Going, going, gone – what happens to antiviral resistance when the selection pressures disappears?

By Jonas Waldenström

Antivirals, antibiotics, antifungals and anthelminthics – over the years we humans have developed a range of pills to kill off the nasty bugs that infect us. However, whenever we take these pills – or feed them to our domestic animals – we potentially reduce their effectiveness. The drugs are very potent, but even more so is evolution, and any mutation that circumvents the action of the drugs has the potential to spread in the pathogen population. The emergence of resistance is not a mere theoretic observation – infections caused by resistant microorganisms are increasing all over the world.

A question to ask is what the fate of such a mutation is once the selection pressure is lifted. Will the mutant have a lower fitness in the absence of the drug? Will it remain in the population, and if so, at what level? There are several examples of resistant bacteria being maintained even when there is little or no selection pressure from antibiotics. In other words, even if we stop taking the pills the bug is still resistant. One of the reasons lies in compensatory mutations in other genes, restoring the fitness of the mutant to that of the wild type. Additionally, many resistance genes in bacteria occur together in plasmids, which means that co-selection can occur between different traits.

A similar arms race occurs in viruses too, just think about HIV and the intense struggles to find good, lasting antivirals (and vaccines). One problem with viruses is that they lack metabolism, which gives fewer potential drug targets. In the case of influenza A viruses (the favorite virus on this blog) the antivirals are so called neuraminidase inhibitors that interferes with the neuraminidase protein and the release of virions from infected cells. The compounds are sold under the brand names Tamiflu and Relenza, and although the effect of them is not staggeringly good, they are still sold in large quantities during seasonal flu epidemics. And in the case of a pandemic with a novel influenza these compounds are among the few available treatment options until a vaccine can be manufactured.

Our twin lab in Uppsala has investigated resistance evolution in influenza viruses the last 5-10 years. Their model system is natural infections in Mallards, where virus can be propagated in different ducks with different exposures to antiviral drugs. Previously the Uppsala team has shown that avian influenza viruses exposed to fairly low concentrations (in the water the ducks drink) of neuraminidase inhibitors rapidly acquire resistance mutations. One particularly interesting mutation that arose in a H1N1 virus is the H274Y mutation in the neuraminidase gene, which is associated with severely reduced sensitivity to Tamiflu. In a new experiment, they followed what happened with a virus carrying this mutation when the concentration of the drug vanes.

This poster isn't related to the topic at all. In fact, it belongs to a Hindi zombie comedy movie. But the title is similar, sort of, to this blog post.

This poster isn’t related to the topic at all. In fact, it belongs to a Hindi zombie comedy movie. But the title is similar, sort of, to this blog post.

So the Tamiflu-resistant influenza A(H1N1) virus with the H274Y mutation was propagated in ten successive generations of experimental ducks under a decreasing drug concentration (oseltamivir carboxylate, the active metabolite in Tamiflu). Samples were taken for detection, sequencing and virus isolation at different time points. Throughout the experiment, the mutant remained the dominant genotype in the virus population, with no visible trend detected even after deep sequencing. Phenotypically, early and late generation viruses were similar in their susceptibility to Tamiflu and Relenza.

Under these conditions, the resistance did not go away. In fact, it did not even decrease in frequency. Ten duck generations may sound like a short time period, but one needs to contemplate that this is not the same as virus generations. In each infected host, the infecting virus genotype cloud will cause an exponential increase of progeny viruses. Given the mutation rate of 10-5 per site and replication cycle it is fairly likely that some viruses may acquire mutations that reverse them to wild type at the 274 position. Or that there were wild type viruses present in small fraction already at the start that may be selected for if their relative fitness was higher than the mutant in the absence of the drug. Ideally, one would like to continue follow the fate of the mutant over an even longer time period, or in tests of competitiveness. However, this study concludes that resistance does not immediately go away once the drug pressure disappears. This open up the possibility for resistance to maintain in the virus population, and possibly also spread by reassortment to other gene constellations.

It is not gone, and not really going either.

Gillman, A., Muradrasoli, S., Söderström, H., Holmberg, F., Latorre-Margalef, N., Tolf, C., Waldenström, J., Gunnarsson, G., Olsen, B. & Järhult, J.D. 2015. Oseltamivir-Resistant Influenza A(H1N1)/NA-H274Y Persists Without Drug Pressure in Infected Mallards. Applied and Enviromental Microbiology, ahead of print.