On the steppes of Askania Nova

The circles are the parts of the field that are irrigated, while the drier ‘corners’ are planted with different crops.

Southern Ukraine. From Zaporizhza to Askania Nova we pass field after field on the straight (but bumpy) road. The fields are huge, bigger than any fields I’ve seen. This is farming on the industrial scale. Once upon a time this was the river bed of one of Europe’s largest river, which deposited a thick layer of soil perfect to till. But it is dry, and without pumping water from Dnepr most of the fields would be steppe.

Johannes Rydström and I have traveled here to meet with Denys Muzika and his team of ornithologists and virologist. Over the course of a week, we try and catch ducks to equip them with GPS loggers that allow us to study migratory connectivity and influenza A virus dispersal. Just a few weeks earlier I was doing similar work in Bangladesh, and the contrasts in temperature, landscape and number of people couldn’t be larger.

Our base is Askania Nova, a pristine steppe reserve in the southern part of the country. It is a popular tourist destination and the site has a very ambitious zoo with large ungulates and birds, and a huge park with a collection of diverse trees. It is a gem and as a birdwatcher the steppe birds are amazing to see, with a constant background of singing Calandra larks.

Our team scouted different wetlands in the area and we tried to capture birds most nights using mist nets and duck calls. Depite our efforts and some amazing wetlands, we were not as successful as we had hoped. But at the end of the trip we can note 9 mallards equipped with loggers, of which one directly migrated to Russia. A big part of the trip was to connect and build for the future, because this is a site of strategic importance for influenza and duck research, on the gateway to Europe on the Caspian/Black Sea flyway.

We will be back.

A male mallard is about to be equipped with a logger. This bird is currently in southern Ukraine. Photo Johannes Rydström.

Several nights we worked in a beautiful steppe lake, putting up mist nets to catch ducks. This is Denys in action. Photo Johannes Rydström.

Sweden-Ukraine Duck Team (Denys, Sasha, Raysa, Jonas and Kolja) Photo Johannes Rydström.

This is me!

Tanguar Haor – a legendary wetland

IMG_0414There’s no business, like duck business

This spring I have been going places. First Bangladesh, and then Ukraine. Both trips connected by ducks, and the hopes of using telemetry to infer migratory connectivity of waterfowl populations and the transmission risk of avian influenza viruses.

Together with our colleagues at IUCN Bangladesh we spent some magnificent weeks in the wetlands of northeastern Bangladesh catching wintering ducks. I am writing up a longer piece of this trip for Birdlife Sweden’s magazine Vår fågelvärld which I hope to share with you in a couple of months. In the meantime, I’d like to refer you to an excellent article by Abida Rahman Chowdhury, a journalist from The Daily Star who visited us in the field in Tanguar Haor – the gem of wetlands in the north. Please read it on this link.

Quack, quack – quack it out! Twelve years of flu research in Mallards!

A good day in the duck trap! Duck trapper Gabriel Norevik is herding the ducks. Photo by Ville Fagerström

A good day in the duck trap! Duck trapper Gabriel Norevik is herding the ducks. Photo by Ville Fagerström

By Jonas Waldenström

It is time to do a field season wrap-up. There are still a few weeks of fieldwork to do, but now it is mainly the everyday routine trapping of ducks that remains. And when I say routine, I mean it. We have run our Mallard disease-monitoring scheme at Ottenby Bird Observatory, Sweden, since 2002. A full dozen years with daily sampling during the field seasons! That is truly remarkable!

If you don’t think 12 years is a long time, then you are likely not a scientist, at least not one working with animals in the wild. The truth is that long time series are rare in biological systems. Very rare. The few that are still running (some for 50+ years) have produced fantastic data, such as the Darwin Finches at the Galapagos island Daphne Mayor, run by Peter and Rosemary Grant since 1973, the St Kilda Soay Sheep project in the UK, the Great Tit population in Wytham Woods outside Oxford, or the Collared Flycatchers of Southern Gotland, Sweden. For flu, there are the sampling schemes of shorebirds at Delaware Bay, and the long-running duck sampling in Alberta by St Jude’s Children Research Hospital – two programs that have shaped our view of flu. But why then, you may ask, are long time series rare? That my friend is an excellent question!

One failed grant application can put a grinding halt to a time series.

One failed grant application can put a grinding halt to a time series.

To start with, funding typically favors shorter projects, roughly 2-4 years long. No research body says ‘cool project, let’s fund it for the next 20 years’, unless it is mega-large projects like CERN (in France/Switzerland), the International Space Station (in orbit), or the Human Genome project (finished). For us mortal researchers, a long time series rely on successful applications in grant cycle, after grant cycle, after grant cycle. This is a major hurdle for long projects. For instance, the Swedish Research Council, one of the main funding bodies in Sweden, turned down 84 % of the proposals in 2013. Thus, it only takes one year with bad luck to put a grinding halt to a time series.

And even if it is the senior researcher(s) who fund the project, it is often the PhD and postdoctoral students that actually run it. The length of a PhD varies (in Sweden it is 4 years) but are fairly short, and postdocs even shorter. When the student has graduated, chances are that the continued fieldwork simply dies, especially if techniques/skills were not shared between staff, or that if no suitable replacement was found. Also, similar to old land-owning dynasties (where a drunken playboy lost the manor house and the estate playing dice), a wrong recruit may effectively spoil a time series.

The staff is always important in any project...

The staff is always important in any project…

Another pitfall is curiosity. Researchers are by and large driven by curiosity, and sometimes the allure of greener pastures elsewhere seems more compelling to pursue, than to dig where you stand yet another year. Consequently, there is a risk that the leading scientist leaves a project that could have grown into an important long-term data series because he/she started to grow bored and restless. Thus, funding is scarce, time changes, people move and priorities shift. And as a result few time series reach a decade.

So how come the Ottenby data series is still running after twelve years?

The project was started by professor Björn Olsen (birder, physician, and the chair of Infectious Diseases at Uppsala University) in 2002. Björn had worked with tick-borne infections, such as Lyme Disease, and gastrointestinal bacteria such as Salmonella and Campylobacter, and when a move to Kalmar Hospital brought him close to Ottenby Bird Observatory it was like pieces of the puzzle just came together. For what can be more of a perfect match for a physician interested in birds than the avian zoonotic pathogen influenza A virus? And to have a field site at the best birding spot in Sweden! Fabulous!

The dismantled duck trap from an earlier trapping period 1960s – 1980 was resurrected and hopes were high that ducks would start to appear. Which they did, but only after a few months of very low trapping numbers, which made everyone wondering whether we would have to cancel the whole thing. Furthermore, funding was initially modest. Agencies thought there were more pressing research fronts – one review of a proposal actually dismissed the value of birds as hosts for influenza at all, as he/she believed minks were the most important hosts… But the work was done, the publications started to come out and brick by brick the flu house was constructed. Funding came in more steadily, and in the last decade we have had grants from the regional councils (FORSS, Sparbanksstiftelsen Kronan), national councils (including the Swedish Research Councils VR and FORMAS), and international councils (EU-FP6, NIAID), plus authorities such as the Swedish Board of Agriculture and the European Commission. So far the grants have come when we needed them, and never too late. It has been close sometimes, but so far so good. Another reasons to why we still are in the business are great collaborations! Already from the start we collaborated closely with Albert D. M. E. Osterhaus and Ron Fouchier from Erasmus MC in Rotterdam – a collaboration that has continued ever since. Other long term research friends are Johan Elmberg in Kristianstad, Åke Lundqvist in Stockholm, Vladimir Grosbois and Nicolas Gaidet at CIRAD, France, and Martin Wikelski at Max Planck in Constance, Germany, as well as Calle Nyqvist and Kalmar Surveillance AB! And many, many more!

A tipping point was when the highly pathogenic avian influenza H5N1 crossed Eurasia and hit Europe with force in the winter 2005/2006. This was almost like a deus ex machina moment, and suddenly the things we did were what everyone wanted. We were at the center – the eye of a hurricane – and delivered data to national and European authorities, helped with risk assessments, answered billions of news reporters, and through out it all continued to do good science and publish quality papers. In those days, Björn could be seen in three, four major newspapers, and national TV in the same day! Crazy times!

Throughout there has been a great team that made it all possible. The Ottenby project has so far directly involved seven PhD students:

  • Anders Wallensten (now at the Swedish Institute for Communicable Disease Control)
  • Neus Latorre-Margalef (now at University of Georgia, USA)
  • John Wahlgren (now at Qiagene in Denmark)
  • Josef Järhult (rising star at Uppsala University)
  • Goran Orozovic
  • Michelle Wille (still in the lab trenches)
  • Daniel Bengtsson (still in the field trenches)

And five postdocs:

  • Elsa Jourdain (now at INRA, France)
  • Gunnar Gunnarsson (now at Kristianstad University, Sweden)
  • Conny Tolf (longstanding king in the lab)
  • Alexis Avril (battling the computer with CMR epidemiology models)
  • Joanne Chapman (defending the innate immune system of ducks)

Lab work has been immense and a large number of hands have helped out during longer and shorter times. Among others: Abbtesaim Jawad, Sara Larsson, Maria Blomqvist, Diana Axelsson-Olsson, Lovisa Svensson, Petra Griekspoor, Jenny Olofsson, Jorge Hernandez, Oskar Gunnarsson, Lorena Grubovic, Anna Schager, and many, many more.

And in the field we talk about more than 40 duck trappers and >33,000 duck trap/retrap occasions! The two most frequent are Stina Andersson and Frida Johnsson that have made several seasons in the duck trap! The list is too long to post here – but I promise to return soon with a ‘best of’ post with statistics of duck trapping and trappers! Simply, without the trappers no science – incredibly important people!

Who knows what the future may bring? Painting by French artist in 1910 imagining what life would be in 2000.

Who knows what the future may bring? Painting by French artist in 1910 imagining what life would be in 2000.

To sum up a long post: we have done well because of fortunate timing, a good study site, great staff in the field and in the lab, good collaborators, and lastly great science! A few weeks ago we learned that we have funding for another three years – hopefully we can get this virus-host time series through adolescence and into adulthood! Time will tell.

In the mean time – shout it out for the ducks! Quack, quack, quack!

*******************************************************************************************************************

If you enjoyed this post, or other posts on this blog, why not follow the blog via email, Feedly or get updates via Twitter by following @DrSnygg?

Lost (and found) in translation

By Jonas Waldenström

We aim for the stars

We aim for the stars

Your mum likely told you not to brag, but I will do it anyway: we have such an awesome research group, and we are on a roll! A rare collection of individuals that fits surprisingly well together. It is like if you randomly twisted Rubik’s cube with your feet in the darkness and it came out all solved. Yes, we are great scientists (and humble), but more importantly we are witty, intelligent, friendly and caring. Good human beings.

Stewing in our pot at the moment we have ingredients from Iraq, Canada, New Zeeland, France, Catalonia, and Sweden (including the God’s forgotten little city of Västervik and the forested no-man’s-land Värmland). This makes for interesting conversations and different perspective on things. And good food. Especially if you like moose.

Some see 'the grandness of nature' in this picture - others see the 500+ kg of meat! (Photo from Wikimedia Commons, Oliver Abels)

Some see ‘the grandness of nature’ in this picture – others see the 500+ kg of meat! (Photo from Wikimedia Commons, Oliver Abels)

I enjoy the coffee discussions, which regularly include parasite life histories, the more gory the better, the weirdness of Swedish people in general, and the faiblesse for putting things in tubes in particular, all untranslatable words and silly signs. And, of course, the ever ongoing friendly bickering between the Kiwi and the Frenchman.

How Swedish can it get? This is smoked raindeer cheese in a tube! (Photo Jo Chapman)

How Swedish can it get? This is smoked raindeer cheese in a tube! (Photo Jo Chapman)

Language is both a barrier and bridge. Even though English is the language we us for communication, our various accents, proficiency and speed create misunderstandings. For instance, a ‘smiling’ and a ‘smelling’ face are two different things. And we do not sample ‘girls’ we sample ‘gulls’. And – very importantly – we put ‘candles’ not ‘condoms’ on cakes.

It is, as we often say in our group ‘largely sufficient’.

How bats in Peru change our view of flu (and it rhymes!)

By Jonas Waldenström

I am the real Bat Man and here to bite y'all

I am the real Bat Man and here to bite y’all

One of the major news in the virology community last year was the publication in PNAS describing a completely new influenza A virus. In line with the taxonomy traditionally used for influenza viruses it got the name H17N10, illustrating that it possessed novel hemagglutinin (H17) and neuraminidase variants (N10). However, it wasn’t the numbers that was the ground breaking news, it was the fact that the virus was detected in a Central American bat, and not in a bird. A tropical bat is very far from the ‘normal’ diversity of influenza A viruses seen in wetland birds and waterfowl. Although bats and ducks both have wings, in evolutionary terms they separate a very, very long time ago in the age of dinosaurs. In fact, there are more differences than similarities between bats and gulls in ecology, physiology and aspects of cellular biology. Hence, the bat flu was a remarkable observation. A real shaker. In one sweep, the whole flu field needed to come with terms that not all viruses are bird viruses.

The initial findings also hinted that the first bat influenza virus was unlikely to be alone. An influenza-iceberg, of sorts, made up of fluffy, winged mammals. This week, a first follow-up was published in PLOS Pathogens. A crew of (mainly American) scientists analyzed samples from bats sampled in the Amazonian parts of Peru in 2010, collected as part of CDC’s tropical pathogen surveys. In total, 114 individuals of 18 bat species were taken out from the freezers and different sample types were screened with a molecular method designed to broadly pick-up the RNA of any influenza A virus. They got one hit from a fecal sample in a single bat! A lucky shot at the Tivoli, given the low sample size. Prompted by this, the authors brought in the big machinery and sequenced the totality of the genetic material in the samples from this poor, long-dead bat and used bioinformatic tools to resolve the genome of the virus that had infected its intestines. When bit by bit was added it became clear that it was indeed a completely new influenza A virus, very different from avian viruses, and similar, but still distinctly different from the earlier H17N10 bat virus. And the name? H18N11 of course!

Please take a close look at the figure below. It shows the phylogenetic relationships of each of the influenza A virus’ eight RNA segments – in black are all ‘non-bat viruses’ and in red the two new bat viruses H17N10 and H18N11. For all the segments coding for ‘internal’ proteins, i.e. those involved in the polymerase machinery or the structural properties of the virus, you see that the two bat viruses are always found in a neat little red outgroup. This signals a long evolution away from other known influenza A viruses. It is a little prematurely to say exactly how long, but the branch lengths indicate that this happened a long time ago.

Phylogenetic trees for the 8 different IAV segements, see http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1003657

Phylogenetic trees for the 8 different IAV segements, see http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1003657

Now look at the hemagglutinin and the neuraminidase trees (HA and NA, respectively). The same pattern is repeated for the NA, but not the HA. In fact, the two novel hemagglutinins are nested within avian hemagglutinins. How can we interpret this? At first this doesn’t make any sense, but one has to remember that influenza viruses don’t evolve in the same way you or me, trees, shrimps or ferns do. Influenza viruses can reassort, meaning that if two viruses of different origin infect the same cell the different RNA segments can be put in new combinations in the resulting virions. Imagine two decks of cards being shuffled, one red and one black, and that each virion randomly consists of a draw of card from the combined shuffled deck, sometimes red, sometimes black, and sometimes mixed.  This is a rapid way in which new variants can arise, and a reason behind the genesis of pandemic flu in humans.

Returning to the bats, it seems that bat and avian viruses have met in a not too distant evolutionary past, and that a HA variant have sailed into the bat influenza gene pool. It will be interesting to see how the picture changes when more bat viruses are sequenced. Has there been one reassortment event, followed by drift and a subsequent separation into H17 and H18? Or, has there been many? Are there, perhaps, avian H17 and H18 to be found in South American birds? What about bats in North America, Europe, Africa and Asia?

One thing we can be sure of is that there are more viruses waiting to be detected and described. One sign of this comes from the current paper. The authors used the sequenced genomes to construct recombinant HA and NA molecules (using fancy virologist tricks) and used these to build assays (ELISAs) where bat sera could be screened for antibodies against the new HA and NA variants. Where the molecular screening yielded one positive bat, the serology approach found 55 of 110 bats showing signs of having been infected with flu earlier in life. This clearly indicates that influenza viruses are widespread in Peruvian bats, and likely in other parts of the world too. Moreover, they found cases of bats with antibodies to one of the recombinant HA or NA, but not to the other, suggesting that are more combinations of HA/NA to be found.

Finally, and perhaps the most interestingly of all results was that the hemagglutinin of bat influenza viruses does not to behave in the same way as avian hemagglutinins. When a virus is to infect a cell it needs the hemagglutinin protein to serve as a key, docking with a sialic acid receptor – the lock – on the cell. If the key and the lock don’t fit infection will not occur. For instance, a major division between human flu and avian flu is the preferred conformation of a galactose residue on the sialic acid receptors. This little difference makes it hard for avian viruses to infect humans, and vice versa. But with bat viruses it seems sialic acid receptors are not used at all! Instead bat HA uses an unknown receptor for cell entry. Holy Moses!

More to follow shortly, I suppose. Major obstacle at present is the lack of a culturing method for bat influenza viruses. Neither cell lines nor eggs have worked so far. Without the means to grow the virus it is very tricky to study it. But there are many clever virologists out there, so it is likely not too far away.

But I still prefer feathers to fur, and will stick with ducks.

Links to articles:

Tong S, Zhu X, Li Y, Shi M, Zhang J, et al. (2013) New World Bats Harbor Diverse Influenza A Viruses. PLoS Pathog 9(10): e1003657. doi:10.1371/journal.ppat.1003657

Tong S, Li Y, Rivailler P, Conrardy C, Castillo DA, et al. (2012) A distinct lineage of influenza A virus from bats. Proc Natl Acad Sci USA 109: 4269–4274.

This flu, that flu, and Tamiflu®

By Jonas Waldenström

Lovely start of the day. Not.

Lovely start of the day. Not.

It is early morning, just before the alarm clock is about the give the wake-up call. You lie in bed. It is an ordinary day, just as all others; a day at work, kids at shool and all other steps on the treadmills that make up our lifes. But hey, what’s this? Your throath is dry, and your nose is warm. On top of that: headache, nausea and fever. Shit – you are sick! Is it the flu? Fuck!

This hypothetic and unfortunate morning scenario is not a rare occurrence. In fact, ever day, a fraction of the human population is down with a disease – caused by bacteria and viruses, and more rarely by fungi or protozoa. However, not all bugs are potent enough to keep you in bed. Many can be cleared by the immune system without you even knowing it. It has been estimated that each of us are at all times on average infected with 12 different viruses. On average! And in, and on, your body there are bacteria waiting for an opportunity to establish infection. We are, in truth, a walking goodie bag for pathogens!

But we are not helpless. The goodie bag is shielded by different lines of defenses in order to protect our cells from invading pathogens. It is just like a battlefield of old times. First line defenses are structural obstacles, such as skin, nails and other solid barriers that keep the bugs out. (There are also behavioral defenses, such as wash your hands, don’t eat poo, stay away from rabid dogs, etc). Then, different excretions of slimy substances, like snot in your nose, mucus in your airways and in your gut – all there to wash the bad guys away. There is also an army of specialized molecules and cells of the immune system patrolling around in our blood and lymphatic systems. Some of these guys are rather non-specific brutes, punching holes in bacterial membranes, others have the function of whistle blowers, recruiting the heavy machinery in forms of macrophages and neutrophils. This perpetual war is constantly waged in our system, each and every day. Even now when I sip my morning coffee and write this blog. Hail the immune system – it is your best friend (and sometimes your worst enemy, but that’s a topic for another day)!

On top of the natural defenses, we humans have invented a range of drugs to help fight diseases. We are best at fighting bacteria, mainly because their metabolism and their cell membranes are so different from ours. This means that we can attack them with substances that are harmful for them, but which actions have negligeble effect on ourselves. Antibiotics are, in other words, a nuke that only kills bacterial cells. For viruses the best defense is vaccination, triggering the memory part of the immune system to produce pathogen-specific defenses. Drugs are usually less effective against viral infections, as the virus particles spend most of the time inside cells, and thereby are harder to nail. Secondly, as viruses do not have a metabolism of their own there are fewer targets for us to aim at.628x471

With that said, there are acutally a few substances that work on viruses. One famous drug is Tamiflu®, or oseltamivir as it also known as, which works against influenza virus infections. If you know Tamiflu by name it is likely due to the attention the bird flu (H5N1) and the swine flu (H1N1) viruses got during the last decade. You might even have taken it yourself during an influenza infection? Oseltamivir is what is called a neuraminidase inhibitor. Simply, it interferes with the catalytic action of the influenza virus’ surface protein neuraminidase (the N part of the name used in classification of subtypes). Neuraminidase proteins help releasing new virions from an infected cell, by scissoring of all the anchorage of the particle with the cell wall, and if this process is halted there will be less spread of viruses and a reduced number of infected cells.

It is not a wonder drug. My physician friends say it reduces symptoms with a day or two in already infected patients. The best effect is seen when the drug is used prophylactic, to reduce the chances of becoming sick. This property is what have made governments all over the world to stockpile it, in the case of a new flu pandemic and the shit hits the fan. A defense of sorts, albeit a rather weak one. But, a weak defense is better than none – as there will take time to generate, and to distribute, a new vaccine in case of a pandemic.

However, a weapon needs to stay sharp, otherwise it looses its use. Many antibiotics are nowadays countered by bacteria with acquired resistance. Trying to fight resistant bacteria with the wrong antibiotic is like fighting windmills with horses. Not very effective. The best way of keeping a weapon sharp is actually not to use it. Keep it on the shelf, and not prescribe it to patients. The same story as for antibiotic resistance goes for Tamiflu – resistant seasonal flu viruses started to appear not long after the drug was first prescribed in larger quantities. With time, the proportion of resistant phenotypes has increased, clearly indicating that there is a selective advantage for human-adapted flu viruses to be resistant to this drug. And that the benefits overweighs any costs associated with this trait. The question is whether the mutations that render the virus resistant occurs also in wild type viruses, those that have not yet crossed species barriers and jumped from birds to mammals and humans? And what mechanisms that induce resistance?

In our zoonotic network, the Uppsala node headed by Dr Josef Järhult focuses on these kinds of questions. In his studies, Josef has shown that the concentration of oseltamivir that can occur in natural waters downstream cities during an outbreak of seasonal influenza is sufficient to cause wild type influenza viruses to evolve resistance. How can this be? Well, to start with not all of the drug is processed by the body, a rather large proportion is excreted in the urine. As the drug is fairly persistent, the treatment in the sewage plant will not affect it and it can thereby reach the natural water column. And in water there are ducks, and in ducks there are influenza viruses. Hence a potential way of making viruses resistant just by peeing out the drug in the toilet!

To study this in detail, Josef, his PhD student Anna Gillman, and colleagues from Uppsala, Umeå and Linnaeus Universities invented an infection model to study natural transmission of influenza viruses in ducks. I like to think of it as the ‘Järhult model’ – it is simple and elegant. What you do is to have a room in which you place two ducks. You infect the ducks in the bill with a suspension of virus. This artifical infection will cause the ducks to be infected with influenza viruses, and viruses will start to replicate in the gastrointestinal tract of the ducks. After a few days, virions are excreted with the feces and it is time to bring in some fresh ducks. In this case the new ducks will be infected by natural transmission via the fecal-oral route, and the backside of forced infections are avoided. By constantly, every two or three days, bring in new and take away old ducks one can passage viruses in a number of duck individuals and study what is happening with the viruses with time. As the main research question is induction of resistance mutations, Josef and coworkers can adjust exposure to Tamiflu in the drinking water, mimicking different environmental settings from the wild.

The 'Järhult model' from PLOS ONE 8(8):e71230

The ‘Järhult model’ from PLOS ONE 8(8):e71230

In a couple of publications, one which came out just a few weeks ago, the team (where our Kalmar ZEE laboratory is partaking) has shown exactly what happens when a wild type low-pathogenic virus is exposed to drug selection pressures. It seems that there is a certain drug concentration treshold that determines whether drug-resistant mutants will take over the virus population or not. Generally, the influenza virus is such a poor proof-reader during replication that loads of mutants are created in every infected cells. By pure numbers, any infected duck has the potential to carry a mutation that affects drug resistance. But, the selection pressure, in the case environmental load of the compound in the water, needs to be strong enough for it to increase in frequency. Actually, classic population biology, but on viruses and not beak length as in the Darwin finches of Galapagos.

Interestingly, resistance could be induced with the same relative ease in two very different types of neuraminidases, representing the two major classes of the proteins: the N1 group that contains the subtypes N1, N4, N5 and N8, and the N2 group that contains the subtypes N2, N3, N6, N7 and N9. The actual mutations are not the same, as these proteins functions slightly differently and have evolved separately for a long time. However, the phenotypic response is similar, and so is the growing take-over of resistant vs susceptible viruses in the virus populaton over time in the experiments. This also suggests that the initial mutation, which changes the neuraminidase function, is backed-up by later compensatory mutations that make sure that fitness is maintained in the mutant virus.

Dear reader, if you are still with me so far down on the page, you may ask what this is all good for. After all, a duck is duck, and a man is a man, and “I ain’t afraid of no virus”. Well, perhaps you should be worried. Sooner or later there will come a new flu pandemic, most likely caused by a virus with a genome partially derived from a duck influenza virus. When that happens we need every gun and man we can muster, and therefore we need to know if the Tamiflu bullet will be explosive or not. What weapons we have should be wielded wisely.

IMG_1261

Links to the papers:

Gillman, A., Muradrasoli, S., Söderström, H., Nordh, J., Bröjer, C., Lindberg, R. H., Latorre-Margalef, N., Waldenström, J., Olsen, B. & Järhult, J. D. 2013. Resistance mutation R292K is induced in influenza A(H6N2) virus by exposure of infected Mallards to low levels of oseltamivir. PLOS ONE 8(8):e71230. doi:10.1371/journal.pone.0071230

Järhult, J.D., Muradrasoli, S., Wahlgren, J., Söderström, H., Orozovic, G., Gunnarsson, G., Bröjer, C., Latorre-Margalef, N., Fick, J., Grabic, R., Lennerstrand, J., Waldenström, J., Lundkvist, Å. & Olsen, B. 2011. Environmental levels of the antiviral oseltamivir induce development of resustance mutation H274Y in influenza A/H1N1 virus in Mallards. PLOS ONE 6(9): e24742. doi:10.1371/journal.pone.0024742

Disease is a property of the individual

Ecologists are obsessed with variation, in any form, the more bizarre, the better. We really love it! But why?

The textbook explanation is that variation among individuals, if heritable, work as a template for selection and thus drives evolution. Without variation, little can change. Evolutionary important variation relates to genetic traits that make the organism better adapted to its environment, a better competitor, more disease resistant, or relates to traits that make him/her more attractive to the other sex, thereby increasing the likelihood of siring offspring.

l_015_04_l

And additional explanation, and sometimes equally important, is that it is fun with variation: an animal may be short or long, have a peculiar nostril shape, vary in the curvature of antlers, or have striking plumage colors. Simply, humans like variation, and the diversity in itself therefore drives curiosity-driven researchers.

This said, when it comes to disease in animals most researchers tend to neglect variation. Disease is commonly treated as a constant; the animal is either infected with parasite X or is not. However, in reality what the researcher denotes as parasite X may actually be a plethora of different pathogen genotypes, all seemingly dressed in the same costume (the phenotype), or sometimes even consist of cryptic species. This is dangerous, as things that look the same in the microscope (or in a conserved gene used for molecular screening) may have fundamental differences in traits that are relevant for infection processes, such as pathogenicity, transmission and virulence. Simply, we may run the risk of not seeing patterns that are there, or jump to the wrong conclusion based on simplified assumptions.

Further, surprisingly often wildlife diseases are treated at the level of the population (especially abundant in veterinary medicine), and not at the level of the individual animal. For instance, prevalence, the proportion of individuals carrying a particular disease at a given time, is much more frequently used than estimates of incidence, which relates to the risk of acquiring infection. In the former you can adhere to a ‘hit and run’ sampling approach, in the latter you need to monitor individuals across time and take repeated samples.

For a long time, actually since 2002, we have studied influenza A virus in a migratory population of Mallards in SE Sweden. We also started at the level of population, describing temporal variation in influenza A virus prevalence in the duck population, and describing differences in prevalence among ages and sexes. And yes, we treated the virus as pathogen X, not at the level of subtype (which there are many of in flu). But with time we have moved to assessing what is happening at the individual level, and how differences among individuals in susceptibility drive disease dynamics, and how disease histories and immunity patterns in turn drive evolution in the virus.

These efforts are starting to pay, and in a paper published this week (http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0061201) we address the issue of individual variation among Mallards in influenza A virus infection risk. The question we asked is how individuals with the same background, in a shared environment with similar exposure to influenza, differ in disease histories and immune responses.

In our monitoring program we use a large duck trap to catch wild ducks. By providing grain we give the birds an incentive to visit the trap, and as additional attraction we have a compartment with lure ducks, that are supposed to get the wild ducks to enter. In this study, we used the lure ducks as a natural infection experiment. Ten immunologically naïve, juvenile Mallards from a farm were placed in the trap and were then followed throughout an autumn season, and then for the next spring, summer and autumn. Fecal samples were collected daily and blood samples approximately every second week. A lot of samples, and collected with a precision that allowed us to give very detailed infection histories for each individual.

cropped-oimg_3582.jpg

In turned out that our study ducks varied tremendously in disease patterns, despite being of the same age, raised in the same farm, sharing the same little experimental enclosure and being exposed to the same environmental variation. All ducks became infected with flu within the first five days of being placed in the trap, but the number of infection days varied tremendously. And so did the number of retrieved virus subtypes, thus different individuals were infected with varying number of virus variants, in this case equal to different infection events.

Furthermore, we got really nice long-term patterns. After the initial primary infections early on in the first autumn, and a number of secondary infections later the same autumn, we recorded only a single infection day the next spring and summer. It wasn’t until the second autumn, when migration of wild ducks started in earnest again, that new infections were seen in the lure ducks. And in this case, no infection was of a subtype the individual had experienced the year before, suggesting very strong and long-lasting homosubtypic immunity.

Individuals also varied profoundly in their immune responses. We measured the humoral immune response, manifested as anti-influenza-antibodies (raised against the conserved nucleoprotein of the virus), across time. Have a look at the figure below; it really shows variation both on a temporal scale, but also at the individual scale, both in patterns and in height of response.

journal.pone.0061201.g004

So what does it tell us? To start with, there is a large difference between individuals in resistance/susceptibility to influenza A virus infection in Mallards. This difference is not only manifested in different infection histories, but also as very variable immune responses. Second, these differences are very likely determined by genetic differences, meaning that there are heritable differences, and thus traits that could be selected for by natural selection. Not all ducks are equal – and this important for our ability to model disease dynamics in this system. Is it really the mean that is important for assessing the transmission probabilities along migration? Perhaps it is the outliers that are driving the processes?

This study is a first step to adress individual variation, and there are already a couple of follow-up publications in the peer-review tube, so we will have opportunities to get back to this topic.

That’s all for now. Live long and prosper – and don’t treat disease simply as a property of the population.

Jonas Waldenström