Pathobiological features of a novel, highly pathogenic avian influenza A(H5N8) virus

Figures & data

Figure 1 Growth kinetics and attachment of viruses in human respiratory tissues ex vivo. Replication of the MDk/W452(H5N8) virus was monitored in human nasal respiratory epithelial (A) and lung (B) tissue explants and corresponding culture supernatants (C and D, respectively) starting at 12 hpi and at 24-h intervals thereafter. Growth kinetics were compared to those of the control HPAI A(H5N1) (En/W149 and MDk/W401) and 2009 pandemic CA/07(H1N1) viruses. The titers shown are means±SD from three independently performed experiments. Immunohistochemical detection of viral NP antigen was performed using nasal and lung tissue blocks incubated with CA/07(H1N1) (E and F), the control strain, or MDk/W452(H5N8) (G and H), the test virus. Arrows indicate positive immunostaining. Scale bars=50 µm. *P<0.05, ***P<0.0001 relative to MDk/W452(H5N8).

Figure 2 Virus receptor-binding specificity assays. (A) Binding affinity of inactivated whole viruses to SA α2,3′-SL-PAA-biotin, SA α2,6′-SL-PAA-biotin or SA α2,6′SLN-PAA-biotin glycans. The results shown are means±SD (mean of three replicates). The dashed lines indicate the limit of detection. (B) HA assays using re-sialylated cRBCs. The results obtained with re-sialylated cRBCs were normalized to the results obtained with untreated cRBCs.

Table 1 Viral tissue titers in experimentally inoculated animals^a

Tissue	MDk/W452(H5N8)							En/W149(H5N1)								MDk/W401(H5N1)
	Chicken												Duck												Mouse												Ferret		Chicken		Duck		Mouse		Ferret		Chicken		Duck		Mouse		Ferret
	3d^b	5d	3d	5d	3d	5d	3d	5d	3d	5d	3d	5d	3d	5d	3d	5d	3d	5d	3d	5d	3d	5d	3d	5d
Lung	6.5 ±0.1^c	5.0±0.2	5.0±0.3	4.0±0.1	2.6±0.3	3.0±0.4	2.0±0.2	2.5±0.3	6.0±0.3	6.5±0.4	6.0±0.3	6.5±0.1	6.0±0.2	6.25±0.2	5.0±0.3	4.0±0.3	5.3±0.1	6.0±0.4	5.5±0.2	6.5±0.3	4.8±0.4	4.1±0.1	3.7±0.1	2.5±0.1
Brain	3.5±0.4	—	—	—	—	2	1±0.1	1.0	4.0±0.2	4.5±0.2	4.0±0.2	5.0±0.2	3.7±0.3	3.0±0.1	—	—	3.3±0.2	4.0±0.3	1.5	4.5±0.2	3.0±0.2	2	—	—
Kidney	5.5±0.2	3.0±0.2	3±0.2	3.5±0.2	—	—	—	—	3.0±0.2	3.0±0.2	3.5±0.2	4.0±0.1	1.5±0.1	1.0±0.1	2.5±0.2	1.5	1.5±0.1	2.0±0.1	2.5±0.2	3.5±0.2	3.5±0.3	2.5±0.1	—	—
Spleen	3.5±0.2	—	3.5±0.1	1.5	—	—	—	1.0±0.1	1.5±0.1	2.0±0.1	3.5±0.1	4.5±0.2	1.5±0.1	1.0±0.1	2.0±0.1	1	2.5±0.2	3.0±0.2	2.3±0.1	4.0±0.1	1.0±0.1	—	—	—
Heart	6.5±0.3	2.5±0.1	5±0.3	2.75±0.1	—	—	—	—	2.5±0.1	3.0±0.1	4.5±0.2	5.5±0.3	2.5±0.2	1.5±0.3	—	—	2.5±0.3	3.0±0.1	3.5±0.2	5.0±0.3	1.5±0.1	1.0±0.2	—	—
Liver	1.5^d	—	3.5±0.2	—	—	—	—	1.5	2.5±0.2	1.5±0.2	4.0±0.2	2.5±0.1	2.5	—	—	—	1.5±0.1	—	3.0±0.1	—	1.5	—	—	—
Colon	2.5±0.1	2.75±0.2	4±0.3	3±0.3	—	—	—	1.5	5.3±0.4	5.5±0.3	6.5±0.3	6.5±0.2	2.0±0.1	1.0±0.1	—	—	5.3±0.4	5.5±0.2	6.5±0.3	6.5±0.3	1.5±0.2	1.0±0.1	—	—

Dashed lines indicate that the tissue was negative for virus detection (lower limit=0.7 log₁₀EID₅₀/g).

a Results were obtained from three animals per group at each time point and are expressed as log₁₀ EID₅₀/g tissue.

b Day.

c Standard deviation titers.

d Positive detection in one animal.

Figure 3 Replication of MDk/W452(H5N8) in mammalian animal models. Virus replication was examined in mice, ferrets, dogs and cats that had been experimentally inoculated intranasally with 10⁶ EID₅₀/mL of designated virus. Mouse lung titers shown are means±SD from three animals (A). Individual nasal wash titers are shown for ferrets inoculated with MDk/W452(H5N8) (B), En/W149(H5N1) (C) or MDk/W401(H5N1) (D). To examine transmission, the inoculated animals were individually paired with a DC and an RD-contact animal (1∶1∶1 setup, triplicate). Three infected dogs (E) and cats (F) were also individually cohoused with contact animals in the same cage at 1 dpi and monitored for virus shedding in nasal swabs. The limit of virus detection was 0.7 log₁₀EID₅₀/mL; titers below that limit are shown as 0.5 log₁₀EID₅₀/mL (dashed lines). An asterisk (*) signifies seroconversion as determined by HI analysis at 21 dpi. Inf, directly infected animal.

Figure 4 Histopathology and immunohistochemical staining for influenza virus antigen in tissues of mouse (A–D) and ferret (E–H) infected with the HPAI A(H5N8) virus. In the lung, the alveolar septum is mildly thickened with increased cellularity (mouse: A and B; ferret: E and F). The influenza viral NP antigen was rarely detected in the lungs and hearts of mice (B and C, respectively) and was infrequently detected in mononuclear phagocytic cells in the lungs and hearts of ferrets (F, arrows; G, arrowheads, respectively). In contrast, in the nasal cavities of mice (D, arrows) and in the intestines of ferrets (H), mononuclear phagocytic cells were commonly immunopositive for influenza virus antigen. Scale bars=50 µm.

Figure 5 Cytokine and chemokine responses in the lungs of infected mice. Concentrations of various cytokines/chemokines in BAL fluids of infected mice at 1, 3, 5 and 7 dpi were measured by protein analysis with the Luminex-based multiplex immunoassay kit (Affymetrix, Santa Clara, CA, USA). The values shown are means±SD (errors bars) from three mouse BAL fluids per time point tested.

Figure 6 Putative generation of the novel HPAI A(H5N8) viruses that caused domestic poultry outbreaks in Korea and Japan in 2014. Genes from top to bottom are PB2, PB1, PA, HA, NP, NA, M and NS. For simplicity, each color represents an individual virus background. Italicized virus names are the Korean strains, and the underlined virus is the isolate we characterized. Virus abbreviations: Dk/Jiangxi/28(H11N9), A/duck/Jiangxi/28/2009(H11N9); Dk/Jiangsu/k1203(H5N8), A/duck/Jiangsu/k1203/2009(H5N8); Dk/Jiangsu/1-15(H4N2), A/duck/Jiangsu/1-15/2011(H4N2); Dk/EC/1111(H4N2), A/duck/Eastern China/1111/2011(H4N2); Dk/Zhejiang/W24(H5N8), A/duck/Zhejiang/W24/2013(H5N8); BDk/Gochang1(H5N8), A/broiler duck/Korea/Gochang1/2014(H5N8; BTl/Donglim3(H5N8), A/Baikal teal/Korea/Donglim3/2014(H5N8); Dk/Buan2(H5N8), A/duck/Korea/Buan2/2014(H5N8); MDk/W452(H5N8), A/mallard duck/W452/2014(H5N8); Ck/Kumamoto/1-7(H5N8), A/chicken/Kumamoto/1-7/2014(H5N8).