The duck genome – and why it is important

It is Friday night, the kids are in bed and my wife is out with her friends. What do you do? Go to bed with a Sci-Fi book? No. Watch TV? No. Sort the laundry? NO!

The answer: I open a beer and read an article on duck genomes!

A happy Pekin duck – the domestic variant of the Mallard – and the most recent bird to have had all its genes sequenced. (From Wikipedia, Marin Winter)

The article was published this week in Nature genetics, and I know at least two of the 51 authors (by the way, it is amazing how many authors there are on genome papers – more people than base pairs sometimes…). I have been waiting for this particular article a long time and have known that it is was on the way. In the pipeline, as they say.

Why so eager? Well, the duck – or more properly termed the Mallard, Anas platyrhynchos – is the main study organism in my lab. The most common duck in Europe, the most widespread duck in the world, the reservoir host of so many influenza A viruses, the most beautiful…. Eh, hmpf, perhaps not the most beautiful bird, but you get the picture – it is an important bird to me. And the duck genome is a treasure trove for us duck researchers; in essence the blueprints of what makes a duck a duck. Some of the base pairs in the genetic code might be coding for that particular trait you are interested, be it plumage, migration directions, or ability to withstand infection. And that’s when you need the blueprint.

The last couple of years, in the aftermath of highly pathogenic H5N1, you often hear the words Mallard and flu together. And it is right: Mallards are an important reservoir host for influenza A viruses. Meaning that they sustain perpetuation of virus subtypes in nature and are important for influenza A virus evolution. And, as you know by now, flu in humans and influenza A virus in birds are linked – thus flu concerns both ducks and men.

The paper of Huang and her 50 academic friends presents the overall genetic architecture of the Mallard genome and put it in relation to earlier bird genomes (chicken, turkey and zebra finch) and genomes from fish and mammals. It gives a tale on events that have occurred on really long time-scales, for instance the rate of gene duplication and gene loss over the last 100 million years. However, for me the most interesting is the second part of the article where they infect duck with highly-pathogenic H5N1 viruses and do what is called transcriptomics to investigate which genes that are affected by infection.

A transcriptome is a deep sequencing of mRNA transcripts, the transcribed genes on their way to the ribosomes to become proteins. By amplifying the RNA in your treated animals (in this case ducks infected with virus) and comparing the number of copies of particular gene mRNAs to untreated animals (in this case ducks not infected with virus) you can make a crude measurement of which genes that are up- or down-regulated upon infection. This can then help you to understand, and pinpoint particular genes with certain functions that may be important for immune processes and pathogenicity.

The wild Mallard – the home of influenza A viruses in their billions. (From Wikipedia, Richard Bartz)

It is a great piece of work. And what I like is that it is the entry point for new studies; it’s like the opening of a highway where we other duck researchers can drive our cars. For my own part, I am extremely interested in the duck immune genes and the list of 150 cytokines, the Toll-like receptors, the defensins and the MHCs will be scrutinized in detail. We are already working on some of those, but now it becomes much easier to make progress.

Having said all these nice things, I do have some objections too. The strongest is how well the infection, and subsequent transcriptomes, reflect the natural situation. Experimental intranasal infections with a high titer of virus is not the natural way of infections, and hence may evoke biased responses, either because of wrong dosage, or because virus ends up in the wrong tissue. It is also important that controls and experimental animals measure the same thing, and in the right tissues. Some additional experiments, involving more animals and natural infections are warranted.  But overall it is a great achievement and staggering amount of work poured down in this paper. Hats off for you – all 51 of you!

The rest of us, we roll up our sleeves and get to work with the blueprint of the duck! Interesting times ahead!

Jonas Waldenström

Full link to the article: http://www.nature.com/ng/journal/vaop/ncurrent/full/ng.2657.html

How do you do, the things that you do, Mr. Flu?

The influenza A virus is a simple little thing. Just eight strands of RNA packed inside an envelope consisting of a host cell membrane. No metabolism, no fuzz, all very simple.

But, but, but – it is not very simple after all. In the flu business we still have large problems understanding many basic things this virus does. For instance, how does it survive in the environment? What governs host shifts? What makes a virus airborne, and what determines virulence properties? It is, in truth, a virus full of surprises.

This week, we published an article in Virology (http://www.sciencedirect.com/science/article/pii/S0042682213002638) on a particular aspect of flu – the way it changes genotype and phenotype through reassortment. Does this sound dull? NO, this is extremely interesting – follow me and see. It is evolution in the fast lane!

One thing that we are interested in in my research group is how influenza A viruses evolve. Being a RNA-virus with a sloppy RNA polymerase (equivalent to the virus’ genome copy machine) it mutates a lot all the time, much more than eukaryotes (animals like you, me, amoebae, fish, insects and whales). Thus, natural selection has a lot of variation to act upon, and hence the virus’ evolutionary trajectories may spurt out in different directions. Sometimes very fast, other times more slowly, when balanced by evolutionary constraints imposed by the host.

Long-term evolution in hominids

Long-term evolution in equines

But the influenza A virus has another tool in its toolbox when it comes to evolutionary change. The reassortment tool. Reassortment starts when two viruses infect the same cell. The cell machinery is hitchhiked and forced into making new copies of the virus genome and making new virus proteins. And we are talking many, many, many copies of virus genomes!

The newly borne RNA segments are embedded with certain viral proteins and migrate to the cell surface. There they are stuffed into small pockets of cell membrane and are released to the outside by the scissor-like protein neuraminidase, that cuts the last anchors binding the virus to the cell surface. Off they go to infect new cells! But, the process of stuffing RNA segments into the virus bag is a rather random process (at least we think it is), which means that RNA segments from one virus can suddenly get in a genome constellation with RNA segments from another virus.

Reassortment of two influenza A viruses in a duck

This means that the new virus progeny can consist of segments (each coding for one, or two proteins; thus having a function) with completely different descent. Different evolutionary histories! This is also the process that has started several of the flu pandemics we have seen in humans during the last century. In this context, it is often talked about the pig as a mixing vessel where flu viruses with human origin can meet viruses with an avian origin, since the pig has receptors on its cells that allows both virus types to enter the cell. Coinfections in the pig could then give rise to reassortant viruses, sometimes with the mammalian transmission adaptions and novel antigenic properties from the avian side. Wham-bam – no prior immunity in the population of humans and loads of sick people! Thus, understanding reassortment is important also for our own health.

The picture below summarizes how drastically the genotype/phenotype could change with reassortment.

Reassortment creates genome constellations where segments may have completely different descent.

But how common is reassortment in nature? We know it should be fairly common, but can we enumerate it? I have previously written a post on individual disease histories and immune responses in Mallards. That article, published in PLoS ONE in April (http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0061201), followed 10 semi-domestic lure ducks in our duck trap at Ottenby for 1.5 years. The Virology article is a follow-up study on the viruses collected from these ducks in 2009.

It is a fairly complex story, and the most devoted readers of this blog should have a look at the original article (if you don’t have access contact me and we’ll sort it out). From the ten Mallards, we managed to retrieve 92 viruses of 15 different subtypes. With a detailed sampling scheme, where each bird was sampled daily, we could really determine the sequence of infection in each duck from primary infections, to secondary infections, and to study how different RNA segments were exchanged among viruses in different hosts across time.

In order to quantify reassortment, we used a combination of phylogeny-based and network-based tools (and we even needed to invent some novel ways of analyzing data, to make it happen). The level of reassortment was extremely high in our population of Mallards, and we estimated that >50% of the viruses were reassortants.

In a way this doesn’t make sense. If segments are exchanged so frequently, we would expect the influenza A virus gene pool to be in a panmictic stage, and that there shouldn’t be any linkage between segments. The solution, we believe, is that there is a strong selection afterwards, in the environment, or in transmission, where certain combinations of segments have a higher fitness. The discrepancy between the structured populations, where certain subtypes such as H4N6 or H1N1 are very common, and others combinations such as H4N1 or H1N6 are very rare, and the promiscuous panmictic reassortment levels suggest very strong selection pressures outside the host cells.

This is also the reason why unicorns are rare.

Jonas Waldenström