Comparison of the pathogenic potential of highly pathogenic avian influenza (HPAI) H5N6, and H5N8 viruses isolated in South Korea during the 2016–2017 winter season

Figures & data

Fig. 1 Growth kinetics and attachment of viruses in human lung tissues ex vivo.

Replication of two Em/W541(H5N6) and CT/W555(H5N8) viruses were monitored in Madin-Darby Canine Kidney (MDCK) cells (a), Normal Human Bronchial Epithelial (NHBE) cells (b), Human lung tissue explants and corresponding culture supernatants (c and d, respectively) starting at 12 and 24 hpi intervals thereafter. Growth kinetics were compared to those of the control 2009 pandemic CA/07(H1N1) virus. The titers shown are means ± SD from three independently performed experiments. (*p < 0.05, **p < 0.01, ^†p < 0.001)

Biological properties of HPAI H5 viruses

Virus Name	H5 Clade	Biological characteristics						Transmission		Reference
		EID50	TCID50	Ck LD50	Dk LD50	IVPI	MLD50	Chicken	Duck
Em/W541 (H5N6)	2.3.4.4	9.2	7.8	3.2	5.5	2.7	5.5	6/6	6/6	[9]
CT/W555 (H5N8)	2.3.4.4	8.9	6.5	4.7	≥6.0	2.9	5.2	6/6	6/6	[11]
MDk/W452 (H5N8)	2.3.4.4	8.9	6.9	2.5	≥6.0	3	5.8	6/6	6/6	[14]
BDk/Gochang1 (H5N8) ^*	2.3.4.6	N/A	N/A	N/A	N/A	3^*	N/A	N/A	N/A	[4]

All values are expressed as log₁₀: EID₅₀, 50% egg infectious dose; TCID₅₀, 50% tissue culture infectious dose; Ck LD₅₀, 50% lethal dose in chickens; Dk LD₅₀, 50% lethal dose in ducks; IVPI, intravenous pathogenic index determined in chickens; MLD₅₀, 50% lethal dose in mice

N/A not applicable

^*As reported by Lee et al. (2014)

Fig. 2 Replication of Em/W541(H5N6) and CT/W555(H5N8) in avian models.

Virus replication was examined in chickens and ducks that had been experimentally inoculated via the intranasal route with 10⁶ EID₅₀/ml of each virus. Oropharyngeal swabs, cloacal swab, and lung titers of chickens are shown for Em/W541(H5N6) (a) and CT/W555(H5N8) (b). Oropharyngeal swabs, cloacal swabs, and lung titers from ducks are shown for Em/W541(H5N6) (c) and CT/W555(H5N8) (d). Mean viral titers (log₁₀ EID₅₀/ml) are shown for each group of birds. The limit of virus detection was 0.7 log₁₀ EID₅₀/ml

Viral tissue titers in experimentally inoculated animals

Tissue^a	Em/W541(H5N6)								CT/W555(H5N8)
	Chicken		Duck		Mice		Ferret		Chicken		Duck		Mice		Ferret
	1d^b	2d	3d	5d	3d	5d	3d	5d	3d	5d	3d	5d	3d	5d	3d	5d
Lung	5.5 ± 0.1^c	5.0 ± 0.2	4.6 ± 0.4	5.0 ± 0.5	4.2 ± 0.3	5.2 ± 0.3	6.0 ± 0.2	5.5 ± 0.3	5.5 ± 0.5^b	4.5 ± 0.2	4.2 ± 0.2	3.5 ± 0.3	3.5 ± 0.3	4.3 ± 0.2	3.5 ± 0.3	3.0 ± 0.2
Brain	2.5 ± 0.4	2.0 ± 0.2	3.8 ± 0.1	3.2 ± 0.4	–	–	3.2 ± 0.1	1.2 ± 0.4	3.3 ± 0.3	–	–	–	0.8 ± 0.5	1.8 ± 0.3	1.0 ± 0.3	–
Kidney	2.5 ± 0.3	3.0 ± 0.4	2.8 ± 0.4	4.5 ± 0.1	1.0 ± 0.1	–	–	–	5.3 ± 0.3	3.3 ± 0.2	4.3 ± 0.2	3.3 ± 0.1	1.2 ± 0.5	1.0 ± 0.5	–	–
Spleen	2.5 ± 0.2	2.3 ± 0.3	1.7 ± 0.4	–	2.7 ± 0.5	–	1.0 ± 0.2	2.0 ± 0.1	3.8 ± 0.4	–	3.5 ± 0.3	1.5 ± 0.2	1.5 ± 0.1		–	–
Heart	3.5 ± 0.3	2.5 ± 0.1	4.4 ± 0.4	5.4 ± 0.4	4.2 ± 0.2	2.7 ± 0.5	–	–	5.5 ± 0.3	3.5 ± 0.1	3.8 ± 0.2	1.8 ± 0.3	1.2 ± 0.2	1.2 ± 0.3	–	–
Liver	3.0 ± 0.2	2.8 ± 0.5	1.5 ± 0.1	–	2.2 ± 0.4	–	–	–	1.8^d	–	4.2 ± 0.2	2.7 ± 0.3	1.2 ± 0.3	1.5 ± 0.2	1.0 ± 0.2	1.0 ± 0.2
Colon	4.5 ± 0.1	4.75 ± 0.2	2.1 ± 0.4	2.7 ± 0.2	–	–	–	–	4.5 ± 0.3	3.75 ± 0.3	2.5 ± 0.1	1.3 ± 0.3	0.8 ± 0.4	1.5 ± 0.3	1.3 ± 0.3	1.0 ± 0.5

Dashed lines indicate negative for virus detection (lower limit = 0.7 log₁₀EID₅₀/g)

^aResults were obtained from three animals per group at each time-point and are expressed as log₁₀ EID₅₀/g tissue

^bDays post infection

^cStandard deviation titers

^dPositive detection in one

Fig. 3 Cytokine and chemokine responses in the lungs of infected mice.

Concentrations of various cytokines/chemokines in BAL fluid from mice at 1, 3, 5, and 7 dpi were measured by protein analysis with the Luminex™ Instrumentation Systems multiplex array reader (Bio-Plex Workstation from Bio-Rad Laboratories). The values shown are means ± SD (error bars) from BAL fluid of three mice per time point tested. (*p < 0.05)

Fig. 4 Histopathology and immunohistochemical staining for influenza virus antigen in mouse (a, b, e, and f) and ferret (c, d, g, and h) lungs at 3 (a, c, e, and g) and 5 dpi (b, d, f, and h) with HPAI Em/W541(H5N6) and CT/W555(H5N8).In the lung, viral antigens are widely presented in the alveolar septum which are mildly thickened with increased cellularity of inflammatory cells and also necrotic cell debris are presented in the lumen (arrows in a, b, e, and f)

Fig. 5 Replication of Em/W541(H5N6) and CT/W555(H5N8) in ferrets experimentally inoculated via the intranasal route with 10⁶ EID₅₀/ml of each virus.

Rectal temperatures and body weights were measured at 1, 3, 5, 7, 9, 11, and 14 dpi (a and b). Individual nasal wash titers are shown for ferrets inoculated with Em/W541(H5N6) and CT/W555(H5N8). To examine transmission, the inoculated animals were individually paired with a direct contact (DC) and a respiratory droplet (RD)-contact animal (1:1:1 setup, triplicate) (c and d). The mean viral titers (log₁₀ EID₅₀/ml) are shown for each group of mammals. The limit of virus detection was 0.7 log₁₀ EID₅₀/ml

Seroconversion in inoculated and contact ferrets

Virus	Exposure	Nasal wash titers (log10 EID50/ml)							Seroconversion (HI titer)
		1dpi	3dpi	5dpi	7dpi	9dpi	11dpi	14dpi
Em/W541 (H5N6)	Inoculated	6.2	5.2	4.2	–	–	–	–	320
	Direct contact	1.3	1.3	-	–	–	–	–	<10
	RD contact	–	–	–	–	–	–	–	<10
	Inoculated	7.3	5.5	5.3	–	–	–	–	640
	Direct contact	2.3	1.3	1.3	–	–	–	–	80
	RD contact	–	–	–	–	–	–	–	<10
	Inoculated	6.7	5.3	4.9	–	–	–	–	320
	Direct contact	2.3	1.3	1.3	–	–	–	–	80
	RD contact	–	–	–	–	–	–	–	<10
CT/W555 (H5N8)	Inoculated	3.3	2.0		–	–	–	–	80
	Direct contact	–	–	–	–	–	–	–	<10
	RD contact	–	–	–	–	–	–	–	<10
	Inoculated	3.8	2.5		–	–	–	–	160
	Direct contact	-	-	-	-	-	–	–	<10
	RD contact	–	–	–	–	–	–	–	<10
	Inoculated	3.8	2.4		–	–	–	–	80
	Direct contact	–	–	–	–	–	–	–	<10
	RD contact	–	–	–	–	–	–	–	<10