What can 1081 influenza viruses tell you?

By Jonas Waldenström

Today we published a major article in a well-respected journal. The reason why I write major is not to brag (although I am very pleased). No, the reason for that epithet is that the paper is based on such a huge long-term effort. In fact, in this paper, ten years of fieldwork, laboratory work, and statistical analyses are boiled down into nine glossy pages!

As frequent readers of this blog probably know, mallards and flu is our main study system. Through repeated captures, samplings and recaptures of ducks at a migratory stopover site we have built very large datasets that we now can analyze for long-term patterns in virus-host interactions. The title of the current paper is: “Long-term variation in influenza A virus prevalence and subtype diversity in migratory mallards in northern Europe”

Influenza A virus prevalence was in part determined by peaks of mallard migration. Photo by Sergey Yeliseev under a CC BY-NC-ND 2.0 license.

What we did was to screen all 22,229 samples collected in the period 2002-2010 for the presence of influenza A virus RNA. Positive samples were then inoculated in eggs in order to obtain virus isolates. After this process, we had a virus bank consisting of 1081 viruses of 74 different subtypes, ranging from H1N1 to H12N3. As you can see from the figures above, influenza virus research is time-consuming and costly, and the travel from sample to RRT-PCR-positive to characterized virus could be described as a negative logarithmic function. It is all about big numbers! You need a lot of samples to get the statistical power to say something about virus ecology and epidemiology at the level of subtypes. You also need to be stubborn as a mule.

There are three major results that I would like to share with you.

First, we were able to fit a model of how influenza A virus varied with season in the sampled mallard population. The resulting figure very neatly shows how the virus starts low in spring, becomes more or less absent during the breeding season, and how it suddenly increases in frequency in August when the first wave of migrating mallards arrive at Ottenby. The August peak is followed by a second peak in October-November, likely consisting of mallards with a Finnish or Russian origin. Actually, the plot looks like a camel!

Influenza A virus prevalence showed two distinct peaks in autumn, one in August and one in October-November.

However, plotting prevalence rates over time has been done before. The strength with our analysis is that it includes and accounts for the variation in prevalence induced by year effects. Mallards are migratory birds, but their timing of migration is rather flexible. In years characterized by mild autumns they arrive late at our study site, and in years with harsh autumns they are early. The final model accounted for approximately half of the variance in prevalence, which is pretty good all considered.

Second, I would like to stress the incredible diversity of subtypes! The two surface proteins hemagglutinin (16 variants) and neuraminidase (9 variants) sit on two different RNA-segments in the genome and can theoretically be combined in 144 different ways, or subtypes as we call them. We found 74 different HA/NA subtypes. In addition, some subtypes are likely not functional, or would have to include a hemagglutinin (like H14 or H15) that is restricted to areas outside Europe. This plethora of genotypes is a world record from a single site. Or to put it in perspective: more than half of the possible subtypes have been found in mallards trapped in our little duck pond on the southern point of the island Öland, in the SW part of the Baltic Sea, in Northern Europe. A speck in the ocean, but a global diversity of viruses.

Further, the 1081 viruses were not evenly distributed on subtypes. Rather, some subtypes were very common, such as the H4N6, the H1N1, or the H2N3 subtypes. Others were rare, including the famous combinations H5N1 and H7N9, both which were only found once, and not in the pathogenic forms known from elsewhere. Interestingly, the high frequency of certain combination, and a low frequency of other combinations despite the HA and NA being common in other virus constellations suggests that some subtypes have low fitness. Consider for instance H4N3 that was found only 5 times, while the H4 hemagglutinin was found in 291 viruses, and the N3 neuraminidase in 116 viruses.

A cute mallard couple. Photo by Chuq Von Rospach under a CC BY-NC-ND 2.0 license

Third, and perhaps most interestingly, we found a heterosubtypic effect at the virus population level. By grouping viruses in classes depending on their HA relatedness we could see that the different virus classes peaked at different times within an autumn. The virus type that was common in early autumn was rare in late autumn and vice versa. Understanding how individual and herd immunity processes affect influenza A virus dynamics in nature is highly warranted, as that would aid our capacity to predict how the virus population could change over time. Viruses in wild birds remain an important pool from which genotypes could be seeded in domestic animals, and even humans.

Finally, I would like to say how incredibly fortunate I am to have had the opportunity to work in such a hard-working and persistent research group. The work we presented today has been collected by a small army of duck trappers, a score of laboratory staff, a handful PhD-students, a couple of postdocs and a quartet of PIs from Kalmar, Uppsala and Rotterdam. And the most important of all was Dr Neus Latorre-Margalef, who carried this publication from start to finish! Well done!

Link to the article:

Latorre-Margalef, N., Tolf, C., Grosbois, V., Avril, A., Bengtsson, D., Wille, M., Osterhaus, A.D.M.E., Fouchier, R.A.M., Olsen, B. & Waldenström, J. 2014. Long-term variation in influenza A virus prevalence and subtype diversity in migratory Mallards in Northern Europe. Proceedings B, online early.

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Pathogen tea bags, golden orioles and climate change

By Jonas Waldenström

Close your eyes and let your mind wander. Try to imagine the most beautiful place you can think of, fill it with scents and colors, let flowers blossom and birds sing, let there be volcanoes and old ruins! And let there be food, rich wonderful food, and wines that make your heart sing! Where would that be? What kind of paradise is that? The answer is simple: an Italian island in April and May! There are few places on this planet that are more endearing than the Mediterranean coast in spring, where the wing beats of history are present in the landscape itself, and where the stunning orioles and bee-eaters make any birder go bananas!

Recently, I wrote a post on how biologists are obsessed with grim, dirty and tedious fieldwork – the worse the better! I poured a lot of wisdom in that text and have received a share of acknowledging nudges from colleagues. However, in science all normal distributions have outliers, be it the trunk length of elephants, dunnock testis size, or the harshness of fieldwork. Thus, admittedly, if there is fieldwork that is grim, there must be fieldwork that is agreeable, perhaps even wonderful. The most extreme outlier is the fieldwork conducted on Italian islands.

The particular island for this story is the island of Capri. This gem is situated in the mouth of the bay of Naples, with Ischia and Proscida as closest neighbors in the north, and Sorrento and the Amalfi coast to the south. Capri has been inhabited for a very long time, perhaps as far back as the Bronze Age. The Roman emperor Augustus started to develop the island, and his successor Tiberius continued and built several villas on Capri. Ever since the Romans, Capri has been the home for the rich and famous – and lately to a few Italian and Swedish researchers. To get to Capri you either have to sail your own yacht, or you take the ferry from Castello d’Ovo in Naples to Marina Grande on Capri. Once ashore, the island is towering high above you, reaching some 580 m in altitude at the highest peak. From Marina Grande you need to transport yourself to the village of Anacapri, on the plateau on the western part of the island.

Villa San Michele is a piece of serene beauty. From Wikimedia Commons

Anacapri is a very pleasant Italian town, with several restaurants and cafés, but we are not stopping yet. The goal for our imaginary travel is just a little further bit ahead. From the piazza you need to walk along Via Capidimonte together with the large crowd of tourists that are heading to Villa San Michele – one of the island’s most famous attractions built by the eccentric Swedish physician Axel Munthe. Through the locked gate in the villa garden we take the winding path to the top of the mountain. There at the summit lies Castello di Barbarossa, the remnants of the legendary pirate Red-beard’s castle. The view is stellar! On one side there is a 300 m fall, and on the other side a more gently declining hillside covered in macchia vegetation. Welcome to Capri Bird Observatory!

Axel Munthe bought the old castle and the mountaintop in order to give birds a shelter during migration. Hunting of birds was a large operation at Capri in those days, and still is in some parts of Italy today. Axel Munthe pitied the animals and took a strong stand against hunting. When he died, the Villa and the mountain were given to the Swedish state and in 1956 the first Swedish ringers visited the island and started the observatory. Since 20-30 years the site is run by Italian ringers as a part of their island project ‘Progetto Piccole Isole’.

Yes, that’s the castle! Photo Magnus Hellström

It is truly the best field site in the world. Not only is it strikingly beautiful, it is also a place where you can do great science. In April and May the island can receive large downfalls of migratory birds, particularly songbirds, that have crossed the Mediterranean Sea over night. At times there are birds everywhere, in all colors and sizes, from Willow Warblers, Whinchats and Tree Pipits to Quails and Scoops Owls. Traditionally, bird ringing has been more commonplace in Northern and Western Europe and the trapping series at Capri is in fact the longest available from the Mediterranean area. A few years ago we collated the data from Capri with similar data from Scandinavian bird observatories and analyzed long-term trends in the timing of migration. This data, published in Science, showed that the tropical migrants arrived earlier to the breeding areas as a response to an earlier spring, but that the major shift was a more rapid migration through Europe, and not an earlier arrival at the Mediterranean region.

The flying banana – the Golden Oriole. During migration you see flocks of this stunning bird at Capri. Image from RSPB

In recent years, a team of Swedish researchers has visited the castle together with the Italian ringers. But this time it is not the birds that are in focus, but the ticks the birds are carrying. Ticks are the ultimate pathogen vector, a cosy teabag for a variety of blood-borne viruses and bacteria, some which are very virulent to humans. Inside the tick the pathogens can survive and get transported to a new host without exposing themselves to the harsh outside environment. Some pathogens can even multiply within the tick, thus utilizing the arthropod as an intermediate host.

After a few field seasons, and the labor of an army of students in the lab, the publications are starting to emanate. Just a week ago, we published one survey of West Nile virus from Mediterranean ticks in Infection Ecology and Epidemiology Journal. This study was mainly a report of negative results, but a year ago we published a paper in Emerging Infectious Diseases on the findings of Crimean-Congo hemorrhagic fever virus (CCHF), and more analyzes are due to be published soon. The ticks are truly pathogen arcs, and birds potentially important for long-distance dispersal of diseases.

Thus, the heritage of Axel Munthe lingers. And in the distance Vesuvio sleeps.

 

Hagman, K., Barboutis, C., Ehrenborg, C., Fransson, T., Jaenson, T.G.T., Lindgren, P-E., Lundkvist, Å., Nyström, F., Waldenström, J., & Salaneck, E. 2014. On the potential roles of ticks and migrating birds in the ecology of West Nile virus. Infection Ecology and Epidemiology 4: 20943

Lindeborg, M., Barboutis, C., Ehrenborg, C., Fransson, T., Jaenson, T.G.T., Lindgren, P-E., Lundkvist, Å., Nyström, F., Salaneck, E., Waldenström, J. & Olsen, B. 2012. Migratory birds, ticks, and Crimean-Congo hemorrhagic fever virus. Emerging Infectious Diseases 12: 2095–2097.

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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

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.

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…

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.

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!

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