Diverse infectivity, transmissibility, and pathobiology of clade 2.3.4.4 H5Nx highly pathogenic avian influenza viruses in chickens

Figures & data

Table 1. Mortality, virus shedding, and seroconversion of chickens inoculated with and contact of clade 2.3.4.4 highly pathogenic avian influenza viruses. Inoculum dose for intrachoanal inoculation included in first column.

Challenge viruses (clade) (titer^a)	Group	Mortality (dead/total) (MDT^b)	Viral shedding^c (positive/total)	Seroconversion^d (positive/total)
A/Ck/VNM/14 (2.3.4.4a) (10^5.7)	Inoculated	9/10 (3.3)	9/10	0/1
	Contact	2/3 (4.5)	2/3	0/1
A/Dk/VNM/15 (2.3.4.4a) (10^5.9)	Inoculated	9/10 (3.2)	9/10	0/1
	Contact	2/3 (4.5)	2/3	0/1
A/TD/DNK/16 (2.3.4.4b) (10^6.1)	Inoculated	10/10 (2.1)	10/10	N/A
	Contact	0/3	0/3	0/3
A/Tk/HUG/16 (2.3.4.4b) (10^6.5)	Inoculated	10/10 (2.1)	10/10	N/A
	Contact	0/3	0/3	0/3
A/Tk/MN/15 (2.3.4.4c) (10^5.9)	Inoculated	8/10 (2.1)	8/10	0/2
	Contact	2/3 (3.5)	2/3	0/1
A/Ck/Hok/16 (2.3.4.4e) (10^6.3)	Inoculated	10/10 (2)	10/10	N/A
	Contact	3/3 (3)	3/3	N/A
A/MD/KOR/16 (2.3.4.4e) (10^6.5)	Inoculated	10/10 (2)	10/10	N/A
	Contact	3/3 (2)	3/3	N/A
Sham (PBS)	Negative control	0/10	0/10	0/10

Note: N/A: not applicable.

^a The viruses were inoculated via the intra-choanal route with approximately 10⁶ EID₅₀ in 0.1 mL of each isolate, and the inoculum was back tittered.

^b MDT, Mean death time.

^c Number of birds shedding detectable viruses by the oral and/or cloacal route in at least one time point / total number of birds. The viral shedding was evaluated and quantified by H5 gene-specific qRT-PCR.

^d Determined in 14 dpi serum samples from surviving chickens by HI assay using homologous antigen.

Figure 1. Egg infectious dose-equivalent titers from the oropharyngeal (OP) and cloacal (CL) swabs collected from chickens inoculated with or in contact with chickens shedding clade 2.3.4.4 highly pathogenic avian influenza viruses. Chickens in contact group were transferred into the isolators with the inoculated birds on 1 dpi. All samples were negative at 7, 10, and 14 dpi and are not shown. The lower detection limit was 10^2.5 50% egg infectious dose (EID₅₀)/mL. Negative RT-PCR samples were treated as 10^2.4 EID₅₀/mL for statistical purposes.

Figure 2. Histopathology and immunohistochemical staining for AIV antigen in tissues of chickens infected with clade 2.3.4.4 highly pathogenic avian influenza viruses, 2 days post-inoculation. Hematoxylin-eosin staining (A-E); immunohistochemistry (insets), virus staining of cells (nuclei – clearly stained circular bodies) in red. A. Lung, A/Ck/VNM/14 (clade 2.3.4.4a). Interstitial pneumonia with lymphoplasmacytic infiltrates. Inset: Lung, same area. Viral antigen in endothelial cells, pneumocytes, mononuclear cells. B. Pancreas, A/TD/DNK/16 (clade 2.3.4.4b). Necrotic pancreatic acinar cells with lymphoplasmacytic infiltrates. Inset: Pancreas, same area. Viral antigen in acinar cells, duct cells, endothelial cells. C. Thymus, A/TD/DNK/16 (clade 2.3.4.4b). Necrosis in medulla and lymphocyte depletion in cortex. Inset: Thymus, same area. Viral antigen in mononuclear cells, thymic epithelium in medullary area, endothelial cells. D. Duodenum, A/Ck/Hok/16 (clade 2.3.4.4e). Focal necrosis with lymphoplasmacytic infiltrates in submucosa. Inset: Duodenum, same area. Viral antigen in enterocytes, endothelial cells, mononuclear cells in lymphoid associated tissue, smooth muscle cells. E. Cecal tonsil, A/MD/KOR/16 (clade 2.3.4.4e). Focal necrosis with lymphoplasmacytic infiltrates in submucosa. Inset: Cecal tonsil, same area. Viral antigen in enterocytes, endothelial cells, mononuclear cells in lymphoid associated tissue.

Table 2. Microscopic lesions and viral antigen distribution in chickens inoculated with clade 2.3.4.4 viruses, 2 days post-inoculation.

	A/Ck/VNM/14 (2.3.4.4a)		A/Dk/VNM/15 (2.3.4.4a)^a		A/TD/DNK/16 (2.3.4.4b)		A/Tk/HUG/16 (2.3.4.4b)		A/Tk/MN/15 (2.3.4.4c)		A/Ck/Hok/16 (2.3.4.4e)		A/MD/KOR/16 (2.3.4.4e)
Tissue	HE^b	IHC^c	HE	IHC	HE	IHC	HE	IHC	HE	IHC	HE	IHC	HE	IHC
Nasal turbinates	++ − ++	+++ + +++	++ ++	+++ +++	++ ++ +++	+++ ++ +++	− +++ +++	+ +++ +++	++ ++ ++	+++ ++ +	++ ++ ++	+++ +++ +++	++ ++ ++	+++ ++ +++
Trachea	− + −	+ ++ −	− −	− −	nt ++ ++	nt ++ ++	− − −	+ − −	− − −	++ − +	− ++ −	+ +++ −	− − ++	+ + ++
Lung	+ +++ +++	++ +++ +++	+++ +++	+++ +++	++ ++ +++	++ +++ +++	+++ +++ +++	+++ +++ +++	+++ ++ +++	++ ++ +++	− − −	− − ++	+ + ++	++ ++ +++
Heart	+++ +++ +++	+++ +++ +++	+++ −	+++ −	+++ − +++	+++ − +++	+++ +++ +++	+++ +++ ++	+++ +++ +++	+++ +++ +++	+++ +++ +++	+++ +++ +++	+++ +++ +++	+++ +++ +++
Brain	++ +++ ++	++ +++ +++	+++ ++	+++ +++	++ − ++	+++ + +++	− + −	++ ++ ++	+ + +	++ + +	++ ++ ++	++ +++ ++	++ ++ +	++ ++ +
Duodenum	− − −	+ +++ +++	− −	+ ++	− − +	+ ++ +++	− − −	++ ++ −	nt − −	nt + −	− + +	++ +++ +++	+ − ++	+++ ++ +++
Ileum	nt − −	nt ++ ++	− −	+ −	++ ++ +	+++ ++ +	− − −	++ − −	nt − nt	nt + nt	− − −	− ++ ++	− − nt	− +++ nt
Cecal tonsil	nt − −	nt ++ ++	− −	+ −	nt nt nt	nt nt nt	− − −	+ ++ ++	nt − −	nt ++ −	− − −	++ +++ −	− +++ −	+++ +++ ++
Pancreas	− − +	+ ++ ++	− −	++ −	++ − ++	++ − +	+ − −	++ ++ −	− − −	− + −	− − +	++ ++ ++	− − −	+ − +
Liver	− − +	− +++ +++	− −	+ −	− − −	+ + ++	− − −	+ − −	− − −	+ − −	+ + +	+++ ++ +++	− − −	++ ++ ++
Spleen	++ ++ ++	++ ++ +++	++ ++	+++ +++	+++ ++ +++	− − +++	+++ +++ +++	+++ +++ ++	++ ++ ++	+++ ++ ++	nt +++ −	nt +++ ++	− +++ +++	++ +++ +++
Thymus	+ +++ +++	++ +++ +++	+++ +++	+++ +++	− +++ +++	+ +++ +++	+++ +++ +++	+++ +++ +++	+++ − +	+++ + ++	− ++ +	− +++ −	− − −	− + +
Cloacal bursa	+ +++ +++	− +++ ++	++ ++	+++ ++	+++ +++ +++	− − +	+++ +++ −	+ − −	+ + −	+ + −	++ − −	++ + +	− +++ −	− ++ +
Kidney	− − ++	− ++ +++	− −	++ +	+ − ++	+ + ++	++ − −	+ + +	− − −	− − −	+ − −	++ − −	− − −	− + −
Skeletal muscle	− − −	− − −	− − −	− − −	− − −	− − −	− − −	− − −	− − −	− − −	− − −	− − −	− − −	− − −

Note: nt: no tissue.

^a One bird from the A/Dk/VNM/15-inoculated group showed no microscopic lesions or IHC staining in any tissue. Because this bird had no virus shedding or PCR-positive tissues, it was considered non-infected and was excluded from the histopathological evaluation.

^b Histopathology score of lesions in hematoxylin-eosin (HE) staining: −: no lesions; +: mild; ++: moderate; +++: severe.

^c Immunohistochemical (IHC) staining: −: no antigen staining; +: infrequent; ++: common; +++: widespread.

Table 3. Microscopic lesions and viral antigen distribution in chickens inoculated with clade 2.3.4.4a-e highly pathogenic avian influenza viruses, 2 days post-inoculation.

Tissue	Lesions	Cell types expressing virus antigen
Nasal cavity	Epithelial cell necrosis and desquamation, rhinitis, sinusitis, mononuclear cell infiltrates, heterophilic rhinitis	Vascular endothelial cells, vascular endothelial cells of mucosal glands, nasal epithelial cells, respiratory epithelial cells, nasal gland epithelium, mononuclear cells
Trachea	Focal necrosis, lymphoplasmacytic infiltrates	Pseudostratified epithelial cells, vascular endothelial cells, sternotrachealis muscle
Lung	Interstitial pneumonia, edema, congestion, necrosis, lymphoplasmacytic infiltrates	Vascular endothelial cells, pneumocytes, mononuclear cells
Heart	Focal necrosis of myocardiocytes	Myocardiocytes, vascular endothelial cells
Brain	Neuronal necrosis, gliosis, chromatolysis of Purkinje cell layer	Neurons, Purkinje cells, ependymal cells, glial cells, vascular endothelial cells
Intestine	Focal necrosis, lymphoplasmacytic infiltrates in submucosa	Villi enterocytes, vascular endothelial cells, mononuclear cells in lymphoid associated tissue, smooth muscle cells
Pancreas	Degeneration of individual pancreatic acinar cells, focal lymphoplasmacytic infiltrates	Acinar cells, duct cells, vascular endothelial cells
Liver	Focal necrosis, lymphoplasmacytic infiltrates	Kupffer cells, hepatocytes, vascular endothelial cells, macrophages
Spleen	Multifocal necrosis, hemorrhages, lymphoid depletion	Mononuclear cells, vascular endothelial cells
Thymus	Focal to multifocal necrosis in medullary area (evident in Hassall's corpuscles), lymphocyte depletion, apoptotic lymphocytes in cortex area	Mononuclear cells, thymic epithelium in medullary area, vascular endothelial cells
Cloacal bursa	Lymphocyte necrosis and apoptosis, lymphocyte depletion, phagocytic hyperplasia	Mononuclear cells, medullary support cells, vascular endothelial cells
Kidney	Focal necrosis of tubular epithelium with lymphoplasmacytic infiltrates	Tubular epithelial cells, glomerular cells, vascular endothelial cells
Skeletal muscle	No lesion	No lesion

Figure 3. Egg infectious dose-equivalent titers in brain (A), heart (B), lung (C) and spleen (D) collected from chickens inoculated with clade 2.3.4.4 highly pathogenic avian influenza viruses at 2 days post-inoculation (n = 3 chickens/virus). Viral titers sharing at least one superscript letter indicate no statistically significant differences (p ≥ 0.05).