Retrospective on working with Chronic Bee Paralysis Virus

Despite having perhaps been first described 2,000 years ago by Aristotle and seen on every continent, Chronic Bee Paralysis Virus (CBPV) is still a poorly understood, and therefore very interesting, emerging RNA virus. Chronic Bee Paralysis itself is a infectious disease that, perversely enough, leads to the collapse of the strongest and most populated honey bee hives during the summer seasons in which they (the bees) are most active. Infected individual display both behavioral and physiological symptoms such as darkened and shiny abdomens, trembling, and loss of flight and a severely infected colony may have mounds of dead or dying rejected individuals pushed outside the hive entrance.

With the rising recognition of bees as keystone pollinators and the threat of substantial losses to commercial apiculture posed by virus, improving understanding of CPBV has many practical implications. CPBV is also of substantial academic interest to virologists since it appears to be an evolutionary intermediate between two other virus families (Nodaviridae and Tobusviridae) and may be the first known virus in new group of RNA viruses.

In the course of the Laidlaw Internship scheme, while working in the Evans Apiary and Laboratory, I tried to interrogate pertinent questions concerning the transmission of CBPV. Originally I hoped to run a few preliminary experiment that would reliably produce infected individuals, inform me about their latency period, and help elucidate the natural route of transmission of CBPV. From this, I hoped to run a population-density experiment in which I would put a portion of infected bees in a large volume of space with a population of uninfected bees and to repeat this with a smaller volume. From this I could compare how congestion of hives effects the rate of mortality, appearance of symptoms and spread of CBPV. CBPV presence could be indicated partially by symptoms but would be confirmed by extracting viral RNA from the samples and utilize reverse transcription and PCR to confirm the presence of CBPV within the bee.

However, rarely does experimental research go just as one hopes. The initial perfunctory preliminary experiments yielded confounding and unexpected results. In my first preliminary experiment, investigating which routes of inoculation (Injection, Abrasion, or Feeding) could reliably produce CBPV-infected newly-emerged bees, symptoms never appeared and molecular analysis indicated that shockingly few bees becoming infected, with only half of injected bees dying at all. Initially, I speculated that age played a role: all of my bees were inoculated 1 day after emerging from their comb whereas the previous coworkers of mine had successfully seen infection when inoculating bees of varying ages. Perhaps younger adult honeybees were actually less susceptible to CBPV than mature bees?

A follow up experiment, in which I had hoped to produce  CBPV-replication curves over time in newly emerged and mature adult bees, however, disproved this. Very few bees in either group became infected at all and the results indicated that younger adult bees actually had much higher concentrations of CBPV than mature bees.

From these unusual results I was forced to conclude that the viral stock I had been given was partially inactivated and that previous experiments were erratic because I could not accurately quantify how much virus I was inoculating my bees with. By the time I had run these experiments and verified all of my molecular analysis, there were only a few weeks left in my internship and I had to forego the hope of answering all of the questions I had set out with. However, I did have time to optimize a new viral stock to be used for future experimentation in CBPV at the Evans bench.

While the course of my research didn’t go as intended, it was far from fruitless. When I had initially stated my goals as a leader, I had wanted to gain confidence in self-direction especially when it came to troubleshooting. Having spent the majority of this internship trying to divine what was amiss with my results, I feel I have aptly developed in this sense. Furthermore, while our undergraduate programs provide us with a wealth of laboratory experience, nothing can quite replace the actual experience of working 9-5 on a lab bench and the skills mastery that comes with it. Often we find ourselves following laboratory protocols but there is a separate aspect of craftsmanship which comes with optimizing those protocols. In the courses of this internship I became substantially more efficient with my sample handling; a injection protocol that once took me half a day is now a matter of a few hours, my RNA extractions have improved 60-fold in their concentration, and I’ve learned, sometimes the hard way, how to save as many reagents as possible without sacrificing quality of results. It’s something akin to the difference between reading whole cook books and spending 2 months constantly in the kitchen. My deepest thanks to  Professor David Evans, Katherine, Luke and Alex for all of their guidance and instruction and, especially, patience.

 

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