Multiple introductions of highly pathogenic avian influenza H5N1 viruses into Bangladesh

Figures & data

Table 1 Avian influenza A (H5N1) virus isolates from live bird markets in Bangladesh from December 2010 to March 2013

Isolate	Clade	Isolation date (mm/dd/yy)	Host species	Sample type	Retail market
A/Ck/BD/9636/10	2.2.2	12/11/10	Chicken	C	1
A/Ck/BD/9675/11	2.2.2	01/18/11	Chicken	F	2
A/Ck/BD/12197/11	2.3.2.1	08/14/11	Chicken	W	1
A/Ck/BD/15078/12	2.3.2.1	01/18/12	Chicken	C	3
A/Ck/BD/15079/12	2.3.2.1	01/18/12	Chicken	OP	3
A/Ck/BD/15083/12	2.3.2.1	01/18/12	Chicken	OP	3
A/Ck/BD/15085/12	2.3.2.1	01/18/12	Chicken	OP	3
A/Env/BD/15105/12	2.3.2.1	01/18/12	Quail	F	4
A/Env/BD/15114/12	2.3.2.1	01/18/12	Quail	F	4
A/Env/BD/15121/12	2.3.2.1	01/18/12	Quail	F	4
A/Env/BD/15126/12	2.3.2.1	01/18/12	Quail	F	4
A/Env/BD/15145/12	2.3.2.1	01/18/12	Quail	F	4
A/Env/BD/15147/12	2.3.2.1	01/18/12	Quail	F	4
A/Env/BD/15165/12	2.3.2.1	01/18/12	Quail	F	4
A/Ck/BD/15210/12	2.3.2.1	01/18/12	Chicken	OP	1
A/Env/BD/15661/12	2.3.2.1	02/08/12	Chicken	F	2
A/Env/BD/16298/12	2.3.2.1	03/31/12	Chicken	W	2
A/Dk/BD/17753/12	2.3.2.1	07/24/12	Duck	OP	5
A/Ck/BD/18061/12	2.3.2.1	09/07/12	Chicken	C	5
A/Ck/BD/18065/12	2.3.2.1	09/07/12	Chicken	C	5
A/Ck/BD/18248/12	2.3.2.1	10/21/12	Chicken	C	5
A/Env/BD/18948/13	2.3.2.1	01/28/13	Duck	W	1
A/Dk/BD/18949/13	2.3.2.1	01/28/13	Duck	W	1
A/Ck/BD/18984/13	2.3.2.1	01/28/13	Chicken	W	2
A/Env/BD/18989/13	2.3.2.1	01/28/13	Chicken	W	2
A/Dk/BD/19097/13	2.3.2.1	02/14/13	Duck	OP	5
A/Dk/BD/19099/13	2.3.2.1	02/14/13	Duck	OP	5
A/Quail/BD/19250/13	2.3.2.1	03/30/13	Quail	OP	4
A/Quail/BD/19254/13	2.3.2.1	03/30/13	Quail	OP	4
A/Env/BD/19338/13	2.3.2.1	03/30/13	Chicken	W	1

Abbreviations: BD, Bangladesh; C, cloacal; Ck, chicken; Dk, duck; Env, environment; F, fecal; OP, oropharyngeal; W, water. The five anonymous retail poultry markets in Dhaka city where the H5N1 viruses were detected, were assigned numbers 1, 2, 3, 4, and 5 respectively.

Figure 1 Samples collected and avian influenza viruses isolated in Bangladesh (November 2008–June 2013). Total number of samples collected was 19 897 (3952 oropharyngeal, 4113 cloacal and 11 832 environmental samples, the last set consisting of 1044 water and 10 788 fecal samples). The number of samples collected by bird species is as follows: chickens, 11 513; ducks, 1616; quail, 1460; pet birds, 2350; lesser whistling ducks, 2196; and unidentified wild waterfowl, 725.

Table 2 Antigenic analysis of H5N1 influenza viruses from Bangladesh in the hemagglutination inhibition assays

H5N1 antigen				α-H5N1 (post-infection ferret antisera)
Reference antigens	Clade	α-BHG/QH/1A	α-Hubei/1	α-BS/HK/1161*	α-CM/HK/5052	α-Ck/BD/15205^§	α-Env/BD/15121*,^§	α-Dk/BD/19097*,^§
RG-A/BAR-HEADED GOOSE/QUNGHAI/1A/2005	2.2	640	80	320	80	80	640	160
RG-A/HUBEI/1/2010	2.3.2.1	40	640	2560	320	640	5120	1280
RG-A/BARN SWALLOW/HONG KONG/1161/2010	2.3.2.1	40	160	5120	640	640	5120	1280
RG-A/COMMON MAGPIE/HONG KONG/5052/2007	2.3.2.1	40	160	1280	640	320	2560	640
A/CHICKEN/BANGLADESH/15205/2012	2.3.2.1	40	80	1280	320	640	2560	2560
A/ENVIRONMENT/BANGLADESH/15121/2012	2.3.2.1	40	80	1280	320	320	2560	1280
A/DUCK/BANGLADESH/19097/2013	2.3.2.1	40	160	2560	640	1280	5120	2560
TEST ANTIGENS
A/CHICKEN/BANGLADESH/9636/2010	2.2.2	640	160	320	80	640	640	320
A/CHICKEN/BANGLADESH/9675/2011	2.2.2	320	80	80	40	320	320	160
A/CHICKEN/BANGLADESH/12197/2011	2.3.2.1	40	80	1280	320	320	2560	640
A/CHICKEN/BANGLADESH/15078/2012	2.3.2.1	40	80	1280	160	320	1280	640
A/CHICKEN/BANGLADESH/15079/2012	2.3.2.1	20	160	640	320	320	2560	640
A/CHICKEN/BANGLADESH/15083/2012	2.3.2.1	40	160	1280	320	320	2560	640
A/CHICKEN/BANGLADESH/15085/2012	2.3.2.1	10	160	640	320	320	1280	640
A/ENVIRONMENT/BANGLADESH/15105/2012	2.3.2.1	10	40	1280	320	320	2560	640
A/ENVIRONMENT/BANGLADESH/15114/2012	2.3.2.1	<10	80	1280	160	320	2560	640
A/ENVIRONMENT/BANGLADESH/15126/2012	2.3.2.1	20	80	1280	160	320	5120	640
A/ENVIRONMENT/BANGLADESH/15145/2012	2.3.2.1	10	160	2560	320	640	5120	1280
A/ENVIRONMENT/BANGLADESH/15147/2012	2.3.2.1	20	80	1280	320	320	5120	1280
A/ENVIRONMENT/BANGLADESH/15165/2012	2.3.2.1	10	160	5120	640	640	5120	1280
A/CHICKEN/BANGLADESH/15210/2012	2.3.2.1	20	160	640	80	640	2560	640
A/ENVIRONMENT/BANGLADESH/15661/2012	2.3.2.1	10	80	1280	320	640	2560	640
A/ENVIRONMENT/BANGLADESH/16298/2012	2.3.2.1	10	160	1280	320	640	2560	1280
A/DUCK/BANGLADESH/17753/2012	2.3.2.1	40	160	1280	320	320	2560	640
A/CHICKEN/BANGLADESH/18061/2012	2.3.2.1	40	40	640	80	320	2560	640
A/CHICKEN/BANGLADESH/18065/2012	2.3.2.1	40	80	640	80	320	2560	640
A/CHICKEN/BANGLADESH/18248/2012	2.3.2.1	10	160	1280	320	640	2560	640
A/ENVIRONMENT/BANGLADESH/18948/2013	2.3.2.1	20	80	1280	320	640	2560	640
A/DUCK/BANGLADESH/18949/2013	2.3.2.1	<10	160	2560	320	640	5120	1280
A/CHICKEN/BANGLADESH/18984/2013	2.3.2.1	20	40	640	40	320	2560	640
A/ENVIRONMENT/BANGLADESH/18989/2013	2.3.2.1	20	40	320	40	160	1280	320
A/DUCK/BANGLADESH/19099/2013	2.3.2.1	<10	40	640	160	320	1280	320
A/QUAIL/BANGLADESH/19250/2013	2.3.2.1	<10	20	160	40	40	320	160
A/QUAIL/BANGLADESH/19254/2013	2.3.2.1	40	10	160	40	40	320	80
A/ENVIRONMENT/BANGLADESH/19338/2013	2.3.2.1	<10	20	320	40	80	640	160

^§indicates sera that have been produced against recent highly pathogenic H5N1 viruses from Bangladesh.

*During serum production, ferrets were boosted with antigen in incomplete Freund’s adjuvant on day 14 post-inoculation.

Table 3 Amino-acid residues characteristic of the H5N1 viruses isolated from Bangladesh

Gene segment	Amino-acid residue	Specific function	H5N1 BD Clade 2.2.2	H5N1 BD Clade 2.3.2.1	Comments
HA	140	Antigenic site	R	N
	141	Antigenic site	S	S
	154	Antigenic site	N	D
	155	Antigenic site	D	N*	Increases virus binding to α-2,6 sialic acid receptors and reduces lethality and systemic spread in mice.^Citation23 A/Ck/BD/15210/12 (2.3.2.1) have HA N155D
	156	Antigenic site	A*	A*/T
	162	Antigenic site	I	K	A/Ck/BD/12197/11 (2.3.2.1) has K162R
	163	Antigenic site	S	S	A/Ck/BD/15079/12 and A/Ck/BD/15083/12 have S163D
	189	Antigenic site	K	R*/G	Increases virus binding to α-2,6 sialic acid receptors.^Citation24 A/Ck/BD/15210/12 (2.3.2.1) has HA R189K
	94		N*	N*/T	Enhances receptor specificity for α-2,6 sialic acid receptors and enhances viral infectivity of A/Vietnam/1203/04 in mammalian cells^Citation25
	227 (226)		M	I*
	14		E	K* (also N, M)	Also in clade 2.3.2.1 isolates from India and Nepal and some Cambodian isolates from clade 1.1
	88		D	G*	Also in clade 2.3.2.1 isolates from India and Nepal
	115		Q	R*	Also in clade 2.3.2.1 isolates from India and Nepal
	446		K	R*	Also in clade 2.3.2.1 isolates from India and Nepal
NA	166		A	V*	Also in clade 2.3.2.1 isolates from India
	374		I	V*	Also in clade 2.3.2.1 isolates from India
PB2	102		N	S*	Also in clade 2.3.2.1 isolates from India
	184		T	A*	Also in clade 2.3.2.1 isolates from India
	317		L	M*	Also in clade 2.3.2.1 isolates from India
	702		K	R*	Molecular marker of human H1N1, H2N2 and H3N2, 1918-pandemic virus, and classical swine isolates. Ck/BD/15079/12, Ck/BD/15083/12, Dk/BD/19097/13 and Dk/BD/19099/13 had 702 K which has avian origin
PB1	363		K	R*	Also in clade 2.3.2.1 isolates from India
	105		N	T*	Also in clade 2.3.2.1 isolates from India
	384		L	I*	Also in clade 2.3.2.1 isolates from India
	515		S	A*	Also in clade 2.3.2.1 isolates from India
	584		R	H*	Also in clade 2.3.2.1 isolates from India
	667		I	T*	Also in clade 2.3.2.1 isolates from India
PB1-F2	71		T	I*	Also in clade 2.3.2.1 isolates from India
	74		T	L*	Also in clade 2.3.2.1 isolates from India
	50		V	D*	Found in H5N1 2.3.2.1 from quail feces (2012), but also in the BD H9N2 PB1-F2 and in A/Indonesia/CDC/1032/2007
PA	399		E	D*	Also in 2.3.2.1 isolates from India
	421		S	I*	In A/Vietnam/1203/2004; increases pathogenicity in mice
NP	373		A	V*	Also in 2.3.2.1 isolates from India, and in A/Indonesia/CDC1032/2007
	433		T	A*	Also in 2.3.2.1 isolates from India
NS	60		A	V*	Also in 2.3.2.1 isolates from India
	77		L	F*	Also in 2.3.2.1 isolates from India and H9N2 and H7N9 from China
	217		D	H
M2	10		P	H	H5N1 2.3.2.1 (2012) isolated from quail feces have H10R

*Information relevant to the particular amino-acid residue is shown in the comment box next to it.

Figure 2 Phylogenetic tree of the HA genes of HPAI H5N1 viruses from Bangladesh, generated by neighbor-joining method in MEGA 5. Numbers at the branches indicate bootstrap values; only values >70 are shown. ▴ indicates Bangladesh isolates clade 2.2.2; ▪ indicates Bangladesh isolates from the A/Hubei/1/10-like lineage of clade 2.3.2.1; • indicates Bangladesh isolates from the A/Hong Kong/6841/10-like lineage of clade 2.3.2.1.

Figure 3 Phylogenetic tree of the NA gene of H5N1 viruses from Bangladesh, generated by neighbor-joining method in MEGA 5. Numbers at the branches indicate bootstrap values; only values >70 are shown. ▴ indicates Bangladesh isolates clade 2.2.2; ▪ indicates Bangladesh isolates from the A/Hubei/1/10-like lineage of clade 2.3.2.1; • indicates Bangladesh isolates from the A/Hong Kong/6841/10-like lineage of clade 2.3.2.1.

Figure 4 Phylogenetic tree of the PB2 gene of H5N1 viruses from Bangladesh, generated by neighbor-joining method in MEGA 5. Numbers at the branches indicate bootstrap values; only values >70 are shown. ▴ indicates Bangladesh isolates clade 2.2.2; ▪ indicates Bangladesh isolates from the A/Hubei/1/10-like lineage of clade 2.3.2.1; • indicates Bangladesh isolates from the A/Hong Kong/6841/10-like lineage of clade 2.3.2.1.

Figure 5 Phylogenetic tree of the M gene of H5N1 viruses from Bangladesh, generated by neighbor-joining method in MEGA 5. Numbers at the branches indicate bootstrap values; only values >70 are shown. ▴ indicates Bangladesh isolates clade 2.2.2; ▪ indicates Bangladesh isolates from the A/Hubei/1/10-like lineage of clade 2.3.2.1; • indicates Bangladesh isolates from the A/Hong Kong/6841/10-like lineage of clade 2.3.2.1.

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