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

Phylogenetic trees for the 8 different IAV segements, see

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.


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