Pathogenic assessment of avian influenza viruses in migratory birds

Figures & data

Table 1. Surface glycoprotein (HA and NA) homologies.

Virus	Subtype	Collection site	Hemagglutinin homology		Neuraminidase homology
			Reference virus	%	Reference virus	%
A/Ab/KoreaW336/2008	H1N1	Mangyeong river	A/EgyptianGs/SouthAfrica/AI1448/2007(H1N8)	97.2	A/Dk/Eastern China/1/2008(H6N1)	98.8
A/Ab/KoreaW228/2007	H1N2	Cheonsoman	A/WDk/Korea/SH29/2006(H1N3)	97.7	A/Ab/Korea/w216/2007(H5N2)	98.9
A/Ab/Korea/W107/2006	H1N3	Pungse	A/Dk/Tsukuba/718/2005(H1N1)	98.6	A/Dk/Eastern China/412/2003(H10N3)	97.8
A/Ab/Korea/W431/2010	H1N8	Mihocheon	A/Gs/Italy/296426/2003(H1N1)	95.6	A/avian/Japan/8KI0162/2008(H3N8)	99.4
A/Ab/Korea/W385/2009	H2N3	Mangyeong river	A/WDk/SH17-1/2008(H2N3)	98.9	A/WDk/SH17-1/2008(H2N3)	99.6
A/Ab/Korea/W180/2007	H2N4	Mihocheon	A/Ml/Sweden/58112/2006(H2N9)	98.3	A/Dk/Tsukuba/20/2007(H8N4)	99.0
A/Ab/Korea/W118/2006	H2N9	Cheonsoman	A/Ml/Sweden/58112/2006(H2N9)	98.5	A/N-Sh/Hong Kong/MPC657/2006(H10N9)	99.2
A/Ab/Korea/CN-3/2005	H3N1	Geumgang	A/Dk/Tsukuba/21/2005(H3N1)	98.6	A/Dk/Tsukuba/718/2005(H1N1)	99.6
A/Ab/Korea/KN-2/2005	H3N2	Mangyeong river	A/En/Korea/ESD11/2003(H3N2)	98.5	A/Ab/Korea/CN-2/2004(H3N2)	99.8
A/Ab/Korea/W146/2006	H3N4	Geumgang	A/Dk/Vietnam/G119/2006(H3N8)	98.1	A/ml/Sweden/24/2002(H8N4)	97.7
A/Ab/Korea/W338/2008	H3N6	Mangyeong river	A/Dk/Vietnam/G119/2006(H3N8)	99.0	A/WDk/Korea/SH5-60/2008(H4N6)	99.6
A/Ab/Korea/KN-4/2005	H3N8	Mangyeong river	A/En/Korea/KCA16/2004(H3N8)	99.9	A/Wbf/Korea/KCA16/2003(H3N8)	99.2
A/Ab/Korea/W340/2008	H4N1	Mangyeong river	A/Av/Japan/8KI0184/2008(H4N6)	99.7	A/Dk/Eastern China/1/2008(H6N1)	99.1
A/Ab/Korea/W319/2008	H4N2	Pungse	A/Dk/Taiwan/wb1101/2006(H4N6)	99.1	A/Ab/Korea/W163/2007(H5N2)	99.8
A/Ab/Korea/W187/2007	H4N4	Geumgang	A/Dk/Mongolia/583/2002(H4N7)	97.3	A/Dk/Tsukuba/20/2007(H8N4)	99.2
A/Ab/Korea/W418/2012	H4N6	Pungse	A/Dk/Thailand/CU-11840T/2011	99.3	A/Dk/Hunan/S11200/2012(H4N6)	99.4
A/Ab/Korea/W120/2006	H5N2	Mihocheon	A/Dk/Shimane/19/2006(H5N2)	99.6	A/Ga/SanJiang/16/2006(H5N2)	99.5
A/Ab/Korea/W346/2009	H5N7	Mangyeong river	A/Dk/Tsukuba/536/2006(H5N2)	98.0	A/Mp/Korea/YJD174/2007(H7N7)	99.1
A/Ab/Korea/W237/2008	H6N1	Geumgang	A/Dk/Guangxi/1157/2006(H6N2)	98.9	A/EmperorGs/Alaska/44063-145/2006(H2N1)	98.5
A/Ab/Korea/W09/2005	H6N2	Cheonsoman	A/Dk/Guangxi/1455/2004(H6N5)	99.6	A/Dk/Tsukuba/9/2005(H5N2)	99.8
A/Ab/Korea/W69/2005	H6N5	Cheonsoman	A/Dk/Guangxi/1455/2004(H6N5)	99.2	A/Dk/Guangxi/1455/2004	99.2
A/Ab/Korea/W72/2005	H6N8	Pungse	A/Dk/Guangxi/585/2005(H6N5)	99.2	A/Sn/Shimane/42/1999(H7N8)	97.6
A/Ab/Korea/W44/2005	H7N3	Cheonsoman	A/WDk/Jiangxi/10179/2005(H7N3)	99.5	A/RSdk/Mongolia/P52/2005(H12N3)	99.3
A/Ab/Korea/W152/2007	H7N7	Mangyeong river	A/Dk/Shiga/B149/2007(H7N7)	99.2	A/Dk/Shimane/18/2006(H7N7)	99.0
A/EM/Korea/W410/2011	H7N9	Mangyeong river	A/Dk/Iwate/301012/2012(H7N1)	99.2	A/northern shoveler/Hong Kong/MPL133/ 2010(H2N9)	99.5
A/Ab/Korea/W141/2006	H8N4	Mangyeong river	A/common teal/Netherlands/1/2005(H8N4)	97.1	A/Dk/Hokkaido/HY57/2005(H9N4)	97.2
A/Ab/Korea/W392/2010	H9N1	Pungse	A/Dk/Thailand/CU-8319T/2010(H9N7)	98.7	A/Ml/Hokkaido/24/2009(H5N1)	99.7
A/Ab/Korea/W408/2012	H9N2	Pungse	A/En/Bangladesh/1041/2009(H9N2)	98.0	A/Ga/Altai/1213/2007(H5N2)	98.7
A/Ab/Korea/W124/2006	H10N2	Mihocheon	A/Ml/Sweden/5812/2005(H10N9)	96.9	A/Ga/SanJiang/16/2006(H5N2)	99.3
A/Ab/Korea/W140/2007	H10N4	Mangyeong river	A/Dk/Hokkaido/W87/2007(H10N2)	99.2	A/Dk/Tsukuba/20/2007(H8N4)	98.2
A/Ab/Korea/W145/2006	H10N9	Geumgang	A/Ml/Sweden/5812/2005(H10N9)	97.1	A/N-Sh/Hong Kong/MPC657/2006(H10N9)	99.2
A/Ab/Korea/W157/2007	H11N2	Geumgang	A/N-Pt/Hong Kong/MPC2085/2007(H11N9)	99.6	A/Ab/Korea/W216/2007(H5N2)	98.2
A/Ab/Korea/W160/2007	H11N9	Geumgang	A/N-Pt/Hong Kong/MPC2085/2007(H11N9)	99.6	A/Dk/Hunan/1590/2007(H6N9)	98.6
A/Ab/Korea/W134/2006	H12N5	Mangyeong river	A/Ml/Sweden/343/2002(H12N5)	97.5	A/Dk/Tsukuba/255/2005(H8N5)	99.0

Closely related viruses were identified through BLAST analysis. Percentage sequence homologies were verified by alignment using DNAStar 5.0 software DNAStar 5.0 (DNAStar, Madison, WI, USA). Ab, aquatic bird; Av, ayan; En, environment; Dk, duck; Mdk, mallard duck; WDk, wild duck; Ml, mallard; N-Sh, northern shoveler; Wbf, wild bird fowl; Ga, garganey; Mp, Magpie; Sn, swan; EmperorGs, emperor goose; RSdk, ruddy shelduck; N-Pt, northern pintail; EgyptianGs, Egyptian goose.

Table 2. Virus replication in chickens.

Virus	Subtype	Mean oropharyngeal/cloacal swab titers (SD)^a			Contact transmission^b
		3 dpi	5 dpi	7 dpi	Day^b	No./total
Ab/Kor/W336/08	H1N1	2.5 (0.3)/–	2.1 (0.1)/–	–	3 DPC	3/3
Ab/Kor/W228/07	H1N2	2.1 (0.2)/–	3.0 (0.1)/–	–	3 DPC	3/3
Ab/Kor/W107/06	H1N3	–	–	–	–	0/3
Ab/Kor/W431/10	H1N8	1.5 (0.5)/–	2.5 (0.1)/–	–	3 DPC	3/3
Ab/Kor/W385/09	H2N3	2.5 (0.7)/–	3.0 (0.3)/–	–	3 DPC	3/3
Ab/Kor/W180/07	H2N4	1.7 (0.1)/–	–	–	–	0/3
Ab/Kor/W118/06	H2N9	–	–	–	–	0/3
Ab/Kor/CN-3/05	H3N1	2.3 (0.3)/–	1.7 (0.7)/–	–	5 DPC	3/3
Ab/Kor/KN-2/05	H3N2	2.5 (0.3)/–	1.7 (0.5)/–	–	5 DPC	3/3
Ab/Kor/W146/06	H3N4	2 (0.5)/–	1.3 (0.3)/–	–	–	0/3
Ab/Kor/W338/08	H3N6	2.5 (0.1)/–	1.3 (0.3)/–	–	–	0/3
Ab/Kor/KN-4/05	H3N8	2.3 (0.3)/–	1.7 (0.5)/–	–	5 DPC	3/3
Ab/Kor/W340/08	H4N1	1.1 (0.6)/–	2.0 (0.1)/–	–	–	0/3
Ab/Kor/W319/08	H4N2	3 (0.3)/ 1.3(0.5)	1.5(0.3)/1.5(0.5)	–	–	0/3
Ab/Kor/W187/07	H4N4	2.7 (0.3)/–	1.7 (0.5)/–	–	–	0/3
Ab/Kor/W418/12	H4N6	3 (0.2)/1 (0.6)	1.5(0.5)/1.5(0.7)	–	–	0/3
Ab/Kor/W120/06	H5N2	1.7 (0.5)/–	–	–	–	0/3
Ab/Kor/W346/09	H5N7	–	–	–	–	0/3
Ab/Kor/W237/08	H6N1	–	–	–	–	0/3
Ab/Kor/W09/05	H6N2	1.5 (0.1)/–	2.0 (0.1)/–	–	5 DPC	1/3
Ab/Kor/W69/05	H6N5	–	–	–	–	0/3
Ab/Kor/W72/05	H6N8	–	–	–	–	0/3
Ab/KorW44/05	H7N3	2.3 (0.3)/–	1.7 (0.7)/–	–	–	0/3
Ab/Kor/W152/06	H7N7	2.0(0.1)/3.0(0.5)	2 (0.3)/2.0 (0.7)	–	3 DPC	3/3
A/EM/Kor/W410/11	H7N9	1.7 (0.5)/–	2 (0.3/–)	–	–	0/3
Ab/Kor/W141/06	H8N4	–	–	–	–	0/3
Ab/Kor/W392/10	H9N1	3(0.3)/ 2.5(0.7)	2.5 (0.3)/ 3 (0.5)	–	–	0/3
Ab/Kor/W408/12	H9N2	1 (0.5)/1.3 (0.3)	1.5(0.3)/0.7(0.7)	–	–	0/3
Ab/Kor/W124/06	H10N2	–	–	–	–	0/3
Ab/Kor/W140/07	H10N4	2.3 (0.3)/–	1.3 (0.7)/–	–	–	0/3
Ab/Kor/W145/06	H10N9	–	–	–	–	0/3
Ab/Kor/W157/07	H11N2	–	–	–	–	0/3
Ab/Kor/W160/07	H11N9	–	–	–	–	0/3
Ab/Kor/W134/06	H12N5	1.3 (0.5)/–	–	–	–	0/3

^a Titers are mean from six chickens and expressed as log₁₀ EID₅₀/ml; dashed lines indicate negative virus detection (limit of <0.75 log₁₀ EID₅₀/ml).

^b Earliest timepoint at transmission.

Transmission to cohoused chickens was also determined by monitoring viral titration of oropharyngeal/cloacal swabs; DPC, days post-contact; –, virus not detected.

Table 3. Virus replication in mouse lungs.

Virus	Subtype	Lung viral titers (SD)^a				Mean Wt. Loss (%)^b	MLD50
		3 dpi	5 dpi	7 dpi	9 dpi
Ab/Kor/W336/08	H1N1	5.1 (0.1)	5.5 (0.3)	4.1 (0.1)	–	10.2	>6.0
Ab/Kor/W228/07	H1N2	4.5 (0.1)	5.5 (0.3)	4.3 (0.1)	–	4.4	>6.0
Ab/Kor/W107/06	H1N3	2.3 (0.5)	3.3 (0.1)	–	–	1.5	>6.0
Ab/Kor/W431/10	H1N8	5.3 (0.3)	5.5 (0.1)	3.1 (0.5)	–	0.7	>6.0
Ab/Kor/W385/09	H2N3	3.7 (0.3)	4.8 (0.1)	2.2 (0.7)	–	1.3	>6.0
Ab/Kor/W180/07	H2N4	3.3 (0.3)	4.5 (0.5)	1.7 (0.3)	–	3.6	>6.0
Ab/Kor/W118/06	H2N9	3.0 (0.5)	3.7 (0.3)	–	–	0.9	>6.0
Ab/Kor/CN-3/05	H3N1	3.3 (0.3)	4.3 (0.3)	2 (0.5)	–	1.8	>6.0
Ab/Kor/KN-2/05	H3N2	3.5 (0.5)	4.7 (0.5)	2.3 (0.3)	–	2.4	>6.0
Ab/Kor/W146/06	H3N4	4.0 (0.5)	5.0 (0.7)	2.5 (0.5)	–	4.3	>6.0
Ab/Kor/W338/08	H3N6	3.5 (0.3)	4.5 (0.3)	1.7 (0.3)	–	3.5	>6.0
Ab/Kor/KN-4/05	H3N8	3.3 (0.3)	4.3 (0.3)	–	–	2.1	>6.0
Ab/Kor/W340/08	H4N1	5.1 (0.1)	4.5 (0.1)	–	–	2.3	>6.0
Ab/Kor/W319/08	H4N2	3.7 (0.3)	4.5 (0.3)	1.5 (0.3)	–	–	>6.0
Ab/Kor/W187/07	H4N4	–	–	–	–	–	>6.0
Ab/Kor/W418/12	H4N6	2.3 (0.1)	3.5 (0.1)	–	–	0	>6.0
Ab/Kor/W120/06	H5N2	3.5 (0.1)	4.5 (0.30)	2.3 (0.5)	–	5.3	>6.0
Ab/Kor/W346/09	H5N7	3.3 (0.1)	1.5 (0.5)	–	–	2	>6.0
Ab/Kor/W237/08	H6N1	6.1 (0.3)	5.1 (0.2)	3.7 (0.7)	–	7	>6.0
Ab/Kor/W09/05	H6N2	4.1 (0.3)	3.1 (0.1)	2.8 (0.5)	–	4.8	>6.0
Ab/Kor/W69/05	H6N5	3.3 (0.5)	4.5 (0.7)	1.3 (0.3)	–	3.2	>6.0
Ab/Kor/W72/05	H6N8	3.0 (0.3)	2.7 (0.5)	–	–	1.1	>6.0
Ab/Kor/W44/05	H7N3	3.3 (0.3)	5.3 (0.3)	1.7 (0.3)	–	5.3	>6.0
Ab/Kor/W152/06	H7N7	3.0 (0.3)	4.5 (0.3)	5.0 (0.7)	2.5 (0.5)	8.0	>6.0
A/EM/Kor/W410/11	H7N9	3.3 (0.3)	3.0 (0.1)	–	–	4.3	>6.0
Ab/Kor/W141/06	H8N4	2.7 (0.5)	2.7 (0.5)	–	–	2.1	>6.0
Ab/Kor/W392/10	H9N1	3.0 (0.1)	3.7 (0.3)	–	–	3.3	>6.0
Ab/Kor/W408/12	H9N2	2.5 (0.3)	3.5 (0.30)	–	–	2.8	>6.0
Ab/Kor/W124/06	H10N2	3.3 (0.7)	4.7 (0.5)	1.7 (0.5)	–	4.3	>6.0
Ab/Kor/W140/06	H10N4	2.1 (0.5)	0.7 (0.5)	–	–	1.2	>6.0
Ab/Kor/W145/06	H10N9	–	–	–	–	–	>6.0
Ab/Kor/W157/07	H11N2	–	–	–	–	–	>6.0
Ab/Kor/W160/07	H11N9	–	–	–	–	–	>6.0
Ab/Kor/W134/06	H12N5	–	–	–	–	–	>6.0

^a Titers are mean lung titers from three mice per time-point and are expressed as log₁₀ EID₅₀/g; dashed lines indicate negative detection (limit <0.75 log₁₀ EID₅₀/g).

^b Data obtained from 10 mice monitored for 14 dpi.

Figure 1. Receptor-binding specificity profiles of H3 (A), H6 (B), H8 (C), H9 (D), H11 (E), and H12 (F) AI virus isolates. Binding affinities of inactivated whole viruses to SA α2,3’-SL-PAA-biotin (left panels) or SA α2,6’SLN-PAA-biotin (right panels) glycans are shown. Results shown are means ± SD (mean of three replicates). Dashed lines indicate the limit of detection. The 2009 pandemic H1N1 virus was used as a positive control for comparing binding preferences for mammalian receptors.

Table 4. Virus replication in the upper respiratory tract of ferrets.

Group	Virus Subtype	Nasal wash titers (log10EID50/ml)				Contact transmission		Seroconversion
		1 dpi	3 dpi	5 dpi	7 dpi	Day^d	No./total	No./total
A^a	Ab/Kor/W336/08 H1N1	3.5 (0.1)	4.4 (0.1)	5.3 (0.1)	–	3DPC	3/3	1/3
	Ab/Kor/KN-2/05 H3N2	3.4 (0.1)	2.5 (0.1)	1.5 (0.5)	–	4DPC	3/3	0/3
B^b	Ab/Kor/W385/09 H2N3	3.5 (0.1)	2.5 (0.1)	2.0 (0.1)	–	–	0/3	0/3
	Ab/Kor/W340/08 H4N1	2.9 (0.2)	3.4 (0.1)	1.4 (0.2)	–	–	0/3	0/3
	Ab/Kor/W418/12 H4N6	2.5 (0.2)	3.0 (0.2)	1.5 (0.1)	–	–	0/3	0/3
	Ab/Kor/W120/06 H5N2	2.5 (0.2)	3.3 (0.1)	2.5 (0.2)	–	–	0/3	0/3
	Ab/Kor/W237/08 H6N1	2.4 (0.2)	3.3 (0.1)	1.9 (0.2)	–	–	0/3	0/3
	Ab/KorW44/05 H7N3	3.0 (0.2)	3.0 (0.2)	2.0 (0.1)	–	–	0/3	0/3
	Ab/Kor/W152/06 H7N7	2.5 (0.2)	3.5 (0.1)	2.5 (0.1)	–	–	0/3	0/3
	A/EM/Kor/W410/11 H7N9	2.5 (0.2)	3.5 (0.1)	2.0 (0.1)			0/3	0/3
	Ab/Kor/W408/12 H9N2	3.5 (0.1)	1.5 (0.5)	–	–	–	0/3	0/3
	Ab/Kor/W140/07 H10N4	2.5 (0.2)	4.0 (0.1)	2.5 (0.2)	–	–	0/3	0/3
C^c	Ab/Kor/W146/06 H3N4	3.6 (0.1)	2.9 (0.2)	2.4 (0.2)	–	3DPC	3/3	0/3
	Ab/Kor/KN-4/05 H3N8	2.5 (0.2)	3.3 (0.1)	1.9 (0.2)	–	–	0/3	0/3
	Ab/Kor/W72/05 H6N8	2.0 (0.2)	2.5 (0.2)	1.9 (0.2)	–	–	0/3	0/3
	Ab/Kor/W141/06 H8N4	2.4 (0.2)	–	–	–	–	0/3	0/3
	Ab/Kor/W392/10 H9N1	3.0 (0.2)	3.6 (0.1)	2.5 (0.2)	–	–	0/3	0/3
	Ab/Kor/W157/07 H11N2	2.5 (0.2)	1.5 (0.5)	–	–	–	0/3	0/3
	Ab/Kor/W160/07 H11N9	3.3 (0.1)	1.5 (0.5)	–	–	–	0/3	0/3
	Ab/Kor/W134/06 H12N5	2.5 (0.2)	–	–	–	–	0/3	0/3

^a Group of viruses that have historically caused human pandemics (i.e. H1N1 and H3N29).

^b Group of viruses of purely avian origin previously detected in mammals such as pigs (H2N3, H4N1, H4N6), minks (H10N4), seals (H7N7), and humans (H5N2, H6N1, H7N3, H7N9, and H9N2).

^c Group of viruses with high binding affinity for α2,6-SAs (over 0.2 O.D) (Figure 1).

^d Earliest timepoint at transmission.

Figure 2. Replication and transmission of AI viruses in ferrets inoculated intranasally with 10⁶ EID₅₀/ml of each virus. Individual nasal wash titers of ferrets inoculated with Ab/Kor/W336/08 (H1N1) (A), Ab/Kor/KN-2/05 (H3N2) (B), and Ab/Kor/W146/06 (H3N4) (C). To examine transmission, the inoculated animals were individually paired with direct contact (DC) animals (1:1 setup). Mean viral titers (log₁₀ EID₅₀/ml) are shown for each group of mammals. The limit of virus detection was 0.75 log₁₀ EID₅₀/ml.