Generation and characterization of recombinant influenza A (H1N1) viruses harboring amantadine resistance mutations

Abstract

The emergence of resistance to amantadine in influenza A viruses has been shown to occur rapidly during treatment as a result of single-amino-acid substitutions at position 26, 27, 30, 31, or 34 within the transmembrane domain of the matrix-(M)-2 protein. In this study, reverse genetics was used to generate and characterize recombinant influenza A (H1N1) viruses harboring L26F, V27A, A30T, S31N, G34E, and V27A/S31N mutations in the M2 gene. In plaque reduction assays, all mutations conferred amantadine resistance, with drug concentrations resulting in reduction of plaque number by 50% (IC(50)s) 154- to 3,300-fold higher than those seen for the wild type (WT). M2 mutants had no impairment in their replicative capacities in vitro on the basis of plaque size and replication kinetics experiments. In addition, all mutants were at least as virulent as the WT in experimentally infected mice, with the highest mortality rate being obtained with the recombinant harboring a double V27A/S31N mutation. These findings could help explain the frequent emergence and transmission of amantadine-resistant influenza viruses during antiviral pressure in the clinical setting.

FIG. 1.

Virus plaque size of recombinant WT A/WSN/33 (H1N1) virus and various M2 mutants. Recombinant WT and M2 mutant (L26F, V27A, A30T, S31N, G34E, and V27A/S31N) viruses generated in the supernatant of transfected 293T cells were propagated in 6-well plates of MDBK cells under 0.8% agarose. Plaques were visualized after 48 h of incubation at 37°C in the absence of drug. Numbers in parentheses indicate the mean plaque diameter in millimeters ± standard deviation determined from four plaques after enlargement (magnification, ×2.5).

FIG. 2.

Replication kinetics of recombinant WT A/WSN/33 (H1N1) virus and various M2 mutants. MDBK cells were infected with the recombinant WT and M2 mutant (L26F, V27A, A30T, S31N, G34E, and V27A/S31N) viruses at an MOI of 0.001. At the indicated times postinfection, supernatants were collected, and virus titers were determined in MDBK cells by use of a standard plaque assay. No significant differences were found when comparisons of mean titers of recombinant mutants with those of the WT virus were made at each time point (Student's t test with Satterthwaite correction). The values represent the means of three experiments with similar results.