Evolution and extensive reassortment of H5 influenza viruses isolated from wild birds in China over the past decade

Figures & data

Figure 1. Geographical locations and bird species involved in the wild bird outbreaks caused by different genotypes of H5 viruses.

Table 1. H5 avian influenza outbreaks in wild birds in China.

Time	Province	No. of bird died	Bird species/order	Virus information
				Name	HA clade	IVPI
May 29, 2010	Tibet (TB)	170	brown-headed gull (BHGL)/charadriiformes (CHA)	BHGL/TB/1/2010(H5N1)	2.3.2.1c	/*
				BHGL/TB/2/2010(H5N1)	2.3.2.1c	/
January 5, 2015	Henan (HeN)	93	whooper swan (WS)/anseriformes (ANS)	WS/HeN/1/2015(H5N1)	2.3.2.1f	2.90
May 27, 2015	Inner Mongolia (IM)	300	black-necked grebe (BNG)/ciconiiformes (CIC)	BNG/IM/2/2015(H5N1)	2.3.2.1f	/
May 28, 2015	TB	415	bar-headed Goose (BHG)/ANS, BHGL/CHA	BHG/TB/3/2015(H5N1)	2.3.2.1f	/
				BHGL/TB/4/2015(H5N1)	2.3.2.1f	/
				BHG/TB/5/2015 (H5N1)	2.3.2.1f	/
June 13, 2015	Jiangsu (JS)	1858	black swan (BS)/ANS, greylag goose (GG)/ANS, green peacock (GP)/galliformes (GAL), common pheasant (CP)/GAL	BS/JS/5/2015(H5N1)	2.3.2.1d	/
				GG/JS/6/2015(H5N1)	2.3.2.1d	3.00
				GP/JS/7/2015(H5N1)	2.3.2.1d	/
				CP/JS/8/2015(H5N1)	2.3.2.1d	/
June 16, 2015	Qinghai (QH)	2361	great black-headed gull (GBHG)/CHA	GBHG/QH/9/2015(H5N1)	2.3.2.1f	2.68
December 3, 2015	Hunan (HuN)	381	golden pheasant (GDP)/GAL, CP/GAL	GDP/HuN/10/2015(H5N6)	2.3.4.4g	/
				CP/HuN/11/2015(H5N6)	2.3.4.4g	2.90
				GP/HuN/12/2015(H5N6)	2.3.4.4g	/
				GP/HuN/13/2015(H5N6)	2.3.4.4g	2.76
				GP/HuN/15/2015(H5N6)	2.3.4.4g	/
				GP/HuN/14/2015(H5N2)	2.3.4.4c	2.54
January 27, 2016	HuN	91	GP/GAL silver pheasant (SP)/GAL, WS/ANS	GP/HuN/1/2016(H5N6)	2.3.4.4f	2.68
				CP/HuN/2/2016(H5N6)	2.3.4.4f	2.74
				SP/HuN/3/2016(H5N6)	2.3.4.4f	/
				WS/HuN/4/2016(H5N6)	2.3.4.4f	/
December 24, 2016	Shanxi (SX)	18	WS/ANS	WS/SX/6/2016(H5N8)	2.3.4.4b	2.56
				WS/SX/7/2016(H5N8)	2.3.4.4b	/
January 3, 2017	HuN	1054	GG/ANS	GG/HuN/1/2017(H5N6)	2.3.4.4e	2.62
January 12, 2017	Hubei (HuB)	99	BS/ANS	BS/HuB/2/2017(H5N8)	2.3.4.4b	/
				BS/HuB/3/2017(H5N8)	2.3.4.4b	/

*:/, not done.

Figure 2. Phylogenetic analysis of H5 wild bird viruses and genotypes. The Maximum Clade Credibility (MCC) tree of the HA gene was constructed by using the package BEAST (v1.8.4). The clades shown in different colours in the tree were annotated by using FigTree (v1.4.4) (a), and the origins of each gene segment are indicated by different coloured bars (b). Phylogenetic trees with complete information are provided as Supporting Figures S1 and S2.

Figure 3. Diagrammatic representation of the formation of different H5 reassortants. Viral particles are represented by coloured ovals containing horizontal bars representing the 8 gene segments (from top to bottom: PB2, PB1, PA, HA, NP, NA, M, and NS). Segments in descendant viruses are coloured according to their corresponding source virus (top) to illustrate gene ancestry through reassortment events. Possible donor viruses are adjacent to arrow tails; arrowheads point to the resulting reassortants. The timeline in the middle indicates the possible time of virus emergence or reassortment events. The tMRCA was estimated by using the Bayesian Markov chain Monte Carlo method in the BEAST v1.8.4 software package.

Table 3. Replication and virulence of different H5 viruses in ducks.

Virus	Replication in organs (mean titre)^a		Virus shedding on different days post inoculation, positive/total (mean titre: oropharynx/cloaca)^b						Death/total		Seroconversion: positive/total (mean HI antibody titre, log2)
			Day 3		Day 5		Day 7
	Lung	Brain	Inoculated	Contact	Inoculated	Contact	Inoculated	Contact	Inoculated	Contact	Inoculated	Contact
GG/JS/6/2015(H5N1)	4.8	5.6	/	/	/	/	/	/	5/5	3/3	/	/
WS/HeN/1/2015(H5N1)	5.0	7.2	5/5 (3.7/1.3)	3/3 (2.7/0.9)	5/5 (2.0/1.5)	3/3 (3.0/0.9)	3/3 (1.2/<)	3/3 (1.6/1.1)	4/5	0/3	1/1 (6.0)	3/3 (3.7)
GBHG/QH/9/2015(H5N1)	6.4	3.3	5/5 (3.4/0.9)	3/3 (4.2/2.3)	0/5	3/3 (2.2/0.9)	0/5	0/3	0/5	0/3	5/5 (6.4)	3/3 (5.0)
GP/HuN/14/2015(H5N2)	5.4	4.3	5/5 (3.8/1.9)	3/3 (1.8/1.6)	5/5 (1.3/0.9)	3/3 (1.6/1.5)	0/5	0/3	0/5	0/3	5/5 (6.2)	3/3 (5.3)
GP/HuN/13/2015(H5N6)	4.8	5.0	5/5 (4.0/2.1)	3/3 (3.1/2.8)	5/5 (2.7/2.4)	3/3 (3.4/1.7)	/	0/3	5/5	0/3	/	3/3 (6.0)
CP/HuN/11/2015(H5N6)	5.6	5.3	5/5 (3.8/2.9)	3/3 (4.3/3.9)	5/5 (2.9/1.9)	3/3 (1.0/0.9)	2/4 (1.3/0.9)	0/3	1/5	0/3	4/4 (6.3)	3/3 (6.0)
CP/HuN/2/2016(H5N6)	5.8	4.8	4/4 (4.6/3.2)	3/3 (5.4/2.4)	1/3 (1.2/0.9)	3/3 (4.8/2.7)	2/2 (1.0/0.9)	2/2 (1.2/1.4)	4/5	1/3	1/1 (7.0)	2/2 (7.5)
GP/HuN/1/2016(H5N6)	4.3	5.0	5/5 (4.0/3.0)	3/3 (3.6/2.8)	1/4 (0/3.8)	3/3 (1.2/0.9)	0/4	0/3	1/5	0/3	4/4 (5.3)	3/3 (5.7)
GG/HuN/1/2017(H5N6)	5.8	5.8	5/5 (4.9/3.3)	3/3 (4.8/2.4)	2/5 (4.4/0.9)	3/3 (3.1/1.6)	/	/	5/5	3/3	/	/
WS/SX/6/2016(H5N8)	4.8	5.8	5/5 (4.4/3.3)	3/3 (4.8/2.0)	5/5 (1.9/0.9)	3/3 (2.8/0.9)	0/5	3/3 (1.8/0.9)	0/5	1/3	5/5 (4.8)	2/2 (4.5)

^aViral titre of all ten organs tested were show in supporting Figure 3.

^bSwabs of survival ducks on day 9 p.i. were also collected, virus was only detected in one contact duck of the WS/HeN/1/2015(H5N1)-inoculated group, with titres of 1.0 and 0.9 in the oropharynx and cloaca, respectively. /, all ducks died; <, virus was not detected in the cloacal swabs.

Table 2. Mutations detected in the H5 wild bird viruses that contributed to increased binding to human-type receptors, virulence in mammals, and resistance to antiviral drugs (blank cell indicates no such mutation at that position).

Virus	Clade	Amino acids in HA that may increase the affinity to human-type receptor (H5 numbering)								Mutations or deletions in different genes that may increase virulence in mice				Mutations in M2 that increase resistance to amantadine and rimantadine
										NA^a	PB2	NS1
		94N	133A	156A	183N	188 I/A	189R/A/D/N/S	192K	235S	Stalk deletion	627K	42S	80–84 deletion	27I/A	31A/N
BHGL/TB/1/2010(H5N1)	2.3.2.1c	N	A				R			Yes		S	Yes
BHGL/TB/2/2010(H5N1)	2.3.2.1c	N	A				R			Yes		S	Yes
WS/HeN/1/2015(H5N1)	2.3.2.1f	N	A							Yes		S	Yes	I
BNG/IM/2/2015(H5N1)	2.3.2.1f	N	A							Yes		S	Yes	I
BHG/TB/3/2015(H5N1)	2.3.2.1f	N	A							Yes		S	Yes	I
BHGL/TB/4/2015(H5N1)	2.3.2.1f	N	A							Yes		S	Yes	I
BHG/TB/5/2015 (H5N1)	2.3.2.1f	N	A							Yes		S	Yes	I
BS/JS/5/2015(H5N1)	2.3.2.1d	N	A			I	A			Yes		S	Yes		N
GG/JS/6/2015(H5N1)	2.3.2.1d	N	A			I	A			Yes		S	Yes		N
GP/JS/7/2015(H5N1)	2.3.2.1d	N	A			I	A			Yes		S	Yes		N
CP/JS/8/2015(H5N1)	2.3.2.1d	N	A			I	A			Yes		S	Yes		N
GBHG/QH/9/2015(H5N1)	2.3.2.1f	N	A	A						Yes	K	S	Yes	I
GDP/HuN/10/2015(H5N6)	2.3.4.4g	N	A	A	N		D	K		Yes		S	Yes
CP/HuN//11/2015(H5N6)	2.3.4.4g	N	A	A	N		D	K		Yes		S	Yes
GP/HuN//12/2015(H5N6)	2.3.4.4g	N	A	A	N		D	K		Yes		S	Yes
GP/HuN/15/2015(H5N6)	2.3.4.4g	N	A	A	N		D	K		Yes		S	Yes
GP/HuN/13/2015(H5N6)	2.3.4.4g	N	A	A	N		D	K		Yes		S	Yes
GP/HuN/14/2015(H5N2)	2.3.4.4c		A	A	N	I	N	K		No		S	No		N
GP/HuN/1/2016(H5N6)	2.3.4.4f	N	A	A	N	A	S	K	S	Yes		S	Yes
SP/HuN/3/2016(H5N6)	2.3.4.4f	N	A	A	N	A	S	K	S	Yes		S	Yes
WS/HuN/4/2016(H5N6)	2.3.4.4f	N	A	A	N	A	S	K	S	Yes		S	Yes
CP/HuN/2/2016(H5N6)	2.3.4.4f	N	A	A	N	A	S	K	S	Yes	K	S	No		N
GG/HuN/1/2017(H5N6)	2.3.4.4e	N	A	A	N		N	K		Yes		S	Yes
WS/SX/6/2016(H5N8)	2.3.4.4b		A	A	N		N	K		No		S	No
WS/SX/7/2016(H5N8)	2.3.4.4b		A	A	N		N	K		No		S	No
BS/HuB/2/2017(H5N8)	2.3.4.4b		A	A	N		N	K		No		S	No
BS/HuB/3/2017(H5N8)	2.3.4.4b		A	A	N		N	K		No		S	No

^aH5N1 viruses had a deletion of 10 amino acids at positions 49–68 of NA (the stalk of N1 NA), and all of the H5N6 viruses had a deletion of 12 amino acids at positions 59–70 of NA (the stalk of N6 NA).

Figure 4. Receptor binding properties of H5 wild bird viruses. Binding properties were analyzed by using two types of receptors, α-2,6-linked SAs (human-type receptors) and α-2,3-linked SAs (avian-type receptors).

Figure 5. Replication and virulence of H5 wild bird viruses in mice. (a) Viral titres in organs of mice that were euthanized on day 3 post-inoculation with 10⁶EID₅₀ of the test virus. (b) MLD₅₀ of each test virus.