Rapid development of resistant
influenza variants after
amantadine treatment is one of the main drawbacks of M2 blockers. On the other hand, the emergence of variants with low susceptibility to the
neuraminidase (NA) inhibitors is limited. In the present study we examined whether combination
therapy with two classes of anti-
influenza drugs can affect the emergence of resistant variants in vitro. We observed that virus yields of human A/Nanchang/1/99 (H1N1), A/Panama/2007/99 (H3N2), and A/Hong Kong/156/97 (H5N1) viruses in MDCK cells were significantly reduced (P<0.005) when the cells were treated with the combination of
amantadine and low doses of
oseltamivir carboxylate (< or =1microM). After five sequential passages in MDCK cells, the M2
protein of viruses cultivated with
amantadine alone mutated at positions V27A and S31N/I. Viruses cultivated with
oseltamivir carboxylate (> or =0.001microM) possessed mutations in the
hemagglutinin (HA)
protein. These variants showed reduced efficiency of binding to
sialic acid receptors and decreased sensitivity to NA inhibitor in plaque reduction assay. Importantly, no mutations in the HA, NA, and M2
proteins were detected when the drugs were used in combination. Our results suggest that
combination chemotherapy with M2 blocker and NA inhibitor reduced the emergence of
drug-resistant
influenza variants in vitro. This strategy could be an option for the control of influenza virus
infection, and combinations with other novel drugs should be explored.