Susceptibility to Neuraminidase Inhibitors and M2 Blockers of Seasonal Influenza Strains Isolated in Bulgaria 2004-2007

M2 blockers and neuraminidase inhibitors (NAIs) are two classes of drugs currently approved for prophylaxis and treatment of seasonal influenza A virus infections. The frequency of antiviral drug resistance has increased dramatically over the last 20 years, therefore monitoring of susceptibility to licensed inhibitors should be an essential component of influenza surveillance and therapy in Bulgaria and worldwide. Classical and molecular techniques were used to evaluate the susceptibility of influenza (H1N1) and (H3N2) strains isolated in Bulgaria 2004-2007 to neuraminidase inhibitors and M2 blockers. IC50 values of rimantadine were determined by CPE inhibition in cell cultures. IC50 of NAIs were evaluated fluorimetrically by neuraminidase susceptibility assay with MUNANA substrate. RT-PCR and sequencing were carried out on gene segments HA, NA and M2 with subsequent phylogenetics analysis using the neighbor-joining method and bootstrap analysis. Of the total 26 influenza strains (H1N1) and (H3N2) analyzed, 22 were sensitive and 4 (two H1N1 and two H3N2) were resistant to rimantadine hydrochloride in CPE inhibition assay. 17 isolates were subjected to a fluorescent assay, which showed the IC50 of zanamivir to range from 1.05 nM to 5.28 nM and that of osletamivir IC50 were - from 0.28 nM to 1.31 nM. Sequencing revealed S31N and V27T mutations in transmembrane region of the M2 protein to confer resistance to adamantanes in the A/Sofia/1250 (H3N2) strain. The virus remained susceptible to neuraminidase inhibitors. In all other viruses analyzed no mutations associated with resistance to either M2 blockers or NAIs were found.