Why are there so many flu viruses?

967259_10151436300376338_1637333899_oThe only thing constant in flu epidemiology is that it is always changing. New subtypes appear, old ones retreat; like a play where actors constantly change masks and costumes. Names are put forward in the press, such as the Mexican flu, which changed to swine flu, which changed to the new flu A/H1N1 (but, of course, the swine flu label stuck). The current evildoers in humans are H1N1 and H3N2. These are seasonal flu viruses, meaning that they circulate predominantly in humans, and only occasionally give infections in other animals. Both of them made the leap from another animal reservoir before becoming human flu viruses, and both, in turn, have once been avian influenza viruses.

Most readers will also remember the ‘bird flu’ virus H5N1. First of all: it still exists, endemic in parts of Asia, and in Egypt. It hasn’t left the scene. This virus is a highly-pathogenic avian influenza virus that cause rare, but often fatal infections in humans. The highly-pathogenic prefix means that it is an efficient poultry killer – with close to 100% mortality in infected chicken flocks. That’s like tossing in a mini nuke, closing the barn door and wait for the explosion. A mean virus, for a chicken.

However, the norm among avian influenza viruses is to be low-pathogenic, only causing mild infections in their hosts. For domestic poultry that equals a mild cold, in wild ducks even less so. Recently, yet another flu actor entered the scene: H7N9. This virus has caused a number of human infections and deaths in China, but contrary to H5N1 has not been associated with die-off of domestic poultry. New costume, new play, but still a deadly mix.

So, there is H1N1, H3N2, H5N1 and H7N9 out there – all with the capacity of infecting humans. Earlier flu pandemics have been caused by yet other viruses, and from studies of poultry workers and veterinarians we know that there are viruses with other H and N letters that can infected humans, but without leading to severe symptoms. Even if the list seems long, it is nothing compared to the total diversity of influenza A viruses. The H and the N are shorts for the two surface proteins hemagglutinin (responsible for attachment to cells, and to invasion) and neuraminidase (responsible for letting new virus progeny leave an infected cell). There are 16 H variants, and 9 N variants and as they are encoded on different genome segments, they can end up in any of 144 possible combinations, or subtypes. More than 100 of these subtypes have been found in ducks, and more than 70 of them have been found in our study population of Mallards at Ottenby, in SE Sweden. Thus, there are many, many more flu viruses out there lurking in the shadows.

But why are there so many viruses? And especially, why so many in Mallards?

In study published last week in PLOS Pathogens, we returned to this question and analyzed infection histories of more than 7,000 Mallards sampled at Ottenby during 8 years! Together these ducks were caught and sampled more than 18,000 times! The repeated capture and recaptures of ducks is a major benefit of our trapping scheme, as it allows us to follow the course of natural infections in different birds. This is a gospel I have been singing in two previous posts on individuals and reassortment, and a topic I am likely to return to. Predictable fellow, yes, yes. But let’s turn back to the subject.

What did we do? Well, we analyzed all cases where we had at least two characterized virus isolates from the same bird in the same season. Then we used this data to investigate how frequent reinfection with a particular subtype was given the first detected subtype and how this depended on time. This sounds rather simple, doesn’t it? In truth it was a rather large statistical undertaking, as the 25 supplementary files tells. The devil is in the detail – in this case in dealing with potential pseudo replication and test assumptions. Anyway, we leave the finer details of the stats for now and instead take a look at the table below. It is a contingency table, where rows and columns relate to H subtype at first and second infection, respectively. This means that the diagonal shows cases where the same subtype was isolated at both occasions. The colors highlight combinations that were either overrepresented (blue), or had a deficiency of cases (red) compared to the expected. The first thing to note is the diagonal, where very few cases of reinfections were noted. In other words, a bird infected with, let’s say, an H4 virus, will have a low probability of being infected with the same subtype again the same autumn. This is called homosubtypic immunity, and not different from what we want to achieve with vaccination in humans. Once you have had it you are immune (at least for some time…).

journal.ppat.1003443.g003 However, we also found a great degree of heterosubtypic immunity, meaning that an infection with one H subtype made reinfections with other related subtypes less frequent than expected. If you check the figure again, you can see that there are patterns to these cases of heterosubtypic immunity. In fact, they follow higher order clustering of hemagglutinin gene relationships, as can be seen in the next figure. H1, H2, H5 and H6 viruses belong to the H1 Clade, and a primary infection with any of these will make it less likely to be reinfected with other viruses of the same clade. The pattern was similar for other clades and was actually also detectable at the H Group level (the highest level of structure).

journal.ppat.1003443.g001

But what does this mean?

It is actually big business. It gives a very strong case for existing selection pressures for hemagglutinin gene diversification. Subtypes as a term predates the genomic area and is based on immune reactions. Typically, a subtype is defined as a group of viruses recognized by the same antisera (antibodies towards a particular virus). Subtypes are well resolved for hemagglutinin and neuraminidase, both in phylogenetic relationships and in responses to antisera. Things match. You would be tempted to think that virus subtypes have diversified until their antigenic properties are different enough for the immune system of the host animal to be unable to treat them with the same set of weapons. For instance, antibodies to an H1 virus shouldn’t interfere with an H2 virus infection.

Here, we show that heterosubtypic immunity is strong for hemagglutinin (but absent for neuraminidase), and that it follows genetic relationships. This means that there is ongoing warfare among hemagglutinin subtypes. If an individual is infected with one subtype, it then becomes harder for other related subtypes to enter and cause reinfections. The strength of this response, and its longevity, will be extremely important for infection dynamics at the population scale and drive which viruses that peak at different times. This is especially interesting in a migratory species like the Mallard, where viruses need to follow their hosts, not in only time, but also in geography. And it means that H subtypes are still diverging. The pace of this divergence would be very interesting to tackle, but will require good time series of influenza genomes (rest assured, we are sequencing like crazy and will return to this subject).

To conclude, our study provides evidence from the field on how natural selection in influenza A virus is driven by host immune processes and that it is evident for the most antigenic protein. The question ‘why’ is therefore dependent on disruptive selection. It also raises a bundle of additional questions. Is the diversification we see in influenza A virus the result of geographic allopatric processes, or through separation in different host species, or is there sympatric diversification going on?

More to do, more to do. This virus will keep us busy for sure.

Jonas Waldenström

Link to the article:

http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1003443

Bubbles and dots – novel ways of perceiving scientific impact

winner-takes-all-2012-07_3

Science is a very competitive business. We compete with our colleagues for positions, grants and tenures. The main currency is publications – the more the better, and in as good journals as possible. (Teaching is often portrayed as being important for your career, but in most cases that are simply not true – just lip service from the system). But how can we measure quality?

Quantity – the number of articles – is one way to show it. This is probably most important in the early part of your career, where each and every publication counts and competition for postdoc money is fierce. But for established scientists this is not as relevant; really, is a scientist with 60 publications better than one with 50? And of course the number of publications is a function of time too, and the old silverback will always win in such comparisons.

Everyone agrees that it should be quality, not quantity that should be most important. But we can’t read everything everyone is publishing – it is simple beyond the realms of possibilities, given the enormous flow of articles in peer-reviewed fora. So how, then, can we put a quality brand on our work? For the last 10 years, the light from the journal Impact Factor has been the beacon to which scientists have set their course. This is an index on how much the average article in a specific journal is cited by other articles in the years that follow. Undoubtedly a very crude measure, and an AVERAGE measure of the journal, not a metric of the specific articles that appears in the journal. (Or in other words: just because an article is published in Nature, it doesn’t need to be a gold nugget.)

Thus science has a huge problem in measuring researcher, article and journal qualities. The quest of publishing in journals with highest possible impact factors, rather than in the journal with the best scope for your study, overloads the peer-review system with an ever-increasing number of reviews.

For individual researchers, the total number of citations, and the arithmetic H2 factor (a value of 3 means the person has 3 articles that have been cited at least 3 times; a value of 23 means the person has 23 articles that have been cited at least 23, etc.) are becoming more and more used.

But impact can also be at the societal level; how well it gets across to the public. The journal family PLOS just released a beta-version of a new article-level metric system that measure a range of factors in articles published in their journals. Quick and easy you can see the number of viewings of a particular article (and all PLOS articles are open access, by the way), the number of downloads, the number of citations in different databases, the social media impact (twitter, Facebook, Wikipedia etc.) and how all these things change over time. You can also play around and compare different articles and journals. A fun exercise, but potentially informative too.

Five hundred PLOS articles matching the keyword 'avian influenza'

Five hundred PLOS articles matching the keyword ‘avian influenza’

The graph above shows the change over time in citations for 500 articles matching the keyword avian influenza. Different journals in different colors, PLOS One in yellow, and the high impact journals PLOS Biology, PLOS Medicine and PLOS Pathogens in green and shades of purple, respectively. And, yes, over time the average article seem to do better in the ‘best’ journals, but the spread in PLOS One is more interesting – with many articles with as good, or better impact than those published in the top-notch journals.

You can also gain insights in where science is made. For instance have a look at where researchers on sexual selection have their headquarters. The dominance of Europe and America is monumental; partly of course due to historic reasons, research infrastructure, funding etc., but likely also because of language (Russians still publish a lot in Russian, Latin American researchers in Spanish, etc.).

Affiliations of researchers on 500 sexual selection papers.

Affiliations of researchers on 500 sexual selection papers.

Speaking about sexual selection. Guess which article that has had highest ALM impact? The dot in the graph below is an article that appeared in PLOS One on fellatio in bats. Perhaps not the most important paper in terms of science, but a curiosity teaser likely picked up by a lot of newspapers. This paper has been cited 6 times, but have more than 9000 shares on social media and 288,000 views at the homepage.

fellatio in batsFinally, what could PLOS do to make it better?

  • It would be awesome if this could be more in Gapminder style, where the user could use combinations of search terms to contrast the results. For instance, if I want to see how well my articles on flu are doing in relation to other articles on flu – how can I do that?
  • It would also be interesting to add journal or keyword-based regression lines.
  • The author institution map is very slow when many articles are chosen. Speed it up please!
  • And of course, it would be nice to see a similar system incorporating other journals too. But, that’s something for the future.

A good initiative!

Jonas Waldenström

The duck genome – and why it is important

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Jonas Waldenström

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

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!

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

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.

Knarrens svanesång – finns det någon ljusning för försommarnattens punkigaste fågel?

En särling i vår fågelfauna. Bild från Wikipedia, av Sergey Yeliseev

En särling i vår fågelfauna. Bild från Wikipedia, av Sergey Yeliseev

Om man har kommit kornknarren in på livet så släpper inte kärleken taget. Aldrig någonsin. Det är en särling i vår fågelfauna. En skränande punkare som taktfast terroriserar natten med sitt raspiga snärpande. Där andra fåglar sjunger vackert eller har utpräglad fjäderdräkt har knarren attityd, hög röst och ett lite argt dinosaurielikt utseende.

Dessvärre går det inget vidare för kornknarren i dagens förändrade jordbruksvärld. Knarren är egentligen en rallfågel, men till skillnad från sina släktingar som är starkt knutna till våtmarker är knarren förtjust i lite torrare marker. Inte helt torra, men en fuktig frisk äng, en vallåker med inslag av diken med högre vegetation, eller en trädaåker är mumma. Den stora strukturomvandling av jordbruket som präglat hela 1900-talet och som fortgår än i dag har inneburit stora förändringar för kornknarren. Åkrar och vallar är nuförtiden torra, dikade och hårt rationaliserade. Vallen i södra Sverige slås redan i maj, ofta precis när knarren har börjat häcka. Skördaren blir som en giljotin på hjul som hackar boet och ungarna, samtidigt som biotopen försvinner för de vuxna fåglarna.

Mannaminnet är kort. Arter kommer och går utan att vi riktigt märker det. Men med knarren finns för de som varit med ett tag en pockande känsla. En tystnad. Tyst när det borde varit ljud. En skriande tystnad. När jag började skåda fågel på allvar under tidigt 1990-tal var kornknarren relativt vanlig på Öland, dels i de allra sydligaste socknarna, dels omkring Löt på norra delen av ön. Runt vandrarhemmet i Ås kunde man höra upp mot 10 knarrar från en och samma plats. Går man tillbaka ytterligare 100 år i tiden var knarren en karaktärsfågel på flera håll i landet. Bonden somnade till ett taktfast snärpande i ängen.

Under 90-talet startade Ottenby fågelstation en WWF-finansierad studie om kornknarr. Under ett antal år fångades knarrar in och förseddes med små radiosändare. Genom sändarna kunde fåglarnas rörelser pejlas i landskapet och kunskap om häckningsframgång med mera lättare studeras. Du som läser dessa rader kanske inte inser hur svårt detta projekt var. Till och börja med sker all fångst på natten, med hjälp av bandspelare och tunna slöjnät. Och inte är knarren lättfångad. Den är klok, rent av pillemariskt smart, och de flesta nätter kunde kanske endast en eller ett par knarrar fångas. Sedan skall deras rörelser följas. Tekniken har tagit elefantkliv på sistone, men på 90-talet var det klassisk radiopejling som gällde. Åka till stället fågeln senast var på. Pejla. Ingen där. Byta frekvens. Pejla. Ingen där. Byta frekvens. Pejla. Åka någon annanstans. Pejla igen. Inse att sändarhelvetet trillat av och att knarren måste fångas in igen. Och hur hittar man ett bo i ett hav av nässlor? Eller äggskal i en slagen vall? Ni förstår att detta var ett projekt för folk med extremt stort tålamod. Alternativt galningar. Kontentan av projektet blev att kornknarrens dystra situation lyftes upp på agendan och att ett antal åtgärder föreslogs.

Nu ca 20 år senare har jag återvänt till Öland. Lite äldre och mer stadd i kassa. Jag har köpt ett hus på sydöstra delen av ön. I knarrarnas gamla fäste. Men nu saknas knarren på de flesta ställen där den förr fanns. Fortfarande sitter det ett par stycken mellan Gräsgård och Össby, men antalet är dykande. Nu för tiden hör man fler vaktlar på en sommar än vad man hör knarrar. Men hjärtat klappar fortfarande för knarren och jag brukar ta ett par cykelturer varje år för att få in rytmen av knarr-snärpandet i blodet. I år har jag inte hunnit. Jobb, ungar och ett hus som skall renoveras. Knarren har fått vänta.

I helgen kom min granne Jan med ett bylte i näven. Redan på håll såg jag ett par långa ben, rödbruna vingar och ett litet hönsahuvud – en kornknarr! Men död. Påkörd av några andra bekanta bara 200 meter från mitt hus.

bild

Sitter och håller knarren i handen. Fortfarande varm. Vingen är bruten, antagligen ryggen också. Lite kornknarrsnor rinner ner på handen. Efter tag med blod i. Blåser den på magen. Jo, magfjädrarna är bortplockade; som isolering till boet, en rynkig ruvfläck tyder på att det är en hona som lagt ägg. En av de sista knarrarna på södra Öland.

Kan inte riktigt släppa det där med knarren. Som sagt: en kärlek som inte släpper. En kliande sårskorpa som måste pillas på. Ringer upp Henrik Ehrenberg som de senaste åren tillsammans med några kamrater tagit upp tråden om knarrarnas liv och leverne. Tekniken har utvecklats och Henrik med flera sätter på små ljusloggrar på benen på knarrarna. Loggern mäter dagsljusets längd och man kan med hjälp av det och en klocka räkna ut var på jordklotet fågeln befunnit sig. En ganska billig grej, men väldigt användbar för vissa fågelarter. Positionerna blir inte helt exakta, men en upplösning på ett par kilometer till ett par mil spelar inte så stor roll om man vill ta reda på var i Afrika kornknarren övervintrar. Kruxet är att man måste återfånga fågeln – något som de faktiskt har lyckats med i år! Skall bli spännande att se resultaten av deras studier!

Ett knippe illegalt skjutna knarrar från Grekland - farorna lägs flyttningen är påtagliga.

Ett knippe illegalt skjutna knarrar från Grekland – farorna lägs flyttningen är påtagliga.

Det blir ett långt samtal med Henrik. Nja, helt bra är det väl inte för knarren. Men det är inte helt nattsvart. I Skåne, Östergötland och andra ställen utanför de gamla kärnområdena (Öland, Gotland och Uppland) har det dykt upp fler knarrar de senaste åren. Och i delvis nya biotoper. Exempelvis finns det rapporter om knarrar från energiskog – så länge det finns gott om nässlor. Men där finns andra faror. Förändrade miljöer under flyttningen. Jakt i Medelhavet. Listan är lång.

Kanske är det inte ett helt kört. Men känslan av att knarren sjunger på sin svanesång är stark. Och försommarnatten riskerar att för alltid förändras.

Jonas Waldenström (på tåget mot Uppsala)

Almost forgot: it is dead duck day

As our blog has the subtitle “of ducks and men” we should not forget to commemorate the ‘dead duck day’: http://moeliker.wordpress.com/the-duck/

This story went viral a couple of years ago and interested readers can find the original published story here: http://www.hetnatuurhistorisch.nl/fileadmin/user_upload/documents-nmr/Publicaties/Deinsea/Deinsea_08/Deinsea_8_15_Moeliker_.pdf

Over and out.

Jonas Waldenström

Graduation day!

Life and education is a winding road (Photo Linus Heedh)

Life and education is a winding road (Photo Linus Heedh)

Today it is graduation day for our Biology Bachelor students! Time for celebration and some sparkling stuff! Sunny weather! Nice clothes! Proud mums and dads!

I have been at Linnaeus University for seven years now, and the last three years more involved in teaching. All students that graduate today have been on my course in Zoonotic Ecology and Epidemiology, and four of them have done their final projects in my research group. I know them and like them – super students! Hats off for all of you, and especially to Anu Hellin, Olivia Borg, Johanna Carlbrand, and Andras Turai!

Their projects ranged from beta-defensin phylogenies in Anseriformes birds (Anu), gammacoronavirus diversity in Mallards (Olivia), Candidatus Neoehrlichia mikurensis presence in biting ticks (Johanna), to comparative studies of screening methods for avian paramyxoviruses (Andras). With some additional analyses much of this data will end up in publications! You really, really have done a great job!

Best wishes for your future where ever you choose to go: Linnaeus University, other universities or out on the labor market! Your future is so bright, you gotta wear shades!

Jonas Waldenström