Plaque Assay for Murine Norovirus
Plaque Assay for Murine Norovirus
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DOI：
10.3791/4297
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发表时间：
2012-08-01
影响因子：
1.2
通讯作者：
Wobus, Christiane E.
中科院分区：
文献类型：
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作者：
Gonzalez-Hernandez, Mariam B.;Cunha, Juliana Bragazzi;Wobus, Christiane E.
鼠诺如病毒（MNV）是唯一在组织培养中有效生长的诺如病毒属成员（1，2）。在MNV-1感染鼠树突状细胞或巨噬细胞期间观察到细胞溶解和细胞病变效应（CPE）（1）。MNV-1的这种特性可用于通过进行噬斑测定来定量给定样品中的感染性颗粒的数量（1）。空斑试验依赖于MNV-1裂解细胞并在融合细胞单层中形成孔的能力，这些孔被称为空斑（3）。多种技术可用于检测组织培养物、收获组织、临床和环境样本中的病毒感染，但并非所有技术都能测量感染性颗粒的数量（例如qRT-PCR）。定量感染性病毒颗粒的一种方法是进行噬斑测定（3），这将在下面详细描述。MNV空斑试验的一种变体是荧光焦点试验，其中MNV抗原在细胞单层中进行免疫染色（4）。该测定可以更快，因为病毒抗原表达先于空斑形成。它也可用于滴定不能形成噬斑的病毒。然而，荧光焦点测定需要除噬斑测定之外的额外资源，例如抗体和显微镜以计数焦点形成单位。感染性MNV也可通过测定50%组织培养感染剂量（TCID 50）进行定量（3）。本试验通过终点滴定法（5）测定50%接种的组织培养细胞中产生CPE所需的病毒量。然而，它的检测限比空斑试验更高（4）。在这篇文章中，我们描述了一种空斑试验方案，可用于有效地确定生物或环境样品中存在的感染性MNV颗粒的数量（1，4，6）。该方法基于含MNV样品的10倍系列稀释液的制备，其用于接种单层容许细胞（RAW 264.7鼠巨噬细胞）。使病毒附着于细胞单层一段给定的时间，然后在用琼脂糖和细胞培养基的混合物覆盖细胞之前吸出。琼脂能够使病毒子代扩散到邻近细胞，同时限制扩散到远处的细胞。因此，受感染的细胞被裂解并在单层中形成称为噬斑的孔。当病毒充分传播时，用染料如中性红、亚甲蓝或结晶紫对细胞染色后，噬斑变得可见。在低稀释度下，每个空斑源自一个感染性病毒颗粒及其后代，后者传播到邻近细胞。因此，计数噬菌斑的数量允许计算未稀释样品（3）中存在的噬菌斑形成单位（PFU）。
Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture (1, 2). Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages (1). This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay (1). The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques (3).Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay (3), which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers(4). This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) (3). This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration (5). However, its limit of detection is higher compared to a plaque assay (4).In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples (1, 4, 6). This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample(3).