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).

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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

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.

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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.

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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.

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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