SARS-CoV-2,动物模型 z-bio 2022-01-15 413

　　Overview of SARS-COV-2:

　　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) called the virus "Severe Acute Respiratory Syndrome Coronavirus 2" (or SARS-CoV-2). Shortly thereafter, the World Health Organization Announced that COVID-19 is 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 disease. Progress. At the same time, it is 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 pathology of COVID-19. Here, we briefly describe related cell lines, organoids and animal models.

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

　　SARS-CoV2 research, an in vitro cell model to understand the life cycle of the virus, and to amplify and isolate the virus for further research, to help develop therapeutic molecules. -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). These are used by the virus to activate its receptors. S protein (SARS-CoV-2 spike protein). SARS-CoV-2 infection experiments using primary human respiratory epithelial cells have shown that it exhibits a cytopathic effect 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 simulate the physiological functions of the human body, nor will they produce low-titer infectious SARS-CoV-2. Despite this limitation, we can learn about the preciousness of virus infection and replication from studies using these cell lines. information. However, Vero cells provide high titer virus particles. For effective SARS-CoV-2 research, cell lines (such as Vero cells) that can easily replicate and isolate the virus are essential. These cells were isolated from the kidney epithelial cells of African savannah monkeys in 1963 and do not produce interferon (IFN) when infected with Newcastle disease virus, rubella virus or other viruses. 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. Due to the lack of interferon, SARS-CoV-2 can replicate in Vero cells. Among a large number of Vero cell clones, VeroE6 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 replication and isolation efficiency of SARS-CoV-2 in VeroE6 cells, Matsuyama et al. used VeroE6 cells overexpressing TMPRSS2. According to the report, the virus RNA copy number in the supernatant of these cells is more than 100 times higher than that of VeroE6 cells, and that VeroE6 cells overexpressing TMPRSS2 were used to isolate higher titers of virus. This shows that you can do it.

　　Organoids: Organoids are composed of different types of cells, 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 many organs such as the kidney, liver, and cardiovascular system. Montaire et al. Studies have shown 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. Prove 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 can lead to the accumulation of bile acids and subsequent liver damage in COVID-19 patients. In addition, the intestine is expected to become another viral target organ. Lammers and Zhou et al. It is reported that human intestinal organoids established by primary intestinal epithelial stem cells support SARS-CoV-2 replication. In addition, Monteil et al. It has also been proved that SARS-CoV-2 can directly infect vascular organoids differentiated from human induced pluripotent stem cells. Varga et al. The accumulation of viral components and inflammatory cells in endothelial cells was confirmed. 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 specific model tissues, they cannot reproduce the systemic symptoms associated with the systemic response of viral infections.

　　Animal model for SARS-CoV-2 research: Only by replicating tissue-specific and systemic virus-host interactions can we understand the complex pathophysiology of the disease. Cell lines and organoids are faster systems to study their interaction with viruses in host cells, but they can only reproduce the symptoms of COVID-19 in specific cell types or organs, respectively. On the other hand, the pathology of COVID-19 can be replicated and observed in animal models in a tissue-specific and systemic manner. Several different animals are used to study diseases and test candidate therapeutic compounds.

　　Little PIN: Zhou and others. SARS-CoV-2 infection experiments were carried out using HeLa cells. It expresses the ACE2 protein in various animals from mice to humans. Interestingly, SARS-CoV-2 can use all ACE2 proteins except mouse ACE2. Therefore, Bao et al. used transgenic mice expressing human ACE2. Researchers found that after SARS-CoV-2 infection, 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 SARS-CoV-2 to S proteins in different membranes, and found that Syrian hamsters may be suitable. After infection, these hamsters exhibited shortness of breath, weight loss, and alveolar damage, accompanied by extensive apoptosis.

　　Large PV: Small animals such as mice and Syrian hamsters are useful for studying SARS-CoV-2 because they reproduce faster, but they are larger and can faithfully reproduce the pathology of human COVID-19. 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, ferrets and cats are recommended when choosing large laboratory animals instead of rodents. Another model that can be used for COVID-19 research is the primate cynomolgus monkey, which 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 formation. Therefore, cynomolgus monkeys may be infected with SARS-CoV-2 and replicate certain human pathologies of COVID-19. Rhesus monkeys have also been used in COVID-19 research, confirming the therapeutic effects of adenovirus vector vaccines, DNA vaccine candidates expressing S protein, and remdesivir. These models may be the best choice for reproducing 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 5 months, COVID-19 has spread rapidly around the world. Even today, the number of infections and deaths are still rising. Currently, there are no preventive treatments or interventions. The only way to contain 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 select suitable cell and animal models for SARS-CoV-2 research, evaluate the advantages and disadvantages of each model, and find a better model.