Comparison of pathogenicity of subtype H9 avian influenza wild-type viruses from a wide geographic origin expressing mono-, di-, or tri-basic hemagglutinin cleavage sites

Table 1 Endoproteolytic cleavage site of the hemagglutinin protein (HACS), in silico prediction and in vitro measurement of furin-mediated cleavage and trypsin dependency of cell culture growth of several H9Nx avian influenza viruses

Viruses^a	Subtypes	Deduced HACS sequences		ProP score^b	PiTou score^c	Quanti-Luc assay^d		Trypsin-independent growth
Viruses^a	Subtypes	Motif-P14 to -P1	Motif type	ProP score^b	PiTou score^c	Avian cells^e	Mammalian cells^f	Trypsin-independent growth
A/duck/Bangladesh/AR132-D1/2016 (BD_132-D1)	H5N1 (positive control)	LATGLRNSPQRERRRKR	Polybasic	*0.771*	*369*	26 ± 0.3	10 ± 0.7	Yes
BD_11749	H9N2	LAIGLRNVPAKSKR	Tribasic	*0.628*	*376*	35 ± 0.3	9 ± 0.6	No
BD_11758				*0.628*	*376*	N/D^g	N/D	N/D
BD_11760				*0.628*	*376*	N/D	N/D	N/D
BD_11787				*0.628*	*376*	N/D	N/D	N/D
IN_117				*0.628*	*376*	N/D	N/D	No
IN_3535				*0.628*	*376*	N/D	N/D	N/D
IN_118	H9N2	LAIGLRNVPARSSR	Dibasic-1	0.344	*366*	11 ± 0.2	6 ± 0.7	No
IN_119				0.344	*366*	N/D	N/D	N/D
IN_3532				0.344	*366*	N/D	N/D	N/D
IN_3533				0.344	*366*	N/D	N/D	N/D
EG_536				0.344	*366*	N/D	N/D	N/D
BD_VP01	H9N2	LAIGLRNVPAKSSR	Dibasic-2	0.063	− 175	N/D	N/D	No
BD_3534	H9N2	LAIGLRNVPAKSSR	Dibasic-2	0.063	− 175	N/D	N/D	N/D
DU_120	H9N2	LAIGLRNVQARSSR	Dibasic-3	0.325	*213*	N/D	N/D	No
MO_166	H9N2	LAIGLRNVHARSSR	Dibasic-4	0.324	*320*	10 ± 0.5	4 ± 0.6	No
DU_121				0.324	*320*	N/D	N/D	No
SA_1885				0.324	*320*	N/D	N/D	N/D
DE_3280	H9N2	LAVGLRNVPAASKR	Dibasic-5	0.375	178	2 ± 0.2	1 ± 0.2	N/D
DE_234	H9N2	LAVGLRNVPAASNR	Monobasic-1	0.054	− 490	0.4 ± 0.1	0.1 ± 0.1	No
DE_143	H9N3			0.054	− 490	N/D	N/D	No
DE_144	H9N3			0.054	− 490	N/D	N/D	No
DE_142	H9N8	LAVGLRNVPAASSR	Monobasic-2	0.054	− 490	N/D	N/D	No
pSelect-BACE^h	Negative control	DKIKGLAGDRGGGSSAAAMV	N/D	< 0.08	− 612	1 ± 0.05	1 ± 0.4	N/D

Viruses are listed according to the HACS sequence type.
Italicized font indicates the basic amino acids.
^aAbbreviations indicate country of origin (two letters) and number of isolate. Full names, origin etc. are presented in Additional file 1.
^bThresholds of software-assisted cleavage prediction of > 0.5 (ProP) and.
^c> 185 (PiTou), respectively, indicate likelihood of intracellular cleavage by furin-like endoproteases (bolditalics).
^b,cSimilar values, within a motif type simply indicate that the same HACS sequence of different viruses has been tested.
^dFold increase of luciferase release from cells expressing a protein construct that links secretable luciferase via the respective cleavage site to a trans-Golgi anchor motif (see Figure 1A).
^eAvian cells—QM-9, DF-1.
^fMammalian cells—A549, ST.
^gN/D—not done/tested/applicable.
^hpSelect-BACE construct lacking any proteolytic cleavage site (used as a negative control in luciferase assay).

ISSN: 1297-9716