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 hominids

Long-term evolution in equines

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

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.

flu 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

Mr and Mrs Borin are on their way back

An explosion in colors

An explosion in colors

It is beginning of May and the air is full of chirping, lovemaking sounds of House Sparrows. And the fluty tunes of newly arrived long-distance migrants! The marked seasonality is really fantastic at our latitude. A few weeks ago we had some late snow, and everyone was desperately longing for spring. And now it is here – slippery and fast as quicksilver! In a few days most trees will have unfolded their leaves, the insect life started to boom and all other animals just try to follow as fast they can.

For the birds, timing is everything, and they just have to get it right. If you are a migrant bird and arrive a bit too late, your chances of getting laid, and thereby producing offspring that survives, are slim. Either the earlier fellows already got the best territories and you are stuck with a shitty little shrub, or, you and your partner started breeding so late that your offspring were born mismatched in relation to the peak of surplus food; simply, the caterpillars and all other juicy insect stuff exploded in abundance while your chicks were still in the eggs.

There is a strict phenology to bird migration, each species adhering to its own itinerary. Some species are always first, some, like the Chiff-chaff, even start to appear when winter is still dominating, and spring is just almoooost is getting there; others are always late to arrive, when you think that spring really is summer. The late ones include some of the species that have travelled the longest, like the Marsh Warbler, the Icterine Warbler, the Swift and others. Species that spend the winter in Sub-Saharan Africa.

One of my favorites – the Garden Warbler – is among those late stragglers. It is not a stunning beauty; no red breast or fancy orbital ring, just shifts of grey, grey-brown, and brownish-grey. Somewhat non-descript, perhaps even drab, or boring (the Latin name is Sylvia borin, not boring, mind you). No, it isn’t very beautiful. But it has a fine voice and a rich, melodious song. The Garden Warbler is acquired taste; the first cigarette doesn’t taste good either, nor the first glass of wine, or the first taste of coriander. But Garden Warblers grow on you. They are really stunning creatures, in more ways than one.

One thing I find exceptionally intriguing is their plasticity in weight. There is nothing on this planet as fat as a fat Garden Warbler. The laws of gravity work hard on any animal that wants to sail the winds. The heavier, the more work is needed to stay in the air. Thus there is a balance on how much cargo you can put aboard and still fly, or where costs exceed benefits. Garden Warblers and other migratory birds use fat as fuel.

Early morning at Amurum, Jos Plateau State, Nigeria, April 2003

Early morning at Amurum, Jos Plateau State, Nigeria, April 2003

My friends and I, particularly Dr Ulf Ottosson, have long been interested in Palearctic migrants in Africa; specifically in Nigeria in West Africa, where many of our Swedish migrants either winter or pass through on their way south or north. I did my master thesis project in Nigeria, at Malamfatori in the extreme NE of the country, just up in the northern Sahel zone. Dry and hot most of the year, but exceedingly inhospitable in April at the end of the dry season. But at the same time a place where many migrants make their last stopover before passing the Sahara desert and the Mediterranean Sea. It is mind-boggling to realize that Whitethroats really choose this site actively for a last gluttony meal before the most challenging leg of the journey. All because of the Salvadora persica bush that fruits in April.

A really fat Garden warbler - the whole belly, tracheal pit and body sides are covered with a layer of fat

A really fat Garden warbler – the whole belly, tracheal pit and body sides are covered with a layer of fat

But this text wasn’t on the Whitethroat (I have to tell you more later, tales of gunshots, rebels and birds). The Garden Warbler doesn’t use the Sahel zone, it starts the spring migration further south, in the Guinea Savanna, some 300-400 km south of Mallamfatori. As they do not stop to forage until they reach the northern shores of the Mediterranean they need to put on a lot of fat. Huge amounts! A Garden Warbler that may weigh 16 g on the breeding grounds may reach 32 g in Nigeria, and it is not unusual that the leanest ones caught on Italian islands after the passage weigh as little as 13-15 g. It cost half of your weight to cross the dessert-Mediterranean Sea barrier. And then you still may need to travel some 3000 km to the breeding grounds. Better eat well and prepare for your journey!

Jonas Waldenström

Ottosson, U., Waldenström, J., Hjort, C. & McGregor, R. 2005. Garden Warbler Sylvia borin migration in sub-Saharan West Africa: phenology and body mass change. Ibis 147: 750-757.

http://onlinelibrary.wiley.com/doi/10.1111/j.1474-919X.2005.00460.x/full

Of chickens, wild birds and men – host specificity in Campylobacter jejuni

Rule number one in the kitchen: be wary of chickens! Improperly handled, this meat may spice up your dish with unwanted avian gut bacteria. The most notorious chicken bug is Campylobacter jejuni – which gives you really, really bad gastroenteritis (or ‘shits’ as most of you would say).

Campylobacter

C. jejuni is a quite common bug. At the poultry flock level, prevalence vary between 0 and 90% depending on which time of the year it is (more in summer months), which country we are talking about (less at northern latitudes), and of course the hygiene level of the farm in question (greasy farms get more bugs). However, nice Campylobacter-free chickens may be soiled with bacterial cells from infected birds during the long winding road from the farm, through the various stations in the abattoir (de-feathering stations, rinsing etc.) and to the retail level and end-consumer.

In our research we have addressed wild birds as hosts for campylobacters. Over the years we have spent a lot of effort to find out which bird species that are carriers of campylobacters, and which that are not. And what kind of differences there are between bacteria from different bird species. Earlier this year, we published a study in Molecular Ecology, where we genotyped a large collection of C. jejuni collected from Sweden, England and Australia. For a full view, down-load it here: http://onlinelibrary.wiley.com/doi/10.1111/mec.12144/full

We used multilocus sequence typing (MLST), which is equal to sequencing parts of seven different housekeeping genes distributed around the bacterial chromosome. Each unique allele gets a number, and the combined row of numbers of the seven loci is used to create a sequence type (ST). A sort of fingerprinting, you could say. And a very handy technique for C. jejuni, as it is one of the most recombinatory bacteria we know of; tree-based methods for inferring relationships don’t work as good on campylobacters.

mec12144-fig-0001

We found two things: First, C. jejuni populations in wild birds have very different genetic structure from C. jejuni in farm animals. In the figure above, you see how all human and food-animal C. jejuni populations cluster together, and where the different wild bird hosts have distinct populations of bacteria with long branch lengths. Second, we found strong patterns of host specificity.

Have a look at the picture again. If you look carefully, you will see that dunlins in Sweden and sharp-tailed sandpipers in Australia have more or less similar C. jejuni, despite huge geographic distances! Same goes for black-headed gulls and silver gulls, very similar to one another, but very different from the waders! And have a look at the blackbird – introduced to Australia by acclimatization societies in the 19th century they seem to have retained similar genotypes of C. jejuni that modern blackbirds have in Europe! Remarkable!

This really tells you of host adaptations – there are certainly differences in the enteric environment of different bird species, and in their diets, but there may also be differences in ecology that affects transmission properties, or survival in the environment. And why is all this important? Well, it says something about the peculiarity of current food animal C. jejuni. In these hosts, C. jejuni are more genetically similar and have a larger host range, suggesting that particular features involved in survival and transmission in the farm environment has caused expansion of particular genotypes.

In the future, identifying these properties are key. Hopefully we could do that with our wild bird campylobacters. But in the mean time, wash your hands and cook your chicken properly.

Jonas Waldenström

http://onlinelibrary.wiley.com/doi/10.1111/mec.12144/full

The Oxford comma – or the perils of a non-English scientist

 

English is today’s leading scientific language. No question about it, what so ever. However, it could equally well have been German, French, or Spanish. In Sweden, science was to a large extent written in German up until the 1940s, but after that well-known geospatial conflicts tended to favour the English language.

English as scientific lingua franca is of course very convenient for all you native speakers out there. You lucky bastards! For the 90% of mankind that are not, it is not as convenient. It is a struggle to find the right words, the right balance, the little magic flow, or Fingerspitzengefühl as the Germans would say (in lack of a proper English word for it); simply, what we want to say do not come naturally, it has to be drawn out, slowly and painfully.

But I enjoy it more and more. English is a fantastic language, not only because it makes conversation possible with other people, but also because it’s great possibilities to alter flow, tempus and direction. A sentence can easily start, but then a little comma can make it float away in a different direction, and then a new comma can make it get back on track, and then again drift, and back, and so forth, etc., etc., etc. Or it may be short. Succinct even. Endless possibilities.

But how shall we non-natives learn to write proper Anglaise? For my own part, I read a lot of novels in English. Mainly Sci-Fi or Fantasy, often thick books, with swords and fire on the cover. Many books. In the beginning it took time, but nowadays I read as quick in English that I do in Swedish. I believe reading is the precursor to writing, so the more you read, the better you will write. And then, of course, you need to practice. This little blog is one attempt for me to write in a more free form, not as constricted and concise as scientific English.

OxfordComma

And then there are the Oxford commas. I fail miserably and repeatedly with those darn Oxford commas. We don’t really use them the same way in Swedish. Last week I asked Michelle Wille, my Canadian PhD-student, to read through a draft manuscript that I was working on. And there they came again, the Oxford commas. Or rather, that I had forgotten to put them in. Again. For you, that like me, tend to forget when, and how, a Oxford comma should be used, please see the picture above. I hope I get them this time, and the next time.

Thanks Michelle, Jo, and others, that read the manuscripts before we submit them – and long live the Oxford commas.

Jonas Waldenström