| Analysis of the Effect of Sphingomyelinase on Rubella Virus Infectivity in Two Cell Lines
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Author:
Date:
2018-09-05
[Abstract] Rubella is a mildly contagious disease characterized by low-grade fever and a morbilliform rash caused by the rubella virus (RuV). Viruses often use cellular phospholipids for infection. We studied the roles of cellular sphingomyelin in RuV infection. Treatment of cells with sphingomyelinase (SMase) inhibited RuV infection in rabbit kidney-derived RK13 cells and African green monkey (Cercopithecus aethiops) kidney-derived Vero cells. Our data further demonstrated that RuV used cellular sphingomyelin and cholesterol for its binding to cells and membrane fusion at the step of virus entry. Detailed protocols of our assays, which assess the effects of SMase treatment on RuV infectivity in RK13 and Vero cells, are described.
[摘要] 风疹是一种轻度传染性疾病，其特征是低风热和由风疹病毒（RuV）引起的麻疹样皮疹。 病毒通常使用细胞磷脂进行感染。 我们研究了细胞鞘磷脂在RuV感染中的作用。 用鞘磷脂酶（SMase）处理细胞抑制兔肾衍生的RK13细胞和非洲绿猴（ Cercopithecus aethiops ）肾源性Vero细胞中的RuV感染。 我们的数据进一步证明RuV在病毒进入的步骤中使用细胞鞘磷脂和胆固醇来结合细胞和膜融合。 描述了我们的测定的详细方案，其评估了SMase处理对RK13和Vero细胞中RuV感染性的影响。
【背景】风疹病毒（RuV）是一种正链RNA病毒，属于 Togaviridae 家族中的 Rubivirus 属。该家族有两个属， Rubivirus 和 Alphavirus 。风疹病毒是属的鞋底构件的风疹病毒，而许多病毒，例如塞姆利基森林病毒（SFV）和辛德毕斯病毒（SINV），是归类于甲病毒属。 RuV是风疹和先天性风疹综合征（CRS）的致病因子。风疹的特征是低烧，麻疹样皮疹和淋巴结肿大。它通常是一种轻微的疾病。然而，CRS是一种严重的疾病。 CRS导致在怀孕早期患有风疹的母亲所生的新生儿出现多器官缺陷。白内障，感音神经性听力损失和心血管缺陷在CRS中很常见。
以前的研究表明，细胞膜脂质作为RuV感染的结合或进入因子（Mastromarino ...
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| Protocol-In vitro T Cell Proliferation and Treg Suppression Assay with Celltrace Violet
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Author:
Date:
2016-01-05
[Abstract] Measurement of the incorporation of radionuclides such as 3H-thymidine is a classical immunological technique for assaying T cell proliferation. However, such an approach has drawbacks beyond the inconvenience of working with radioactive materials, such as the inability of bulk radionuclide incorporation measurements to accurately quantitate T cell divisions, and an inability to combine proliferation analyses with simultaneous evaluation of the expression of cellular markers in divided cells. By labeling T cells with reactive dyes such as CFSE, Celltrace Violet, and others that are partitioned equally between daughter cells at each cell division, one can relatively easily track generations of proliferated cells and their expression of various molecules by flow cytometry.
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[摘要] 放射性核素如3 H胸腺嘧啶核苷的掺入的测量是用于测定T细胞增殖的经典免疫学技术。然而，这种方法具有超出使用放射性材料的不便之处的缺点，例如体放射性核素结合测量不能准确地定量T细胞分裂，以及不能结合增殖分析与同时评价分裂的细胞标记物的表达细胞。通过用诸如CFSE，Celltrace Violet等活性染料标记T细胞，在每个细胞分裂的子细胞之间平均分配T细胞，可以通过流式细胞术相对容易地追踪增殖细胞的代和它们的各种分子的表达。 > FoxP3 + 调节性T细胞（Treg）是免疫耐受的关键介质，其功能评价是表征许多免疫模型的重要步骤（Rudensky，2011）。基于其表面表达的CD25（Treg：CD4 + CD25 + sup/+），已经分离了经典的CD4 + Treg和常规或"应答者"T细胞， ，Tresp：CD4 + CD25 - sup/- ）。然而，我们和其他人已经注意到，CD4 + CD25 - 细胞群表达FoxP3转录因子并具有抑制功能。因此，我们利用转基因FoxP3-EGFP小鼠促进基于EGFP（并因此FoxP3）表达的抑制基因和应答者群体的活性纯化。在这里我们提出我们适应的协议，用于测定调节性T细胞抑制Celltrace紫罗兰标记响应T细胞。
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| Measuring Genetic Robustness in Vesicular Stomatitis Virus
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Author:
Date:
2014-03-20
[Abstract] Genetic robustness is the ability of a genome to incorporate mutations with the result of no fitness changes. Thus, more robust viruses have an increased neutral mutation rate. This property is particularly important in RNA viruses due to their high mutation rates. The most direct way of measuring robustness in vesicular stomatitis virus (VSV) is to carry out clonal analysis of populations: randomly isolating individual VSV strains (plaques), measuring the fitness of each one and generating fitness distributions (Novella et al., 2010). A second possibility is to carry out multiple replicates of repeated plaque-to-plaque passages, determining fitness in progeny populations and generating fitness distributions (Novella et al., 2010). Depending on the expected differences, ...
[摘要] 遗传稳健性是基因组掺入突变的能力，没有适应度变化的结果。因此，更强壮的病毒具有增加的中性突变率。由于它们的高突变率，这种性质在RNA病毒中特别重要。测量水泡性口炎病毒（VSV）的稳健性的最直接的方法是进行群体的克隆分析：随机分离个体VSV株（噬斑），测量每个VSV株的适合度并产生适应度分布（Novella等人， ，2010）。第二种可能性是进行重复的斑块到斑块通道的多次重复，确定后代群体中的适合度并产生健身分布（Novella等人，2010）。根据预期的差异，前者可能需要数百个测定，而后者可能需要数十次测定。第三种方法包括增加分析中的群体的突变率以扩大可能存在的任何差异，并且代替测量适合度，测量存活率（Novella等人，2013）。该方法的一个警告是生存的变化也可以通过聚合酶保真度的变化来解释。因此，重要的是进行互补实验，在这种情况下量化突变频率。
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