Pathogenesis and genetic characteristics of novel reassortant low-pathogenic avian influenza H7 viruses isolated from migratory birds in the Republic of Korea in the winter of 2016–2017

Figures & data

Number of avian influenza viruses (AIVs) isolated from wild birds in the Republic of Korea between 2010 and March 2017

Year	No. of total samples	No. of AIV-positive samples (prevalence^a; %)	No. of AIV-positive samples for H7 subtypes
			H7N1	H7N2	H7N3	H7N4	H7N6	H7N7	H7N8	H7N9	Subtotal (proportion^b; %)
2010	6789	56 (0.8)				1		2	1		4 (7.1)
2011	7156	48 (0.7)			1			1		11	13 (27.1)
2012	7889	42 (0.5)	5								5 (11.9)
2013	10,029	22 (0.2)	3					3			6 (27.3)
2014	13,228	137 (1.0)		3				1			4 (2.9)
2015	19,533	145 (0.7)						11			11 (7.6)
2016	9538	140 (1.5)						12		1	13 (9.3)
2017	3224	110 (3.4)	2	2	1		1	42			48 (43.6)
Sub-total	77,386	700 (0.9)	10	5	2	1	1	72	1	12	104 (14.9)

^a[(Number of AIV-positive samples)/(number of total samples)] × 100 (%).

^b[(Number of H7 viruses)/(number of AIV-positive samples)] × 100 (%).

Fig. 1 Phylogenetic analysis of H7 influenza A viruses in Eurasia.

a Phylogenetic tree of the HA genes of 41 H7 influenza A viruses, representative of 107 viruses isolated in the Republic of Korea between 2010 and 2017 (red lines), in addition to all 2963 H7 sequence >1600 nt from viruses isolated in Eurasia that were available from the Global Initiative on Sharing All Influenza Data (GISAID). Evolutionary analyses were conducted in RAxML. Red lines denote the H7 viruses isolated in South Korea. b Maximum-likelihood phylogenetic tree of a subset of group B and C H7 genes. Tree stability was determined by bootstrap analysis with 1000 replicates, and bootstrap values >70% are displayed above the branch nodes. Evolutionary analyses were conducted in MEGA6. For the H7 viruses isolated in the Republic of Korea in this study, those isolated during 2016–2017 are indicated by dots, and those isolated at other times are indicated by triangles. Other viral sequences were obtained from GISAID. Ck chicken, Dk duck, Kr Korea, WBF wild bird feces. c Phylogenetic tree of NA genes of 33 H7N7 influenza A viruses isolated in the Republic of Korea between 2010 and 2017 (red lines) and all 505 N7 sequences >1400 nt from viruses isolated in Eurasia that were available from GISAID. Evolutionary analyses were conducted in RAxML. d Maximum likelihood phylogenetic tree of a subset of group 1 and 2 N7 genes. Tree stability was determined by bootstrap analysis with 1000 replicates, and bootstrap values >70% are displayed above the branch nodes. Evolutionary analyses were conducted in MEGA6. For the H7 viruses isolated in the Republic of Korea in this study, those isolated during 2016–2017 are indicated by dots, and those isolated at other times are indicated by triangles. Other viral sequences were obtained from GISAID. Ck chicken, Dk duck, Kr Korea, WBF wild bird feces

Fig. 2 Evolution of avian influenza virus H7 subtypes in Eurasia.

An HA nucleotide-sequence phylogeny was inferred using the SRD06 partitioned-substitution model, an uncorrelated lognormal relaxed clock, and a Bayesian skyline coalescent model in BEAST v1.8.1 with a chain length of 100 million. A genome-constellation analysis was performed by generating gene clusters for each segment using a 97% nucleotide identity cutoff. Cluster assignments are represented by one colored box for each gene segment, creating a genome constellation for each virus. Coloring between the columns is independent, and the total number of colors in a column reflects the number of clusters generated for that gene segment. Ck chicken, Dk duck, Kr Korea, WBF wild bird feces

Fig. 3 Proposed evolutionary pathways leading to the generation of novel-reassortant H7 viruses.

Influenza A viruses are represented by ovals containing horizontal bars for the eight gene segments (from top to bottom, PB2, PB1, PA, HA, NP, NA, MP, and NS). Different colors represent different viral lineages. Solid ovals represent virus strains isolated from wild and domestic birds, and dotted ovals represent hypothetical viral strains. The timescale is indicated on the left side, and the different phases of evolution are indicated on the right side. The labels below the viral ovals are identifiers for corresponding viruses isolated in this study. Ck chicken, Dk duck, Kr Korea, WBF wild bird feces, Gs goose

Fig. 4 Locations of isolation of H7-subtype low pathogenicity (LP) influenza A viruses (IAVs) from wild bird habitats in the Republic of Korea during the winter of 2016–2017.

The light green and blue circles indicate the locations where the G1 (a) and G2 (b) subgroups of the H7 LPIAVs were isolated from wild bird habitats, and the associated collection dates are indicated. The IAVs are represented by ovals containing horizontal bars for the eight gene segments (from top to bottom, PB2, PB1, PA, HA, NP, NA, MP, and NS). The different colors represent different virus lineages. Genotypes G1 and G2 were subgroups within cluster II of group C of H7 LPIAVs

Replication and transmission of representative H7N7 viruses in 4-week-old specific-pathogen-free chickens

Isolate	Group	Sample	Swab viral titers (log10EID50/ml)^a					Seroconversion (HI titer)^b
			3 dpi	5 dpi	7 dpi	10 dpi	14 dpi
A/mandarin Dk/Kr/H539-3/2016 (H7N7)	Challenged	OP	4/5 (2.0 ± 1.6)	3/5 (1.8 ± 1.7)	1/5 (0.2 ± 0.5)	0/4	0/4	4/4 (64, 256, 256, 8)
		CL	3/5 (2.2 ± 2.4)	4/5 (3.6 ± 2.6)	4/5 (4.2 ± 2.5)	2/4 (1.6 ± 2.0)	2/4 (1.6 ± 2.5)
	Direct contact	OP	2/3 (1.0 ± 1.1)	2/3 (1.9 ± 2.0)	0/3	1/3 (1.4 ± 2.4)	1/3 (0.4 ± 0.7)	3/3 (8, 16, 32)
		CL	1/3 (0.7 ± 1.2)	1/3 (1.4 ± 2.4)	2/3 (3.5 ± 3.2)	1/3 (1.4 ± 2.4)	1/3 (1.7 ± 3.0)
A/mallard/Kr/H982-6/2017 (H7N7)	Challenged	OP	0/5	0/5	0/5	0/5	0/5	0/5
		CL	0/5	0/5	0/5	0/5	0/5
	Direct contact	OP	0/3	0/3	0/3	0/3	0/3	0/3
		CL	0/3	0/3	0/3	0/3	0/3

CL cloacal, dpi days post-infection, EID₅₀ 50% egg infective dose, Dk duck, HI hemagglutination inhibition, Kr Korea, OP oropharyngeal

Values shown are (number of infected birds)/(number of inoculated birds) in the challenge group, and (number of infected birds)/(number of naïve birds) in the direct-contact group. Values in parentheses are virus titer (log₁₀EID₅₀/ml), mean ± standard deviation. The inoculation dose for chickens was 10⁶ EID₅₀/0.1 ml

^aMean viral titers from OP and CL swabs were calculated by the Reed and Muench method.

^bSera were collected from chickens at 14 dpi. Seroconversion was confirmed by HI assay.

Virus titers in tissues from 4-week-old specific-pathogen-free chickens challenged with representative H7N7 isolates

Isolate	Tissue
	Trachea	CT	Lung	Kidney	Spleen	Brain	Pancreas
A/mandarin Dk/Kr/H539-3/2016 (H7N7)	3/4 (2.2 ± 1.5)	3/4 (3.1 ± 2.1)	2/4 (1.6 ± 1.9)	2/4 (2.9 ± 3.3)	2/4 (1.4 ± 1.6)	2/4 (1.6 ± 1.8)	3/4 (4.4 ± 3.2)
A/mallard/Kr/H982-6/2017 (H7N7)	0/3	0/3	0/3	0/3	0/3	0/3	0/3

EID₅₀ 50% egg infective dose, CT cecal tonsil, Dk duck, Kr Korea

Values shown are (number of birds with virus in the tissue)/(number of inoculated birds). Values in parentheses are virus titer (log₁₀EID₅₀/g). The virus titer is the mean ± standard deviation of the samples. The inoculation dose for chickens was 10⁶ EID₅₀/0.1 ml

Fig. 5 Histopathological changes and immunostaining of tissues infected with A/mandarin duck/Korea/H539-3/2016 (H7N7).

Photomicrographs of hematoxylin and eosin (H&E)-stained and immunohistochemically stained tissue sections collected 4 days postinfection from chickens challenged with A/mandarin duck/Korea/H539-3/2016 (H7N7) virus. a Chickens challenged with the H7N7 virus showing severe hemorrhaging (h) and congestion (c) in lung tissue (H&E stain). b Infected chickens showing focal necrosis and mononuclear-cell infiltration (m) in cerebral tissue (H&E stain). c Infected chickens showing mild perivascular cuffing (arrow) in cerebral tissue (H&E stain). d IAV NP antigens were immunostained in alveolar macrophages (arrows) in lung tissue. e IAV antigens were immunostained in macrophages of red pulp (arrows) in spleen tissue. f IAV antigens were immunostained in cerebral tissue neurons (arrows)