传播<em> Homalodisca coagulata病毒01</em>通过<em> Homalodisca vitripennis</em>细胞培养
Summary
在这里，我们提出了一个协议来传播Homalodisca vitripennis细胞和HoCV-1 体外 。培养基从HoCV-1阳性培养去除和RNA的提取，每24小时为168小时。细胞生存力进行定量通过台盼蓝染色法。整个病毒粒子中提取的后感染。提取的RNA进行定量通过qRT-PCR。
Abstract
玻翅神枪手（Homalodisca vitripennis）是整个美国西南部发现了一个非常vagile和杂食性昆虫。这些昆虫是Xylella难养（十难养）的主要载体，木质部限细菌是葡萄皮尔斯病（PD）的致病因子。皮尔斯病是造成经济损失;因此，H vitripennis已成为病原体管理战略目标。认定为Homalodisca coagulata病毒01（HoCV-01）一个dicistrovirus已在阁下的死亡率增加有关vitripennis人群。由于宿主细胞需要HoCV-01的复制，细胞培养为目标的复制是后勤和生物农药生产经济价值的统一环境。在本研究中，对于H的大规模繁殖系统通过组织培养vitripennis细胞发达，Providing病毒复制的机制。 HoCV-01是从整个身体的昆虫中提取并用于接种培养的幽门vitripennis细胞在不同的水平。将培养基除去，每24小时为168小时，萃取，用定量RT-PCR分析的RNA。细胞用台盼蓝染色并计数用光学显微镜来量化细胞生存能力。全病毒颗粒中提取多达96小时感染后，为确定为总细胞培养崩溃发生之前的时间点。细胞也进行荧光染色，并用共聚焦显微镜研究上的F-肌动蛋白的附接和细胞核完整性病毒活性观看。本研究的结论是H。 vitripennis细胞能够被培养，并用于批量生产HoCV-01在一个合适的水平，使生产的生物农药。
Introduction
玻翅神枪手（Homalodisca vitripennis Germar 1821）已被确定为Xylella难养 （ 十难养 ）的主要载体，小道消息（PD）的皮尔斯病的病原在北美1。昆虫种群的管理已经迅速成为研究的重点，以打击在加州和整个美国南部这个毁灭性的问题的葡萄种植业。属于家人正意义上说，单链RNA病毒Dicistroviridae，Homalodisca coagulata病毒01（HoCV-01）已经在野生H.确定vitripennis人口和证实会增加死亡率在这些人群2-4，同时降低昆虫的抗杀虫剂。
方法的发展，有效地后方感染H。 vitripennis在实验室环境到成年一直很困难，因为<eM>小时。 vitripennis有需要多种寄主植物5-8不同阶段的特定营养需求。特定设备是必需的，以后部活H. vitripennis在美国;因此，细胞培养物是更经济，可行的替代方案，以及用于HoCV-01检测和复制2,9日益重要。而基本的方法用于建立H的细胞培养vitripennis进行了描述，这些方法还没有被用于商业化生产的生物控制剂，如病毒2。
以下程序的总体目标是生产出高浓度HoCV-01适合于利用作为生物控制剂。病毒复制需要一个活细胞，这就是为什么成功的培育和优化H. vitripennis文化是产生病毒的盈利水平的进步至关重要。
Protocol
Representative Results
Discussion
关于农业侵入物种的大量涌入上升的担忧引发了新的方法来对出现的害虫和病原体防御的需求增加。疾病预防和管理的重点包括病原载体的管理，是本研究的主要目标。经济学中生产这种类型的生物农药来管理病原载体的农业，因为在实际应用程序需要大量的广大地区，但以较低的成本12的决定起着至关重要的作用。利用细胞培养物的研究和开发的实践中已经变得越来越普遍，因此，本研…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We would like to thank the Texas Pierce’s Disease Research and Education Program and USDA-APHIS for their funding support for this project. We would also like to thank Hema Kothari at the University of Texas Health Science Center at Tyler for her assistance with confocal microscopy.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Corning cell culture flasks | Sigma Aldrich | CLS430168 | Surface area 25 cm2, canted neck, cap (plug seal) |
| Olympus DP30BW, IX2-SP, IX71 | Olympus | Inverted microscope and camera | |
| Trypsin-EDTA solution | Sigma Aldrich | T4049 | 0.25%, sterile-filtered, BioReagent, suitable for cell culture, 2.5 g porcine trypsin and 0.2 g EDTA • 4Na per liter of Hanks′ Balanced Salt Solution with phenol red |
| Greiner CELLSTAR multiwell culture plates | Sigma Aldrich | M8937 | 48 wells (TC treated with lid) |
| DETCA | Sigma Aldrich | 228680 | Sodium diethyldithiocarbamate trihydrate |
| Corning bottle-top vacuum filter system | Sigma Aldrich | CLS431206 | Cellulose acetate membrane, pore size 0.45 μm, membrane area 54.5 cm2, filter capacity 500 mL |
| Brij 52 | Sigma Aldrich | 388831 | Polyethylene glycol hexadecyl ether |
| Phosphate buffer solution | Sigma Aldrich | P5244 | Recieved as 100mM diluted to 10mM with sterile water |
| TRIzol LS | Life Technologies | 10296-028 | |
| Agarose | Sigma Aldrich | A5304 | For electrophoresis |
| Ethidium bromide | Sigma Aldrich | E7637 | BioReagent, for molecular biology, powder |
| QIAquick | Qiagen | 28704 | |
| QuantiTect qRT-PCR kit | Qiagen | 204243 | |
| 4% paraformaldehyde | Sigma Aldrich | P6148 | Reagent grade, crystalline |
| PBS | Sigma Aldrich | P5368 | Phosphate buffered saline |
| Triton X-100 | Sigma Aldrich | X100 | |
| Bovine serum albumin (BSA) | Sigma Aldrich | A2153 | |
| Rhodamine red-conjugated phalloidin | Life Technologies | R415 | Rhodamine phalloidin is a high-affinity F-actin probe conjugated to the red-orange fluorescent dye, tetramethylrhodamine |
| DAPI | Sigma Aldrich | D9542 | |
| ProLong Gold Antifade Reagent | Life Technologies | P36934 | |
| LSM510 Meta Confocal System | Carl Zeiss | ||
| LSM Zen 2007 Software | Carl Zeiss | ||
| Grace’s Insect medium (supplemented, 1X) | Sigma Aldrich | G8142 | H2G+ leafhopper medium component |
| L-histidine monohydrate | Sigma Aldrich | H8125 | H2G+ leafhopper medium component |
| Medium 199 (10X) | Sigma Aldrich | M4530 | H2G+ leafhopper medium component |
| Medium 1066 (1X) | Sigma Aldrich | C0422 | H2G+ leafhopper medium component |
| Hank’s Balanced Salts (1X) | Sigma Aldrich | 51322C | H2G+ leafhopper medium component |
| L-Glutamine (100X) | Sigma Aldrich | G3126 | H2G+ leafhopper medium component |
| MEM, amino acid mix (50X) | Sigma Aldrich | 56419C | H2G+ leafhopper medium component |
| 1 M MgCl solution | Sigma Aldrich | M8266 | H2G+ leafhopper medium component |
| Pen-Strep (w/ Glutamine) | Sigma Aldrich | G6784 | H2G+ leafhopper medium component |
| Nystatin | Sigma Aldrich | N6261 | H2G+ leafhopper medium component |
| Gentamycin | Sigma Aldrich | 46305 | H2G+ leafhopper medium component |
| Dextrose | Sigma Aldrich | D9434 | H2G+ leafhopper medium component |
| Fetal Bovine Serum | Sigma Aldrich | F2442 | H2G+ leafhopper medium component |
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