Table 1.

Cleavage site motifs of European and Non-European H9N2 viruses from 1966 to 2019.

	Motif (P1/P1´)	European H9N2 (total 91)		Non-European H9N2 (total 2926)
		No	%	No	%
1	ASGR/G	2	2.4	13	0.4
2	ASAR/G	6	7.3	0	0.0
3	ASNR/G	11	13.4	3	0.1
4	RSSR/G	9	11.0	2384	81.5
5	ASDR/G	39	47.6	70	2.4
6	ASKR/G	4	4.9	0	0.0
7	RSKR/G	3	3.7	6	0.2
8	VSDR/G	1	1.2	20	0.7
9	TSNR/G	2	2.4	0	0.0
10	TSGR/G	3	3.7	16	0.5
11	ISGR/G	1	1.2	2	0.1
12	ISDR/G	1	1.2	0	0.0
13	RSGR/G			3	0.1
14	RSNR/G			35	1.2
15	ASDK/G			1	0.0
16	VSNR/G			7	0.2
17	VSGR/G			7	0.2
18	VSSR/G			9	0.3
19	ASYR/G			7	0.2
20	ISNR/G			3	0.1
21	KSSR/G			172	5.9
22	ISSR/G			1	0.0
23	KASR/G			2	0.1
24	RYSR/G			2	0.1
25	RASR/G			21	0.7
26	RLSR/G			13	0.4
27	RSRR/G			7	0.2
28	GSSR/G			3	0.1
29	RPSR/G			2	0.1
30	RFSR/G			9	0.3
31	RSTR/G			2	0.1
32	RSSK/G			4	0.1
33	RCSR/G			1	0.0
34	RSIR/G			1	0.0
35	KSKR/G			96	3.3
36	RSNK/G			1	0.0
	Total	82	100.0	2926*	100.0

A total of 2926 non-European viruses were retrieved from GISAID and GenBank on 21–01-2020. Three sequences from chickens (2 from Iran in 2007 and 1 from Egypt in 2013) had RSNR/R, RSNK/R and KSSR/A motifs assuming wrong or unusual sequences in the HA2 (i.e. underlined R or A). Only motifs No. 6, 7, 27 and 35 (highlighted in grey) encode dibasic or multibasic HACS. Using reverse genetics, we generated six recombinant viruses with polymorphism at position P2 resembling the first 6 motifs (no. 1 to 6).