In vitro passaging of a pandemic H1N1/09 virus selects for viruses with neuraminidase mutations conferring high-level resistance to oseltamivir and peramivir, but not to zanamivir

Pandemic H1N1/09 viruses with the neuraminidase H274Y mutation have emerged in untreated patients or following oseltamivir therapy or prophylaxis. There have been no reports yet of zanamivir-resistant H1N1/09 viruses in previously healthy patients. We wanted to determine whether we could select for neuraminidase mutations conferring high-level resistance to zanamivir by in vitro passage of the virus. We also wanted to investigate if passaging in a combination of zanamivir and oseltamivir could prevent the emergence of the H274Y mutation. An H1N1/09 virus was passaged in cell culture in increasing concentrations of either zanamivir or a combination of zanamivir and oseltamivir. Passage in zanamivir selected a virus with N146S neuraminidase and G158E haemagglutinin mutations. The neuraminidase mutation only reduced drug susceptibility by 2-fold in enzyme assays. The haemagglutinin mutation conferred drug dependence and drug resistance in cells to oseltamivir and zanamivir and reduced binding to red blood cells. After four passages in zanamivir and oseltamivir, virus with a D198G neuraminidase mutation was selected with around 10-fold reduced susceptibility to oseltamivir, zanamivir and peramivir in the enzyme assay. Further passaging selected a virus with both D198G and H274Y mutations that was highly resistant to oseltamivir and peramivir, but not zanamivir. All mutations affected growth in cell culture and decreased affinities of the neuraminidases for substrate. We did not select a virus with a neuraminidase mutation conferring high-level resistance to zanamivir. Dual exposure to zanamivir and oseltamivir was not sufficient to prevent selection of the H274Y mutation.