Table 2.

Identification of the molecular determinant responsible for the NA-mediated binding of the recombinant A/Victoria/361/2011(H3N2) virus.

NA amino acid at residue				Infectivity (log10 IU/ml)	Hemagglutination titer (HAU/50 μl)				NA-mediated binding
					Turkey RBCsa		Guinea pig RBCs
150	258	329	439		− Oseb	+ Ose	− Ose	+ Ose
R	K	T	R	8.1 ± 0.3	128	<2	512	2	Yes
H	K	T	R	7.8 ± 0.5	128	<2	512	2	Yes
R	E	T	R	7.6 ± 0.1	128	<2	512	2	Yes
R	K	N	R	7.6 ± 0.2	128	<2	512	2	Yes
R	K	T	T	8.2 ± 0.1	<2	<2	<2	2	No
R	K	T	A	7.5 ± 0.1	<2	<2	<2	<2	No
R	K	T	I	Not rescued	-	-	-	-	-
R	K	T	K	7.6 ± 0.2	128	<2	512	<2	Yes
R	K	T	P	Not rescued	-	-	-	-	-
R	K	T	S	7.7 ± 0.1	<2	<2	<2	<2	No
R	K	T	T	8.2 ± 0.1	<2	<2	<2	<2	No

Recombinant PR8-based A(H3N2) viruses containing the NA from A/Victoria/361/2011 were generated by reverse genetics. Unique NA amino acid sequence at positions 258, 329, and 439 were sequentially reverted to wild-type sequences by site-directed mutagenesis. In addition, other naturally occurring mutations (boldface) at residue 439, identified from NA sequences deposited in GISAID, were also examined for their role in NA-binding. Dash lines (–) indicate no data to show as these variants failed to be rescued.

^aRBCs, red blood cells.

^bOseltamivir, 20 nM.