Replication and pathogenic potential of influenza A virus subtypes H3, H7, and H15 from free-range ducks in Bangladesh in mammals

Figures & data

Antigenic characterization by the hemagglutination inhibition assay of H3 viruses isolated from free-range ducks in Bangladesh

	Antisera
	North American	Eurasian				Australian	Bangladeshi
Reference virus	IN/0392 H3N2	Hong Kong/1/68	Ukraine/1/63	Shantou/1283/2001	Bulgaria/61/2008	South Australia/55/2014	A/duck/BD/26920/2015 H3N6
A/swine/Indiana/0392/2011 H3N2	2560	<10	<10	40	<10	<10	<10
A/Hong Kong/1/68 H3N2	20	10,240	2560	10,240	640	<10	2560
A/duck/Ukraine/1/63 H3N8	<10	160	40	1280	80	<10	160
A/duck/Shantou/1283/2001 H3N8	<10	160	80	640	40	<10	160
A/muleduck/Bulgaria/61/2008 H3N2	<10	80	40	320	80	<10	320
A/South Australia/55/2014 H3N2	<10	20	<10	<10	<10	160	<10
A/Aichi/2/68 H3N2	<10	2560	1280	2560	160	<10	2560
Test virus
A/duck/Bangladesh/26920/2015 H3N6	<10	80	80	640	80	<10	640
A/duck/Bangladesh/26948/2015 H3N6	<10	80	40	320	40	<10	320
A/duck/Bangladesh/26918/2015 H3N6	<10	80	80	640	80	<10	640

Homologous titers are bold underlined

Antigenic characterization by the hemagglutination inhibition assay of H7 viruses isolated from free-range ducks in Bangladesh

	Antisera
	North American		Eurasian
Reference viruses	A/Canada/RV444/2004	A/chicken/Saskatchewan/HR10/2007 H7N3	A/equine/Prague/56 H7a	A/FPV/Rostock H7b^a	A/Netherlands/219/2003 H7N7	Anhui/1/2013 H7N9	A/duck/Bangladesh/24692/2015 H7N1	A/duck/Bangladesh/26980/2015 H7N9
RG-A/Canada/RV444/2004 H7N3	80	40	<10	5120	40	20	640	1280
A/equine/Prague/56 H7a	40	10	320	5120	40	10	160	160
RGA/Netherlands/219/2003 H7N7	40	20	<10	2560	20	10	320	640
RG-A/Anhui/1/2013 H7N9	80	40	<10	5120	320	80	1280	1280
A/seal/MA/1/80 H7N7	80	40	<10	5120	20	20	640	640
A/Ruddy turnstone/NJ/65/85 H7N3	40	10	<10	1280	10	10	320	320
Test virus
A/duck/Bangladesh/24692/2015 H7N1	40	20	<10	2560	10	10	320	640
A/duck/Bangladesh/24694/2015 H7N1	40	40	<10	2560	20	20	320	160
A/duck/Bangladesh/24705/2015 H7N1	80	20	<10	5120	20	10	320	80
A/duck/Bangladesh/24706/2015 H7N1	40	20	<10	2560	10	20	320	80
A/black-tailed godwit/Bangladesh/24734/2015	40	20	<10	2560	10	10	320	160
H7N5
A/duck/Bangladesh/26980/2015 H7N9	80	40	<10	2560	20	10	320	320
A/duck/Bangladesh/26992/2015 H7N9	40	20	<10	2560	10	10	320	80
A/duck/Bangladesh/27042/2015 H7N9	40	20	<10	2560	20	10	320	160

Homologous titers are bold underlined

^a Goat hyperimmune sera

Fig. 1 Percent weight loss and survival and lung titration in DBA/2J mice infected with 10⁶ EID₅₀ of selected isolates from free-range ducks and wild birds.

a Daily weight loss was calculated as the percentage of weight relative to that at 0 dpi, and mean weight loss for each group is shown. b Mortality was recorded based on actual death or killing at 25% weight loss, according to our protocol. For each virus isolate, five mice were infected. c Lung tissues were collected at 3 dpi, and viral titers were determined by EID₅₀ assays. Error bars indicate standard deviation, and the dotted line indicates the lower limit of detection of the infectious virus

Pathogenicity in DBA/2J mice infected with avian viruses isolated from free-range ducks and wild birds

Influenza strain	Subtype	Survival (AUC)	Weight loss (AUC)	Survival score^a	Weight loss score^b	Final score^c	PI
A/duck/Bangladesh/26918/2015	H3N6	1400	1469.07	0.800	0.210	1.010	0
A/duck/Bangladesh/26920/2015	H3N6	1400	1342.33	0.800	0.192	0.992	1
A/duck/Bangladesh/26948/2015	H3N6	1400	1447.08	0.800	0.207	1.007	0
A/duck/Bangladesh/24692/2015	H7N1	640	646.24	0.366	0.092	0.458	4
A/black-tailed godwit/Bangladesh/24734/2015	H7N5	1400	1422.82	0.800	0.203	1.003	0
A/duck/Bangladesh/26980/2015	H7N9	1100	1051.84	0.629	0.150	0.779	2
A/duck/Bangladesh/24697/2015	H15N9	1400	1402.67	0.800	0.200	1.000	0
PBS		1400	1454.62	0.800	0.208	1.008	0

AUC area under the curve, PBS phosphate-buffered saline, PI pathogenicity index

^a Survival score was calculated as 0.86 (survival AUC/maximum AUC)

^b Weight loss score was calculated as 0.26 (weight loss AUC/maximum AUC)

^c Total pathogenicity score was the sum of survival and weight loss scores

Replication kinetics of H7 AIVs in lung and extrapulmonary tissues in mice

Virus	PI	Subtype		Viral titers (log10 EID50/mL)^a
				Day
				2	4	6	8
A/duck/Bangladesh/24692/2015	4	H7N1	Lung	3/3 (5.67 ± 0.52)	3/3 (5.42 ± 0.88)	3/3 (6.42 ± 0.14)	3/3 (6.17 ± 0.38)
			Heart	2/3 (2.13 ± 0.88)	2/3 (3.00 ± 0.71)	3/3 (2.92 ± 1.13)	2/3 (2.63 ± 0.18)
			Liver	3/3 (2.67 ± 0.52)	1/3 (4.25)	1/3 (2.50)	2/3 (2.50 ± 1.41)
			Intestine	—	—	—	—
			Brain	2/3 (2.13 ± 0.18)	2/3 (1.88 ± 0.53)	1/3 (3.75)	3/3 (2.67 ± 0.52)
A/duck/Bangladesh/26980/2015	2	H7N9	Lung	3/3 (5.50 ± 1.00)	3/3 (5.88 ± 0.53)	3/3 (5.92 ± 0.38)	3/3 (5.25 ± 0.75)
			Heart	2/3 (3.38 ± 1.59)	—	1/3 (2.00)	1/3 (1.75)
			Liver	2/3 (2.50 ± 0.35)	—	2/3 (2.63 ± 1.24)	—
			Intestine	—	—	—	—
			Brain	2/3 (2.25 ± 0.71)	—	1/3 (1.50)	2/3 (2.13 ± 0.18)
A/black-tailed godwit/Bangladesh/24734/2015	0	H7N5	Lung	2/3 (2.88 ± 2.2)	3/3 (2.67 ± 1.01)	3/3 (3.42 ± 1.01)	3/3 (1.83 ± 0.58)
			Heart	—	—	—	—
			Liver	—	—	—	—
			Intestine	—	—	—	—
			Brain	—	—	—	—

^a The lower limit of detection of infectious virus was <0.75 log₁₀ EID₅₀

Fig. 2 Pulmonary lesions and virus spread in the lung of mice infected with A/duck/Bangladesh/24692/2015 H7N1 (I A–I), A/duck/Bangladesh/26980/2015 H7N9 (II A–I), and A/black-tailed godwit/Bangladesh/24734/2015 H7N5 (III A–I) at 2, 4, and 8 dpi

. Mouse lungs were fixed in 10% neutral buffered formalin and stained with hematoxylin-eosin (HE), subjected to immunohistochemical (IHC) staining with anti–NP antiserum, or analyzed by histomorphometry (magnification: ×40 HE, ×20 IHC, and ×2 histomorphometry). In the histomorphometry images, the total lung areas examined are outlined in green; areas of active infection with antigen-positive cells are shown in red

Fig. 3 Replication and transmission of H7 viruses in mallard ducks.

Viral titers in cloacal (a, b) and oropharyngeal (c, d) swabs of mallard ducks infected with A/duck/Bangladesh/24692/2015 H7N1 and A/duck/Bangladesh/26980/2015 H7N9 viruses. Virus shedding of both donor and contact ducks was monitored, and swabs were collected at 3, 5, and 7 dpi and dpc. The dotted line indicates the lower limit of detection of the infectious virus. dpi days post inoculation, dpc days post contact

Comparison of amino acid sequences and IVPI of viruses isolated from free-range ducks in Bangladesh

Strain	Subtype	IVPI	HA			NA			PB2			PB1-F2	NS1
			HA cleavage site	Q226L	G228S	E119V	H275Y	R292K	E627K	D701N	G309D	N66S	C-terminal motif
A/duck/Bangladesh/26920/2015	H3N6	0.0	PEKQTR↓GLF	Q	G	E	H	R	E	D	G	S	ESEV
A/duck/Bangladesh/26948/2015	H3N6	0.05	PEKQTR↓GLF	Q	G	E	H	R	E	D	G	S	ESEV
A/duck/Bangladesh/26974/2015	H3N6	ND	PEKQTR↓GLF	Q	G	E	H	R	E	D	G	S	ESEV
A/duck/Bangladesh/26918/2015	H3N6	0.0	PEKQTR↓GLF	Q	G	E	H	R	E	D	G	N	ESEV
A/duck/Bangladesh/24694/2015	H7N1	0.31	PETPKGR↓GLF	Q	G	E	H	R	E	D	G	N	ESEV
A/duck/Bangladesh/24692/2015	H7N1	0.0	PETPKGR↓GLF	Q	G	E	H	R	E	D	G	S	ESEV
A/duck/Bangladesh/24706/2015	H7N1	ND	PETPKGR↓GLF	Q	G	E	H	R	E	D	G	S	ESEV
A/duck/Bangladesh/24705/2015	H7N1	1.04	PELPKGR↓GLF	Q	G	E	H	R	E	D	G	S	ESEV
A/black-tailed godwit/Bangladesh/24734/2015	H7N5	0.0	PELPKGR↓GLF	Q	G	E	H	R	E	D	G	N	ESEV
A/duck/Bangladesh/26980/2015	H7N9	0.0	PELPKGR↓GLF	Q	G	E	H	R	E	D	G	S	ESEV
A/duck/Bangladesh/26992/2015	H7N9	0.35	PELPKGR↓GLF	Q	G	E	H	R	E	D	G	S	ESEV
A/duck/Bangladesh/27042/2015	H7N9	0.0	PELPKGR↓GLF	Q	G	E	H	R	E	D	G	S	ESEV
A/duck/Bangladesh/24704/2015	H15N9	ND	PEKTHTR↓GLF	Q	G	E	H	R	E	D	D	N	ESEV
A/duck/Bangladesh/24697/2015	H15N9	0.0	PEKTHTR↓GLF	Q	G	E	H	R	E	D	G	N	ESEV

IVPI intravenous pathogenicity index, ND not done