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


3 thoughts on “This flu, that flu, and Tamiflu®

    • The doses are not/will not be evenly distributed in time and space. Already now, some countries, for instance Japan, are chewing Tamiflu in excess every seasonal flu season, while other countries are not using much at all.

      Anyway, a lot of people and a lot of drugs. Some of the antibiotics we use in health and agriculture are also persistent and may invoke resistance in bugs for a long time.

  1. Pingback: Going, going, gone – what happens to antiviral resistance when the selection pressures disappears? | Zoonotic Ecology and Epidemiology

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