动物造模,动物模型 z-bio 2022-09-09 327

　　SARS-COV-2 Overview:

　　In December 2019, China confirmed pneumonia of unknown etiology. The Chinese Center for Disease Control and Prevention (CCDC) has identified a novel coronavirus infection as the cause of the pneumonia. The World Health Organization (WHO) named the disease "2019

　　Novel coronavirus disease (or COVID-19) and the International Committee on Taxonomy of Viruses (ICTV) named the virus “Severe Acute Respiratory Syndrome Coronavirus 2” (or SARS-CoV-2). -19 is a rapidly evolving pandemic. As of May 26, 2020, a total of 5,406,282 people have been infected with COVID-19 and an estimated 343,562 people have died worldwide. Several clinical trials are currently underway to prevent or intervene in disease progression. At the same time, it is equally important to conduct basic research on SARS-CoV2 to support the effective development of therapeutics.

　　For this, models that can faithfully replicate the behavior of the virus and replicate the pathology of COVID-19 are needed. Here, we briefly review relevant cell lines, organoids, and animal models.

　　Cell Lines and Organoids for SARS-CoV2 Research:

　　For SARS-CoV2 research, in vitro cellular models are critical for understanding the viral life cycle, amplifying and isolating the virus for further research, and preclinical evaluation of therapeutic molecules. This section lists 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 infection in specific human tissues.

　　Cell lines: In humans, airway epithelial cells highly express receptors that bind SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), which the virus uses to initiate the S protein (the spike protein of SARS-CoV-2). SARS-CoV-2 infection experiments using primary human airway epithelial cells have been found to be cytopathic at 96 hours post-infection. However, human primary airway epithelial cells are expensive and do not proliferate indefinitely. Several immortalized cell lines, such as Caco-2, Calu-3, HEK293T, Huh7, have been used in SARS-CoV-2 infection experiments. These cell lines cannot accurately mimic human physiological conditions and produce low titers of 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 provided high titers of viral particles. For effective SARS-CoV-2 research, a cell line, such as Vero cells, that can easily replicate and isolate the virus is essential. The cells, isolated in 1963 from the renal epithelial cells of an African green monkey, did not produce interferon (IFN) when infected with Newcastle disease virus, rubella virus and other viruses. A 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. . IFN deficiency enables SARS-CoV-2 replication in Vero cells. Among the numerous Vero cell clones, Vero E6 is the most commonly used cell for replicating and isolating SARS-CoV-2 because these cells highly express ACE2 in the membrane domain. In this clone, however, the expression level of TMPRSS2, the receptor that the virus uses to initiate the S protein (the spike protein of SARS-CoV-2), is rather low. To enhance the replication and isolation efficiency of SARS-CoV-2 in Vero E6 cells, Matsuyama et al. used Vero E6 cells overexpressing TMPRSS2. reported that the viral RNA copy number in the culture supernatant of these cells was more than 100-fold higher than that in Vero E6 cells, suggesting that higher titers could be isolated using Vero E6 cells overexpressing TMPRSS2. Virus.

　　Organoids: Organoids are composed of a variety of cell types that mimic the physiology of human organs. Because of their ability to replicate themselves, organoids 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 affects multiple organs including the kidneys, liver, and cardiovascular system. Suzuki and Han et al. generated human bronchial organoids or human lung organoids, respectively, for SARS-CoV-2 research. They show that organoids allow SARS-CoV-2 infection and that the antiviral effects of COVID-19 candidate therapeutic compounds, including Camostat, can be assessed. In addition to lung damage caused by pneumonia, SARS-CoV-2 affects multiple organs including the kidneys, liver and cardiovascular system. Monteil et al. showed that supernatants from SARS-CoV-2-infected kidney organoids efficiently infect Vero E6 cells, indicating that kidney organoids can produce infectious progeny virus. Additionally, Zhao et al. have demonstrated that human hepatic ductal organoids 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 responsible for bile acid accumulation and subsequent liver damage in COVID-19 patients. Furthermore, the gut 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 supported SARS-CoV-2 replication. In addition, Monteil et al. also demonstrated that SARS-CoV-2 can directly infect human induced pluripotent stem cell-differentiated vascular organoids. Varga et al. demonstrated accumulation of viral components and inflammatory cells within endothelial cells. Taken together, these two studies demonstrate that SARS-CoV-2 infection can directly lead to inflammation in multiple organs. But while organoids could replicate the pathology of COVID-19 in the specific tissues they modeled, they were unable to replicate the systemic symptoms associated with the systemic response to viral infection.

　　Animal models for SARS-CoV-2 research: The complex pathophysiology of the disease can only be understood through replicating tissue-specific and systemic virus-host interactions. Although cell lines and organoids are faster systems for studying viruses and their interactions inside host cells, they can only replicate COVID-19 symptoms 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 the disease and test candidate therapeutic compounds.

　　Small animals: Zhou et al. performed SARS-CoV-2 infection experiments using HeLa cells, which express the ACE2 protein in a variety of animals ranging 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 team found that after SARS-CoV-2 infection, the mice lost weight, viral replication and interstitial pneumonia were found in the lungs. In the search for an alternative small animal model, we performed 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. Following infection, these hamsters exhibited shortness of breath, weight loss, and alveolar damage with extensive apoptosis.

　　Large animals: While small animals like mice and Syrian hamsters are beneficial for studying SARS-CoV-2 because they reproduce faster, larger animal models are preferred to faithfully replicate human COVID-19 pathology . Kim et al. reported non-fatal acute bronchiolitis in a ferret model lung. Another study showed that SARS-CoV-2 can replicate in ferrets and cats, but not pigs, chickens and ducks. Based on these findings, ferrets and cats are recommended when choosing large laboratory animals over rodents. Another model that can be used for COVID-19 research is the primate cynomolgus monkey, which is currently the closest in pathophysiology to humans. Rockx et al. compared MERS-CoV, SARS-CoV, and SARS-CoV-2 using cynomolgus monkeys. While MERS-CoV primarily infects type II pneumocytes, both SARS-CoV and SARS-CoV-2 infect both type I and type II pneumocytes. Damage to type I pneumocytes leads to pulmonary edema and hyaline membrane formation following SARS-CoV-2 infection. Thus, cynomolgus monkeys can infect 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 efficacy of adenovirus vector vaccines, DNA vaccine candidates expressing the S protein, and remdesivir. While these models may be the best at replicating virus-human-host interactions, the major limitation is the low and slow reproductive rate of cynomolgus and rhesus monkeys. Therefore, experiments with transgenic mice and Syrian hamsters can be performed first.

　　Conclusion: During 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 increasing. Currently, there is no preventive treatment or intervention. 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 urgently needed. While multiple clinical trials are currently underway, preclinical studies in vitro and in model organisms are also required to understand the virus and test the safety and efficacy of therapeutics. We believe that this overview will assist researchers in selecting appropriate cellular and animal models for SARS-CoV-2 research, as well as in evaluating the strengths and weaknesses of each model and discovering better models.