For molecular biologists, cell cultures are tools that are just as important as a hive tool is to a beekeeper. A beekeeper can still manage their hives without a hive tool using some other device in its place, but it’s awkward and inefficient compared to the tool that’s made for the job. The value of cell culture is that it allows us to control abiotic (e.g. temperature, humidity) and biotic (e.g. genetic differences, infections) variables that are otherwise difficult or impossible to control in the hive itself. This improved experimental control gives us a more detailed view of how bees respond to pressures like infections or toxin exposure. An article recently published in Scientific Reports  describes how a rare honey bee cell culture was used to delve into the ecology of honey bee viruses during infection.

It’s common knowledge now that viruses are a significant threat to honey bee health. One of the most widely publicized viruses is the Israeli acute paralysis virus (IAPV) which was one of the first shown to be significantly correlated with colony losses in 2006. Since then, the relationship between the varroa mite and virus transmission has also been uncovered, and characteristics of other common viruses (SBV, KBV, DWV, BQCV) have been investigated individually. But the viruses rarely occur on their own – realistically, co-infection of multiple viruses at the same time is the norm. Rather than carrying out their lives in a vacuum, the viruses actually are in constant competition for hosts and resources. This raises the question: in a simultaneous co-infection, does one virus out-compete the rest?

Development of the varroa mite, starting with egg and ending at the deutonymph crysalis stage. The mite infests young brood cells, feeding on the bee’s hemolymph (blood) and transmitting viruses in the same way mosquitos transmit malaria to humans.

The new Scientific Reports paper first investigates what happens when a cell culture is infected with a mix of DWV, SBV, BQCV and IAPV all at the same time. An ideal experiment would start with an inoculum made of equal proportions of the viruses, but the authors were limited to a mixture dominantly composed of SBV (98%) with the remaining 2% split between the other three. To first show that this inoculum was infectious, it was mixed with sugar water and fed to live bees to observe their mortality. Indeed, the inoculated bees experienced > 30% higher mortality than bees that received either boiled inoculum (killing any viable viruses) or plain sugar water. Remarkably, they found that the proportion of SBV viruses rapidly decreased (to 2-3%) while IAPV instead became the overwhelmingly dominant virus (at 95%). As corroborating evidence, they observed very similar results when the cell culture was infected, rather than live bees. This tells us that IAPV is a formidable competitor against the other three viruses, but whether this is because it uses resources more effectively, has a faster life cycle, or some other reason is not clear.

To further delve into infection dynamics, the authors performed the same experiment but this time with an inoculum where KBV was the dominant component (78%) followed by SBV (14%), IAPV (7.6%), BQCV (0.3%) and DWV (0.1%). This time, instead of IAPV rising to dominate, KBV instead remained as the dominant infection. Furthermore, the level of IAPV infection achieved from this mixture was in the order of 1000x less than the first inoculum mixture, despite beginning with about 8x more virus.

A major difference the researchers discovered between the bee infections and cell culture infections was that although the proportion of IAPV viruses increased over time, the virus that consistently dominated in the cell culture was actually DWV. They discovered that this was not because DWV behaved differently in culture than in live bees, but because the cells actually had an underlying, persistent DWV infection to begin with. In other species, sometimes infection with one virus will prevent further infection with different viruses. This would make sense, because from the virus’s point of view, it’s advantageous to not let any other competitors in which will require the same resources that it does. We don’t know why DWV doesn’t cause this kind of competition inhibition, but we speculate that there might be some advantage to DWV of co-infection that we are yet unaware of.

A honey bee worker with Deformed Wing Virus. Image from Prof. Stephen Martin, University of Salford.

Collectively, these results show that virus interactions form a complex micro-ecosystem within the host that has been previously under-appreciated. The experiments hint that the impact of one virus also depends on what other viruses are present and there may be beneficial virus interactions in addition to conventional competition. Although results can be difficult to interpret, these multivariate experiments are realistic scenarios and may be more informative than traditional single-pathogen studies.

An adaptation of this article also appears in the open access BeesCene Spring 2016 magazine.