Congratulations to Fadi Al Naji who recently completed the minor corrections to his PhD. thesis entitled “Isolation and analysis of recombinants from mixed virus infections of poliovirus using next generation sequencing (NGS) and bioinformatics“. Fadi was registered in the School of Life Sciences, University of Warwick and remained there to complete his studies after the rest of the Evanslab moved to St. Andrews. His external examiner was Professor Glyn Stanway, University of Essex (on the left below). Fadi is the good-looking one in the middle.
Just back from the excellent Europic 2016 meeting in Switzerland where Kirsten Bentley “knocked their socks off” with a talk on her recent analysis of the role of sequence identity and RNA structure in genetic recombination in enteroviruses.
Here’s what our friends in the Cameron and Vignuzzi labs tweeted …
With thanks to Caroline, Urs and Laurent for running a great meeting …
The fidelity of the virus polymerase influences the rate of genetic recombination between viruses coinfecting the same cell. We used cell-based and new, biochemically-defined, assays to demonstrate that the viral polymerase is necessary and sufficient for the strand-transfer event of RNA virus recombination. Furthermore, the fidelity of the polymerase is critical in determining the efficiency with which recombination occurs; low fidelity polymerases exhibit high recombination rates, and vice versa.
The paper is published in Nucleic Acids Research:
Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination
Andrew Woodman; Jamie J. Arnold; Craig E. Cameron; David J. Evans
Nucleic Acids Research 2016; doi: 10.1093/nar/gkw567
Genetic recombination in single-strand, positive-sense RNA viruses is a poorly understand mechanism responsible for generating extensive genetic change and novel phenotypes. By moving a critical cis-acting replication element (CRE) from the polyprotein coding region to the 3′ non-coding region we have further developed a cell-based assay (the 3′CRE-REP assay) to yield recombinants throughout the non-structural coding region of poliovirus from dually transfected cells. We have additionally developed a defined biochemical assay in which the only protein present is the poliovirus RNA dependent RNA polymerase (RdRp), which recapitulates the strand transfer events of the recombination process. We have used both assays to investigate the role of the polymerase fidelity and nucleotide turnover rates in recombination. Our results, of both poliovirus intertypic and intratypic recombination in the CRE-REP assay and using a range of polymerase variants in the biochemical assay, demonstrate that RdRp fidelity is a fundamental determinant of recombination frequency. High fidelity polymerases exhibit reduced recombination and low fidelity polymerases exhibit increased recombination in both assays. These studies provide the basis for the analysis of poliovirus recombination throughout the non-structural region of the virus genome and provide a defined biochemical assay to further dissect this important evolutionary process.
One of our colonies in the bee shed swarmed last week. The swarm ended up clustering around the entrance to the hive it had ‘escaped’ from. It was captured and rehoused successfully. The swarm in the picture is up to 5cm deep in places and probably contains 10-15,000 worker bees … and a single queen bee.
Swarming is the natural way that a honey bee colony ‘reproduces’. The old queen and all of the older foragers leave the hive to establish a new colony. The remaining workers raise a new queen from an egg or young larva in the original hive, so generating two colonies from one. Swarming usually occurs in late Spring or early Summer.
Not as unconnected as you might think. The most numerous photosynthetic organisms on earth – the cyanobacteria – are infected by viruses (cyanhophages). Some of these cyanhophages carry components of the photosynthetic machinery and are thought to contribute to host cell photosynthesis. In a recent study on which we collaborated we show that virus-infected cyanobacteria are inhibited in their ability to fix CO2 (in contrast to uninfected cyanobacteria) whereas photosynthetic electron transport is unaltered. The cyanhophages therefore redirect photosynthesis to support phage development.
These results also have implications for our understanding of global warming. The reduction in CO2 fixation in the marine environment, as a consequence of these cyanophage infections, may be as much as 10%. The global warming calculations are based on assumptions of carbon fixation levels being directly linked to photosynthetic activity. We show that that this is incorrect and that CO2 fixation is likely overestimated in marine environments.
The full abstract of the manuscript “Viruses Inhibit CO2 Fixation in the Most Abundant Phototrophs on Earth” by Puxty et al., is shown below.
Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus are the most numerous photosynthetic organisms on our planet. With a global population size of 3.6 × 1027, they are responsible for approximately 10% of global primary production. Viruses that infect Prochlorococcus and Synechococcus (cyanophages) can be readily isolated from ocean waters and frequently outnumber their cyanobacterial hosts. Ultimately, cyanophage-induced lysis of infected cells results in the release of fixed carbon into the dissolved organic matter pool. What is less well known is the functioning of photosynthesis during the relatively long latent periods of many cyanophages. Remarkably, the genomes of many cyanophage isolates contain genes involved in photosynthetic electron transport (PET) as well as central carbon metabolism, suggesting that cyanophages may play an active role in photosynthesis. However, cyanophage-encoded gene products are hypothesized to maintain or even supplement PET for energy generation while sacrificing wasteful CO2 fixation during infection. Yet this paradigm has not been rigorously tested. Here, we measured the ability of viral-infected Synechococcus cells to fix CO2 as well as maintain PET. We compared two cyanophage isolates that share different complements of PET and central carbon metabolism genes. We demonstrate cyanophage-dependent inhibition of CO2 fixation early in the infection cycle. In contrast, PET is maintained throughout infection. Our data suggest a generalized strategy among marine cyanophages to redirect photosynthesis to support phage development, which has important implications for estimates of global primary production.
Puxty, R.J., Millard, A.D., Evans, D.J. and Scanlan, D.J. (2016) Current Biology http://dx.doi.org/10.1016/j.cub.2016.04.036
Slow motion video of bees entering and leaving a colony in the bee shed in our research apiary … look out for the head-on collision early in the video on the left hand side of the frame.
Our snappily-titled manuscript “The Iflaviruses Sacbrood virus and Deformed wing virus evoke different transcriptional responses in the honeybee which may facilitate their horizontal or vertical transmission” has just been published in PeerJ. We analysed changes in the transcriptome following infection with deformed wing virus (DWV) and sacbrood virus, or DWV alone. We propose that the difference in expression we observed of the honeybee immune genes induced by SBV and DWV may be an evolutionary adaptation to the different predominant transmission routes used by these viruses .
On Thursday 14th January I’m talking at an evening meeting of Fife Beekeepers Association about the biology of Deformed Wing Virus and how our understanding of the virus should help devise more rational integrated pest management strategies. This is the first of several outreach events planned for 2016 in which our BBSRC-funded research on honeybee viruses will be discussed.
Not long now until the beekeeping season starts and we can get on with our planned field studies 🙂
Note This post was first published on The Apiarists’ blog.
During previous research on deformed wing virus (DWV) biology and its transmission by Varroa I’ve moved known Varroa-free colonies (sourced from a region of the UK which the mite has yet to reach and maintained totally mite-free) into apiaries in the countryside. Within 2-3 weeks Varroa was detectable in sealed brood, showing that mite infestation occurs very readily. I know other researchers who have made very similar observations. Where do these mites come from?
They’re not all ‘your’ bees
The obvious source would be the phoretic mites transported on workers ‘drifting’ from nearby infested colonies, or on drones which are known to travel quite long distances and may be accepted by almost any colony. If you want to see how frequent this is try marking a few dozen drones with a dab of paint. To avoid confusion use the colour used to mark queens next year. There are unlikely to be 4+ year old queens in the apiary and the drones will all perish before the end of the current season. Over the next few days and weeks the drones will appear in adjacent colonies, and some will likely leave the apiary and be accepted in your neighbours colonies.
Beekeepers are usually aware that colonies at the ends of rows often ‘accumulate’ bees that have drifted when returning to the hive. In shared association apiaries some crafty beekeepers will site their colonies at the ends of rows to take advantage of the ‘generosity’ of other colonies. However, many beekeepers probably do not appreciate the extent to which drifting occurs. Pfeiffer and Crailsheim report (1998) that 13-42% of the population of a colony are ‘alien’ i.e. have drifted from adjacent hives, depending upon the time of season. Remember that drifting occurs in both directions simultaneously, so the overall numbers of bees in a colony may not be adversely affected (or boosted). In other studies Sekulja and colleagues report (2014) that ~1% of marked bees drifted between colonies over a three day observation window. Interestingly, American foulbrood (AFB) infected bees drifted slightly more than uninfected bees. Spread of foulbroods during drifting is one reason the bee inspectors check nearby apiaries when there is an outbreak. These studies were all on workers where drifting primarily occurs during orientation flights before the bees become foragers. Drones drift two to three times more than workers (Free, 1958).
The likelihood of drifting must be closely related to the separation of hives and apiaries. Although workers will forage up to 2-3 miles from the hive I suspect the proportion of bees that drift this distance is small to undetectable. Drones are known to fly up to about five miles to reach drone congregation areas for queen mating and are accepted by all colonies. I’ve regularly found drones appearing in (relatively) isolated mini-nucs. I’m not aware of studies that have formally tested drifting between apiaries (though it is reported in passing in the Sekulja et al., 2014 paper referenced above).
Consequences of drifting
So, your hives contain workers and drones from many nearby colonies, and you can only really be sure that they’re all “your” bees if you live – as the sole beekeeper – on an isolated island. Not only does your neighbour generously exchange bees with you, he or she also kindly shares the phoretic mites those bees are carrying, the viral payload the bees and mites are infected with and – if you’re really unlucky – the Paenibacillus larvae spores responsible for causing AFB infection (and vice versa of course).
There are lessons here that should inform the way we conduct our integrated pest management to maintain healthy colonies.
This post provides background information for an article (“Viruses and Varroa: Using our current controls more effectively” by David Evans, Fiona Highet and Alan Bowman) in the December 2015 issue of Scottish Beekeeper, the monthly magazine for members of the Scottish Beekeepers Association.
More later …
We now have signs informing passers-by that our research apiary is now operational and has resident bees.