Assessing potential pathogenicity of novel highly pathogenic avian influenza (H5N6) viruses isolated from Mongolian wild duck feces using a mouse model

Figures & data

Figure 1. Sampling locations of the Mongolia A/MN/H5N6/2018-19 indicated with red circles (A/duck/Ugii lake/#66/2018/H5N6; A/duck/Khunt lake/#500/2019/H5N6), Mongolia/H5N6/2020 indicated with yellow circles (A/Whooper swan/Mongolia/24/2020/H5N6; A/Swan goose/Mongolia/02/2020/H5N6), and Xinjiang, China viruses indicated with violet circle (A/goose/Xinjiang/12.24_WLMQXL003-C/2018/H5N6-mixed). These sampling sites were close to the site where the novel H5N6/2020 isolates were collected with both sites being separated by about 90 km. Map was edited from Google Maps; March, 15, 2022 (https://www.google.com/maps/d/edit?hl=en&mid=1ZZ4NSJSmmXZo5lbR5GidJWsy422SceyE&ll=28.563846898417626%2C85.17260164694872&z=4).

Table 1. Closest relatives to our viral nucleotide sequences from A/MN/H5N6/2018-19 in the GenBank databases and GISAID.^a

Gene segment	Closest relative	Homology	GenBank ID
PB2	A/chicken/Xinjiang/12.24_WLMQXL006-O/2018 (mixed)	99.74%	MW106970.1
	A/chicken/Guangdong/7.20_DGCP022-O/2017	99.08%	MW104088.1
PB1	A/goose/Xinjiang/12.24_WLMQXL004-O/2018 (mixed)	99.82%	MW110193.1
	A/chicken/Guangdong/7.20_DGCP022-O/2017 (mixed)	99.74%	MW104088.1
PA	A/goose/Xinjiang/12.24_WLMQXL004-C/2018 (mixed)	99.77%	MW110193.1
	A/chicken/Guangdong/7.20_DGCP022-O/2017 (A/H0)	99.49%	MW104088.1
HA	A/goose/Xinjiang/12.24_WLMQXL003-C/2018 (mixed)	99.65%	MW110187.1
	A/chicken/Guangdong/7.20_DGCP022-O/2017 (mixed)	98.71%	MW104088.1
NP	A/goose/Xinjiang/12.24_WLMQXL004-O/2018 (mixed)	99.80%	MW110193.1
	A/chicken/Guangdong/7.20_DGCP022-O/2017 (mixed)	99.47%	MW104088.1
NA	A/chicken/Xinjiang/12.24_WLMQXL006-O/2018 (mixed)	99.78%	MW106970.1
	/duck/Hunan/11.30_YYGK62E3-OC/2017 (A/H5N6)	99.06%	MW108138.1
M	A/goose/Xinjiang/12.24_WLMQXL003-C/2018 (mixed)	100.00%	MW110187.1
	A/duck/Guangdong/7.20_DGCP036-O/2017(H5N6)	99.74%	MW097501.1
NS	A/goose/Xinjiang/12.24_WLMQXL004-O/2018 (mixed)	99.85%	MW110196.1
	A/chicken/Guangdong/7.20_DGCP022-O/2017(mixed)	99.56%	MW104088.1

^a Last accessed 12 November 2021 NCBI, https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi; GISAID, https://platform.epicov.org/epi3/frontend#384e22. HA, hemagglutinin; NA, neuraminidase; MP, matrix protein; NP, nucleoprotein; NS, nonstructural protein; PA, acidic polymerase; PB1, basic polymerase 1; PB2, basic polymerase 2.

Figure 2. The HA and NA gene maximum clade credibility tree for the A/MN/H5N6/2018-19 viruses was constructed using the BEAST 1.10.4 software package (https://beast-dev.github.io/beast-mcmc). Node bars indicate a 95% posterior density for each node height and the coloured of each branch indicate its posterior probability. Both H5N6 isolates reported in this study are shown in orange, the human H5 in red and the MN/H5N6/2020 in blue. The tMRCA is indicated at the bottom of the tree, and was estimated using the Bayesian Markov chain Monte Carlo method in BEAST 1.10.4.

Table 2. Date of isolation for the most recent common ancestor for the HPAIVs H5N6 Mongolia/ 2018–2019 and Xinjiang/2018 viruses when using the surface gene as the comparator.

Gene segment	tMRCA^a Mean (Moth & Year)	tMRCA 95% HPD^# (Moth & Year)	Posterior probability
A/wildDuck/MN/H5N6/2018-19 and A/Xinjiang/12.24_WLMQXL/2018 viruses
HA	MAY.2018	MAR.2018 – JUL.2018	1
NA	JUL.2018	MAR.2018 – AUG.2018	0.99
A/wildDuck/MN/H5N6/2018-19
HA	SEP.2018	JUL.2018 – SEP.2018	1
NA	AUG.2018	JUL.2018 – SEP.2018	0.99

^a tMRCA, time of most recent common ancestry. tMRCA represents the most likely timing for a common ancestral node; ^#HPD, highest posterior density; HA, hemagglutinin; NA, neuraminidase; Posterior probability suggest a high corresponding confidence measure for phylogenetic accuracy when calculated as a value close to 1.

Table 3. Molecular characteristics of the HPAI A/MN/H5N6/2018-19 isolates identified in this study.

Virus	Host	Clade 2.3.4.4	PB2		PB1-F2	HA^a							NA^b	NS
			627	701	Expression	Cleavage site	94	133	156	222	223	224	Stalk deletion 58–68	Deletion 80–84
1	Duck	h	E	D	NO	RERRRKR↓G	N	A	A	Q	R	G	Yes	Yes
2	WS	h	E	D	NO	RERRRKR↓G	N	A	A	Q	R	G	Yes	Yes
3	Human	h	E	D	NO	RERRRKR↓G	N	A	A	Q	R	G	Yes	Yes
4	Human	h	K	D	NO	RERRRKR↓G	N	A	A	Q	R	G	Yes	Yes
5	Human	h	K	D	NO	RERRRKR↓G	N	–	A	Q	G	G	Yes	Yes
6	Human	e	K	D	NO	RERRRKR↓G	N	A	T	Q	R	G	Yes	Yes
7	Human	clade 0	K	D	NO	RERRRKKR↓G	N	S	T	Q	S	G	Yes	No

^a H5, numbering.

^b N6, numbering; WS, Whooper swan; 1, HPAI MG/H5N6/2018-19 isolates; 2, A/Whooper Swan/Mongolia/24/2020 (H5N6); 3, A/Chongqing/00013/2021(H5N6); 4, A/Guangxi/31906/2018 (H5N6); 5, A/Jiangsu/32888/2018 (H5N6); 6, A/Guangzhou/39715/2014 (H5N6); 7, A/Hong Kong/483/1997 (H5N1).

Figure 3. Comparison of H5N6 Mongolia 2019 isolate pathogenicity and Human isolate pathogenicity in a mouse model. Each group of five 6-week-old mice were inoculated via the intranasal route with between 10¹ and 10⁶ EID₅₀ per mouse for each virus in each mouse and then evaluated for survival and body weight change over a 14 day period DPI. A-D, Subclade 2.3.4.4 h A/duck/Khunt lake/#500/2019(H5N6); B-E, Subclade 2.3.4.4e RG A/Guangzhou/39715/2014 (H5N6); C-F, Clade 0 RG A/Hong Kong/483/1997 (H5N1).

Table 4. HI evaluations of HPAI A(H5N6)/Mongolia /2019 influenza viruses using chicken antiserum.

Clade	HA inhibition titre (dilution factor) in antiserum against clades
	0	2.3.4.4e	2.3.4.4c	2.3.4.4h
0	≥2048	≤16	64	64
2.3.4.4e	128	1024	512	128
2.3.4.4c	128	512	1024	16
2.3.4.4h	128	32	128	≥2048

HA, Hemagglutinin. Homologous titres are boldface and underlined

Clade 0: A (H5N1)/Hong Kong/483/1997; subclade 2.3.4.4e: A(H5N6)/Guangzhou/39715/2014.

Subclade 2.3.4.4c: A (H5N6)/Duck/Foshan/41/2019; subclade 2.3.4.4h: A(H5N6)/Mongolia /2019.