SARS-CoV-2,动物模型 z-bio 2021-12-23 418

　　SARS-COV-2 overview:

　　The Chinese Center for Disease Control and Prevention (CCDC) has determined that a new type of coronavirus infection is the cause of this pneumonia. The World Health Organization (WHO) named the disease "2019

　　"New Coronavirus Disease" (or COVID-19) and the International Committee for Classification of Viruses (ICTV) named the virus "Severe Acute Respiratory Syndrome Coronavirus 2" (or SARS-CoV-2). Soon after, WHO 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. A number of clinical trials are currently underway to prevent or intervene in the progression of the disease. At the same time, it is also important to conduct basic research on SARS-CoV2 to support the effective development of therapeutic drugs.

　　For this, a model that can faithfully replicate viral behavior and replicate the pathology of COVID-19 is needed. Here, we briefly reviewed related cell lines, organoids and animal models.

　　Cell lines and organoids used in SARS-CoV2 research:

　　For SARS-CoV2 research, in vitro cell models are essential for understanding the life cycle of the virus, amplifying and isolating the virus for further research, and conducting preclinical evaluation of therapeutic molecules. This section lists the cell lines used to replicate and isolate SARS-CoV-2, as well as organoids that can be used to examine 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), which are used by the virus to activate the S protein (SARS-CoV-2's spike protein) receptor. It has been found that the SARS-CoV-2 infection experiment using primary human airway epithelial cells has a cytopathic effect at 96 hours after infection. However, human main airway epithelial cells are expensive and do not proliferate indefinitely. Several immortal cell lines, such as Caco-2, Calu-3, HEK293T, Huh7, have been used in SARS-CoV-2 infection experiments. These cell lines cannot accurately simulate human physiological conditions and produce low-titer infectious SARS-CoV-2. Despite this limitation, valuable information about virus infection and replication can be learned from studies using these cell lines. However, Vero cells provide high titers of virus particles. For effective SARS-CoV-2 research, a cell line that can easily replicate and isolate the virus, such as Vero cells, is essential. These cells were isolated from the kidney epithelial cells of an African green monkey in 1963 and did not produce interferon (IFN) when infected with Newcastle disease virus, rubella virus and other viruses. The 9 Mbp 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. The lack of IFN allows SARS-CoV-2 to replicate in Vero cells. Among the numerous Vero cell clones, Vero E6 is the most commonly used cell to replicate and isolate SARS-CoV-2 because these cells highly express ACE2 in the membrane domain. However, in this cloned cell, the expression level of TMPRSS2, the receptor used by the virus to activate the S protein (the spike protein of SARS-CoV-2), is quite low. In order to enhance the replication and isolation efficiency of SARS-CoV-2 in Vero E6 cells, Matsuyama et al. used Vero E6 cells overexpressing TMPRSS2. The report states that the viral RNA copy number in the culture supernatant of these cells is more than 100 times higher than the viral RNA copy number in Vero E6 cells, indicating that the use of Vero E6 cells overexpressing TMPRSS2 can isolate higher titers. virus.

　　Organoids: Organoids are composed of a variety of cell types, which can simulate the physiological conditions of human organs. Because organoids have the ability to replicate themselves, they are also suitable models 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 produced 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. Research by Monteil et al. showed that the supernatant of SARS-CoV-2 infected kidney organoids can effectively infect Vero E6 cells, indicating that kidney organoids can produce infectious progeny viruses. In addition, Zhao et al. have demonstrated that human liver duct organoids can allow SARS-CoV-2 infection and support replication. Interestingly, viral infection impairs the bile acid transport function of bile duct cells. This effect may be the cause of bile acid accumulation and subsequent liver damage in COVID-19 patients. In addition, the intestine is expected to be another viral target organ. Lamers and Zhou et al. reported that human intestinal organoids established from primary intestinal epithelial stem cells support SARS-CoV-2 replication. In addition, Monteil et al. also proved that SARS-CoV-2 can directly infect vascular organoids differentiated by 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 replicate the pathology of COVID-19 in the specific tissues they model, they cannot replicate the systemic symptoms associated with the systemic response to viral infections.

　　Animal model for SARS-CoV-2 research: Only by replicating tissue-specific and systemic virus-host interactions can the complex pathophysiology of the disease be understood. Although cell lines and organoids are faster systems for studying viruses and their interactions within host cells, they can only replicate 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 an animal model in a tissue-specific and systemic manner. Several different animals were used to study the disease and test candidate therapeutic compounds.

　　Small animals: Zhou et al. used HeLa cells to carry out SARS-CoV-2 infection experiments, which express the ACE2 protein of a variety of 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. The research team 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 ACE2 of different membranes to the S protein of SARS-CoV-2, 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 animals: Although small animals like mice and Syrian hamsters are useful for studying SARS-CoV-2 because they reproduce faster, but to faithfully replicate human COVID-19 pathology, larger 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. Rockx et al. used cynomolgus monkeys to compare MERS-CoV, SARS-CoV and SARS-CoV-2. Although MERS-CoV mainly infects type I lung cells, 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 hyaline membrane formation. Therefore, cynomolgus monkeys can be infected with SARS-CoV-2 and replicate some of the human pathology 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. Although these models may be the best at replicating virus-human-host interactions, the main limitation is that the reproduction rate of cynomolgus and rhesus monkeys is low and slow. Therefore, experiments with genetically modified mice and Syrian hamsters can be performed first.

　　Conclusion: In the past five months, COVID -19 has spread rapidly around the world. Even now, the number of people infected and the number of deaths is still increasing. Currently, there is no preventive treatment or intervention method. The only way to control the epidemic and reduce the associated loss of life is to change people’s behaviors, such as isolation and social distance. Treatment strategies for prevention and/or intervention are imminent. Although a number of clinical trials are currently underway, preclinical studies on in vitro and model organisms are also needed to understand the virus and test the safety and effectiveness of therapeutic drugs. We believe that this overview will help researchers select suitable cell and animal models for SARS-CoV-2 research, and will help assess the advantages and disadvantages of each model and discover better models.