labeled with podoplanin/PDPN) and type 2 alveolar cells (AT2, labeled with surfactant protein C/SPC), and virus S Protein and hACE2. SARS-CoV-2 S protein mainly co-localizes with CC10 and hACE2, indicating that CC10-positive Clara cells are the main target cells of SARS-CoV-2 in the airway. In addition, immunostaining of hACE2-infected mouse brain sections showed that the expression of viral S protein could also be detected in neurons, astrocytes and microglia. Taken together, these results indicate that, compared with WT mice, hACE2 mice are highly sensitive to nasal SARS-CoV-2 infection and have sustained strong viral RNA replication in lung Clara cells, trachea and brain.

　　 hACE2 mice develop interstitial pneumonia after being infected with SARS-CoV-2:

　　 To detect whether SARS-CoV-2 infected hACE2 mice have pathological features similar to COVID-19, the lung tissues of SARS-CoV-2 infected animals were subjected to histopathological examination, immunohistochemistry and immunofluorescence staining. Compared with SARS-CoV-2 infected WT mice, H&E staining showed that both young and old hACE2 mice developed interstitial pneumonia, which was manifested by inflammatory cell infiltration, thickening of alveolar septum, and obvious vascular system damage. More alveolar epithelial cell lesions and focal hemorrhage were observed in old mice. IHC staining results showed that compared with WT mice, SARS-CoV-2 infection can induce the infiltration of neutrophils (Neu+) and macrophages (CD68+) in aged hACE2 mice. In addition, immunofluorescence co-staining showed that SARS-CoV-2 directly infected CD68-positive macrophages in the lungs, resulting in significant apoptosis (C-Casp3 +) in aged hACE2 mice. Luminex cytokine analysis showed that SARS-CoV-2 infection led to increased cytokine production in elderly hACE2 mice, including eosinophil activating chemokine, G-CSF, IFN-γ, IL-9 and MIP-1β, but in young people The reaction of mice after infection is weak. Therefore, hACE2 mice not only continued viral replication, but also developed lung pathology after SARS-CoV-2 infection.

　　 Inoculation of SARS-CoV-2 via intragastric route established productive infection in hACE2 mice:

　　 SARS-CoV-2 is mainly spread through droplets and close contact between people. However, some people infected with COVID-19 have gastrointestinal symptoms such as diarrhea, abdominal pain, and vomiting. Viral RNA has been detected in the stool of COVID-19 patients, suggesting that SARS-CoV-2 may be transmitted orally through stool. Here, we are trying to study whether SARS-CoV-2 can establish productive infection in hACE2 mice through the intragastric route. After SARS-CoV-2 gavage, all animals received 5dpi daily monitoring and tissue processing. Three hACE2 mice inoculated with SARS-CoV-2 by intragastric administration showed no clinical symptoms, but high levels were detected in the trachea (2.9×106 copies/g) and lung (3.2×106 copies/g) of 2 mice. Viral RNA is equivalent to animals infected by nasal route. Importantly, SARS-CoV-2 S protein expression was also detected in the lung airways of infected hACE2 mice. In addition, interstitial inflammation and thickening of the alveolar compartment were observed in hACE2 mice infected with SARS-CoV-2. The results showed that SARS-CoV-2 inoculation into the stomach of hACE2 mice can also establish productive infection and cause pathological changes in the lungs.

　　 Discussion: In this study, we generated a stable humanized mouse strain, which was fused with the tdTomato reporter gene, and the expression of hACE2 was controlled by IRES. Further characteristics showed that the expression of mACE2 in homozygous mice was completely replaced by hACE2, and the expression of hACE2 in the lung and small intestine reached a peak. In the past, several hACE2 transgenic mice have been produced with different strategies and used to simulate SARS-CoV infection. Generally, the lung pathology caused by SARS-CoV in transgenic mice is related to the expression level of lung hACE2. Therefore, this transgenic mouse was studied to mimic SARS-CoV-2 infection. Compared with this transgenic mouse model, our hACE2 mouse model has several advantages. First, the hAce2 gene was inserted into the GRC m38.p6 site on the X chromosome to replace mAce2, and there was no endogenous mACE2 expression in homozygous hACE2 mice. Since transgene insertion occurs randomly, the integrity of the landing gene may be affected. Secondly, the tissue distribution of hACE2 in our mouse model is consistent with the clinical manifestations of COVID-19 patients, and high levels of hACE2 expression have been detected in the lungs. Interestingly, recent analysis based on the results of single-cell sequencing of human tissues shows that the kidney, heart, esophagus, bladder and ileum also represent high-abundance tissues, while the liver, spleen, small intestine, ovary and brain are the hACE2-rich tissues in our model. organization. This inconsistency between humans and mice deserves further study. Third, the introduction of tdTomato gene can monitor the expression of hACE2 in real time. All these ideal physiological characteristics and the stable heredity and phenotype of hACE2 mice prompted us to test their application in SARS-CoV-2 infection and replication. Our challenging experiments on SARS-CoV-2 clearly show that hACE2 mice are highly sensitive to SARS-CoV-2 infection by intranasal inoculation, and that SARS-CoV-2 at 4×105 pfu can easily cause respiratory tract (lung and tracheal) ) And virus replication in the brain. The viral RNA load in the lung reached 108 copies/g, which was significantly higher than that of hACE2 transgenic mice. Since few patients with COVID-19 have a nervous system infection, the presence of viral RNA in the brain is somewhat unexpected. The transgenic model of SARS-CoV infection also confirmed the chemotaxis of the virus to the brain, which is consistent with our findings. The underlying mechanism of SARS-CoV or SARS-CoV-2 invading the brain and the relationship with the severity of the disease have yet to be determined. Further characterization showed that the CC10 + Clara cells of the airway are the main target cells of SARS-CoV-2 in our model. Specific proteases in Clara cells, such as tryptase Clara, may help the effective cleavage of the S protein, thereby enhancing the ability of the virus to enter the cell. In addition, CD68+ macrophages in the alveoli were also infected by SARS-CoV-2, leading to severe apoptosis. Interestingly, a large number of cells that do not express hACE2 are also infected with SARS-CoV-2. How SARS-CoV-2 invades these cells is also worthy of further study. Overall, our results provide the first line of evidence showing the specific primary target cells of SARS-CoV-2 in mouse lungs. Most importantly, all hACE2 mice nasally inoculated with SARS-CoV-2 developed interstitial pneumonia, which is characterized by inflammatory cell infiltration, thickening of the alveolar septum, and unique vascular system damage, which summarizes most Clinical characteristics of COVID-19 patients. Early epidemiological findings indicate that advanced age will lead to an increase in the mortality rate of COVID-19 patients, and the results we obtained from the hACE2 model well reflect this finding. Older hACE2 mice (30 weeks old) lost weight after SARS-CoV-2 infection, while young hACE2 mice (4.5 weeks old) and wild-type mice maintained weight gain during the observation period. It is worth noting that although both young and old mice maintain similar virus replication in the lungs, more severe pathological changes and enhanced cytokine responses were observed in old hACE2 mice.

　　 There are several pieces of evidence supporting the potential fecal-oral transmission of SARS-CoV-2. First, gastrointestinal symptoms have appeared in many COVID-19 patients, and virus shedding in stool has been reported. Secondly, it was found that ACE2 is highly expressed in epithelial cells of the gastrointestinal tract. In vitro studies have also shown that SARS-CoV-2 can infect multiple cells in the gastrointestinal tract. Third, the experience of SARS and MERS supports human coronaviruses to maintain their viability under the environmental conditions necessary for oral transmission of feces. The recent data obtained from SARS-CoV-2 showed similar stability to SARS-CoV, indicating that similar transmission routes are feasible. Our results from the hACE2 mouse model provide evidence to support this hypothesis. Due to facility limitations, we are unable to collect and quantify fecal samples from each animal. Importantly, the presence of high levels of viral RNA and active viral proteins proved that intragastric inoculation of SARS-CoV-2 caused productive infection in the respiratory tract of hACE2 mice. However, our preliminary results indicate that SARS-CoV intragastric infection is not as effective as intranasal infection: the challenge dose of the intragastric model is 10 times higher than that of the intranasal model, but it was only found in one of the 3 animals tested lung infection. The viral RNA load and protein expression are also lower than the intranasal vaccination model. Whether SARS-CoV-2 gastric infection actually occurs in the human clinical environment remains to be confirmed. The potential mechanism of SARS-CoV-2 survival in the gastrointestinal environment and ultimately invading the respiratory tract of our mouse model is worthy of further study. "The results show that hACE2 mice are highly susceptible to infection after SARS-CoV-2 inoculated into the nose and stomach, and a large amount of virus replication can be seen in lung Clara cells and macrophages. The pathological changes observed in elderly hACE2 mice are more similar to those observed in COVID-19 patients. This article provides a hACE2 mouse animal model for studying the spread and pathogenesis of SARS-CoV-2 and understanding the unexpected clinical manifestations of SARS-CoV-2 in humans. This model also has certain value in detecting vaccines and treatments against SARS-CoV-2.