BACKGROUND: The influenza neuraminidase plays a critical role in the spread of the influenza A and B viruses. Through the cleavage of terminal sialic acid from glycoconjugates, it facilitates the elution of progeny virions from infected cells and prevents their self-aggregation. OBJECTIVES: Our objective was to study the impact of mutations at framework sites not under direct selective pressure in the neuraminidase active site. STUDY DESIGN: In the A/Moscow/10/99 (H3N2) virus background, viruses containing mutations in NA framework residues (E119D, R156K, W178L, S179A, D198N, I222L, E227G, H274Y, E277G, N294D, and E425G) were constructed by reverse genetics. After several passages on MDCK cells, fluorimetric assays were conducted to assess the neuraminidase activity and sensibility to the neuraminidase inhibitors (IC50). RESULTS: Among the viruses detectable through the phenotypic tests, R156K, I222L, H274Y, N294D and E425G viruses presented a NA activity between 70% and 100% of the A/Moscow/10/99 wild type one. The D198N and the E119D mutations decreased seriously in NA activity compared to the wild-type (>10-fold). The I222L mutation reduced susceptibility to oseltamivir (18-fold). CONCLUSION: With the exception of one mutation, framework mutations on N2 background do not induce resistance. Nevertheless they tend to decrease slowly the sensitivity to one or the other inhibitors.
BACKGROUND: The influenza neuraminidase plays a critical role in the spread of the influenza A and B viruses. Through the cleavage of terminal sialic acid from glycoconjugates, it facilitates the elution of progeny virions from infected cells and prevents their self-aggregation. OBJECTIVES: Our objective was to study the impact of mutations at framework sites not under direct selective pressure in the neuraminidase active site. STUDY DESIGN: In the A/Moscow/10/99 (H3N2) virus background, viruses containing mutations in NA framework residues (E119D, R156K, W178L, S179A, D198N, I222L, E227G, H274Y, E277G, N294D, and E425G) were constructed by reverse genetics. After several passages on MDCK cells, fluorimetric assays were conducted to assess the neuraminidase activity and sensibility to the neuraminidase inhibitors (IC50). RESULTS: Among the viruses detectable through the phenotypic tests, R156K, I222L, H274Y, N294D and E425G viruses presented a NA activity between 70% and 100% of the A/Moscow/10/99 wild type one. The D198N and the E119D mutations decreased seriously in NA activity compared to the wild-type (>10-fold). The I222L mutation reduced susceptibility to oseltamivir (18-fold). CONCLUSION: With the exception of one mutation, framework mutations on N2 background do not induce resistance. Nevertheless they tend to decrease slowly the sensitivity to one or the other inhibitors.