Name: preparation of virus-enriched inoculum for oral infection of honey bees (apis mellifera)
Uploaded: 2020-08-26T15:00:00
Description: Watch this Scientific Journal Video about Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees Apis mellifera at JoVE.com

We would like to thank Dr. Julia Fine for her ideas and discussion during the protocol creation process, as well as Dr. Cassondra Vernier for her helpful comments throughout editing. These materials contributed towards projects that were supported in part by the Foundation for Food and Agriculture Research, under grant ID 549025.

1. Mass bee extraction option 1: larval self-removal
<ol>
	<li>Cage a honey bee queen on an empty, drawn-out Langstroth frame and return her to her colony. Allow the queen to lay eggs on this frame for 24 h.
	<ol>
		<li>Check the frame after 24 h to ensure most of the comb cells contain newly laid eggs. Depending on the queen and colony, eggs are sometimes not laid very well in the first 24 h. If this occurs, allow for an additional 24 h and adjust the time as necessary.</li>
	</ol>
	</li>
	<li>After the 24 h egg-layin...

Here we have outlined methods detailing every step of the virus amplification and inoculum stock preparation process, including larvae collection and virus propagation, extraction, and concentration, as well as viral treatment in the form of cage-feeding experiments. These methods allow for production of semi-pure virus particles (Figure 4), the effectiveness of which can be consistently be quantified by dose-response mortality testing for viruses that are lethal to adults (<strong class="xf...

Honey bees (Apis mellifera) play a critical role in the modern global agricultural landscape but are currently suffering from a combination of biotic and abiotic stressors, including pesticide exposure, poor forage, parasites, and pathogens1,2. One of the most important pathogens of concern are viruses, many of which are vectored by another of the major honey bee stressors, the parasitic Varroa mite (Varroa destructor). These viruses can cause an array of negative effects in honey bees including reduced brood survival, developmental defects, and paralysis that can lead to total hive collapse both before and after overwintering periods3,4,5. Although there have been promising advances in the development of technologies used to combat virus infection6,7,8,9, the dynamics by which many viruses propagate, spread, and interact within a honey bee or colony are still poorly understood5,10. Understanding the basic biology of honey bee and virus interactions and their relationships with other environmental factors is critical for developing effective virus management techniques.
However, studying honey bee-virus interactions poses challenges with numerous known and unknown factors complicating the process. These include interactions with diet11,12, pesticide exposure13, and bee genetic background14,15. Even when focusing on virus infection alone, complications are common because honey bee populations, both managed and wild, always have some degree of background virus infection, though often without manifesting acute symptoms16,17, and the effects of virus coinfection are not well understood18. This has made the study of honey bee virus effects difficult to disentangle.
Many honey bee virus studies have used circumstantial virus infections to look for interactions with other stressors, observing how background infections change with other treatments12,19,20,21. While this approach has been successful at identifying important effects, especially discovering how pesticide or dietary treatments affect virus levels and replication, inoculation with a virus treatment of known content and concentration is critical for experimental testing of virus infection dynamics. Even so, separating experimental treatment from background infection can also pose challenges. In field studies, researchers have differentiated strains of deformed wing virus (DWV) to provide evidence for virus transmission from honey bees to bumble bees22, but using this approach would be difficult within honey bees alone. Virus infectious clones are a powerful tool, not just for tracking infection23,24,25 but for reverse genetics studies of honey bee viruses and for virus-host interaction research26,27,28. However, in most instances, infectious clones are still required to fulfill the infection cycle inside cells to produce particles. Such particles are preferred as inocula for experimental treatments because their infectivity is higher than the naked viral RNA and inoculation with encapsidated genomes mimics a natural infection.
The production of pure, uncontaminated honey bee virus inocula (wild-type virus strains or those derived from infectious clones) also pose challenges. These are primarily due to the difficulties in obtaining a reliable, continuously-replicating, virus-free honey bee cell line to produce pure-strain viruses29,30. While some cell lines have been produced, these systems remain imperfect; still, there is hope a viable cell line can be produced29, which would allow for finer control of virus production and investigation. Until such a line becomes widely available, most virus production protocols will continue to rely on the use of in vivo virus production and purification18,31,32,33,34. These approaches involve identifying and purifying virus particles of interest (or producing an infectious clone) and using them to infect honey bees, usually as pupae. The pupae are injected with the target virus and then sacrificed, and further particles are extracted and purified. However, because no bees are virus-free to begin with, there is always some degree of contamination from traces of other viruses in any such concentrate, and, therefore, great care must be taken in choosing bees with a low likelihood of background infections. Further, methods for removing the pupae from the comb cells for use in these protocols33 are very labor intensive and can induce stress in the bees, limiting production by these means18,32. Here, we report an alternative method that allows for large scale removal of larvae with little labor and less mechanical stress on the bees.
Once pupae are obtained and injected with the starting virus inoculum, they must be incubated to provide the virus time to replicate. Subsequently, produced virus particles can be processed into a form usable to infect experimental bees. There are several simple methods to achieve this, including using a crude homogenate35,36 or hemolymph generated from virally infected bees as a source of infection37. These methods are effective but run into a greater chance of introducing unknown variables from the background substrate (e.g., other factors in the dead bee homogenates). Additionally, it is desirable to concentrate the particles if an experiment requires giving a large, known dose of a virus in a short period of time. Therefore, for better control, it is preferable to use methods that allow for some level of purification and concentration of the virus particles. Generally, a series of precipitation and centrifugation steps will result in the removal of almost all possible non-target virus material33.
After producing this concentrated inoculum, it is beneficial to quantify the viral titers (qPCR) and characterize it with in vivo bioassays to test its viability and ability to cause mortality, as well as to corroborate that increased virus titers are obtained after infection. This can be achieved through injection experiments (either into pupae or adults) or feeding experiments (into larvae or adults). While all these approaches are possible, feeding to groups of adult bees in a cage is often the fastest and simplest. The cage assay method is also widely used for testing various other treatments on bees including pesticide toxicity38, ovary development39, and nutritional influence on behavior40,41 and, therefore, can form a good basis for experiments linking virus infection with other factors42.
Here we describe a reliable method for producing large quantities of semi-pure, highly-enriched virus particles without using an expensive ultracentrifuge, including a method for removing pupae that reduces labor and mechanical stress on the bees and a highly repeatable, high-throughput bioassay for testing viral infection and effects. By tightly controlling the purity of the viral inocula, investigators are able to reduce variation in honey bee viral response relative to other viral inoculation methods. Furthermore, the bioassay can screen for viral effects at a small groups level using highly repeatable experimental units before scaling to field-realistic settings, which is far more labor intensive to manage. In combination, these two methods provide the necessary tools for studies that can help improve our overall understanding of honey bee-virus physiological interactions.

<table>
	<tbody>
		<tr>
			<td>10% bleach solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24:1 chloroform:isoamyl alcohol</td>
			<td>SigmaAldrich</td>
			<td>C0549</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cages for bioassay</td>
			<td></td>
			<td></td>
			<td>Dependent on experimental setup</td>
		</tr>
		<tr>
			<td>Combitips Advanced 0.1 mL</td>
			<td>Eppendorf</td>
			<td>30089405</td>
			<td>Optional (if no injector appartus is available)</td>
		</tr>
		<tr>
			<td>Containers for larval self-removal</td>
			<td></td>
			<td></td>
			<td>Should measure roughly 19&#34; x 9-1/8&#34; (Langstroth deep frame dimensions)</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td>Blunt, soft forceps for larval separationl; blunt, hard forceps for pupal excision</td>
		</tr>
		<tr>
			<td>Fume hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td></td>
			<td></td>
			<td>Capable of maintaining 34 &#186;C and 50% relative humidity</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Fisher Scientific</td>
			<td>06-666</td>
			<td>Any absorbent wipe will work</td>
		</tr>
		<tr>
			<td>Medium-sized weight boats</td>
			<td></td>
			<td></td>
			<td>Serve as inoculum trays</td>
		</tr>
		<tr>
			<td>Microcon-100kDa with Biomax membrane</td>
			<td>MilliporeSigma</td>
			<td>MPE100025</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrile gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>SigmaAldrich</td>
			<td>P5119</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol 8000 (PEG)</td>
			<td>SigmaAldrich</td>
			<td>1546605</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated benchtop centrifuge</td>
			<td></td>
			<td></td>
			<td>Capable of 15,000 x g</td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td></td>
			<td></td>
			<td>Capable of 21,000 x g</td>
		</tr>
		<tr>
			<td>Repeater M4 Multipipette</td>
			<td>Eppendorf</td>
			<td>4982000322</td>
			<td>Optional (if no injector appartus is available)</td>
		</tr>
		<tr>
			<td>RNAse Away</td>
			<td>ThermoFisher</td>
			<td>7000TS1</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse-free water</td>
			<td>SigmaAldrich</td>
			<td>W4502</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TES</td>
			<td>SigmaAldrich</td>
			<td>T1375</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of virus-enriched inoculum for oral infection of honey bees (apis mellifera)

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.

Successfully following the protocols (Figures 1) for pupal injection and viral extraction should produce large quantities of virus particles. However, sampling and injecting pupae sourced from a variety of colonies at multiple time points maximizes the chances of acquiring target virus with low contamination. The dynamics by which viruses replicate and interact with one another within a honey bee is not well understood; coupled with the likelihood for preexisting infection, there is no guarantee that the...

Watch this Scientific Journal Video about Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees Apis mellifera at JoVE.com

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees Apis mellifera

Here we describe two protocols: first to propagate, extract, purify, and quantify large quantities of honey bee non-enveloped virus particles, including a method for removing honey bee pupae and second to test the effects of viral infection using a highly repeatable, high-throughput cage bioassay.

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees (Apis mellifera)

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 ...

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