协议侦办主机组织分布，传播模式和效果上浓核病毒的主机健身的棉铃虫
Summary
在这里，我们提出了一个协议，调查宿主组织分布，传输模式和效果上的浓核病毒的宿主健身鳞翅目物种内，棉铃虫。该协议也可以用于研究其他经口传播的病毒和它们的昆虫宿主之间的相互作用。
Abstract
许多新的病毒已经在用新一代测序技术的动物宿主被发现。此前，我们报道了互惠病毒， 棉铃虫浓核病毒（HaDV2），在鳞翅目的物种，棉铃虫， 棉铃虫 （棉铃虫）。在这里，我们描述了目前用于研究HaDV2其主机上的效果的协议。首先，我们从单一的养殖对建立自由HaDV2，棉铃虫落。然后，我们口服接种用含有HaDV2-过滤的液体以产生两个菌落用该遗传背景一些新生儿幼虫后代：一个HaDV2感染，其他未感染的。一个协议到HaDV2感染和-uninfected个体还提出之间比较生命表参数（ 例如，幼虫，蛹和成虫期和繁殖力），作为用于确定HaDV2的宿主组织分布和传输效率的协议。这些协议をULD也适用于研究在其昆虫宿主，特别是鳞翅目的主机其他口头传播病毒的影响。
Introduction
在过去的几十年中，测序技术的发展，如下一代测序（NGS）提供了便利的许多新的DNA和RNA病毒，特别是非致病性病毒，而且先前已知的病毒1，2，3新颖菌株的发现， 4，5，6，7，8，9，10，11，12。在模式生物果蝇 ，已使用的宏基因组技术13检测超过20个新的部分病毒基因组。许多病毒序列，包括新颖的病毒，也已在其它昆虫，如蜜蜂，米识别osquitoes，亚洲柑橘木虱，蜻蜓，和多个鳞翅目种类14，15，16，17，18，19，20，21。
在未来，可以预计将有更多的新病毒会使用这些先进技术的昆虫被发现;因此，我们对病毒与宿主相互作用的理解可能会相应地改变6,9。例如，病毒与宿主相互作用被认为是比以前认为的更加复杂，因为许多新的病毒被定义为互惠的合作伙伴，而不是严格的病原体22。例如，在Dysaphis plantaginea的共生浓核病毒DplDNV诱导翅变形和增加流动性，有利于主机的扩散以及病毒23。此外，互惠病毒已与关于哺乳动物健康，干旱和植物的耐寒性，和细菌感染24的碰撞描述。塞内卡山谷病毒-001被证明介导选择性细胞毒性对肿瘤细胞具有神经内分泌癌特征25。 A型肝炎病毒感染抑制丙型肝炎病毒的复制，并可能导致丙型肝炎26恢复。疱疹病毒潜伏期赋予免受细菌感染27共生保护。人内源性逆转录病毒HERV-W的包膜糖蛋白诱导对脾坏死病毒28的细胞抗性。从内生真菌弯孢热耐受性病毒（CThTV）参与这种真菌和热带恐慌草之间的互惠互动REF“> 29。因此，新发现的病毒和其宿主之间的相互作用的知识应该产生自己的生物学和管理的新观点。然而，新的病毒，尤其是隐蔽病毒，显示典型的急性感染无明显体征，有很少被查处，我们需要一个管道和协议来调查他们的主人的新发现的病毒的影响。
此前，我们已经报道的棉铃虫， 棉铃虫的流行新monosense浓核病毒棉铃虫浓核病毒（HaDV2），并提出了HaDV2和棉铃虫30,31之间的互惠关系的证据。在本文中，我们将介绍实验室实验方案进行详细研究HaDV2和棉铃虫宿主之间的相互作用。这里介绍的协议也可能是研究人员对第r高度相关OLE的其他口服传播的病毒，特别是在鳞翅目害虫。
Protocol
1. HaDV2无棉铃虫殖民地的建设后棉铃虫幼虫（ 棉铃虫 ）上以受控的生长室或人工气候室中的人工饲料32在25±1℃，14小时光照/ 10小时黑暗和60％相对湿度。 通过使用覆盖有棉纱布每对塑料笼子（10厘米的高度，直径5cm），保证良好的通风，并作为一个产卵基板帮助新eclosed蛾的单对配合。为了提高获得无病毒菌落的可能性，利用至少30对单对交配。用10％的糖?…
Representative Results
从分别HaDV2 -自由（ 图3A）的父母后代饲养作为NONINF应变。我们使用相同的DNA模板，这表明DNA模板质量良好（ 图3B）成功地扩增肌动蛋白基因。此外，随机选择的8个后代也是免费HaDV2（ 图3C），HaNPV（ 图3D），并沃尔巴克氏体 （ 图3E）的。再一次，我们的结论是，后代的DNA样品的质量好，因为?…
Discussion
在过去的几十年中，以昆虫病毒相互作用的大多数研究都集中在蜜蜂健康34，35，36，人类疾病37的载体，植物病毒38，和一些昆虫致病病毒有很大的潜力作为生物控制剂39。很少有人注意支付给隐蔽病毒的昆虫，特别是鳞翅目害虫。在这里，我们提出了一个协议的隐蔽病毒与…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由中国国家重点基础研究发展计划（编号2013CB127602）和中国国家自然科学基金（31321004号）的创新研究群体科学基金的支持。
Materials
| 24-well plate | Corning | 07-200-740 | Multiple suppliers available. |
| DNA extraction kit | TIANGEN | DP304-03 | Multiple suppliers available. |
| thermal cycler | Veriti; Applied Biosystems | 4375786 | |
| PBS | Corning | 21-040-CV | |
| 0.22 µm membrane filter | Millipore | SLGS025NB | |
| pEASY-T Cloning Vector | TransGen, Beijing, China | CT301-02 | |
| Tweezers | IDEAL-TEK | 2.SA | |
| Premix Ex Taq (Probe qPCR) | Takara | RR390A | |
| Probes | Invitrogen | Custom order | |
| Primers | Invitrogen | Custom order | |
| microspectrophotometry NanoDrop 2000c | Thermo scientific | not available | |
| 7500 Real-Time PCR system | Applied Biosystems | not available | |
| stereomicroscope SZX-16 | Olympus | not available | |
| sucrose | Multiple suppliers available. | ||
| vitamin complex | Multiple suppliers available. |
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Cite This Article
Yang, X., Xu, P., Graham, R. I., Yuan, H., Wu, K. Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm. J. Vis. Exp. (122), e55534, doi:10.3791/55534 (2017).