动物造模,动物模型,SARS-CoV-2 z-bio 2021-06-08 1020

　　SARS-COV-2 Summary:

　　In December 2019, China was diagnosed with pneumonia of unknown cause. The Chinese Center for Disease Control and Prevention (CCDC) has identified the new coronavirus infection as the cause of this pneumonia. The World Health Organization (WHO) named the disease "2019

　　"New Coronavirus Disease" (or COVID-19), the International Committee for Classification of Viruses (ICTV) named the virus "Severe Acute Respiratory Syndrome Coronavirus" 2" (or SARS-CoV-2). Soon thereafter, the World Health Organization declared that COVID-19 was a rapidly developing pandemic. As of May 26, 2020, a total of 5,406,282 people worldwide have been infected with COVID-19, and an estimated 343,562 people have died. Many clinical trials are currently underway to prevent or intervene in the progression of the disease. At the same time, it is also important to carry out basic research on SARS-CoV2 to support the effective development of therapeutic drugs.

　　For this, we need a model that can faithfully reproduce the behavior of the virus and reproduce the pathophysiology of COVID-19. The following is a brief review of relevant cell lines, organoids and animal models.

　　The cell lines and organoids used in the SARS-CoV2 study:

　　SARS-CoV2 research, in vitro cell model to understand the virus life cycle, amplify and isolate the virus for further research, and treat it. Helping to develop molecular use clinical evaluation is essential. This section lists the cell lines used to replicate and isolate SARS-CoV-2 and the organoids that can be used to study the effects of SARS-CoV-2 on specific human tissue infections.

　　Cell line: In humans, airway epithelial cells highly express receptors that bind to SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), and viruses act as receptors. Start using S protein (SARS-CoV-2 spike protein). SARS-CoV-2 infection experiments using human primary respiratory epithelial cells showed that cytopathic effects occurred 96 hours after infection. However, human main airway epithelial cells are expensive and will not proliferate indefinitely. Several immortalized cell lines, such as Caco-2, Calu-3, HEK293T and Huh7, have been used in SARS-CoV-2 infection experiments. These cell lines cannot accurately mimic human physiological conditions and produce low-titer infectious SARS-CoV-2. Despite this limitation, valuable information about viral infection and replication can be learned from studies using these cell lines. However, Vero cells provide high titers of virus particles. In order to effectively study SARS-CoV-2, cell lines that can easily replicate and isolate the virus (such as Vero cells) are essential. These cells were isolated from the kidney epithelial cells of African green monkeys in 1963 and do not produce interferon (IFN) when infected with viruses such as Newcastle disease virus and rubella virus. The 9 Mbps homozygous deletion on chromosome 12 results in the loss of the type I interferon (IFN-I) gene cluster and the cyclin-dependent kinase inhibitor gene. Without interferon, SARS-CoV-2 can replicate in Vero cells. Among many Vero cell clones, Vero E6 is the most commonly used cell to replicate and isolate SARS-CoV-2. This is because these cells highly express ACE2 in the membrane domain. However, in these cloned cells, the expression level of TMPRSS2, the receptor used by the virus to activate the S protein (SARS-CoV-2 peplomer), is very low. In order to improve the efficiency of SARS-CoV-2 replication and isolation in VeroE6 cells, Matsuyama et al. used VeroE6 cells overexpressing TMPRSS2. It is reported that the virus RNA copy number in the supernatant of these cells is more than 100 times higher than that of VeroE6 cells, and VeroE6 cells overexpressing TMPRSS2 can be used to isolate higher titers of viruses.

　　Organoids: Organoids are composed of a variety of cell types, which can simulate the physiological state of human organs. Organoids have the ability to replicate themselves, making them a good model for large-scale screening in drug discovery and disease research. In addition to lung damage caused by pneumonia, SARS-CoV-2 also affects many organs such as the kidney, liver, and cardiovascular system. Suzuki and Han et al. respectively created human bronchial organs or human lung organs for SARS-CoV-2 research. They showed that organoids allow SARS-CoV-2 infection and can evaluate the antiviral effects of COVID-19 candidate therapeutic compounds, including Camostat. In addition to lung damage caused by pneumonia, SARS-CoV-2 also affects multiple organs such as the kidney, liver, and cardiovascular system. The study by Monteil et al. showed that the supernatant of kidney organoids infected by SARS-CoV-2 can effectively infect VeroE6 cells, proving that kidney organoids can produce infectious progeny viruses. In addition, Zhao et al. showed that human liver ductal organoids can infect SARS-CoV-2 and support replication. Interestingly, viral infection can impair the bile acid transport function of bile duct cells. This effect may be the cause of the accumulation of bile acids and subsequent liver damage in COVID-19 patients. The intestine is also expected to become another virus-targeted organ. Lamers and Zhou et al. reported that human intestinal organoids established by primary intestinal epithelial stem cells support SARS-CoV-2 replication. In addition, Monteil et al. demonstrated that SARS-CoV-2 can directly infect vascular organoids differentiated from human induced pluripotent stem cells. Varga et al. confirmed the accumulation of viral components and inflammatory cells in endothelial cells. In summary, these two studies show that SARS-CoV-2 infection can directly cause inflammation of multiple organs. However, although organoids can reproduce the pathology of COVID-19 in the specific tissues they model, they cannot reproduce the systemic symptoms associated with the systemic response of viral infections.

　　Animal model for SARS-CoV-2 research: Only by reproducing tissue-specific and systemic virus-host interactions can the complex pathophysiology of the disease be understood. Cell line. Organoids are faster system cells that study their interaction with viruses in the host, but they can only replicate the symptoms of COVID-19 in specific cell types or tissues. On the other hand, the pathology of COVID-19 can be replicated and observed in animal models in a tissue-specific and systemic manner. Disease studies and candidate therapeutic compounds are tested using several different animals.

　　Small animals: Zhou et al. conducted SARS-CoV-2 infection experiments with HeLa cells, and expressed ACE2 protein in a variety of animals from mice to humans. Interestingly, SARS-CoV-2 is a mouse, and all ACE2 proteins except ACE2 can be used. Therefore, Bao et al. used transgenic mice expressing human ACE2. After being infected with SARS-CoV-2, the researchers found that the mice lost weight, virus replication and interstitial pneumonia were found in the lungs. In the process of searching for alternative small animal models, we conducted molecular docking studies on the binding of ACE2 to SARS-CoV-2 S protein in different membranes, and found that Syrian hamsters may be suitable. After infection, these hamsters exhibit shortness of breath, weight loss, alveolar damage, and extensive apoptosis.

　　Large animals: Small animals such as mice and Syrian hamsters reproduce faster, which is conducive to the study of SARS-CoV-2, but the larger ones are conducive to the faithful reproduction of human COVID-19 pathology, and animal models are preferred. Kim et al. reported non-fatal acute bronchiolitis in the lungs of a ferret model. Another study showed that SARS-CoV-2 can be replicated in ferrets and cats, but not in pigs, chickens, and ducks. Based on these findings, large experiments are recommended when selecting animals, not rodents, ferrets and cats. Another model that can be used to study COVID-19 is the primate cynomolgus monkey. It is currently the closest to humans in pathophysiology. ockx et al. used cynomolgus monkeys to compare MERS-CoV, SARS-CoV and SARS-CoV-2, MERS-CoV mainly infects type II lung cells, while SARS-CoV and SARS-CoV-2 both infect type I and type II Lung cells. After SARS-CoV-2 infection, type I lung cells are damaged, leading to pulmonary edema and vitreous membrane formation. Therefore, cynomolgus monkeys can be infected with SARS-CoV-2 and replicate some human pathologies of COVID-19. Rhesus monkeys have also been used in COVID-19 research and have been shown to have therapeutic effects on adenovirus vector vaccines, DNA vaccine candidates expressing S protein, and remdesivir. These models may be the best choice for replicating virus-human-host interactions, but the main limitation is the low and slow reproduction rate of cynomolgus and rhesus monkeys. Therefore, it is possible to experiment with genetically modified mice and Syrian hamsters first.

　　Conclusion: In the past five months, COVID-19 has spread rapidly around the world. Even now, the number of infections and deaths is still increasing steadily. Currently, there are no preventive treatments or interventions. The only way to control the epidemic and reduce the associated loss of life is to change people’s behavior, such as isolation and social distancing. Treatment strategies for prevention and/or intervention are imminent. Many clinical trials are currently underway, but preclinical trials in vitro and model organisms are also needed to understand the virus and test the safety and effectiveness of therapeutic agents. We believe that this review will help researchers choose appropriate cell and animal models for SARS-CoV-2 research, evaluate the advantages and disadvantages of each model, and discover better models.