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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Looking back on 2013 (part I): A dozen publications, some zombies, but no pandemics

By Jonas Waldenström

Post-apocalyptic dawn will have to wait some time more

A year goes by so fast, and soon it is time to close the book on 2013. One thing we can conclude, at least, is that there was no apocalypse, and no end-of-humanity pandemic. However, there have been some worrying notes on new emerging pathogens in 2013. On top of the list of concern we find the MERS coronavirus in the Middle East, and the H7N9 low-pathogenic influenza virus in China. Neither of them has caused many human casualties, nor are they common or widespread. No, it is not what they do, but what they potentially could do that worries the disease world. The MERS virus is related to SARS – a deadly viral pathogen that in 1997 jumped from bats, to civets, and further to humans, and which was on the brink of causing a pandemic before it was fortunately contained and stopped. The other bad guy, the H7N9 influenza virus, carries novel antigenic properties to which the human population lacks immunity; thus, if it becomes adapted to spread between people (and not as today, between infected poultry and humans) it could turn into pandemic flu. Both viruses face strong guilt by association, you can say. These pathogens, and others, are like butterflies, fluttering in and out of detection. Worrisome echoes on the radar screens at WHO and CDC. They are also good examples to why field biology is needed in medicine: we need to track reservoirs of diseases, new and old, and we need to understand how diseases evolve. And that’s exactly what we try to do in ZEE. (In sale pitch jargon: we are the good guys!)

So what happened in ZEE during 2013? In this and other posts we will give you a hint of what we did, and how things went.

Publications. 2013 has been a productive year for the ZEE group! More than a dozen publications were published from Linnaeus University (plus a bunch from Uppsala). Most of these are available freely and you can reach them by following the links below. This year was also the year when this blog was launched! A motto we have is to provide popular accounts on the science we do. Thus, for some of the publications there is a link to a blog post in the list below. Read them – lots of fun!

1. Tolf, C., Wille, M., Haidar, A-K., Avril, A., Zohari, S. & Waldenström, J. Prevalence of avian paramyxovirus type 1 in Mallards during autumn migration in the western Baltic Sea region. Virology Journal 10: 285  [Ebola, Chikungunya and Newcastle – of places, names and Mallard viruses] http://bit.ly/IToOVG

2. Gillman, A., Muradrasoli, S., Söderström, H., Nordh, J., Bröjer, C., Lindberg, R.H., Latorre-Margalef, N., Waldenström, J., Olsen, B. & Järhult, J. 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. [This flu, that flu, and Tamiflu®] http://bit.ly/1gw2roa

3. Safi, K., Kranstauber, B., Weinzierl, R., Griffin, L., Rees, E., Cabot, D., Cruz, S., Proaño, C., Takekawa, J. Y., Waldenström, J., Bengtsson, D., Kays, R., Wikelski, M. & Bohrer, G. Flying with the wind: scale dependency of speed and direction measurements in modelling wind support in avian flight. Movement Ecology 1: 4. [As the Mallard flies] http://bit.ly/19mFVtC

5. Wille, M., Tolf, C., Avril, A., Latorre-Margalef, N., Bengtsson, D., Wallerström, S., Olsen, B. & Waldenström, J. 2013. Frequency and direction of reassortment in natural influenza A virus infection in a reservoir host. Virology 443: 150-160. [How do you do, the things that you do, Mr Flu?] http://bit.ly/18JpZkp

6. Latorre-Margalef, N., Grosbois, V., Wahlgren, J., Munster, V.J., Tolf, C., Fouchier, R.A.M., Osterhaus, A.D.M.E., Olsen, B. & Waldenström, J. Heterosubtypic immunity to influenza A virus infections in Mallards may explain existence of multiple virus subtypes. PLoS Pathogens 9(6):  e1003443. [Why are there so many flu viruses?] http://bit.ly/IUiwoM

7. van Toor, M. L., Hedenström, A., Waldenström, J., Fiedler, W., Holland, R.A., Thorup, K. & Wikelski, M. Flexibility of continental navigation and migration in European mallards. PLoS ONE 8(8): e72629. [Perdeck revisited – or how does a Mallard know its way?] http://bit.ly/1904O0h

8. Tolf, C., Latorre-Margalef, N., Wille, M., Bengtsson, D., Gunnarsson, G., Grosbois, V., Hasselquist, D., Olsen, B., Elmberg, J. & Waldenström, J. 2013. Individual variation in influenza A virus infection histories and long-term immune responses in Mallards. PLoS ONE 8(4): e61201. [Disease is a property of the individual] http://bit.ly/1ebcGOz

9. Hellgren, O., Wood, M. J., Waldenström, J., Hasselquist, D., Ottosson, U., Stervander, M. & Bensch, S. 2013. Circannual variation in blood parasitism in a sub-Saharan migrant passerine bird, the garden warbler. Journal of Evolutionary Biology 26: 1047-1059.

10. Griekspoor, P., Colles, F.M., McCarthy, N.D., Hansbro, P.M., Ashhurst-Smith, C., Olsen, B., Hasselquist, D., Maiden, M.C.J. & Waldenström, J. 2013. Marked host specificity and lack of phylogeographic population structure of Campylobacter jejuni in wild birds. Molecular Ecology 22: 1463-1472. [Of chickens, wild birds and men – host specificity in Campylobacter jejuni] http://bit.ly/1bCcFeu

11. Griekspoor, P., Olofsson, J., Axelsson-Olsson, D., Waldenström, J. & Olsen, B. 2013. Multilocus Sequence Typing and FlaA sequencing reveal the genetic stability of Campylobacter jejuni enrichment during coculture with Acanthamoeba polyphaga. Applied and Environmental Microbiology 79: 2477-2479.

12. Hernandez, J., Johansson, A., Stedt, J., Bengtsson, S., Porczak, A., Granholm, S., Gonzalez-Acuna, D., Olsen, B., Bonnedahl, J. & Drobni, M. 2013. Characterization and comparison of Extended-Spectrum β-Lactamase (ESBL) resistance genotypes and population structure of Escherichia coli isolated from Franklin’s gulls (Leucophaeus pipixcan) and humans in Chile. PLoS ONE 8(9): e76150. [Travel the world – can antibiotic resistant bacteria hitchhike with migratory birds?] http://bit.ly/19mF2kV

13. Olofsson, J., Axelsson-Olsson, D., Brudin, L., Olsen, B. & Ellström, P. 2013. Campylobacter jejuni actively invades the amoeba Acanthamoeba polyphaga and survives within non-digestive vacuoles. PLoS ONE 8(11): e78873. [doi:10.1371/journal.pone.0078873]. [Good morning Mr Amoeba, may I come in?] http://bit.ly/1cqPybs

We study ducks, and they study us.

Staff and students. A year is also a quarter of a PhD time span, and half of a master student’s time. This means that there are many comings and goings in a research group over time. This year, one PhD left the nest and graduated, and four are due in 2014. No new PhD student started, but there were three babies born, thereby boosting the current ZEE children count to more than 10, enough for a football team!

Two former PhD students got a flying start: Dr Neus Latorre-Margalef is on a postdoc in Athens, Georgia, funded from the Swedish Research Council (VR), and Dr Josef Järhult in Uppsala got a huge researcher grant from VR to build up his own group! Fantastic news!

After finishing her MSc last year, Anna Schager got a PhD position in Italy in the spring. Olivia Borg and Anu Helin made their honors’ degree in the lab and then moved to Uppsala for MSc studies, while Johanna Carlbrand and Andras Turai stayed on with MScs at Linnaeus University.

With no real apocalypse in sight, 2013 instead became the zombie year, a trend that culminated with the WWZ movie. If you want to prepare for the coming zombie apocalypse, the author and blogger Colin M. Drysdale has a range of tips for you, including how to make projectile weapons with toilet brushes. You never know, such a skill may come in handy one day when the undead are going for your entrails. Next week we will get back on the-end-of-year-theme and present the best and the worst links/papers/topics of 2013! Cheers!

Perdeck revisited – or how well does a Mallard know its way?

By Jonas Waldenström

At this time of the year the air is full of migrating birds. Some, as cranes or geese with their conspicuous formations are easily spotted with the naked eye, while other birds, including most smaller songbirds, fly at altitudes where you need a scope to see them. But you can often hear them; each species has its own tune, and an experienced ear can tell them apart on call alone.

The question “how do they find their way” is as old as the field of ornithology itself. Generally, migration wouldn’t be possible without some sort of compass; a way of telling the bird in which direction to move. It has been shown that birds may use the sun, the stars, and the earth’s magnetic field for assessing their heading. And in some species also visible cues, a sort of map sense from previous travels, or even olfactory cues (a posh word for smelling where home is). As the vast majority of birds migrate without the guidance of their parents (which seems reserved to some flock-living species), a juvenile bird must be born with not only the tools to assess where it is, but also a sense of where it should go.

One of the pioneering fathers of ornithology was the Dutch professor Albert Christiaan Perdeck. He made one of the first real tests on how birds can sense where they are going, and how they can adjust the course if they get out of track. In order to test this he wanted to do a displacement study, where birds should be experimentally transported to a novel site, far from the catching site. As this study was conducted in the 1950s, in the pre-gadget era of ornithology, he needed a species that he could catch in large quantities, and where ring recovery data could be collected. His choice of study animal was the European Starling Sturnus vulgaris, a common farmland bird in most of Northern Europe. Starlings in autumn can aggregate in huge flocks, sometimes consisting of several thousand individuals, and was thus a good target species for Perdeck.

With a remarkable enthusiasm, the team caught and ringed thousands of starlings. Some were released at the ringing site in the Hague, while the other half were transported with airplanes to Switzerland and released. After some time the ring recoveries started to come in, and the results were extremely interesting. It seemed as the young starlings had a vector compass, as the birds that were transported south stayed on the same heading as they had when they were caught. But instead of ending up in Holland, the young starlings ended up way south, sometimes even on the Iberian peninsula. I wrote ‘young’ deliberately, as there was a clear age effect. Where the juvenile birds continued on the same vector, the adult starlings compensated for the displacement, changed course and headed to the original winter quarters. Adult birds are more experienced, and in the starling case they were able to adjust to the circumstances and get back on the right track. A quite remarkable feat – some of my colleagues cant find their way to the university canteen without a helper…

Spurred by the old studies (classics, you could say) and the advancement of new tracking tools we conducted a similar experiment with Mallards. The study was a collaborative effort with scientists from Sweden, Germany, the UK and Denmark (with the lead from Professor Martin Wikelskii at the Max Plank Institute for Ornithology, in Constance, Germany). Today’s gadgets can do stuff Perdeck could only dream about. During two autumn seasons, we caught juvenile Mallard females at Ottenby – our beloved duck field site – and equipped a total of 76 birds with satellite GPS transmitters. Half of the ducks were released at Ottenby, and the other half were transported in a private airplane to Lake Constance in southern Germany and released there. The tags had solar panels and, in the best of circumstances, had the potential to send data for at least two years; providing highly accurate GPS fixes at several times a day.

However, the best of circumstances is not often met in nature. The tags on the birds in Ottenby had problems with the lack of sunshine during Swedish late autumn and winter, and many of them just went offline. But a fair number of tags delivered data on movements both in autumn/winter and in spring, when birds headed to their breeding grounds. Contrary to the Perdeck’s starlings, our displaced Mallards did not continue migration in autumn; they stayed in the Lake Constance region. Of the Mallards released at Ottenby, some continued migration to the general wintering area of our study population, that is Denmark and Germany, south to The Netherlands.

After the winter: “most of the translocated ducks headed straight north-north-east, as if heading towards Ottenby, with one duck going as far as northern Sweden. Three of the transported ducks, however, first headed in a more easterly direction and turned northwards when reaching the longitudes of the area the control birds migrated to. It is unclear how these birds decided when to turn north, but the movement trajectories could be interpreted as if individuals had noticed that they were in the wrong place and then corrected for the southward translocation. Based on the observation that this second group of transported ducks ended up in their potential natural breeding grounds, and the first group had a more northerly heading than the control group, we conclude that mallards, just like the starlings from Perdeck’s original experiment, can correct for translocation during the spring season following the experiment.”

Thus, there was quite large differences between individuals in the translocated group, from those that seemed to take the shortest route north to Ottenby in spring, to those that followed a eastern direction (and then going north), more in the direction of what they should have had if the stayed in the normal wintering grounds: a flexibility in continental navigation and migration.

The article is open access and can be found here.

Badrul Hasan presented his thesis on antimicrobial resistance in Bangladesh wildlife

I spent the day in Uppsala – the old student town where I once did my undergraduate studies in Biology. It was good to be back, to meet some great friends and collaborators, and – of course – to attend the thesis defense of Badrul Hasan!

A proud Dr Hasan!

Badrul came to Sweden from Bangladesh four years ago and have spent time both with us at Linnaeus University and at Uppsala University. It was great to see him present his thesis – calm, confident and skilled!

So what has Badrul done? The topic of the thesis is very timely – antimicrobial resistance in wildlife. By sampling wild birds, domestic poultry and humans in different areas of Bangladesh, Badrul tries to connect the various sources of resistance in an epidemiological framework.

Sometimes the thesis is akin to a horror-story! The situation in Bangladesh is alarming, to say the least – with widespread occurrence of multidrug resistance, beta-lactamase producing Escherichia coli more or less wherever you stick your sampling swab, and unrestricted use (or rather misuse) of antibiotics in agriculture and aquaculture. A ticking bomb.

Indian House Crows doing what they are best at – searching for food in waste

In one of Badrul’s studies, he investigated the carriage of resistant bacteria in Indian House Crows at two hospital campuses. The crows were thriving, feeding on waste put out in the yard – a mixture of surgical leftovers, patient’s food and normal garbage. And the antibiotic resistant bacteria were thriving too. The close connection between humans and crows is evident, and there aren’t any closed doors between indoors and outdoors. What we select for in agriculture and in human medicine in terms of resistant bacteria are the very same infections that later will be close to impossible to treat when our children’s children get sick.

Or quoting Badrul:

“Every morning you wake up in bed, you hear a crow”

Some links to Badrul’s published papers:

The thesis as pdf

Antimicrobial Drug–Resistant Escherichia coli in Wild Birds and Free-range Poultry, Bangladesh

High prevalence of antibiotic resistance in pathogenic Escherichia coli from large- and small-scale poultry farms in Bangladesh.

Dissemination of NDM-1.