Biological Characteristics Of2009Pandemic H1N1with H274Y And Synergistic Mutations In The Neuraminidase Protein

PART IESTABLISHING THE METHOD FOR SURFACEPRESENTING NEURAMINIDASE OF INFLUENZA A VIRUSObjective To establish the appropriate platforms for neuraminidasesurface expression.Methods The surface NA expression was obtained by transfecting byrecombinant NA constructors with specific tag-labels or live virus infection.The functional activity was measured by the fluorescent assay. Theirbindings to the Abs were detected by flow cytometry.Results The surface NAs presenting on the yeast-displaying system andeukaryotic expression system exhibited functional NA activities as the NA atthe surface of virus-infected cells which showed affinities to Ab1,2,3. Thesame bindings to Ab1and3were found in the surface NA expressed byeukaryotic expression system while minor binding was observed in the yeastdisplayed-NA. Conclusion The infected cells are suitable for the surface presenting ofNA and Ab1and Ab3can be used to detect the surface expression of NA. PART ⅡBIOLOGICAL CHARACTERISTICS OF2009PANDEMICH1N1WITH H274Y AND SYNERGISTIC MUTATIONS INTHE NEURAMINIDASE PROTEINObjective To determine the biological characteristics of2009pandemic H1N1with H274Y and synergistic mutations in neuraminidaseprotein.Methods Reverse genetics and site-directed mutagenesis were used toproduce the recombinant A/California/04wild-type virus and sevenvariants (H274Y, I222R, I222K, I222T, I222R+H274Y, I222K+H274Y,I222T+H274Y). The functional activity was measured by the fluorescentassay. NA surface expression was detected by flow cytometry. We didkinetics experiments with MDCK cells to determine the replicativecapacity of variants.Results Compared to wild type, the single I222T mutant wasmarginally resistant to oseltamivir without impaired viral fitness, NAsurface expression and total activity. The I222K/R mutation conferred amultidrug resistance phenotype to two NAIs but compromised viral fitness.These three mutations had a synergistic effect on the oseltamivir resistancein the presence of H274Y mutation, while I222K/R+H274Y mutants werealso resistant to zanamivir. Conclusion In conclusion, I222R/K/T mutations of NA conferredsynergistic effects on the NAI-resistance of A(H1N1)pdm09with variedfitness and NA enzymatic properties.