我们感谢的Wobus实验室为重要的意见和建议。 CEW的实验室工作是由美国密歇根大学的启动资金，职业生涯发展的资助NIH / NIAID区域卓越中心的生物防御和新发传染病研究计划（RCE），五区“大湖的RCE（NIH奖1-U54-AI-057153）和NIH R01 AI080611。 MBG-H。实验免疫学研究院（NIH T32 A1007413-16）和微生物发病的分子机制（NIH T32 A1007528）培训补助金是由美国密歇根大学的。 JBC是由德PessoalCoordenação的AperfeiçoamentoNIVEL的高级（CAPES），巴西利亚，巴西。

1。培养的巨噬细胞株RAW 264.7 <ol><li>维护的RAW 264.7细胞（ATCC，目录＃TIB-71）在DMEM-10介质，它由低内毒素胎牛血清（&lt;10 EU / ml）的10％（体积/体积）的高葡萄糖DMEM中，10 mM HEPES的，100 U / ml青霉素，100微克/毫升链霉素，1mM的非必需氨基酸，2mM的L-谷氨酰胺。细胞通常保持在175厘米2组织培养瓶中含有35毫升每个烧瓶中的介质，并在37℃和5％CO 2中的组织培养箱中。然而，可用于任何规模的烧瓶中的介质的体积，是适当的大小将烧瓶。 </li><li>分裂细胞：吸去旧媒体，加入10毫升的新鲜DMEM-10介质的细胞，然后用细胞刮刮的细胞，从瓶底。接着，成均匀的溶液重悬细胞通过绘制到10ml移液管的细胞，并有力地挤压的细胞，通过移液管尖端公关烧瓶底部essed对。重复此动作的至少3倍，使细胞不再聚集在一起。验证产生单细胞悬浮液，通过光学显微镜。然后，1毫升（1:10稀释或〜1×10 7细胞） - 2毫升（1:5的比例进行稀释，或〜2×10 7个细胞）的细胞悬浮液转移到一个新的175 cm 2的烧瓶中，使终体积媒体35毫升。 </li><li>拆分单元格时，他们几乎是汇合（〜1×10 8个细胞total/175厘米2瓶）：每三天开始用1:5稀释1:10稀释，或每两天开始。前拆分单元格，使用光学显微镜检查细胞形态。大多数细胞看起来应该圆形的，并没有被激活。活化细胞有颗粒和/或延长，细长的形态与附属物。不要让这些细胞的细胞长满通常不形成斑块。跟踪的传代次数和频繁启动细胞解冻下通道等分。 （我们使用通道30作为一个截止）。 </li></ol> 2。感染RAW 264.7细胞与MNV接种物<ol><li>种子的RAW 264.7细胞到6孔培养板（3.5厘米直径）在密度为1×10 6活细胞/ ml的DMEM-10介质中，并加入2毫升该悬浮液中，以各孔。是很重要的细胞均匀地分发通过摇动板手的至少10倍，或通过使用〜10分钟的摆动装置，用于在井中。不要旋转板，因为这会导致细胞聚集在井的中心。将板成组织培养箱中（在37℃和5％CO 2）。让细胞在37℃连接过夜或至少4小时细胞应该是60 - 80％汇合为斑块检测，并均匀分布在整个井。 </li><li>第二天，制备的病毒接种物，它可以是来自MNV-感染的细胞在组织培养中的或从均质化的组织或MNV感染的小鼠的粪便样品。当使用豌豆大小的组织样本，PI欧洲经委会在2毫升的带螺旋帽的管含有无菌硅珠用组织匀浆器（ 例如 MagnaLyser;罗氏）在1毫升的DMEM-10的组织均化。对于粪便样品，不超过3的粪便颗粒应在1毫升介质匀浆。所有的样品，然后冻结（在-80℃）和解冻的前一次执行的噬斑分析。 </li><li>准备的病毒接种物的10倍稀释液，在完全DMEM-5培养基的DMEM /高葡萄糖，5％（体积/体积）的低内毒素牛胎儿血清（&lt;10 EU /毫升），10mM的HEPES，100，它由U / ml青霉素，100μg/ ml链霉素，1mM的非必需氨基酸，2mM的L-谷氨酰胺。 </li><li>十倍系列稀释液在24孔培养板中制备：用于分配成多个井1.35毫升媒体的中继器移液管，在10 -1稀释由混合媒体和1.35毫升，0.15毫升含病毒样品，并然后加入0.15毫升10 -1稀释到1.35毫升的介质中，以使在10 -2 dilution和等等。重要的是要改变提示每次做出一个新的稀释。可以使用多通道移液管，使多个样品的稀释液，在同一时间有两个秘诀嵌合到一个孔的24孔板转移的总体积为0.15毫升，每孔（参见图3A）。 </li><li>一个典型的稀释范围内的组织匀浆和粪便的含量是10 -1〜10 -3。然而，从这些样品中的斑块往往比较小，相比那些从组织文化样本。此外，在某些情况下，粪便样品1:100的稀释度，需要充分地稀释，满分任何有毒成分的粪便，可能会扰乱细胞单层，从而阻碍了对噬斑计数能力。组织培养的溶胞产物的稀释范围取决于感兴趣的时间点上，在病毒的生命周期期间。在感染的高峰期，可能需要的稀释液上去〜10 -9。 </li><li>制备系列稀释后，标记的6 - 孔板含RAW 264.7细胞单层（2.1节），样品名称和稀释电镀。一盘的时间，取出所有介质刷出或吸它。紧接着添加0.5毫升稀释样品到好，然后重复重复的井，然后再进入到下一个稀释。一旦所有3稀释液被加入到一个板，倾斜板用手来回，以确保所有的细胞已被覆盖。处理一次，以确保电池不会干出的一盘。 </li><li>后向每孔中加入0.5毫升的稀释液中，堆叠板直立，它们在室温下的1小时孵育。因为音量加入到各孔不足以完全覆盖单层，板需要轻轻来回倾斜手每10-15分钟，或对上的摆动装置（〜18每分钟的振荡）下。这可以防止干燥的细胞。 </li></ol> 3。低熔点琼脂糖（SeaPlaque）覆盖准备<ol><li>计算的量的叠加之前所需的总体积，板孵育1小时完成。需要的体积是2ml /孔或12 ml/6-well板。 ，准备琼脂糖（参见3.2节）和媒体（参见3.3节）分别。 </li><li>为了制备琼脂糖，暂停3克SeaPlaque琼脂糖中的总体积为100毫升的玻璃瓶中，用蒸馏水（3％w / v的）。高压灭菌20-30分钟。 （如果琼脂糖已经前手，重新熔融琼脂糖在微波制备。）重要的是平衡至42℃下，在使用前，在水浴中，因为，当琼脂糖是太热，它会杀死细胞SeaPlaque琼脂糖。确保水位是等于或高于琼脂糖的水平，以避免不希望的凝固。 </li><li>为了准备对媒体表示：100毫升2个的MEM媒体，其中包括2×MEM，10％（体积/体积）的低内毒素牛胎儿血清（&lt;10 EU /毫升），10mM的HEPES，100 U / ml青霉素，100μg/ ml链霉素，4 mM的L-谷氨酰胺。平衡至37℃下在水浴中的媒体。 </li><li> SeaPlaque琼脂糖和2倍的MEM培养基在无菌瓶一起立即按1:1的比例混合，然后再重叠感染的细胞单层。如果超过200毫升覆盖是必要的，分割成多瓶量，并保持在37°C水浴中，直到准备使用。 </li><li>在培养1小时（参见2.7节），吸接种，每口井。慢慢加入2毫升的叠加到各孔的边缘，将移液管尖，对各孔的壁。最多5块板可以同时被处理，无细胞干燥。 </li><li>前板直立组织培养孵化器，允许将覆盖约10分钟，在室温下固化。温育平板48小时在37℃下，在5％CO 2。 </li><li>后潜伏期，斑块肉眼依稀可见，所以检查未染色板存在的斑块。如果没有斑块是可见的，额外的4小时孵化，并再次检查。然而，最大的孵化时间不得超过72小时。 </li></ol> 4。可视化斑块中性红染色<ol><li>为了可视化斑块，中性红染色溶液制备的每一个97毫升的1x PBS（组织培养级，Mg 2 +的-通过加入3毫升的中性红（0.33％重量/体积的DPBS; Sigma公司，目录＃N2889），的Ca 2 + -免费Gibco公司，产品编号10010）。计算所需的实验：12毫升中性红染色溶液所需的每个6孔板的中性红染色溶液的体积。然后，向每孔加入2毫升。虽然有些斑块检测的协议要求琼脂糖插头，可以从水井中删除，在该协议中的中性红染色液直接添加到覆盖。 </li><li>经过一个小时的别加入1 mL PBS，在37°C，如果斑块是明显的中性红染色解决方案仍然在井。如果斑块是不是很明显，染色，继续为另一个HR。继续孵化，直到斑块是明显的。 （注：是不是最佳的染色超过3小时，如果3小时后的染色可见阳性对照样品没有斑块，斑块检测没有正常工作。）染色完成后，吸出中性红染色液，确保琼脂糖插件的不被打扰，然后继续计数的斑块。 </li><li>将板倒置的灯箱和标志计数的斑块上的一个点，以避免重复计数，计数斑块。选择稀释井斑被清楚地分开计算斑块（ 即无视觉证据的斑块的融合在一起）。如果可能的话，在两次稀释噬斑计数。重要的是要注意，斑块大小之间可能会发生变化MNV菌株，病毒接种物，并依赖于RAW 264.7细胞在斑块检测的条件。 </li><li>如果没有斑是在一个良好可见，无论有没有病毒样品中存在的或病毒的噬斑分析的检测极限下的量。在这种情况下，各孔染色类似的颜色为红色，其他含斑块井。可替代地，没有斑时，也观察到有太多的病毒颗粒存在于一个给定的稀释。这导致裂解整个单层和水井出现橙色/黄色。 </li><li>计算病毒滴度。添加在两口井在一个单一的稀释斑块数，乘以稀释倍数（ 即 1毫升，如果用0.5毫升）2口井被感染。这将产生在您的接种体积为1毫升的噬斑形成单位（PFU）的量。例如，在图4中的10 -2稀释，一个阱（标记为“II”）的有14个噬菌斑和其他井（“V”标示）有17个斑块。因此，病毒滴度是14X10 + 17x10 = 3100（3.1×10 3）PFU /毫升。 </li></ol> 5。代表性的成果示意性地在图1中所概述的，用噬斑分析。传染性MNV-1颗粒可以被量化图2A示出了与一个单层的RAW 264.7细胞在感染前，而图2B示出了表示由罗马数字I，II的三个可见斑块和III在一个井。测定的各个步骤通过F. 图3A示出在图3A示出制备的含病毒样品的10倍稀释系列， 图3B示出了重复的6孔板的孔中的稀释度转移图图3C示出的摆动装置，用于孵化RAW 264.7细胞的接种物，在室温下1小时， 图3D示出了细胞与SeaPlaque重叠：MEM混合物。 图3E示出了在室温下，以允许叠加到固化的板，而图3F示出了被染色的细胞用0.01％中性红溶液48小时购买。染色后细胞1-3小时，并吸移的中性红染色溶液，斑块是可见的，并可以被计算（ 图4）。 <img alt="图1" src="/files/ftp_upload/4297/4297fig1.jpg" /> 图1。MNV的牌匾分析协议的示意图。 <img alt="图2" src="/files/ftp_upload/4297/4297fig2.jpg" /> 图2。阱单层感染前和后形成的斑块的代表图像。 A）的RAW 264.7细胞中，培养过夜，并在光学显微镜下在20倍的放大倍率成像。 B）的细胞，48小时后，用0.01％中性红溶液染色r的感染，并在4倍放大倍率的光学显微镜下观察。罗马数字I，II，III表示可见斑。 <img alt="图3" src="/files/ftp_upload/4297/4297fig3.jpg" /> 图3不同的空斑测定步骤的代表图像。甲）MNV-1接种物在10倍稀释液中制备。 B）接种物被添加到重复的井的细胞单层。 C）的细胞和接种物温育1小时，在室温下摇动。 D）的重叠细胞SeaPlaque琼脂糖和2x MEM培养基的1:1混合物中。 E）的板在室温下温育10分钟，以允许覆盖层固化。 F）染色的细胞，中性红染色液，感染后48小时。 <img alt="图4" src="/files/ftp_upload/4297/4297fig4.jpg" /> 图4。MNV-1的形式斑块的细胞一元醇艾尔斯。这里展示的是代表斑块的检测板，感染后48小时，1小时的潜伏期后，中性红染色液染色显示斑块。板显示重复的3个10倍稀释液的孔中。威尔斯标有罗马数字I和IV对应到10-1稀释，II和V对应到10-2稀释，III和VI对应到10-3稀释。下面的样本显示，病毒滴度（见第4.5节的详细的计算）。

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没有利益冲突的声明。

空斑法MNV-1的方法，这里介绍的是量化传染病MNV颗粒的一种方式。通过测定图3中示出的步骤之后，可以得到重现性的病毒滴度。检测测定的极限取决于所用的起始稀释。当如上所述用1:10稀释的样品，开始检测的空斑测定的极限是10 pfu的（ 即，1斑块可见在10 -1稀释）。由于每个斑块代表一个单一的病毒，噬斑分析也可以被用来净化无性系种群MNV采摘孤立的斑块和它们传播如前所述1。此外，也可以使用斑块纯化，以分离单个病毒人口从混合病毒种群。用空斑法为MNV感染的检测的一个限制是，并非所有的的MNV菌株形成斑块4。然而，它可能是能够克服inabiliTY的一些MNV株，从动物中分离，形成斑块，通过连续传代，这些病毒在组织培养7。的噬斑分析的另一种方法是测量通过TCID 50技术3，4的感染性颗粒。该测定中量化所需的病毒的量在50％的接种组织培养细胞中的端点稀释和1周〜为MNV 4完成后产生CPE。除了 ​​慢比空斑测定，TCID 50测定也是不那么敏感（检测限= 200 TCID 50 /毫升）由于组织样本，以RAW 264.7细胞4的毒性。 虽然整个协议在协议的关键步骤，下面的部分提供了一个总结，以方便排除故障。在协议中最关键的一步是确保整个检测，支持病毒复制，RAW 264.7细胞保持活力。这可以在每个阶段的试验中通过光学显微镜进行监测。细胞存活率确保在两个方面。首先，应注意不要让细胞干出来的，而处理板。因此，板接种一次一个，轻摇感染期间，并应该保持关闭时，它们并没有被处理。二，添加到细胞中的解决方案应该是平衡〜37℃此外，它是非常重要的RAW 264.7细胞介质中含有低内毒素血清（&lt;10 EU /毫升），这限制了活化细胞，以维持他们的整体健康。此外，我们观察到细胞时，通道30或更高的故障率较高的斑块检测。虽然这可能会有所不同从实验室到实验室，它是重要的，包括阳性对照，以确保重现性滴度，尤其是当使用较高的通道的RAW 264.7细胞（ 例如，一个样品与公知的病毒滴度）。要限制使用更高的传代细胞，最好是冻结小瓶早期帕萨GE细胞在收到RAW 264.7细胞经常从冷冻瓶，并开始一个新的文化。从低传代细胞培养也将是有帮助的，当细胞表现出改变的特点，如不遵守，细胞形态变化（ 例如从圆形到细长的传播），或已检测支原体污染。另外重要的一点要注意的是，以确保枪头之间改变样品在稀释。这将确保精确的连续稀释，防止样品间的交叉污染。协议，其中可以再次使用相同的移液管尖中的一个步骤是，当相同的样品的系列稀释液添加到井。在这种情况下，应该开始从最摊薄接种至少，大力移液管向上和向下时制定了一个新的稀释。 空斑法协议是可以改变的了若干修改。一个修改型式中，可以当t这里没有足够的细胞接种井一式两份，每个稀释度接种只是单一的。然而，由于接种物的体积为0.5毫升，然后空斑数需要乘以2的因数正常化pfu /毫升。噬斑分析也可以适于与任何其他的贴壁细胞线是能够支持MNV复制使用，这已经描述了小鼠小胶质细胞的BV-2细胞系8。可以实现其它的修改，已经描述了为开发对其他病毒的噬斑分析协议的适应。 MNV的情况下，下面的修改已经被成功实施，使用甲基纤维素，而不是海斑块琼脂糖9，结晶紫染色的细胞或亚甲基蓝的，而不是中性红10，11。 总体而言，该协议可以很容易地被修改为所需的量化其它噬斑形成的病毒或用于超视距ER病毒导致溶骨性感染的RAW 264.7细胞，使其成为一个有用的工具，量化，一般的感染性病毒颗粒。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> DMEM /高糖</td><td> Hyclone公司</td><td> SH30243.02 </td></tr><tr><td> 2个MEM </td><td> Gibco公司</td><td> 11935 </td></tr><tr><td> 100倍的青霉素和链霉素</td><td> Hyclone公司</td><td> SV30010 </td></tr><tr><td> 10毫米的非必需氨基酸</td><td> Hyclone公司</td><td> SH30238.01 </td></tr><tr><td> 1M HEPES </td><td> Hyclone公司</td><td> SH30237.01 </td></tr><tr><td> 200mM的L-谷氨酰胺（100x）的</td><td> Hyclone公司</td><td> SH30034.01 </td></tr><tr><td>胎牛血清</td><td> Gibco公司，胎牛血清</td><td> 10437，SH30070.02 </td></tr><tr><td>海斑块琼脂糖</td><td>龙沙</td><td> 50100 </td></tr><tr><td>中性红0.33％ </td><td>西格玛</td><td&gt; N2889 </td></tr><tr><td> 1X PBS </td><td> Gibco公司</td><td> 10010 </td></tr><tr><td> 1.0毫米氧化锆/二氧化硅珠</td><td> BioSpec产品</td><td> 11079110z </td></tr><tr><td> 35型变速摇杆</td><td> Labnet </td><td> S2035 </td></tr><tr><td>麦格纳Lyser仪器</td><td>罗氏公司</td><td> 03358968001 </td></tr><tr><td> RAW 264.7细胞株</td><td> ATCC </td><td> TIB-71 </td></tr><tr><td>组织培养箱</td><td>三洋</td><td> MCO-18AIC </td></tr></tbody></table>

plaque assay for murine norovirus

小鼠诺罗病毒（MNV）是诺如病毒属的唯一成员，有效地生长在组织培养1，2。细胞裂解和细胞病变效应（CPE）的过程中观察到MNV-1感染的小鼠树突状细胞或巨噬细胞1。这个MNV-1的属性可以用于量化一个给定的样品中，通过进行空斑测定1感染颗粒的数目。噬斑分析依赖于MNV-1的能力在汇合的单层细胞，它们被称为斑块3裂解细胞，以形成孔。 多种技术可用于检测病毒感染的组织培养，收获组织，临床和环境样品，但不是所有的措施的感染性颗粒的数目（ 例如，定量RT-PCR）。量化感染性病毒颗粒的方法之一是进行空斑测定3，这将在下面详细描述的。对的MNV斑块检测的一种变化是氟escent重点检测，，MNV抗原免疫组化染色在细胞单层4。此法可以更快，因为病毒抗原的表达斑块形成之前。它也是有用的滴定病毒无法形成噬斑。但是，荧光焦点检测超出那些噬斑分析需要额外的资源，如抗体和显微镜计数聚焦形成单位。传染性MNV也可以进行量化，通过确定50％组织培养感染剂量（TCID 50）3。该测定中，测量所需的病毒的量在50％的接种组织培养细胞中通过终点滴定5产生CPE。然而，其检出限为高于一个噬斑分析4。 在本文中，我们描述一个噬斑分析，可以用来有效地确定的数目传染性MNV颗粒中存在的生物或环境样品1，4，6的协议。这种方法是巴sed的10倍系列稀释液，这是用来允许细胞（RAW 264.7小鼠巨噬细胞）接种到单层的的MNV含样品的制备。病毒允许附加到对于一个给定的时间内的细胞单层，然后吸气之前覆盖琼脂糖和细胞培养基的混合物与细胞。琼脂使子代病毒的蔓延到邻近的细胞同时限制扩散到较远的位于细胞。因此，受感染的细胞裂解和在单层的形式孔称为斑块。足够的病毒传播后，斑块成为可见下面的染料，如中性红，亚甲基蓝，或结晶紫染色的细胞与。每个斑块在低稀释液，源于一个感染性的病毒颗粒和它的后代，蔓延到邻近的细胞。因此，计数噬斑的数量，允许一个计算空斑形成单位（PFU）的未稀释的样品3中存在。

Watch this Scientific Journal Video about Plaque Assay for Murine Norovirus at JoVE.com

Plaque Assay for Murine Norovirus

在这里，我们描述了一种方法来量化小鼠诺罗病毒（MNV），这是唯一的，有效地复制在细胞培养中的诺罗病毒的感染性颗粒。噬斑分析MNV小鼠的巨噬细胞的趋利用，可适于使用的生物或环境样品，含有MNV。

对小鼠诺沃克类病毒的空斑法

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 ...

Research

JoVE Journal

Immunology and Infection

54.5K 次观看.  University of Michigan, Ann Arbor. 在这里，我们描述了一种方法来量化小鼠诺罗病毒（MNV），这是唯一的，有效地复制在细胞培养中的诺罗病毒的感染性颗粒。噬斑分析MNV小鼠的巨噬细胞的趋利用，可适于使用的生物或环境样品，含有MNV。

视频: Plaque Assay for Murine Norovirus

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。第三部分。病毒检测用RT-qPCR的

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

科研

环境科学

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. 
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. 
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

反向遗传学感染小鼠诺沃克病毒介导的恢复

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

免疫与感染

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

病毒浓度的测定通过空斑试验:用传统的和新型覆盖系统

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

定量微中和试验的优化

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

替代体外测定病毒衣壳结构完整性的方法

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Assays for the Specific Growth Rate and Cell-binding Ability of Rotavirus

Rotavirus is the main etiological factor for infantile diarrhea. It is a double-stranded (ds) RNA virus and forms a genetically diverse population, known as quasispecies, owing to their high mutation rate. Here, we describe how to measure the specific growth rate and the cell-binding ability of rotavirus as its phenotypes. Rotavirus is treated with trypsin to recognize the cell receptor and then inoculated into MA104 cell culture. The supernatant, including viral progenies, is collected intermittently. The plaque assay is used to confirm the virus titer (plaque-forming unit: pfu) of each collected supernatant. The specific growth rate is estimated by fitting time-course data of pfu/mL to the modified Gompertz model. In the assay of cell-binding, MA104 cells in a 24-well plate are infected with rotavirus and incubated for 90 min at 4 &#176;C for rotavirus adsorption to cell receptors. A low temperature restrains rotavirus from invading the host cell. After washing to remove unbound virions, RNA is extracted from virions attached to cell receptors followed by cDNA synthesis and reverse-transcription quantitative PCR (RT-qPCR). These protocols can be applied for investigating the phenotypic differences among viral strains.

Here we present two protocols, one for measuring the specific growth rate and the other for the cell-binding ability of rotavirus using the plaque assay and RT-qPCR. These protocols are available for confirming the differences in phenotypes between rotavirus strains.

轮状病毒的特定生长速率及细胞结合能力的测定

Rotavirus is the main etiological factor for infantile diarrhea. It is a double-stranded (ds) RNA virus and forms a genetically diverse population, known ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

使用流细胞测定量化人类诺如病毒样颗粒与准细菌结合

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

Plaque Assay: A Method to Determine Viral Titer as Plaque Forming Units (PFU)

Source: Tilde Andersson1, Rolf Lood1 
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund, Sweden
Viruses that infect prokaryotic organisms, called bacteriophages or simply phages, were identified in the early 20th century by Twort (1) and d&#39;H&#233;relle (2) independently. Phages have since been widely recognized for their therapeutic value (3) and their influence on human (4), as well as global, ecosystems (5). Current concerns have fueled a renewed interest in the use of phages as an alternative to modern antibiotics in treatment of infectious disease (6). Essentially all phage research relies on the ability to purify and quantify viruses, also known as a viral titer. Initially described in 1952, this was the purpose of the plaque assay (7). Decades and multiple technological advancements later, the plaque assay remains one of the most reliable methods for determination of viral titer (8).
Bacteriophages subsist by injecting their genetic material into host cells, hijacking the machineries for production of new phage particles, and eventually causing the host to release numerous progeny virions through cell lysis. Because of their minute size, bacteriophages cannot be observed using solely light microscopy; therefore, scanning electron microscopy is required (Figure 1). Additionally, phages cannot be cultivated on nutritional agar plates like bacteria, as they need host cells to prey on.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10514/10514fig1.jpg" /> 
Figure 1: The morphology of a bacteriophage, here exemplified by an E. coli phage, can be studied using scanning electron microscopy. Most bacteriophages belong to Caudovirales (tailed bacteriophages). This particular phage has a very short tail structure and an icosahedral head, placing it in the family of Podoviruses.
The plaque assay (Figure 2) is based on incorporation of host cells, preferentially in log-phase growth, into the medium. This creates a dense, turbid layer of bacteria able to sustain viral growth. An isolated phage can subsequently infect, replicate within, and lyse one cell. With each lysed cell, multiple adjacent ones become immediately infected. Several cycles in, a clear zone (a plaque) can be observed in the otherwise turbid plate (Figure 2B/Figure 3A), indicating the presence of what was initially a single bacteriophage particle. The number of plaque forming units per volume (i.e. PFU/mL) of a sample, can thus be determined from the number of plaques generated.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10514/10514fig2.jpg" /> 
Figure 2: Testing for plaque forming units (PFU) is a common method for determining the number of bacteriophages in a sample. (A) The base of a sterile Petri dish is covered with an appropriate solid nutrient medium, followed by a mixture of soft media, susceptible host cells and a dilution of the original bacteriophage sample. Note that the phage-suspension could, in some cases, also be evenly spread-out across the surface of already solidified soft agar. (B) Growth of the host bacteria forms a lawn of cells in the top agar layer. Bacteriophage replication generates clear zones, or plaques, caused by host cell lysis.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/10514/10514fig3.jpg" /> 
Figure 3: Results from PFU-testing show multiple plaques generated by bacteriophages. Due to lysis of susceptible host cells, plaques can be seen as clearing zones in the bacterial lawn, either with (A) full clearance, or (B) partial re-growth caused by generation of resistant bacteria (or possibly by temperate phages in the lysogenic cycle).
Certain temperate phages can adopt what is referred to as a lysogenic lifecycle, in addition to the formerly described lytic growth. In lysogeny, the virus assumes a latent state through incorporation of its genetic material into the genome of the host cell (9), often conferring resistance to further phage infections. This is sometimes revealed through a slight clouding of the plaque (Figure 3B). It is worth noting however, that plaques can also appear blurred due to re-growth of bacteria that have evolved resistance to the phage independent of previous phage infections.
Viruses can attach, or adsorb, to only a limited range of host bacteria (10). Host ranges are further limited by intracellular anti-viral strategies such as the CRISPR-Cas system (11). The resistance/sensitivity towards specific phages displayed by bacterial subgroups has historically been used to categorize bacterial strains into different phage types (Figure 4). Although the effectiveness of this method has now been surpassed by novel sequencing techniques, phage typing can still provide valuable information about bacteria-phage interactions, for instance, facilitating the design of a phage cocktail for clinical usage.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/10514/10514fig4.jpg" /> 
Figure 4: Phage sensitivity of different bacterial strains. Soft agar plates with Cutibacterium acnes strain (A) AD27 and (B) AD35, were spotted with 21 different C. acnes bacteriophages. Only phage 11 were able to infect and kill AD27 while strain AD35 showed sensitivity towards all phages. This technique, termed phage typing, can be used to divide bacterial species and strains into different subgroups based on phage susceptibility.

Determining Viral Titer as Plaque Forming Units PFU

斑块测定:确定病毒性皮石为斑块形成单位（PFU）的方法

Source: Tilde Andersson1, Rolf Lood1
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 ...

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An Assay to Quantify the Binding of Human Norovirus VLPs to Intestinal Bacteria

Source: Madrigal, J. L., et al. <a href="https://app.jove.com/v/61048">Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry.</a> J. Vis. Exp. (2020).
This video demonstrates an assay for quantifying human norovirus virus-like particles (VLPs) binding to human intestinal bacteria. The bacterial suspensions are mixed with the VLPs and incubated to allow the formation of VLP-bacteria complexes. The complexes are then labeled with fluorophore-tagged VLP-specific antibodies, followed by flow cytometry-based visualization and quantification to identify the percentage of VLP-bound bacteria.

定量人诺如病毒 VLP 与肠道细菌结合的测定法

1. VLP-bacteria Attachment Assay
CAUTION: Human norovirus VLPs are a biosafety level (BSL)-2 hazard, and all work involving VLPs should be performed in a ...