Very few is known about the role of the gut virus population (the virome) in health and disease of honey bees. Our lab developed the NetoVIR protocol to purify viral particle from biological samples and to deep sequenced them using Illumina NGS technology. Using this approach, our lab recently published a paper describing a diverse set of bacteriophages associated with honey bees, and several more papers on eukaryotic honey bee viruses are in preparation. However, this type of research is hampered by the inherent introduction of biases in the viral composition after viral purification and random amplification before Illumina sequencing. Furthermore, the interpretation of the roles of identified novel bacteriophages in health and diseases, is hampered by many factors including the lack of good sequence databases and knowledge about their bacterial host.
In the current project, we will
- test and validate different approaches to obtain a more quantitative virome protocol;
- Link novel bacteriophages to their host using bacterial 16S data, as well as novel tools developed in the framework of VIROINF;
- use these optimized wetlab and in silico protocols to study the role of bacteriophages in honey bee health, using in vitro and in vivo approaches.
For the first aim we will use: (a) approaches to spike in known amounts of viruses into the samples as internal controls, (b) methods to accurately count virus particles by flow cytometry (in collaboration with NIOZ) and (c) test Minion sequencing technology on unamplified extracted viral nucleic acids.
For the second aim we will collaborate closely with ESR 13, ESR 12 and Baseclear for the improvement of the existing bioinformatics analyses and interpretation of the viral NGS data. Their expertise in bacteriophage and eukaryotic virus identification from metagenomics datasets, respectively, and the tools they will develop will be very important for the success of this project.
The third objective of this project is to provide an in depth in silico and in vivo characterization of the bacteriophage population infecting the most important members of the honeybee gut microbiota, as well as studying their role in honey bee health and development. For the sample we have access to samples collected during the European B-GOOD project, headed by Prof. Dirk de Graaf. Both the bacteriome (16s) and the virome (shotgun) inhabiting the digestive tract of these honey bees will be characterized. Furthermore, bacteria will be isolated and characterized from the crop, midgut and hindgut of the collected honeybees, and finally these bacterial isolates will be used to isolate bacteriophages followed by their genome sequencing and functional gene annotation. Depending on the available time, we may also start with inoculation experiments of life honey bees.