使用流细胞测定量化人类诺如病毒样颗粒与准细菌结合
Summary
该协议的目标是量化真核病原体人类诺如病毒与细菌的结合。在进行初始病毒-细菌附件测定后，流细胞测定用于检测种群内的病毒结合细菌。
Abstract
共生细菌已广为人知，可影响真核病毒的感染。病原体和宿主微生物群之间的直接结合是改变许多这些病毒的感染的原因。因此，描述病毒-细菌结合的性质是阐明细菌改变病毒感染机制所需的基本步骤。对于人类诺如病毒，共性细菌可增强B细胞感染。病毒直接与这些细菌结合，表明这种直接相互作用涉及感染增强机制。多种技术可用于量化细菌和病毒之间的相互作用，包括放射性标记病毒的闪烁计数和聚合酶链反应 （PCR）。这两种方法都需要使用活病毒，可能需要在实验室中生成。目前，人类诺如病毒的成熟体外培养系统都不足以产生高度浓缩的病毒种群。代替活病毒，病毒样颗粒（VLP）被用来描述诺如病毒和细菌之间的相互作用。本文介绍一种流细胞测定方法，使用病毒特异性抗体来量化VLP与克阴性和克阳性细菌结合。只包含细菌和等感控制，可以优化测定，以减少背景抗体结合，并准确量化VLP附件对所测试的细菌。高VLP：细菌比导致VLP与细菌群的较大百分比结合。但是，当 VLP 数量减少时，细菌结合的百分比也会降低。最终，这种方法可用于未来的实验，阐明规范诺如病毒的特定条件和结构成分：细菌相互作用。
Introduction
人诺病毒（HuNOV）是全世界胃肠道疾病的主要原因，每年造成6.85亿例感染和超过20万例死亡。与其他肠道病毒一样，共性细菌的存在已证明能增强该病原体及其代理病毒——鼠诺如病毒22、33的感染。也有相互矛盾的报告，细菌可以抑制感染人类诺如病毒44，5，6。5,6对于几种病毒，病毒和细菌之间的直接相互作用似乎是影响病毒感染的机制的基础22，7，8，9，10，7,8,9,10并且通过电子显微镜证明，人类诺如病毒直接结合到细菌的表面11，12。,12因此，描述这些相互作用对于确定细菌影响病毒感染的机制至关重要。这种特性从量化病毒结合开始，这些细菌物种是宿主微生物群77、12、1312,13的组成部分。这些附件测定不仅揭示了与细菌结合的病毒数量，还有助于确定这种相互作用对病毒健康与生存的影响。
为了量化病毒附着，传统上使用的方法包括基于PCR的检测，量化病毒基因组12或产生放射性标记病毒，并使用闪烁计数来量化病毒颗粒77，8，9，13。8,9,13使用这些方法通常需要获得高数量病毒储存和体外培养技术，从而产生它们。虽然人类诺如病毒的几个培养系统现在存在2,2，14，15，,15没有一个支持所需的强大的复制，以产生这些高度集中的种群，限制或消除使用PCR和闪烁计数来量化人类诺如病毒/细菌相互作用。
为了规避这个问题，病毒状粒子（VlPs）可以作为活病毒的代用品来研究人类诺如病毒和细菌之间的相互作用16，17。16,VLP 是非传染性颗粒，与它们衍生的病毒非常相似。在人类诺如病毒的情况下，这些颗粒是由VP1（以及VP2）蛋白质的表达产生的，这种蛋白质自我组装，创造出缺乏遗传物质（即诺如病毒的RNA）的完整病毒帽。这些VlPs具有很好的特征，在结构和抗原上与野生病毒相似，它们来源于18、19、20、21、22、23。18,19,20,21,22,23因此，VLP是研究人类诺如病毒和共性细菌表面相互作用的理想代用品。鉴于VLP缺乏遗传物质，基于PCR的测定不能用于量化病毒结合。先前曾描述过一种基于抗体的流细胞测定方法，能够以半定量的方式检测与细菌结合的低水平VLP。该方法经过优化，使人类诺如病毒VLP与克阴性和克阳性共性细菌16结合进行精确定量。
Protocol
Representative Results
Discussion
量化肠道病毒与细菌结合的能力是阐明这些细菌改变病毒感染机制的关键第一步。本文所述的方法经过优化，以测量人类诺如病毒VLP与E.氯环酶（克阴性细菌）和L.gasseri（一克阳性细菌）的相互作用，但可以适应任何哺乳动物病毒和感兴趣的细菌。虽然VLP是活病毒的理想替代品，用于附件测定，这些粒子可以很容易地量化使用流细胞测定，P粒子也被用来检查?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们要感谢苏托努卡·巴哈尔和香奈儿·莫斯比-豪德鲁普对书面手稿的批判性审查，以及阿方索·卡里略在生成细菌标准曲线方面的帮助。这项工作部分由国家卫生研究所（R21AI140012）赠款和佛罗里达大学粮食和农业科学研究所的种子赠款资助。
Materials
| 5ml Polystrene Round-Bottom Tubes with Cell-Strainer Cap | Corning | 352235 | After antibody staining, sample are transferred into tubes for flow cytometry analysis. |
| Agar | Sigma | A7002 | Used for media preparation |
| AnaeroPack | Thermo Scientific | R681001 | Anaerobic gas pack used for culture of Lactobacillus gasseri |
| BD FacsDiva software | |||
| BD LSR Fortessa flow cytometer | |||
| Bovine Serum Albumin | Fisher Bioreagents | BP1605 | Used for flow cytometry |
| Flow Cytometry Stain Buffer (FCSB) | BD Biosciences | 554657 | Used for flow cytometry |
| Mouse IgG2b kappa Isotype Control (eBMG2b), PE, eBioscience | Thermo Fisher Scientific | 12473281 | Isotype control. This antibody is purchased in the conjugated form from the manufacturer. |
| MRS Powder | BD Biosciences | 288130 | Used for media preparation and to culture Lactobacillus gasseri. |
| Norovirus capsid G2 Monoclonal Antibody (L34D) | Thermo Fisher Scientific | MA5-18241 | Norovirus GII antibody. This antibody is only available in the unconjugated form and thus must be fluorescently conjugated prior to use in the outlined flow cytometry assays. In this protocol, PE was the chosen fluor, however, other fluorescent molecules can be chosen as best suits the flow cytometer being used by the researcher. |
| Norovirus GII.4 VLP | Creative Biostructure | CBS-V700 | human norovirus virus like particle, VLPs were generated using the baculovirus system and resuspended in phosphate buffered saline with 10% glycerol. The authors performed independent nanosight tracking analysis to determine the particle concentration of the VLPs. The concentration is approximately 1011 VLPs per milliliter. Based on the protein concentration of the VLPs, approximately 200 particles are added per bacterium in VLP attachment assays. |
| PBS 10X | Fisher Bioreagents | BP665 | Dilute to 1X prior to use. |
| SiteClick R-PE Antibody Labeling Kit | Thermo Fisher Scientific | S10467 | Conjugation kit used for labling of unconjugated antibody. |
| Sodium Chloride | Fisher Scientific | S271 | Used for media preparation |
| Tryptone | Oxoid | LP0042 | Used for media preparation |
| Tube Revolver | ThermoFisher Scientific | 88881001 | Used in virus:bacterium attachment assay. Set to max speed (40 rpm). |
| Yeast Extract | BD Biosciences | 212750 | Used for media preparation |
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