Molecular analysis of amantadine-resistant influenza A (H1N1 pdm09) virus isolated from slum dwellers of Dhaka, Bangladesh.

Influenza is a highly contagious viral infection associated with excessive hospitalizations and deaths throughout the world. Continuous antigenic shift and drift is not only responsible for this devastating effect of influenza but also causes ineffectiveness of antiviral drugs and vaccines. In this study, we investigated the effectiveness of ribavirin, oseltamivir, and amantadine drugs in vitro against nine influenza A isolates collected during June 2012-August 2013 from different slums in Dhaka city. The effectiveness of these drugs was determined by measuring the inhibition of virus-induced cytopathic effect on MDCK cells through MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Our data showed that all nine influenza isolates (6 H1N1 pdm09 and 3 H3N2 subtypes) were completely susceptible to ribavirin (The 50% effective concentrations, EC50 3.0 µg/ml) and oseltamivir (EC50 0.35 µg/ml). When influenza A infection was challenged with amantadine drug, eight out of nine isolates (88%) demonstrated susceptibility to amantadine drug (EC50 0.30 µg/ml) while one H1N1 pdm09 isolate exhibited higher EC50 value (>10 µg/ml) beyond the cell tolerance level of drug (>5 µg/ml). Genetic analysis of transmembrane matrix protein 2 (M2), which is a target for the amantadine drug and vital for viral replication, showed a substitution of amino acid at position 31(S31 N) of that amantadine-resistant isolate indicating the possible reason of amantadine drug resistance.