ABSTRACT
This study aimed to investigate the drug susceptibility of wild-type and mutant avian influenza A (H7N9) virus neuraminidase (NA) to oseltamivir and zanamivir. Codon optimized DNA of H7N9 (A/ Hangzhou/1/2013) NA was synthesized and constructed into the pcDNA3.1/His vector (NA(H7N9-WT)). Mutant NA(H7N9-H274Y) and NA(H7N9-R292K) plasmids were constructed by directed mutagenesis PCR using NA(H7N9-WT) plasmid as the template followed by sequencing. NA plasmids were transfected into 293T cells and cell lysates containing NAs were collected 48 h post-transfection. Wild-type and mutant NAs were analyzed by Western blotting and their activities were tested by the 4-MUNANA-based assay. All three NAs were expressed and enzymatic activities were confirmed. The effects of oseltamivir and zanamivir on all three NAs were then tested. It showed that the half maximal inhibitory concentrations (IC50s) of oseltamivir carboxylate on NA(H7N9-WT), NA(H7N9-H274Y) and NA(H7N9-R292K) were 1.6 nM, 15.1 nM, and > 1 000 nM with fold changes of 9 and > 625, respectively. The IC50 values of zanamivir on NA(H7N9-WT), NA(H7N9-H274Y), and NA(H7N9-R292K) were 1.1 nM, 1.4 nM, and 38.0 nM with fold changes of 1.3 and 34, respectively. These results indicated that oseltamivir and zanamivir could significantly inhibit NA(H7N9-WT). NA(H7N9-R292K) showed high-level resistance to both drugs (34-fold and 625-fold) and NA(H7N9-H274Y) was sensitive to both (1.3-fold and 9-fold). These results indicated that both oseltamivir and zanamivir could be used for patients infected with the H7N9 virus. However, when patients carried the H7N9 virus with a NA R292K mutation, other medications would be preferred over oseltamivir or zanamivir.