Evolution of an avian H5N1 influenza A virus escape mutant

Table 1 Oligonucleotides used for amplification of the H5N1 genes

Locus	Name	Primer sequence	Length	Amplicon size (bp)	Location	Ref.
N1	N1-F	ATGAATCCAAATCAGAAG	18	1350	21-38	[38]
	N1-R	TGTCAATGGTGAATGGCAAC	20		1346-1365
PB2	B2-F	GAGGCGATCTGAATTTCG	18	986	1256–1273	[39]
	B2-R	TATGCTAGAGTCCCGTTTCC	20		2222–2241
PB1	B1F	AGCGAGGAGTATCTGTGAGA	20	601	774–793	[40]
	B1R	TTCCCTCATGATTCGGTGCA	20		1356–1375
PA	PA-F	ATGAAGAGAGCAGGGCAAGA	20	868	491-510	This study
	PA-R	CAATGGGATACTTCCGCTGT	20		1339-1358
NP	NP-F	TGCTTGCCTGCTTGTGTGTA	20	665	823-842	[39]
	NP-R	TACTCCTCTGCATTGTCTCCGA	22		1466-1487
M	M-F	CCCTCAAAGCCGAAATCGCGCA	22	875	56-77	[40]
	M-R	TGCTGTTCCTGCCGATACTCTTCCC	25		906-930
NS	NS-F	CACTGTGTCAAGCTTTCAGG	20	798	23-42	[39]
	NS-R	TCTCTTGCTCCACTTCAAGC	20		786-805

Table 2 Comparison of amino acid signatures in selected genes of avian and human strains to Egyptian H5N1 strains

Gene	Residue	Avian1	Human	Egyptian H5N1	A/CK/Egypt/F10/2009
PB2	475	L214M1	M839L3	L52M2	L
	588	A203/T6/V6	I835/V3/A2	A53/T1	A
	613	V212/A3	T816/I16/A8/V1	V54	V
	627	E196/K19	K838/R2/E1	K48/E6	K
	674	A204/S6/T2/G2/E1	T836/A2/I2/P1	A54	A
PB1	327	R147/K3	K766/R66	R58	R
	336	V142/I8	I773/V59	V58	V
PA	225	S213C1	C829S10	S	S
	268	L214	I827K11	L	L
	356	K212X1R1	R827K11	K	K
	382	E208D5V1	D824E11V2N1	E	E
	404	A214	S828A9P1	A	A
	409	S189N24I1	N830S7I1	S77N1	S
NP	283	L372/P1	L7/P643	L61	L
	293	R371/K2	R28/K622	R60/K1	R
	305	R369/K4	K636/R14	R61	R
	313	F371/I1/L1	Y642/F8	F61	F
	357	Q368/K4/T1	K44/R8/Q1	Q61	Q
	372	E357/D15/K1	D630/E23	E61	E
	422	R373	K630/R23	R61	R
	442	T372/A1	A629/T23/R1	T61	T
	455	D373	E630/D22/T1	D60/E1	D
M1	115	V856/I2/L1/G1	I981/V9	V88	V
	121	T840/A19/P1	A988/T2	T88	T
	137	T859/A1/P1	A974/T12	T88	T
M2	11	T434/I11/S2	I911/T44	T90	T
	20	S471/N13	N926/S29	S90	S
	57	Y481/C1/H1	H913/Y33/R2/Q1	Y90	Y
NS2	70	S453/G21/D1	G903/S2	S61	S

¹Avian and human amino acid signatures in different viral genes of influenza A viruses as previously determined[20]. Numerical superscripts refer to the number of strains that possess those residues.

Table 3 Amino acid site residues associated with virulence in mammals in comparison with Egyptian isolates

GeneGene	Site	Residue1		Egyptian	A/CK/	Ref.
		Virulent	Avirulent	H5N1 isolates	Eg/F10/09
PB2	627	K	E	K	K	[11,27]
	701	N	D	D	D	[28]
PB1	317	I	M/V	M/V	M	[11,27]
PA	127	I	V	V	N.I.2	[25]
	336	M	L	L	L	[25]
M2	64	S/A/F	P	S	L	[17]
	69	P	L	P	P	[17]
NS1	42	S	A/P	S	S	[29]
	92	E	D	D	D	[27]
	97/92	E	D	E	E	[23]
	127	N	T/D/R/V/A	T/I	I	[30]
	189	N	D/G	D	D	[25]
	195	T/Y	S	S	S	[31]
NS2	31	I	M	M	M	[25]
	56	Y	H/L	H	H	[25]

¹Virulent and non virulent amino acid residues refer to the ability of the virus to replicate in mammals as determined by Lycett et al[17].

²N.I.: Not included since it is not flanked by the primers used in the current study.