Hsieh Edward M, Carrillo-Tripp Jimena, Dolezal Adam G
Department of Entomology, University of Illinois;
Department of Microbiology, Center for Scientific Research and Higher Education of Ensenada (CICESE).
J Vis Exp. 2020 Aug 26(162). doi: 10.3791/61725.
Honey bees are of great ecological and agricultural importance around the world but are also subject to a variety of pressures that negatively affect bee health, including exposure to viral pathogens. Such viruses can cause a wide variety of devastating effects and can often be challenging to study due to multiple factors that make it difficult to separate the effects of experimental treatments from preexisting background infection. Here we present a method to mass produce large quantities of virus particles along with a high throughput bioassay to test viral infection and effects. Necessitated by the current lack of a continuous, virus-free honey bee cell line, viral particles are amplified in vivo using honey bee pupae, which are extracted from the hive in large volumes using minimally stressful methodology. These virus particles can then be used in honey bee cage bioassays to test inocula viability, as well as various other virus infection dynamics, including interactions with nutrition, pesticides, and other pathogens. A major advantage of using such particles is that it greatly reduces the chances of introducing unknown variables in subsequent experimentation when compared to current alternatives, such as infection via infected bee hemolymph or homogenate, though care should still be taken when sourcing the bees, to minimize background virus contamination. The cage assays are not a substitute for large-scale, field-realistic experiments testing virus infection effects at a colony level, but instead function as a method to establish baseline viral responses that, in combination with the semi-pure virus particles, can serve as important tools to examine various dimensions of honey bee-virus physiological interactions.
蜜蜂在全球具有重大的生态和农业意义，但也面临着各种对蜜蜂健康产生负面影响的压力，包括接触病毒病原体。此类病毒可引发各种各样的毁灭性影响，而且由于多种因素使得难以将实验处理的效果与预先存在的背景感染的影响区分开来，因此对其进行研究往往具有挑战性。在此，我们提出一种大量生产病毒颗粒的方法以及一种高通量生物测定法，用于测试病毒感染及影响。鉴于目前缺乏连续的、无病毒的蜜蜂细胞系，病毒颗粒在体内利用蜜蜂蛹进行扩增，这些蛹采用压力最小的方法从蜂巢中大量提取。然后，这些病毒颗粒可用于蜜蜂笼养生物测定，以测试接种物的活力以及各种其他病毒感染动态，包括与营养、农药和其他病原体的相互作用。使用此类颗粒的一个主要优点是，与当前的替代方法（如通过受感染蜜蜂的血淋巴或匀浆进行感染）相比，它大大降低了在后续实验中引入未知变量的可能性，不过在获取蜜蜂时仍应谨慎，以尽量减少背景病毒污染。笼养试验并非用于替代在蜂群层面测试病毒感染影响的大规模、符合田间实际情况的实验，而是作为一种建立基线病毒反应的方法，与半纯病毒颗粒相结合，可作为研究蜜蜂 - 病毒生理相互作用各个方面的重要工具。
