Highly pathogenic avian influenza (H5N1) in ...

Highly pathogenic avian influenza (H5N1) in Myanmar, 2006-2010

Pont Pont Mon, Jiradej Lapkuntod, Min Thein Maw, Bundit Nuansrichay, Sujira Parchariyanon, Thanawat Tiensin, Than Htun, et al. Archives of Virology Official Journal of the Virology Division of the International Union of Microbiological Societies ISSN 0304-8608 Arch Virol DOI 10.1007/s00705-012-1411-y

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Author's personal copy Arch Virol DOI 10.1007/s00705-012-1411-y

ORIGINAL ARTICLE

Highly pathogenic avian influenza (H5N1) in Myanmar, 2006-2010 Pont Pont Mon • Jiradej Lapkuntod • Min Thein Maw • Bundit Nuansrichay • Sujira Parchariyanon • Thanawat Tiensin • Than Htun • Pawin Padungtod • Wantanee Kalpravidh • Kyaw Sunn • Murray Maclean • Alongkorn Amonsin

Received: 25 April 2012 / Accepted: 30 May 2012 Ó Springer-Verlag 2012

Abstract Highly pathogenic avian influenza (HPAI) virus subtype H5N1 was first reported in Myanmar in 2006. In this study, we have characterized 6 HPAI (H5N1) viruses recovered from 2007-2010 as well as three additional available nucleotide sequences representing Myanmar AI outbreaks. Phylogenetic analysis showed that the Myanmar viruses belong to HPAI (H5N1) clades 7, 2.3.2 and 2.3.4. The result suggested that the HPAI (H5N1) viruses recovered from Myanmar had been introduced into the country by multiple introductions. Genetic analysis of the viruses confirmed the HPAI characteristics of the viruses.

P. P. Mon J. Lapkuntod A. Amonsin (&) Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand e-mail: [email protected] M. T. Maw K. Sunn Research and Disease Control Division, Livestock Breeding and Veterinary Department, Yangon, Myanmar B. Nuansrichay S. Parchariyanon Department of Livestock Development, National Institute of Animal Health, Bangkok, Thailand T. Tiensin Department of Livestock Development, Bureau of Disease Control and Veterinary Services, Bangkok, Thailand T. Htun M. Maclean Avian Influenza Integrated Control Program, Food and Agriculture Organization of the United Nations, Yangon, Myanmar P. Padungtod W. Kalpravidh Food and Agriculture Organization of the United Nations, Regional Office for Asia and the Pacific, Bangkok, Thailand

Introduction Highly pathogenic avian influenza (HPAI) virus subtype H5N1 has been reported in several countries in Europe, the Middle East, Africa and Asia [15, 19]. In Myanmar, an HPAI (H5N1) outbreak was first reported in 2006. There have been five waves of HPAI (H5N1) outbreaks during 2006-2011. The first wave of HPAI outbreaks was reported in March-April 2006 in commercial poultry farms in central Myanmar, including 13 townships in the Sagaing and Mandalay Divisions. This outbreak was the largest in Myanmar, with more than 500 poultry farms infected and approximately 650,000 poultry succumbing to the infection or culled. The second wave occurred in the Yangon and Bago divisions and Mon State, Lower Myanmar, in February-March, 2007. Commercially bred chickens as well as backyard chickens were affected in 10 townships. Over 50 farms were infected, and nearly 100,000 poultry were destroyed. The third wave occurred in November-December, 2007 in Eastern Shan State and affected backyard poultry farms. After that, there were no further HPAI outbreaks in Myanmar until the end of 2009. However, in early February 2010, there was a report of unusual poultry deaths in one commercial poultry farm in Mayangone Township in Yangon Division. The Livestock Breeding and Veterinary Department (LBVD, Yangon, Myanmar) investigated the affected poultry farm and collected specimens for HPAI diagnosis. Laboratory tests were conducted at Yangon Veterinary Diagnostic Laboratory (YVDL) for early detection and rapid outbreak response. Upon confirmation of HPAI (H5N1), culling the poultry of the infected farm, movement restriction, and disinfection were conducted according to the contingency plan per OIE recommendations [13, 14]. In addition, a second case was reported

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Author's personal copy P. P. Mon et al.

on February 16, 2010 from the Yaekyieai compound in Mingalardon Township, Yangon Division. In total, six backyard chickens died, and 180 birds were destroyed. The third case occurred in commercial layer farms in Yinmarbin Township, Monywa, in the Sagaing Division, from February to March, 2010. The objectives of this study were to consolidate the epidemiological information on HPAI (H5N1) outbreaks in Myanmar since 2006, especially the 2010 outbreaks and to characterize the HPAI (H5N1) viruses recovered from the outbreaks by whole-genome sequencing.

Materials and methods HPAI (H5N1) viruses A total of six HPAI (H5N1)-positive samples (tissue, swabs and allantoic fluid) were obtained from Yangon Veterinary Diagnostic Laboratory (YVDL) for virus isolation. The viruses characterized in this study represent the second, third, and fourth waves of HPAI (H5N1) outbreaks in Myanmar from 2007-2010. In addition, the nucleotide sequences of the viruses representing the first and second waves of HPAI (H5N1) outbreaks were retrieved from the GenBank database. Isolation and identification of HPAI (H5N1) virus Initial diagnostic work in this study was conducted in a biosafety level 2 (enhanced) laboratory at the Yangon Veterinary Diagnostic Laboratory (YVDL). The viruses were isolated using specific antibody-negative embryonated chicken eggs following OIE recommendations [20]. The swab or ground tissue suspension was inoculated into the allantoic sacs of 9- to 11-day-old chicken eggs and incubated at 37 °C. The inoculated eggs were examined every 12 hours before harvesting the allantoic fluid. The allantoic fluid was confirmed virus positive by hemagglutination activity (HA test) and kept at -80 °C until needed. The viral RNA was extracted from the allantoic fluid using a QIAamp Viral RNA Minikit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. One-Step RT-PCR was performed using a one-step RT-PCR kit (QIAGEN) with H5N1-specific primers as described previously [1]. The viral RNA isolated from the specimens was then reverse-transcribed into cDNA, which was then transported to the laboratories of the National Institute of Animal Health, Thailand, and Department of Veterinary Public Health, Chulalongkorn University, Thailand, for further genetic characterization.

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Whole-genome sequencing of HPAI (H5N1) viruses All eight gene segments of the viruses were amplified using specific primers for each gene and clade of the viruses. Oligonucleotide primer sequences were either obtained from previous reports [11] or newly designed using the Primer 3 program (v 0.4.0) (primer sequences are available upon request). The amplification products were purified using a QIAquick Gel Extraction Kit (QIAGEN). The DNA sequencing reaction was performed using Big Dye Terminator V. 3.0 Cycle Sequencing Ready Reaction (ABI, Foster City, CA) using an ABI-Prism Genetic Analyzer (Perkin Elmer, Norwalk, CT). Nucleotide sequences were assembled and aligned using the DNASTAR program (DNASTAR, Madison, WI). Whole-genome nucleotide sequences of all six HPAI (H5N1) viruses were then submitted to the GenBank database under accession numbers JQ936684-JQ936731. Phylogenetic and genetic analysis of HPAI (H5N1) viruses Phylogenetic analysis of six HPAI (H5N1) viruses was performed by comparing the nucleotide sequences of each virus gene with those of the viruses representing common clades found in Southeast Asia. Phylogenetic analysis was carried out using the program MEGA 4.1 (Tempe, AZ, USA) applying neighbor-joining algorithm with the Kimura 2-parameter model [24]. A Bayesian tree was also generated to confirm the NJ tree using the MrBayes V.3.1.2 with 1 million generations and the sampling frequency of the 500th generation. The substitution model setting was the general time-reversible model (GTR) with a proportion of invariable sites and a gamma-shaped distribution of rates across sites [12]. The tree topology was confirmed by bootstrap analysis with 1,000 replicates and posterior probability from BMCMC analysis. Genetic analysis of the viruses was conducted to identify nucleotide similarities of each virus gene compared to the viruses of common clades using the MegAlign program (DNASTAR). The deduced amino acid sequences of each virus gene were aligned and analyzed for genetic characteristics and virulence determinants.

Results In early 2010, the fourth wave of an AI outbreak was reported in central Myanmar. At least three separate locations (Mayangone and Mingalardon townships in Yangon Division and Yinmarbin Township in Sagaing Division) were affected by HPAI outbreaks in commercial poultry farms and backyard poultry (Fig. 1 and Table 1). Three HPAI (H5N1) viruses isolated from each township

Author's personal copy HPAI (H5N1) in Myanmar

representing the fourth wave of the HPAI outbreaks were subjected to virus characterization. In addition, three HPAI (H5N1) viruses from 2007 representing the second and third waves of AI outbreaks in Myanmar were also included for genetic characterization. The viruses were isolated from chickens (n = 5) and guinea fowl (n = 1) in the areas of the eastern (n = 1), central (n = 1) and southern (n = 4) regions of Myanmar (Table 2). It should be noted that South Myanmar has a high density of chicken, duck and quail farms. Whole genome sequences of HPAI (H5N1) viruses from Myanmar To study the genetic relatedness and genetic diversity of the viruses, whole-genome sequencing was performed on six HPAI (H5N1) viruses recovered from Myanmar during 2007-2010. In addition, HA gene sequences of the Myanmar viruses were also included in the analyses. Analysis of nucleotide sequence identity of each virus gene showed that the 2010 Myanmar virus (Chicken/Yangon/254/10) display profound genetic similarity to most Myanmar isolates from the second, third, and fourth waves (clade 2.3.4),

with nucleotide sequence identity greater than 98 %. These results suggest common genetic drift, with less than 3 % genetic change in influenza viruses isolated in Myanmar and no evidence of genetic reassortment of recent 2010 H5N1 viruses. However, the HA gene of one isolate (Sagaing/295) displays lower nucleotide sequence identity (95.3 %). Similarly, the HA gene of one isolate, Pyigyitagon/204, from the first outbreak displays lower nucleotide sequence identity (94.1 %) (Table 3). Phylogenetic analysis and clades of HPAI (H5N1) viruses from Myanmar The nucleotide sequences of HPAI (H5N1) (n = 42) from 12 different countries were retrieved from the GenBank database and included for phylogenetic analysis. Based on phylogenetic analysis of the HA gene, Myanmar viruses characterized in the study (n = 6) clustered with the viruses of clade 2 (either 2.3.2 or 2.3.4). The H5N1 viruses of clade 2.3.4 were responsible for the second, third, and fourth waves of AI outbreaks in Myanmar, while one 2010 isolate from the fourth wave (Sagaing/295) clustered with viruses of clade 2.3.2. On the other hand, one Myanmar

1st wave

3rd wave

Sagaing Mandalay (March/April 2006)

India China

- chicken/Mandalay/204 (clade 7,

Eastern Shan (November/December 2007) - chicken/Shan/2626 (clade 2.3.4,

)*

)

Laos

Bangladesh

Thailand

2nd wave 4th wave Yangon Bago Mon (February/March 2007)

Yangon Sagaing (February/March 2010)

- guinea fowl/Yangon/834 (clade 2.3.4, - chicken/Yangon/1023 (clade 2.3.4, - quail/Yangon/866 (clade 2.3.4,

) )

)*

- chicken/Yangon/517 (clade 2.3.4,


)


)

- chicken/Sagaing/295 (clade 2.3.2,

)

)*

*characterized in previous study

Fig. 1 Map of Myanmar showing the locations of the four waves of HPAI outbreaks

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Author's personal copy P. P. Mon et al. Table 1 Epidemiological information about HAPI (H5N1) outbreaks in Myanmar Wave

Month/year

Location (division/state)

Type

Description

1

8th March-April 2006

Sagaing Division (5 townships)

Primarily commercial poultry farms and backyard poultry

Affected over 500 poultry and quail farms and

Commercial farms and backyard poultry

Affected over 50 farms and *100,000 poultry died or were destroyed

*650,000 poultry died or were destroyed

Mandalay Division (8 townships) 2

28th February-October 2007

Yangon Division (6 townships) Bago Division (3 townships) Mon State (1 township)

3 4

November-December 2007

Eastern Shan State (2 townships)

Backyard chickens and ducks

27th February-March 2010

Yangon Division (2 townships)

Commercial farms and backyard poultry

Affected mostly backyard poultry *34,000 poultry died or were destroyed Yangon Division; affected a commercial poultry farm and backyard chickens *20,000 poultry died or were destroyed

Sagaing Division (1 township)

Sagaing Division; affected a commercial poultry farm *15,000 poultry died or were destroyed

Table 2 List of HPAI (H5N1) virus isolates analyzed in this study Wave

Year

Isolate

Location

Host

Source

Clade

GenBank accession number

2*

2007

A/guinea fowl/Yangon/ 834/2007

North Okkalapa police station, Yangon

Guinea fowl

Allantoic fluid

2.3.4

JQ936692-99

2*

2007

A/chicken/Yangon/ 1023/2007

Ko Kyaw Minn Htay, Htauk Kyant, Yangon

Layer chicken

Allantoic fluid

2.3.4

JQ936716-23

3*

2007

A/chicken/Shan/2626/ 2007

Mong Phyat, Eastern Shan

Backyard chicken

Allantoic fluid

2.3.4

JQ936724-31

4*

2010

A/chicken/Yangon/182/ 2010

Yaekyieai Compound, Mingalardon, Yangon

Backyard chicken

Allantoic fluid

2.3.4

JQ936700-07

4*

2010

A/chicken/Yangon/254/ 2010

Mayangone, Yangon

Layer chicken

Allantoic fluid

2.3.4

JQ936684-91

4*

2010

A/chicken/Sagaing/295/ 2010

U Zaw Myint Htun, Thayatkan Village, Yinmarbin, Sagaing

Layer chicken

Tissue grind

2.3.2

JQ936708-15

1**

2006

A/chicken/Pyigyitagon/ 204/2006

Pyigyitagon, Mandalay

Chicken

N/A

7

AB474081

2**

2007

A/quail/Mingalardone/ 866/2007

Mingalardone, Yangon

Quail

N/A

2.3.4

AB474084

2**

2007

A/chicken/Hmawbi/ 517/2007

Hmawbi

Chicken

N/A

2.3.4

AB474082

* H5N1 viruses characterized in this study ** H5N1 viruses recovered from Myanmar available in the GenBank database

H5N1 virus (Pyigyitagon/204) retrieved from GenBank that was responsible for the first AI outbreak belonged to clade 7 (Fig. 2). Our results indicated that AI outbreaks in Myanmar had been caused by HPAI (H5N1) viruses of different clades (clades 2.3.2, 2.3.4, and 7), suggesting multiple introductions of the viruses. Geographically, clade

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2.3.2 and clade 7 HPAI (H5N1) viruses were responsible for AI outbreaks in central Myanmar (Mandalay and Sagaing). On the other hand, clade 2.3.4 caused AI outbreaks throughout the country, especially in the south (Yangon and Eastern Shan) (Fig. 1). Phylogenetic analysis of the NA gene also showed that HPAI (H5N1) viruses clustered

Author's personal copy HPAI (H5N1) in Myanmar Table 3 Comparison of the gene segments of A/chicken/Yangon/254/2010 (clade 2.3.4) to those of H5N1 isolates from Myanmar Strains

Clade

% Nucleotide identity Region of comparison (bp) PB2 gene

PB1 gene

PA gene

HA gene

NP gene

NA gene

M gene

NS gene

(58-2225)

(47-2193)

(40-2081)

(7-1662)

(29-1460)

(43-1340)

(160-887)

(50-690)

A/goose/Gaungdong/1/1996

0

92.7

92.9

92.7

94.6

92.5

91.0

96.0

67.4

A/chicken/Vietnam/830/2004 A/chicken/Indonesia/7/2003

1 2.1

96.6 96.0

96.7 97.2

92.4 91.9

95.6 95.8

97.6 98.0

95.2 95.9

97.8 97.9

96.1 96.3

A/whooper swan/Mongolia/3/2008

2.2

96.5

95.7

91.7

95.0

97.2

95.9

97.9

95.2

A/goose/Guangxi/3316/2005

2.3.2

91.1

95.7

92.1

96.3

98.3

95.8

97.8

93.9

A/chicken/Thailand/CU-K2/2004

1

96.6

96.6

92.5

95.8

97.7

94.8

97.8

96.3

A/chicken/Thailand/NP-172/2006

2.3.4

98.1

98.0

98.0

97.2

97.5

98.2

98.5

97.7

A/chicken/shanxi/2/2006

7

95.4

95.4

93.8

93.2

96.8

93.5

95.3

96.4

A/chicken/Pyigyitagon/204/2006

7

-

-

-

94.1

-

-

-

-

A/quail/Mingalardone/866/2007

2.3.4

-

-

-

97.3

-

-

-

-

A/chicken/Hmawbi/517/2007

2.3.4

-

-

-

97.5

-

-

-

-

A/guinea fowl/Yangon/834/2007

2.3.4

98.2

97.3

97.6

97.3

98.1

95.4

98.4

97.7

A/chicken/Yangon/1023/2007

2.3.4

98.2

97.6

97.5

97.5

98.0

95.2

98.5

97.7

A/chicken/Shan/2626/2007

2.3.4

98.9

98.2

98.7

98.4

99.0

98.8

98.9

97.7


2.3.4

99.3

98.9

99.4

98.7

98.8

98.7

98.2

98.4


2.3.4

100

100

100

100

100

100

100

100

A/chicken/Sagaing/295/2010

2.3.2

96.4

96.8

96.3

95.3

96.5

95.4

94.9

96.3

into either clade 2.3.2, 2.3.4 or 7, similar to the HA gene (Fig. 3). Similar findings were obtained by phylogenetic analysis of the six internal genes (data not shown). Genetic analysis of HPAI (H5N1) viruses from Myanmar Genetic analysis of the HA gene revealed that all six Myanmar viruses display multiple basic amino acids at the HA cleavage site (position 320-331), indicating highly pathogenic characteristics (Table 4). It should be noted that two Myanmar viruses of clade 2.3.4 (Mingalardone/866 and Hmawbi/517) contain fewer basic amino acids, similar to clade 2.3.4 viruses from Thailand (Thailand/NP-1720). However, the viruses caused large numbers of deaths in the poultry population in Myanmar. Amino acid residues at the receptor-binding site (Q222 and G224) were found in all Myanmar viruses, indicating preferential binding of the viruses to a 2,3 linkages of avian cell-surface receptors [7]. One potential N-linked glycosylation site was predicted at amino acid positions 154-156 of the HA1 protein in most Myanmar viruses. This site was only detected in the Myanmar viruses of clade 2.3.4 and clade 7 and in the viruses from Thailand and Vietnam (clade 1) [1, 2]. On the other hand, the HPAI (H5N1) virus of clade 2.3.2 (Sagaing/295) does not contain a potential glycosylation site at positions 154-156. Analysis of amino acids under

positive selection pressure at distinct positions showed conserved amino acids at positions 83, 86, 129, and 175, while some amino acid changes were observed at positions 138, 140 and 141 (Table 4). Analysis of the NA gene revealed that the 20-aminoacid deletion in the NA stalk region (positions 49-68) has existed in HPAI (H5N1) viruses since 2003. It is known that the 20-amino-acid deletions indicate the adaptation of the viruses from wild aquatic birds to domestic poultry [16]. In this study, none of the Myanmar viruses displayed oseltamivir-resistant amino acids at positions E119, H275, R293 and N295 of the NA gene [17, 18, 23]. Similarly, analysis of the M2 protein revealed that none of the Myanmar viruses contained any amino acids associated with amantadine resistance. Amino acids at positions L26, V27, A30, S31, and G34 indicated amantadine sensitivity of the six Myanmar H5N1 viruses, while the clade 1 viruses from Thailand and Vietnam show amantadine resistance characteristics at positions I26 and N31 of the M2 gene (Table 4) [6, 9, 10]. Analysis of the NS1 protein showed that the Myanmar viruses exhibit a 5-amino-acid deletion at positions 80-84, similar to all HPAI (H5N1) viruses analyzed since 1997. Virulence determinants associated with NS1 proteins were also analyzed, including amino acid position 92 and the carboxyl-terminus. For example, the mutation at position D92E of the NS gene is associated with high virulence in mammalian species [21].

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Author's personal copy P. P. Mon et al. Fig. 2 Phylogenetic tree of the HA gene. The phylogenetic analysis was generated using the neighbor-joining algorithm with the Kimura 2-parameter model. The Bayesian tree was generated to confirm the NJ tree with 1 million generations. Bootstrap values from 1,000 replicates and posterior probability from BMCMC analysis were calculated to confirm the tree topology and are shown in parentheses (NJ/ BMCMC). The Myanmar viruses characterized in a previous study and this study are indicated by square boxes and triangles, respectively

Our result showed that all Myanmar viruses contain aspartic acid (D) at position 92 of the NS1 protein. However, the ESEV motif was observed in all Myanmar isolates. Analysis of the PB2 gene revealed that amino acids within the virulence determinants (PB2-627 and PB2355) have not been substituted in any Myanmar viruses, indicating lower virulence in mammalian species [8, 22]. Analysis of PB1 showed that the amino acid mutation at

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position 66 in the PB1-F2 gene, which plays a crucial role in the severity of pandemic 1918 influenza A virus, was not present in Myanmar H5N1 viruses [4]. Genetic analysis of amino acids associated with host preference did not reveal any amino acids involved in hostrange specificity in Myanmar viruses. The M gene of Myanmar viruses contains human-virus-like amino acids (V28) and avian-virus-like amino acids (E16 and L55),

Author's personal copy HPAI (H5N1) in Myanmar Fig. 3 Phylogenetic tree of the NA gene. Phylogenetic analysis was done using the neighborjoining algorithm with the Kimura 2-parameter model. A Bayesian tree was generated to confirm the NJ tree, with 1 million generations. Bootstrap values from 1,000 replicates and posterior probability from BMCMC analysis were calculated to confirm the tree topology and are shown in parentheses (NJ/BMCMC). The Myanmar viruses characterized in this study are indicated by triangles

while the PB2 protein contains avian-virus-like amino acids (A119, A661, V667 and K702). The NP and PA proteins also display avian-virus-like amino acids at NPL136 and PA-S409, respectively (Table 4).

Discussion In Myanmar, the first wave of HPAI (H5N1) outbreaks was reported in March 2006 in eight townships (Amarapura, Kyake Se, Chanmyatharsi, Chanayetharzan, Pyigyitagon, Maharaungmyay, Aungmyaytharzan and Singaing) in Mandalay Division, Central Myanmar, and in five townships

(Ziegone, Kantbalu, Ye U, Shwebo and Monywa) in Sagaing division, central Myanmar. Commercial poultry farms were primarily affected. Although the outbreaks were first reported in Mandalay, the data from trace-back investigations suggested that the infection had started in Shwebo Township in Sagaing Division one month prior to the outbreaks in Mandalay. The information from outbreak investigations revealed that the dealers from Mandalay transported the infected birds from Shwebo to Mandalay and sold them to local markets. In addition, it is known that Mahananda Lake in Shwebo is a major habitat of several migratory bird and wild waterfowl species. Although there are many duck-raising areas around Mandalay, no outbreak

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Author's personal copy P. P. Mon et al. Table 4 Genetic analysis of the gene segments of HPAI (H5N1) viruses isolated from Myanmar Virus

Clade

HA HA Cleavage site

Receptorbinding site

N-linked glycosylation site

Antigenic site E

Antigenic site A

320-331

222

224

154-156

83

86

129

138

140

141

175

Q

G

(NSA)

A

A

S

H

R

S

L


0

TPQRERRRKKRG

A/chicken/Vietnam/830/2004

1

SPQRERRRKKRG

Q

G

NNT

A

V

L

Q

K

S

L

A/chicken/Indonesia/7/2003

2.1

SPQRESRRKKRG

Q

G

(NSA)

A

A

S

Q

K

S

L


2.2

SPQGERRRRKRG

Q

G

(DNA)

I

A

S

Q

R

S

L


2.3.2

SPQRE- RRRKRG

Q

G

(NDA)

A

A

L

Q

N

S

L


1

SPQRERRRKKRG

Q

G

NST

A

V

L

Q

K

S

L


2.3.4

SPLRE- RRRKRG

Q

G

NNT

A

A

S

Q

T

P

L


7

APQREGGRRKRG

Q

G

NNT

A

A

-

L

K

P

L


7

TPQREGRRKKRG

Q

G

NNT

A

A

L

L

K

P

L


2.3.4

YPLRE- KRRKRG

Q

G

NNT

A

A

L

Q

T

P

L


2.3.4

YPLRE- KRRKRG

Q

G

NNT

A

A

L

Q

T

P

L


2.3.4

YPLI RERRRKRG

Q

G

NNT

A

A

L

Q

T

P

L


2.3.4

YPLREKGRRKRG

Q

G

NNT

A

A

L

Q

T

P

L


2.3.4

SPLRERRRKKRG

Q

G

NNT

A

A

L

Q

T

P

L


2.3.4

SPLRERRRRKRG

Q

G

NNT

A

A

L

L

T

P

L


2.3.4

SPLREKRRKKRG

Q

G

NNT

A

A

L

L

T

P

L


2.3.2

SPQRERRRKKRG

Q

G

(DNA)

A

A

L

Q

N

S

L

Virus

Clade

NA

M gene

NA stalk region

Oseltamivir resistance

49-68

119

275

293

Amantadine resistance 295

26

27

30

31

Human/avian like 34

16

28

55


0

No deletion

E

H

R

N

L

V

A

S

G

E

V

L


1

20-aa deletion

E

H

R

N

I

V

A

N

G

E

V

L

A/chicken/Indonesia/7/2003

2.1

20-aa deletion

E

H

R

N

L

V

A

N

G

E

V

L


2.2

20-aa deletion

E

H

R

N

L

V

A

S

G

E

V

L


2.3.2

20-aa deletion

E

H

R

N

L

V

A

S

G

E

V

L


1

20-aa deletion

E

H

R

N

I

V

A

N

G

E

V

L


2.3.4

20-aa deletion

E

H

R

N

L

V

A

S

G

E

V

L


7

20-aa deletion

E

H

R

N

L

V

A

N

G

E

V

L


7

-

-

-

-

-

-

-

-

-

-

-

-

-


2.3.4

-

-

-

-

-

-

-

-

-

-

-

-

-


2.3.4

-

-

-

-

-

-

-

-

-

-

-

-

-


2.3.4

20-aa deletion

E

H

R

N

L

V

A

S

G

E

V

L


2.3.4

20-aa deletion

E

H

R

N

L

V

A

S

G

E

V

L


2.3.4

20-aa deletion

E

H

R

N

L

V

A

S

G

E

V

L


2.3.4

20-aa deletion

E

H

R

N

L

V

A

S

G

E

V

F


2.3.4

20-aa deletion

E

H

R

N

L

V

A

S

G

E

V

L


2.3.2

20-aa deletion

E

H

R

N

L

V

A

S

G

E

V

I

Virus

Clade

NS1 gene

NP gene

PB2

PB1

PA

Virulence

Virulence

H/A-like

Amino acid deletion

Virulence determinant

H/A-like

H/A-like

80-84

92

Carboxyl terminal

136

199

661

667

702

627

355

198

409


0

No deletion

D

ESEV

M

A

A

V

K

E

K

K

S


1

5-aa deletion

D

ESEV

L

A

A

V

K

E

R

K

S

123

Author's personal copy HPAI (H5N1) in Myanmar Table 4 continued Virus

Clade

NS1 gene

NP gene

PB2

Amino acid deletion

Virulence determinant

H/A-like

H/A-like

80-84

92

136

199

Carboxyl terminal

661

667

702

PB1

PA

Virulence

Virulence

H/A-like

627

198

409

355


2.2

5-aa deletion

D

ESKV

L

A

A

V

K

K

R

K

S


2.3.2

5-aa deletion

D

ESEV

L

A

A

V

K

E

K

K

S


1

5-aa deletion

D

ESEV

L

A

A

V

K

E

R

K

S


2.3.4

5-aa deletion

D

ESEV

L

A

A

I

K

E

R

K

S


7

5-aa deletion

D

ESEV

L

A

A

V

K

E

R

K

S


7

-

-

-

-

-

-

-

-

-

-

-

-


2.3.4

-

-

-

-

-

-

-

-

-

-

-

-


2.3.4

-

-

-

-

-

-

-

-

-

-

-

-


2.3.4

5-aa deletion

D

ESEV

L

A

A

V

K

E

R

K

S


2.3.4

5-aa deletion

D

ESEV

L

A

A

V

K

E

R

K

S


2.3.4

5-aa deletion

D

ESEV

L

A

A

I

K

E

R

K

S


2.3.4

5-aa deletion

D

ESEV

L

A

A

V

K

E

R

K

S


2.3.4

5-aa deletion

D

ESEV

L

A

A

V

K

E

R

K

S


2.3.2

5-aa deletion

D

ESEV

L

A

A

V

K

E

R

K

S

was reported in any duck farms. The outbreaks started on March 8 and lasted until the end of April 2006. In total, 650,000 birds from 545 commercial poultry and quail farms perished or were culled. The specimens were confirmed as HPAI (H5N1) by RT-PCR in LBVD, Myanmar, and sent for genome sequencing to Japan and Thailand. Nucleotide sequences of the virus (A/chicken/Pyigyitagon/204/2006; GenBank AB474081) were analyzed for clade identification and designated as clade 7. Based on phylogenetic analysis, it should be noted that the first wave of HPAI (H5N1) outbreaks in Myanmar was caused by viruses of one HPAI (H5N1) clade (clade 7) [5]. The second wave started on February 28, 2007 in five townships (Hlaingtharyar, Mingalardon, North Okkalapa, Hmawbi and Htauk Kyant) in Yangon Division. Prior to the outbreaks, unusual deaths of wild birds such as crows were observed in Southern Yangon District in the vicinity of a creek irrigated from the Bago-Sittaung Canal, which originates from the Moeyungyi wetland. The Moeyungyi wetland is a place to which several migratory bird species migrate annually. Approximately 65,812 birds, including ducks from seven commercial farms as well as backyard farms, were culled. Although the outbreak was not as large as the first wave, it lasted longer than the first wave, with some follow-up cases in one township (Insein) in Yangon division, three townships (Bago, Thanapin and Latpadan) in Bago Division, and one township (Thanphyuzayat) in Mon state. The outbreaks lasted until October 2007. It should be noted that Thanapin Township in Bago Division is a high-density duck-raising area. Ducks in the area were

found H5N1 seropositive during the 2006 post-outbreak surveillance (data not shown). Therefore, ducks and migratory birds could be potential sources of virus transmission. Previously, HPAI (H5N1) isolates from the second wave were characterized, and nucleotide sequences were deposited in the database (A/chicken/Hmawbi/517/ 2007, A/guineafowl/N.Okkalarpa /834/2007, A/quail/Mingalardone/866/2007; GenBank AB474082-AB474084). In this study, two additional HPAI (H5N1) viruses have been characterized (Yangon/834 and Yangon/1023). The results of phylogenetic analysis showed that all Myanmar viruses from the second wave belong to clade 2.3.4. From November to December 2007, the third wave of AI outbreaks affected two townships (Kyaing Tong and Mong Phyat) in Eastern Shan that border on Thailand and China. Based on the outbreak investigations, dealers regularly transported day-old-ducks from Mongla, South China, and distributed the birds to farmers in Eastern Shan areas. However, potential source of the viruses could not be unambiguously identified. As there were not many commercial poultry farms in the area, the outbreaks could be controlled within a short period. Approximately 33,568 birds were destroyed during the outbreaks. In this study, one HPAI (H5N1) virus (Shan/2626) was characterized and designated as a clade 2.3.4 virus. It should be noted that there were no reports of HPAI outbreaks in Myanmar during 2008-2009 [5]. On February 27, 2010, a commercial poultry farm situated in Mayangone Township, Yangon Division, was affected by HPAI (H5N1). Approximately 113 out of 2,300

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Author's personal copy P. P. Mon et al.

birds died. Upon confirmation of HPAI (H5N1), all poultry and poultry products were destroyed. During the same period, a second case was reported in backyard chickens in Yaekyieai Compound, Mingalardon Township, Yangon Division. Only six birds were affected, and approximately 170 birds were culled. In addition, there was an outbreak in commercial poultry farms in Yinmarbin Township, Sagaing Division, in early March 2010, with 214 birds dead and approximately 13,500 birds destroyed. In this study, three HPAI (H5N1) isolates from the 2010 outbreaks were characterized. Phylogenetic analysis showed that two viruses from Yangon Division (Yangon/182 and Yangon/ 254) belong to clade 2.3.4, while one virus from Sagaing Division (Sagaing/295) belongs to clade 2.3.2. From our experience with HPAI (H5N1) outbreaks in Myanmar, we found that most outbreaks could be controlled within a short period and did not spread widely, based on surveillance of symptoms. Routine practices were strictly implemented. For example, an ‘‘infected zone’’ within a 1-km radius of the affected area was defined in which restriction of movement, culling of poultry, disinfection of poultry products, and closing of live-bird markets were enforced. Intensive clinical surveillance was also conducted during outbreaks in affected townships. Likewise, post-outbreak surveillance was performed 21 days after the last case of the outbreak. In addition, surveillance of high-risk areas was conducted at 6-month intervals. Targeted high-risk areas were also identified, including poultry-populated areas, duck-raising areas, migratory flyways, wet markets and live-bird markets, wetlands and neighboring areas, and previously infected areas. In addition, during HPAI (H5N1) outbreaks, the government of Myanmar raised public awareness through newspapers, boards, posters, television in cooperation with local and international non-governmental organizations (OIE, FAO, WHO, Care Myanmar, etc.). The Livestock Breeding and Veterinary Department (LBVD) offers training on prevention and control of HPAI at the divisional, district and township levels at regular intervals. Despite the large economic losses, there was no compensation policy in Myanmar. However, the Ministry of Livestock and Fisheries managed to support affected farmers with payments in kind and loans for restocking. In this study, whole-genome characteristics of six HPAI (H5N1) viruses were elucidated and analyzed. The nucleotide sequences of the viruses were compared with those of HPAI (H5N1) viruses previously isolated from Asia and Southeast Asia. Based on phylogenetic analysis, multiple clades of viruses (clades 7, 2.3.4 and 2.3.2) were observed, suggesting multiple introductions of the HPAI (H5N1) viruses in Myanmar. Based on genetic analysis, the HA gene of all six isolates possesses multiple basic amino acids at the HA cleavage site, indicating HPAI characteristics of

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the virus. Receptor-binding-site analysis indicated that the Myanmar viruses preferentially bind to avian cell-surface receptors [7]. Analysis of the NA and M genes suggested susceptibility of the Myanmar viruses to oseltamivir and amantadine [6, 9, 17, 23]. Analysis of virulence determinants of the viruses showed low-pathogenic characteristics. For example, a substitution of glutamic acid to lysine in E627 encoded by the PB2 gene increases the virulence of viruses in mice [8, 22]. Similarly, aspartic acid (D) was found in all Myanmar isolates at NS1-92. Mutation of this amino acid correlates with high virulence of the viruses in mammals [21]. It should be noted that the Myanmar viruses display both human- and avian-virus-like characteristics at positions PA-409, NP-136, M-16, M-28, M-55 [3]. In summary, this study has provided epidemiological information and a-genome characterization of the HPAI (H5N1) viruses recovered from Myanmar, which extends our knowledge about HPAI (H5N1) circulating in Myanmar and Southeast Asia. Especially, additional information on nucleotide sequences of Myanmar viruses is available in the influenza databases. Acknowledgment We would like to acknowledge the Food and Agriculture Organization of the United Nations for the technical support to the project and post-graduate scholarship support to PPM. We would like to thank the Ministry of Livestock and Fisheries, Livestock Breeding and Veterinary Department, Myanmar. We also would like to thank Chulalongkorn University for support to the Emerging and Re-emerging Infectious Diseases in Animals, Research Unit. This work was also supported in part by grants given to AA by National Research University Project of Thailand, Office of the Higher Education Commission (HR1155A) and National Research Council of Thailand (NRCT). Conflict of interest of interest.

The authors declare that they have no conflict

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