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Avian Pathology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cavp20

Molecular evolution of H5N1 highly pathogenic avian influenza viruses in Bangladesh between 2007 and 2012 a

b

a

M. E. Haque , M. Giasuddin , E. H. Chowdhury & M. R. Islam

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Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh b

National Reference Laboratory for Avian Influenza, Bangladesh Livestock Research Institute, Savar, Bangladesh Published online: 01 Apr 2014.

To cite this article: M. E. Haque, M. Giasuddin, E. H. Chowdhury & M. R. Islam (2014) Molecular evolution of H5N1 highly pathogenic avian influenza viruses in Bangladesh between 2007 and 2012, Avian Pathology, 43:2, 183-194, DOI: 10.1080/03079457.2014.898244 To link to this article: http://dx.doi.org/10.1080/03079457.2014.898244

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Avian Pathology, 2014 Vol. 43, No. 2, 183–194, http://dx.doi.org/10.1080/03079457.2014.898244

ORIGINAL ARTICLE

Molecular evolution of H5N1 highly pathogenic avian influenza viruses in Bangladesh between 2007 and 2012 M. E. Haque1, M. Giasuddin2, E. H. Chowdhury1 and M. R. Islam1* 1

Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh, and National Reference Laboratory for Avian Influenza, Bangladesh Livestock Research Institute, Savar, Bangladesh

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In Bangladesh, highly pathogenic avian influenza (HPAI) virus subtype H5N1 was first detected in February 2007. Since then the virus has become entrenched in poultry farms of Bangladesh. There have so far been seven human cases of H5N1 HPAI infection in Bangladesh with one death. The objective of the present study was to investigate the molecular evolution of H5N1 HPAI viruses during 2007 to 2012. Partial or complete nucleotide sequences of all eight gene segments of two chicken isolates, five gene segments of a duck isolate and the haemagglutinin gene segment of 18 isolates from Bangladesh were established in the present study and subjected to molecular analysis. In addition, full-length sequences of different gene segments of other Bangladeshi H5N1 isolates available in GenBank were included in the analysis. The analysis revealed that the first introduction of clade 2.2 virus in Bangladesh in 2007 was followed by the introduction of clade 2.3.2.1 and 2.3.4 viruses in 2011. However, only clade 2.3.2.1 viruses could be isolated in 2012, indicating progressive replacement of clade 2.2 and 2.3.4 viruses. There has been an event of segment re-assortment between H5N1 and H9N2 viruses in Bangladesh, where H5N1 virus acquired the PB1 gene from a H9N2 virus. Point mutations have accumulated in Bangladeshi isolates over the last 5 years with potential modification of receptor binding site and antigenic sites. Extensive and continuous molecular epidemiological studies are necessary to monitor the evolution of circulating avian influenza viruses in Bangladesh.

Introduction Avian influenza viruses are type A influenza viruses in the family Orthomyxoviridae, which cause mild to severe infection in different avian species and are designated as low-pathogenic avian influenza virus and highly pathogenic avian influenza (HPAI) virus (Alexander, 2000). The influenza virus genome consists of eight segments of singlestranded negative-sense RNA. The enveloped virus particles have two surface glycoproteins, haemagglutinin (HA) and neuraminidase (NA). Type A influenza viruses are divided into many subtypes based on the HA and NA proteins they carry on their surface. To date, 18 different types of HA (H1 to H18) and 11 NA (N1 to N11) have been recognized (Fouchier et al., 2005; Tong et al., 2012, 2013). All HPAI viruses belong to either the H5 or H7 subtype. The recent panzootic of H5N1 HPAI that started in the Far East and Southeast Asia towards the end of 2003 has devastated the poultry industry across the eastern hemisphere. The ancestor of this virus is thought to have emerged in China in 1996 and 1997 (Claas et al., 1998; Xu et al., 1999; Li et al., 2004). Since then the virus has evolved into many genetic clades (clade 0 to clade 9), which are defined on the basis of HA gene sequences. Clade 2 viruses have further evolved into many subclades of second, third and fourth order (WHO/OIE/FAO Evolution Working Group, 2012).

HPAI is now considered deeply entrenched in poultry of several countries including Bangladesh (FAO, 2011). The first outbreak of H5N1 HPAI in Bangladesh was reported in 2007 (Biswas et al., 2008; Islam et al., 2008) and since then as many as 549 outbreaks have been reported to OIE as of October 2013 (OIE, 2013). HPAI in Bangladesh has an apparent seasonal pattern, each wave of outbreaks starting usually in the late autumn, reaching the peak in the spring and then declining gradually (Ahmed et al., 2011). There have so far been seven human cases of H5N1 avian influenza in Bangladesh with one death (WHO, 2013). Following the initial spread of clade 2.2 H5N1 HPAI virus in Bangladesh in 2007, there has been new introduction of clade 2.3.2.1 and clade 2.3.4 virus in 2011 (Ahmed et al., 2012; Islam et al., 2012; Hoque et al., 2013). Lowpathogenic H9N2 viruses are also circulating in poultry farms of Bangladesh (Negovetich et al., 2011; Parvin et al., 2011; Shanmuganatham et al., 2013). Stamping out has been the national policy in Bangladesh in combating H5N1 HPAI. However, high human and poultry density, heterogeneous structure of poultry industry and inadequate capacity of veterinary services hindered the success of stamping out. Recently, vaccination against H5N1 has been introduced on a trial basis. According to the current international legislation, H9N2 is still not considered notifiable.

*To whom correspondence should be addressed. Tel: +88 9167401. Fax: +88 9161510. E-mail: [email protected] (Received 13 October 2013; accepted 28 January 2014) © 2014 Houghton Trust Ltd

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Like other RNA viruses, avian influenza viruses are very prone to mutation and rapid evolution, leading to antigenic drift, adaptation to mammalian host, development of antiviral resistance, and so forth. The segmented genome of influenza viruses also favours re-assortment or

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recombination resulting from concurrent infection with more than one subtype of the virus. Such re-assortments are feared to be the cause of possible emergence of new pandemic influenza virus. Swine-origin 2009 pandemic H1N1 influenza virus (Smith et al., 2009) and the avian-

A/chicken/Bangladesh/362/2007 A/chicken/Bangladesh/376/2007 A/chicken/Bangladesh/BL-141/2008 A/chicken/Bangladesh/382/2007 A/chicken/Bangladesh/490/2007 A/chicken/Bangladesh/BL-4/2007 A/chicken/Bangladesh/364/2007 A/chicken/Bangladesh/363/2007 A/chicken/Bangladesh/531/2007 A/chicken/Bangladesh/FDIL(G)502/2007 A/chicken/Bangladesh/FDIL(G)500/2007 A/chicken/Bangladesh/FDIL(G)514/2007 82 A/chicken/Bangladesh/380/2007 A/chicken/Bangladesh/BL-156/2008 A/chicken/Bangladesh/BL-195/2008 A/chicken/Bangladesh/FDIL(J)32/2007 A/chicken/Bangladesh/BL-149/2008 A/chicken/Bangladesh/BL-144/2008 A/chicken/India/WB-NIV2811/2008 A/chicken/Bangladesh/394/2007 A/chicken/Bangladesh/L-152/2008 A/chicken/Bangladesh/11rs1984-4/2010 A/chicken/Bangladesh/11rs1984-24/2011 A/chicken/Bangladesh/11rs1984-20/2011 A/chicken/Bangladesh/11rs1984-35/2011 99 81 A/Bangladesh/3233/2011 A/chicken/Bangladesh/11rs1984-23/2011 A/chicken/Bangladesh/11rs1984-29/2011 96 A/chicken/Bangladesh/11rs1984-28/2011 A/chicken/Bangladesh/11rs1984-21/2011 95 A/chicken/Bangladesh/11rs1984-36/2011 A/chicken/Bangladesh/11rs1984-27/2011 A/chicken/Bangladesh/1151-10/2010 A/chicken/West Bengal/155505/2009 A/chicken/Bangladesh/BL-410/2008 A/chicken/Bangladesh/BL-415/2008 81 A/chicken/Bangladesh/BL-409/2008 A/chicken/Bangladesh/BL-416/2008 A/chicken/Bangladesh/BL-418/2009 A/chicken/Bangladesh/1151-11/2010 99 A/chicken/Bangladesh/11rs1984-7/2010 98 A/chicken/Bhutan/248006/2010 A/chicken/Bangladesh/11rs1984-6/2010 69 80 A/chicken/Bangladesh/1151-9/2010 A/chicken/Bangladesh/11rs1984-10/2010 A/chicken/Bangladesh/11rs1984-9/2010 A/chicken/Bangladesh/11rs1984-8/2010 86 86 82 A/chicken/Bngladesh/BL-470/2010 92 A/chicken/Bangladesh/11rs1984/26/2011 A/chicken/Bangladesh/11rs1984-44/2011 98 A/chicken/Bangladesh/11rs1984-5/2010 A/Bangladesh/207095/2008 79 A/chicken/Bangladesh/BL-165/2008 A/chicken/Bangladesh/BL-411/2008 [Clade 2.2] A/goose/Qinghai/59/2005 [Clade 2.5] A/ck/Korea/es/2003 [Clade 2.1.1] A/ck/Indon./BL/2003 [Clade 2.1.2] A/ck/Indon./BBPV1-534/2006 92 87 [Clade 2.1.3] A/ck/Indon./CDC25/2005 [Clade 2.4] A/ck/Zhengzhou/1/2002 [Clade 1] A/ck/Thailand/2/2004 82 [Clade 9] A/Ck/Henan/wu/2004 [Clade 8] A/ck/Henan/12/2004 94 [Clade 5] A/dk/Guangxi/1378/2004 [Clade 6] A/dk/Hubei/wg/2002 [Clade 0] A/Gs/Guangdong/1/1996 97 [Clade 3] A/ck/HK/879.1/2001 [Clade 4] A/ck/Guiyang/846/2006 74 65 [Clade 7] A/dk/Yunnan/5133/2005 [Clade 2.3.3] A/dk/Guiyang/3009/2005 [Clade 2.3.4] A/ck/Fujian/9821/2005 A/chicken/Yangon/254/2010 98 A/chicken/Bangladesh/11rs1984-30/2011 99 A/chicken/Bangladesh/11rs1984-37/2011 99 [Clade 2.3.1] A/dk/Hunan/127/2005 [Clade 2.3.2] A/dk/Yunnan/5820/2005 [Clade 2.3.2.1] A/chicken/Nepal/2-53/2010 A/chicken/Bangladesh/12VIR-7140-7/2012 A/chicken/Bangladesh/12VIR-7140-19/2012 A/chicken/Bangladesh/12VIR-7140-16/2012 A/chicken/Bangladesh/12VIR-7140-14/2012 A/chicken/India/03CL488/2011 99 A/chicken/Bangladesh/11rs1984-18/2011 A/chicken/Bangladesh/12VIR-7140-10/2012 A/duck/Bangladesh/D-1/2011 A/chicken/Bangladesh/12VIR-7140-5/2012 A/chicken/Bangladesh/12VIR-7140-8/2012 99 A/chicken/Bangladesh/11rs1984-43/2011 A/chicken/Bangladesh/11rs1984-34/2011 A/crow/Bangladesh/11rs1984-11/2011 A/crow/Bangladesh/11rs1984-12/2011 A/crow/Bangladesh/11rs1984-13/2011 A/crow/Bangladesh/11rs1984-14/2011 A/crow/Bangladesh/11rs1984-15/2011 A/chicken/Bangladesh/11rs1984-45/2011 A/chicken/Bangladesh/12VIR-7140-3/2012 94 A/chicken/Bangladesh/12VIR-7140-2/2012 A/chicken/Bangladesh/12VIR-7140-6/2012 A/chicken/Bangladesh/12VIR-7140-1/2012 A/chicken/Bangladesh/11rs1984-40/2011 A/chicken/Bangladesh/11rs1984-19/2011 A/chicken/Bangladesh/11rs1984-22/2011 A/chicken/Bangladesh/11rs1984-17/2011 A/chicken/Bangladesh/11rs1984-16/2011 A/chicken/Bangladesh/11rs1984-33/2011 A/chicken/Bangladesh/12VIR-7140-13/2012 A/chicken/Bangladesh/12VIR-7140-9/2012 A/chicken/Bangladesh/12VIR-7140-18/2012 A/chicken/Bangladesh/12VIR-7140-4/2012 A/chicken/Bangladesh/12VIR-7140-15/2012 A/chicken/Bangladesh/12VIR-7140-17/2012 93 A/chicken/Bangladesh/12VIR-7140-11/2012 95 A/chicken/Bangladesh/12VIR-7140-12/2012 0.01

Figure 1. HA H5 clade assignment of Bangladeshi H5N1 HPAI viruses isolated between 2007 and 2012. A total of 88 Bangladeshi isolates and representative strains of different clades were used to create the maximum likelihood evolutionary tree. Bootstrap values (1000 replication) above 60% are shown next to the nodes. Circle, clade 2.2; square, clade 2.3.4; triangle, clade 2.3.2.1; closed symbols, isolates from Bangladesh; open symbols, isolates from neighbouring countries.

Evolution of H5N1 HPAI in Bangladesh 185

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origin 2013 H7N9 influenza virus (Gao et al., 2013) are two recent examples of segment re-assortment events of influenza viruses causing public health concerns. At the time of submission of this manuscript, Mondal et al. (2013) presented a molecular analysis of Bangladeshi HPAI virus isolates from 2007 to 2011 based on sequences available in public databases, and Monne et al. (2013) reported an event of re-assortment between H5N1 and H9N2 avian influenza viruses in Bangladesh. In the present study we generated partial or complete sequence data of 21 additional, not previously analysed, Bangladeshi isolates and performed molecular analysis of H5N1 avian influenza viruses of Bangladesh isolated during the period from 2007 to 2012. This study presents further insight into the molecular evolution of HPAI viruses in Bangladesh and provides evidence of progressive replacement of clade 2.2 viruses by newly introduced clade 2.3.2.1 viruses.

Materials and Methods Virus isolates. A total of 21 H5N1 avian influenza virus isolates were subjected to molecular characterization in this study (Supplemental Tables 1 and 2). The viruses were selected randomly from isolates that were obtained from outbreaks in different parts of Bangladesh at different time points during the period from 2007 to 2012. They were propagated in embryonated chicken eggs. The allantoic fluid was harvested, stored in aliquots at −70°C and used in the subsequent study.

Amplification of partial or full-length genome segments by reverse transcriptase-polymerase chain reaction. Partial H5 (545 base pairs) and N1 (343 base pairs) gene fragments were first amplified from all 21 HPAI virus isolates for reconfirmation of the virus identity. The partial H5 gene segment was sequenced for HA clade determination. Full-length amplification of all eight segments of two isolates and five segments of another isolate was then generated and sequenced. RNA was extracted from the isolates using the Qiagen RNA extraction kit (Qiagen, Hilden, Germany). The partial H5 and N1 gene fragments were amplified with the Qiagen one-step reverse transcription-polymerase chain reaction (RT-PCR) kit using the primers H5-155F (5′-ACA CAT GCY CAR GAC ATA CT-3′) and H5-699R (5′-CTY TGR TTY AGT GTT GAT GT-3′) for the H5 gene fragment (Lee et al., 2001) and N1-580-607F (5′-TGAAGT ACA ATG GCA TAA TAA CWG ACA C-3′) and N1-891-918R (5′-CAC TGC ATA TAT ATC CTA TTT GAT ACT CC-3′) for the N1 gene fragment (WHO, 2007), respectively. The RT-PCR products were analysed by electrophoresis on 1.5% agarose gel stained with ethidium bromide. For amplification of full-length genome segments from selected isolates, a two-step RT-PCR was followed. First-strand cDNA was synthesized from extracted RNA using Uni-12 primer (Hoffmann et al., 2001) and the ImProm II First Strand cDNA Synthesis Kit (Promega, Madison, WI, USA) as per the manufacturer’s instructions. Synthesized first-strand cDNA was used as the template for amplification of different full-length gene segments by PCR using gene-specific primers (Hoffmann et al., 2001). PCR products were analysed by electrophoresis on 1% agarose gel stained with ethidium bromide.

Cloning and sequencing. The PCR products were purified with the Wizard® SV Gel and PCR Clean-Up System (Promega) and were subjected to direct sequencing with respective PCR primers. Alternatively, the cleaned PCR products were cloned by the TA cloning method using the pGEM®-T Easy Vector System Cloning Kit and chemically competent E. coli JM109 (Promega). The plasmid DNA was isolated and purified with EZ Spin Column (Bio Basic Inc., New York, NY, USA) and the cloned cDNA was sequenced with universal primers. If needed, overlapping primers (C. Tosh, High Security Animal Disease Laboratory, Bhopal, India, personal communication, 2012) were used to sequence longer gene segments. Sequencing was carried out from a commercial laboratory.

Molecular and phylogenetic analysis. The partial and full-length sequence data generated in the present study were subjected to molecular analysis. These included complete or nearly complete full-length coding sequences of all eight segments of two chicken isolates and five segments of a duck isolate, and the partial HA gene sequence of 18 isolates. All sequences have been deposited into GenBank. In addition to the sequences generated in the present study, full-length sequence data of other Bangladeshi H5N1 avian influenza virus isolates that are available in the GenBank were downloaded and used in the analysis. Apart from Bangladeshi isolates, strains representing different genetic lineages of each segment (Zhao et al., 2008) as well as strains representing persistent genotypes (Z, V and G) of HPAI viruses (Duan et al., 2008) were included in the phylogenetic analyses. In the case of the HA gene, representative strains from neighbouring countries such as India, Nepal, Bhutan and Myanmar were also included in the phylogenetic analysis. Names of the isolates and accession numbers of the sequences generated in the present study or retrieved from GenBank are presented in Supplemental Tables 1 and 2. Evolutionary analyses were conducted with the program MEGA5 (Tamura et al., 2011). The evolutionary history was inferred using the maximum likelihood method based on the Tamura–Nei model (Tamura & Nei, 1993). For construction of phylogenetic trees, the program obtained initial tree(s) for the heuristic search automatically by applying neighbour-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach, and then selecting the topology with a superior loglikelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. A bootstrap resampling (1000 replications) was used to assess the robustness of individual nodes of the phylogeny, incorporating the maximum likelihood substitution model defined above. Any sequence appearing as a distinct outgroup was subjected to a BLAST (Basic Local alignment Search Tool) search in the GenBank database to look for its closest neighbours. The deduced amino acid alignment table for each gene segment was thoroughly examined for any substitution that might be of biological significance.

Results and Discussion Phylogenetic analysis. The HA gene sequences of 109 Bangladeshi isolates were analysed, and included 91 full-length or nearly full-length HA gene sequences and 18 partial HA gene sequences. A phylogenetic tree constructed with full-length HA gene sequences of 88 Bangladeshi isolates and the representative strain of each clade and selected strains of neighbouring countries is presented in Figure 1. The Bangladeshi isolates were broadly divided into three clusters belonging to clades 2.2, 2.3.4 and 2.3.2.1. Year-wise distributions of different clades are presented in Table 1. The first outbreak of HPAI in poultry from Bangladesh was recorded in February 2007. Since then the HPAI has become well entrenched in Bangladesh poultry with regular outbreaks. In contrast to Table 1. Distribution of HA clades among Bangladeshi isolates of H5N1 HPAI viruses isolated between 2007 and 2012.

HA clade

Year

Total number of outbreaksa

Number of isolates sequencedb

2.2

2.3.4

2.3.2.1

2007 2008 2009 2010 2011 2012 Total

64 225 35 30 169 23 546

15 14 3 13 41 23 109

15 14 3 13 15 – 60

– – – – 3 – 3

– – – – 23 23 46

a

As reported to OIE. Includes both partial and full-length sequences.

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M. E. Haque et al. A/ck/Bangladesh/12VIR-7140-2/2012(H5N1) A/ck/Bangladesh/12VIR-7140-3/2012(H5N1) A/ck/Bangladesh/12VIR-7140-6/2012(H5N1) A/ck/Bangladesh/12VIR-7140-12/2012(H5N1) A/ck/Bangladesh/12VIR-7140-15/2012(H5N1) A/ck/Bangladesh/12VIR-7140-4/2012(H5N1) 97 A/ck/Bangladesh/12VIR-7140-11/2012(H5N1) 99 A/ck/Bangladesh/12VIR-7140-17/2012(H5N1) A/ck/Bangladesh/12VIR-7140-5/2012(H5N1) A/ck/Bangladesh/12VIR-7140-8/2012(H5N1) A/ck/Bangladesh/12VIR-7140-14/2012(H5N1) A/ck/Bangladesh/12VIR-7140-18/2012(H5N1) A/ck/Bangladesh/12VIR-7140-19/2012(H5N1) 100 A/ck/Bangladesh/12VIR-7140-1/2011(H5N1) A/ck/Bangladesh/12VIR-7140-7/2012(H5N1) 93 A/ck/Bangladesh/12VIR-7140-13/2012(H5N1) A/ck/Bangladesh/12VIR-7140-16/2012(H5N1) A/ck/Bangladesh/12VIR-7140-10/2012(H5N1) 100 [Genotype V] A/ck/HK/947/2006(H5N1) [Genotype Z] A/BHG/Qinghai/65/2005(H5N1) A/ck/Bangladesh/BL-4/2007(H5N1) 100 A/Bangladesh/207095/2008(H5N1) 100 A/ck/Bangladesh/1151-10/2010(H5N1) A/Bangladesh/3233/2011(H5N1) 89 A/ck/Bangladesh/1151-9/2010(H5N1) 82 A/ck/Bangladesh/1151-11/2010(H5N1) A/ck/Bangladesh/BL-470/2010(H5N1) 98 [Genotype G] A/munia/HK/2454/2006(H5N1) A/gs/Guangdong/1/1996(H5N1) 100 A/ck/Hubei/wj/1997(H5N1) A/dk/Yokohama/aq10/2003(H5N1) A/HK/156/1997(H5N1) A/ck/Hubei/wl/1997(H5N1) 96 99

(a)

PB2

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100 0.02

(b)

PB1

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84

A/ck/Bangladesh/12VIR-7140-11/2012(H5N1) A/ck/Bangladesh/12VIR-7140-17/2012(H5N1) A/ck/Bangladesh/12VIR-7140-15/2012(H5N1) A/ck/Bangladesh/12VIR-7140-4/2012(H5N1) A/ck/Bangladesh/12VIR-7140-5/2012(H5N1) A/ck/Bangladesh/12VIR-7140-8/2012(H5N1) A/ck/Bangladesh/12VIR-7140-6/2012(H5N1) A/ck/Bangladesh/12VIR-7140-2/2012(H5N1) 99 A/ck/Bangladesh/12VIR-7140-3/2012(H5N1) A/ck/Bangladesh/12VIR-7140-18/2012(H5N1) A/ck/Bangladesh/12VIR-7140-14/2012(H5N1) A/ck/Bangladesh/12VIR-7140-10/2012(H5N1) 100 A/ck/Bangladesh/12VIR-7140-1/2011(H5N1) A/ck/Bangladesh/12VIR-7140-19/2012(H5N1) 100 A/ck/Bangladesh/12VIR-7140-13/2012(H5N1) A/ck/Bangladesh/12VIR-7140-12/2012(H5N1) 99 [Genotype V] A/ck/HK/947/2006(H5N1) 100 [Genotype G] A/munia/HK/2454/2006(H5N1) [Genotype Z] A/BHG/Qinghai/65/2005(H5N1) A/ck/Bangladesh/BL-4/2007(H5N1) 100 A/Bangladesh/207095/2008(H5N1) 98 A/ck/Bangladesh/1151-9/2010(H5N1) A/ck/Bangladesh/1151-11/2010(H5N1) 98 A/ck/Bangladesh/1151-10/2010(H5N1) A/Bangladesh/3233/2011(H5N1) 100 A/gs/Guangdong/1/1996(H5N1) A/ck/Hubei/wh/1997(H5N1) A/dk/Anyang/AVL-1/2001(H5N1) A/ck/Bangladesh/VP01/2006(H9N2) A/environment/Bangladesh/9457/2010(H9N2) 100 A/ck/Bangladesh/12VIR-7140-16/2012(H5N1) 100 A/ck/Bangladesh/12VIR-7140-7/2012(H5N1) 99 98

0.01

Figure 2. Maximum likelihood evolutionary trees based on (a) PB2, (b) PB1, (c) PA, (d) NP, (e) NA, (f) MP and (g) NS genes of Bangladeshi H5N1 HPAI isolates. Sequences representing different genetic lineages of each gene and genotypes W, V and G were included in the analysis. Two closely related H9N2 isolates are also included in the analysis on PB1 gene (2b). Bootstrap values (1000 replication) above 60% are shown next to the nodes. Closed circle, HA clade 2.2; closed triangle, HA clade 2.3.2.1; closed square in the case of PB1 tree, H9N2 virus.

Southeast and Far East Asia, the virus that caused initial outbreaks in Bangladesh belonged to clade 2.2 (Ahmed et al., 2012; Islam et al., 2012; Hoque et al., 2013). Between 2007 and 2010, the clade 2.2 virus from a single introduction circulated in Bangladesh (Islam et al., 2012), although the virus evolved into several genetic groups (Hoque et al., 2013). In 2011, viruses of clade 2.3.2.1 and clade 2.3.4 were also detected along with clade 2.2 viruses (Islam et al., 2012; Hoque et al., 2013; Mondal et al.,

2013). In the present study, the phylogenetic tree based on the HA gene (Figure 1) revealed at least two subgroups of clade 2.2 viruses, one being dominated by the isolates of 2007 and 2008 and the other containing isolates from 2009 to 2011 and a few isolates from 2008. Among the three selected Indian isolates, one clustered with clade 2.3.2.1 viruses and the other two grouped with the two subgroups of clade 2.2 viruses. The isolate from Bhutan, Nepal and Myanmar clustered with clade 2.2, clade 2.3.2.1 and clade

Evolution of H5N1 HPAI in Bangladesh 187

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99 99

A/ck/Bangladesh/12VIR-7140-2/2012(H5N1) A/ck/Bangladesh/12VIR-7140-3/2012(H5N1) A/ck/Bangladesh/12VIR-7140-6/2012(H5N1) A/ck/Bangladesh/12VIR-7140-10/2012(H5N1) A/ck/Bangladesh/12VIR-7140-15/2012(H5N1) A/ck/Bangladesh/12VIR-7140-4/2012(H5N1) A/ck/Bangladesh/12VIR-7140-11/2012(H5N1) A/ck/Bangladesh/12VIR-7140-17/2012(H5N1) A/ck/Bangladesh/12VIR-7140-1/2011(H5N1) A/ck/Bangladesh/12VIR-7140-7/2012(H5N1) A/ck/Bangladesh/12VIR-7140-12/2012(H5N1) A/ck/Bangladesh/12VIR-7140-14/2012(H5N1) A/ck/Bangladesh/12VIR-7140-18/2012(H5N1) 100 A/ck/Bangladesh/12VIR-7140-5/2012(H5N1) A/ck/Bangladesh/12VIR-7140-8/2012(H5N1) 100 A/ck/Bangladesh/12VIR-7140-13/2012(H5N1) A/ck/Bangladesh/12VIR-7140-16/2012(H5N1) A/ck/Bangladesh/12VIR-7140-19/2012(H5N1) [Genotype V] A/ck/HK/947/2006(H5N1) A/gs/Guangdong/1/1996(H5N1) [Genotype G] A/munia/HK/2454/2006(H5N1) [Genotype Z] A/BHG/Qinghai/65/2005(H5N1) 100 A/ck/Bangladesh/BL-4/2007(H5N1) 100 A/Bangladesh/207095/2008(H5N1) 94 99 A/ck/Bangladesh/1151-10/2010(H5N1) A/Bangladesh/3233/2011(H5N1) 93 A/ck/Bangladesh/1151-9/2010(H5N1) 93 A/ck/Bangladesh/1151-11/2010(H5N1) 72 A/chicken/Bangladesh/BL-470/2010 95 A/ck/Hubei/wl/1997(H5N1) A/HK/156/1997(H5N1) A/dk/Anyang/AVL-1/2001(H5N1) 99

(c) PA

68 0.01

(d)

NP

A/ck/Bangladesh/12VIR-7140-13/2012(H5N1) A/ck/Bangladesh/12VIR-7140-18/2012(H5N1) A/ck/Bangladesh/12VIR-7140-19/2012(H5N1) A/dk/Bangladesh/D-1/2011(H5N1) A/ck/Bangladesh/12VIR-7140-14/2012(H5N1) A/ck/Bangladesh/12VIR-7140-12/2012(H5N1) A/ck/Bangladesh/12VIR-7140-5/2012(H5N1) A/ck/Bangladesh/12VIR-7140-8/2012(H5N1) A/ck/Bangladesh/12VIR-7140-7/2012(H5N1) A/ck/Bangladesh/12VIR-7140-6/2012(H5N1) A/ck/Bangladesh/12VIR-7140-2/2012(H5N1) A/ck/Bangladesh/12VIR-7140-3/2012(H5N1) A/ck/Bangladesh/12VIR-7140-1/2011(H5N1) 99 A/ck/Bangladesh/12VIR-7140-10/2012(H5N1) A/ck/Bangladesh/12VIR-7140-11/2012(H5N1) A/ck/Bangladesh/12VIR-7140-17/2012(H5N1) 98 A/ck/Bangladesh/12VIR-7140-15/2012(H5N1) A/ck/Bangladesh/12VIR-7140-4/2012(H5N1) 78 A/ck/Bangladesh/12VIR-7140-16/2012(H5N1) [Genotype V] A/ck/HK/947/2006(H5N1) 99 [Genotype G] A/munia/HK/2454/2006(H5N1) [Genotype Z] A/BHG/Qinghai/65/2005(H5N1) A/ck/Bangladesh/BL-4/2007(H5N1) 99 A/Bangladesh/207095/2008(H5N1) 99 99 A/Bangladesh/3233/2011(H5N1) A/ck/Bangladesh/1151-10/2010(H5N1) 98 A/ck/Bangladesh/1151-9/2010(H5N1) A/ck/Bangladesh/1151-11/2010(H5N1) A/ck/Bangladesh/BL-470/2010(H5N1) A/gs/Goungdong/1/1996(H5N1) A/ck/Hubei/wl/1997(H5N1) A/ck/Hebei/718/2001(H5N1) 99 A/ck/Hubei/wj/1997(H5N1) 65 82

0.01

Figure 2.

(continued)

2.3.4 viruses of Bangladesh, respectively. This finding would suggest that regional spread of the virus might have taken place across the national border. Interestingly, the present study revealed that all 23 isolates of 2012 belonged to clade 2.3.2.1 and there was no detection of any clade 2.2 or clade 2.3.4 virus in 2012 (Table 1). An extensive surveillance will be needed to confirm whether clade 2.2 and clade 2.3.4 viruses have disappeared from Bangladesh. The reason for this progressive replacement of previously circulating clade 2.2 viruses by newly introduced clade 2.3.2.1 viruses is not clear. The clade 2.3.2.1 viruses might have some special advantages over the viruses of other clades in terms of enhanced transmissibility and/or tenacity.

Until 2011, clinical outbreaks of HPAI with clade 2.2 viruses in Bangladesh were almost restricted to chickens. On the contrary, clade 2.3.2.1 viruses were found to cause clinical disease in ducks and quails as well as to cause mortality in crows (Islam et al., 2012) in addition to outbreaks in chickens. Gradual replacement of the dominating clades or genotypes of H5N1 in the course of time has been reported earlier in China and Vietnam (Duan et al., 2008; Nguyen et al., 2012). Phylogenetic trees based on PB2, PB1, PA, NP, NA, MP and NS gene segments are presented in Figure 2a to g). These trees broadly followed the pattern similar to the tree derived with HA gene sequences of clade 2.2 and clade

188

M. E. Haque et al. A/ck/Bangladesh/12VIR-7140-12/2012(H5N1) A/ck/Bangladesh/12VIR-7140-7/2012(H5N1) A/ck/Bangladesh/12VIR-7140-1/2011(H5N1) A/dk/Bangladesh/D-1/2011(H5N1) A/ck/Bangladesh/12VIR-7140-14/2012(H5N1) A/ck/Bangladesh/12VIR-7140-10/2012(H5N1) A/ck/Bangladesh/12VIR-7140-16/2012(H5N1) A/ck/Bangladesh/12VIR-7140-13/2012(H5N1) A/ck/Bangladesh/12VIR-7140-19/2012(H5N1) 85 A/ck/Bangladesh/12VIR-7140-15/2012(H5N1) A/ck/Bangladesh/12VIR-7140-18/2012(H5N1) A/ck/Bangladesh/12VIR-7140-4/2012(H5N1) 99 98 A/ck/Bangladesh/12VIR-7140-11/2012(H5N1) 99 A/ck/Bangladesh/12VIR-7140-17/2012(H5N1) A/ck/Bangladesh/12VIR-7140-8/2012(H5N1) 86 A/ck/Bangladesh/12VIR-7140-5/2012(H5N1) A/ck/Bangladesh/12VIR-7140-6/2012(H5N1) A/ck/Bangladesh/12VIR-7140-2/2012(H5N1) 99 93 98 A/chicken/Bangladesh/12VIR-7140-3/2012 68 A/ck/Bangladesh/BL-543/2011(H5N1) [Genotype V] A/ck/HK/947/2006(H5N1) 93 [Genotype G] A/munia/HK/2454/2006(H5N1) [Genotype Z] A/BHG/Qinghai/65/2005(H5N1) A/ck/Bangladesh/BL-4/2007(H5N1) 99 A/Bangladesh/207095/2008(H5N1) A/ck/Bangladesh/1151-10/2010(H5N1) 98 A/Bangladesh/3233/2011(H5N1) 88 A/ck/Bangladesh/1151-9/2010(H5N1) 66 A/ck/Bangladesh/BL-470/2010(H5N1) A/ck/Bangladesh/1151-11/2010(H5N1) 98 A/ck/Bangladesh/BL-499/2011(H5N1) A/gs/Guangdong/1/1996(H5N1) A/ck/Hebei/718/2001(H5N1) A/HK/156/1997(H5N1) 99

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(e)

NA

0.02

(f)

MP

A/ck/Bangladesh/12VIR-7140-5/2012(H5N1) A/ck/Bangladesh/12VIR-7140-8/2012(H5N1) A/ck/Bangladesh/12VIR-7140-11/2012(H5N1) A/ck/Bangladesh/12VIR-7140-17/2012(H5N1) A/dk/Bangladesh/D-1/2011(H5N1) A/ck/Bangladesh/12VIR-7140-1/2011(H5N1) A/ck/Bangladesh/12VIR-7140-18/2012(H5N1) A/ck/Bangladesh/12VIR-7140-13/2012(H5N1) A/ck/Bangladesh/12VIR-7140-19/2012(H5N1) A/ck/Bangladesh/12VIR-7140-12/2012(H5N1) A/ck/Bangladesh/12VIR-7140-4/2012(H5N1) A/ck/Bangladesh/12VIR-7140-2/2012(H5N1) A/ck/Bangladesh/12VIR-7140-3/2012(H5N1) 98 A/ck/Bangladesh/12VIR-7140-15/2012(H5N1) A/ck/Bangladesh/12VIR-7140-16/2012(H5N1) A/ck/Bangladesh/12VIR-7140-7/2012(H5N1) 61 A/ck/Bangladesh/12VIR-7140-6/2012(H5N1) A/ck/Bangladesh/12VIR-7140-14/2012(H5N1 A/ck/Bangladesh/12VIR-7140-10/2012(H5N1) [Genotype V] A/ck/HK/947/2006(H5N1) 99 [Genotype G] A/munia/HK/2454/2006(H5N1) [Genotype Z] A/BHG/Qinghai/65/2005(H5N1) A/ck/Bangladesh/BL-4/2007(H5N1) A/ck/Bangladesh/1151-10/2010(H5N1) 99 A/Bangladesh/3233/2011(H5N1) 99 93 A/Bangladesh/207095/2008(H5N1) A/ck/Bangladesh/1151-9/2010(H5N1) 73 A/dk/Bangladesh/5749/2010(H5N1) A/ck/Bangladesh/1151-11/2010(H5N1) A/ck/Bangladesh/BL-470/2010(H5N1) A/gs/Goungdong/1/1996(H5N1) A/ck/Hubei/wh/1997(H5N1) A/HK/156/1997(H5N1) A/ck/Hubei/wl/1997(H5N1) 79

0.01

Figure 2.

(continued)

2.3.2.1 viruses. The viruses of HA clade 2.2 clustered with genotype Z, whereas the viruses of HA clade 2.3.2.1 belonged to genotype V. With the exception of the NS gene, all genome segments of these genotype Z and genotype V viruses were related to the gs/Gd (A/goose/Guangdong/1/ 1996) lineage, while the NS gene was more closely related to the HK/156 (A/Hong Kong/156/1997) lineage. A significant deviation was noticed in the phylogenetic tree based on PB1 gene sequences, where two H5N1 isolates of clade 2.3.2.1—namely A/ck/Bangladesh/12VIR7140-16/2012(H5N1) and A/ck/Bangladesh/12VIR-7140-7/ 2012(H5N1)—formed a separate branch (Figure 2b). The BLAST search in GenBank revealed that the PB1 gene of

these two atypical Bangladeshi H5N1 isolates was closely related to that of Bangladeshi and Indian H9N2 low-pathogenic avian influenza isolates of Eurasian lineage and “Quail/HK/G1/97-like” sublineage. When the most closely related H9N2 isolate A/environment/Bangladesh/ 9457/2010(H9N2) (GenBank accession number KC757902) and the earliest H9N2 isolate from Bangladesh A/ck/ Bangladesh/VP01/2006(H9N2) (GenBank accession number KC986288) were included in the phylogenetic tree, they clustered with the atypical H5N1 Bangladeshi isolates (Figure 2b). Our observation corroborates the findings of Monne et al. (2013), who also reported this event of reassortment. However, in their study the place for this re-

Evolution of H5N1 HPAI in Bangladesh 189

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(g)

NS

A/dk/Bangladesh/D-1/2011(H5N1) A/ck/Bangladesh/12VIR-7140-19/2012(H5N1) A/ck/Bangladesh/12VIR-7140-14/2012(H5N1) A/ck/Bangladesh/12VIR-7140-12/2012(H5N1) A/ck/Bangladesh/12VIR-7140-1/2011(H5N1) A/ck/Bangladesh/12VIR-7140-13/2012(H5N1) A/ck/Bangladesh/12VIR-7140-18/2012(H5N1) A/ck/Bangladesh/12VIR-7140-15/2012(H5N1) A/ck/Bangladesh/12VIR-7140-4/2012(H5N1) A/ck/Bangladesh/12VIR-7140-11/2012(H5N1) A/ck/Bangladesh/12VIR-7140-17/2012(H5N1) A/ck/Bangladesh/12VIR-7140-16/2012(H5N1) A/ck/Bangladesh/12VIR-7140-6/2012(H5N1) A/ck/Bangladesh/12VIR-7140-2/2012(H5N1) 99 A/ck/Bangladesh/12VIR-7140-3/2012(H5N1) A/ck/Bangladesh/12VIR-7140-7/2012(H5N1) A/ck/Bangladesh/12VIR-7140-5/2012(H5N1) 85 A/ck/Bangladesh/12VIR-7140-8/2012(H5N1) A/ck/Bangladesh/12VIR-7140-10/2012(H5N1) [Genotype V] A/ck/HK/947/2006(H5N1) 97 [Genotype G] A/munia/HK/2454/2006(H5N1) [Genotype Z] A/BHG/Qinghai/65/2005(H5N1) A/ck/Bangladesh/BL-4/2007(H5N1) 98 A/Bangladesh/207095/2008(H5N1) 97 A/ck/Bangladesh/1151-11/2010(H5N1) A/ck/Bangladesh/BL-470/2010(H5N1) 91 A/ck/Bangladesh/1151-9/2010(H5N1) A/ck/Bangladesh/1151-10/2010(H5N1) 88 A/Bangladesh/3233/2011(H5N1) A/HK/156/1997(H5N1) A/gs/Guangdong/1/1996(H5N1)

0.05

Figure 2.

(continued)

assortment event remained obscure as no Bangladeshi H9N2 sequence was available for analysis at that time and the most closely related H9N2 strain was from India. The present study reveals that the most closely related H9N2 isolate was from Bangladesh, so the re-assortment most probably took place in Bangladesh. The significance of this re-assortment is not known. Further surveillance is needed to explore whether these re-assortants have already disappeared or established in poultry of Bangladesh. Genetic re-assortment between H5 and H9 viruses has also been reported elsewhere (Zhao et al., 2012). Molecular analysis of nucleotide and deduced amino acid sequences. Deduced amino acid sequences of the HA gene of 88 Bangladeshi isolates were aligned and, based on sequence similarities and divergence, 15 representative sequences having different amino acid substitution patterns were selected. These 15 representative isolates included nine out of 51 clade 2.2 isolates, two out of two clade 2.3.4 isolates, and four out of 35 clade 2.3.2.1 isolates. Alignment of the deduced amino acid sequences from residue 1 to 550 (H5 numbering) of these 15 isolates is shown in Figure 3. A total of 12 and 14 amino acid substitutions appeared to be the signature residues for clade 2.2 and clade 2.3.2.1 viruses, respectively. The two clade 2.3.4 viruses, although they shared several substitutions with either clade 2.2 or clade 2.3.2.1 viruses, still had eight clade-specific substitutions. Nucleotide sequence divergences within and between the clades are shown in Table 2. Among the three clades, clade 2.3.2.1 appeared to be the most divergent having nucleotide divergence up to 9.2% from clade 2.3.4 and 9.6% from clade 2.2 viruses. Up to 2.4% and 2.8% nucleotide divergences were observed within clade 2.3.2.1 and clade 2.2 viruses, respectively. Two isolates of clade 2.3.4 were also 1.5% divergent from each other at nucleotide level. These data would suggest that the viruses are continuously evolving.

Amino acid substitutions were observed throughout the protein sequence, which involved glycosylation sites, the HA cleavage site, the receptor-binding site and antigenic sites. Common mutations in the HA of different Bangladeshi isolates are summarized in Table 3. Mutation hotspots are also indicated on the three-dimensional diagram of a monomeric unit of the HA molecule (Figure 4). To be consistent with published literature, H5 numbering was followed for glycosylation sites, the HA cleavage site and the antigenic site, while H3 numbering was followed for the receptor binding site. Six well-conserved potential glycosylation sites were found in the HA of all Bangladeshi isolates: 11NST, 23NVT, 165NNT, 286NNS, 484NGT, and 543 NGS. All of the clade 2.3.2.1 isolates acquired an additional glycosylation site 140NSS through R140N mutation (Figure 3 and Table 3). Similarly, the clade 2.3.4 isolates also acquired an additional glycosylation site, 154 NNT, through two consecutive mutations, D155N and A156T (Figure 3 and Table 3). Ten out of 51 clade 2.2 isolates and two out of 35 clade 2.3.2.1 isolates acquired another glycosylation site, 236NDT, through A238T mutation (Figure 3 and Table 3). The surface glycoprotein HA plays a crucial role in virus attachment and subsequent invasion into the cell. Deletion or appearance of a glycosylation site may thus influence the infectivity and antigenicity of a virus (Schulze, 1997). All clade 2.2 viruses had a 321PQGERRRKKR*GLF333 motif at the HA cleavage site, except two isolates that had either Q322K or R325K mutation (H5 numbering). Clade 2.3.4 and clade 2.3.2.1 viruses had two deletions at the cleavage site, and the motive for clade 2.3.4 viruses was ALREKRRK*GLF and that for clade 2.3.2.1 viruses was PQRERRRK*GLF. All isolates maintained multiple basic amino acids at the cleavage site as a characteristic of high pathogenicity. The conserved residues at the base of the receptor binding site 98Y, 153W, 183H and 195Y (H3 numbering)

M. E. Haque et al.

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190

Figure 3. Alignment of deduced amino acid sequences of the HA protein from 15 representative Bangladeshi isolates of H5N1 HPAI viruses. Positions of glycosylation sites and the receptor binding site are indicated with bars.

were present in all isolates, as expected. All isolates had Q and G at positions 226 and 228 (222 and 224 in H5 numbering) as well as E and G at positions 190 and 225 (186 and 221 in H5 numbering), indicating that the viruses Table 2. Nucleotide sequence divergence (%) between and within different clades of H5N1 HPAI viruses isolated in Bangladesh.

Clade 2.2 Clade 2.3.4 Clade 2.3.2.1

Clade 2.2

Clade 2.3.4

Clade 2.3.2.1

0.0 to 2.8 5.8 to 8.0 6.7 to 9.6

1.5 7.2 to 9.2

0.0 to 2.4

are primarily specific for avian type receptor (α-2,3-linked sialic acid) (Stevens et al., 2006). However, amino acid substitutions were noticed in the 190 helix and 130 and 220 loops (Table 3 and Figure 4). Based on analysis of escape mutants, three antigenic sites have been suggested for H5N1 HA (Kaverin et al., 2002); site 1 from residues 136 to 141, site 2 from residues 152 to 153, and site 3 from residues 125 to 129 (H5 numbering) (Kaverin et al., 2002; Stevens et al., 2006). In the present study, several amino acid substitutions have been observed among Bangladeshi isolates at these sites (Table 3 and Figure 4). These mutations would very probably result in antigenic diversity among the viruses of three different clades. However, cross-

Evolution of H5N1 HPAI in Bangladesh 191 Table 3. Molecular differences in HA of different H5N1 HPAI isolates from Bangladesh.

Amino acid residues/substitutionsa

Feature Glycosylation site Conserved sites (H5 numbering) Non-conserved sites (H5 numbering)

HA Cleavage site (H5 numbering)

11

NST, 23NVT, NSS 154 NNT 236 NDT

165

NNT,

321

190

EQTRLYQNP198

190

EQTKLYQNP198 EQIQLYQNP198 135 VSSA138 190

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130 loop (H3 numbering)

135

220 loop (H3 numbering)

221

VSAA138 SKVNGQSG228

221

SKINGQSG228 PKINGQSG228 98 Y, 153W, 183H, 195Y 221

Base (H3 numbering) Antigenic site Site 1 (H5 numbering)

136

PYQGRS141 PYQGRP141 136 PYQGNS141 136 SYQGNS141 136 PYLGTP141 152 KK153 125 HEASS129 125 HKASS129 125 HEASL129 136

Site 2 (H5 numbering) Site 3 (H5 numbering)

NNS,

PQGERRRKKR*GLF333 PKGERRRKKR*GLF333 321 PQGEKRRKKR*GLF333 321 ALREKRRK− −*GLF333 321 PQRERRRK− −*GLF333 321

Receptor binding domain 190 helix (H3 numbering)

286

140

125

HETSL129

484

NGT,

Presence in Bangladeshi isolates 543

NGS

All Bangladeshi isolates All (35) isolates of clade 2.3.2.1 All (2) isolates of clade 2.3.4 10 out of 51 isolates of clade 2.2 2 out of 35 isolates of clade 2.3.2.1 49 out of 51 isolates of clade 2.2 1 out of 51 isolates of clade 2.2 1 out of 51 isolates of clade 2.2 All (2) isolates of clade 2.3.4 All (35) isolates of clade 2.3.2.1 All (35) isolates of clade 2.3.2.1 22 out of 51 isolates of clade 2.2 28 out of 51 isolates of clade 2.2 All (2) isolates of clade 2.3.4 All (51) isolates of clade 2.2 All (2) isolates of clade 2.3.4 All (35) isolates of clade 2.3.2.1 All (51) isolates of clade 2.2 All (2) isolates of clade 2.3.4 31 out of 35 isolates of clade 2.3.2.1 3 out of 35 isolates of clade 2.3.2.1 Conserved in all Bangladeshi isolates 48 out of 51 isolates of clade 2.2 3 out of 51 isolates of clade 2.2 34 out of 35 isolates of clade 2.3.2.1 1 out of 35 isolates of clade 2.3.2.1 All (2) isolates of clade 2.3.4 All (88) Bangladeshi isolates 50 out of 51 isolates of clade 2.2 1 out of 51 isolates of clade 2.2 All (35) isolates of clade 2.3.2.1 1 out of 2 isolates of clade 2.3.4 1 out of 2 isolates of clade 2.3.4

a

Amino acid substitutions are indicated in italic font.

neutralization or cross-haemagglutination inhibition tests using homologous and heterologous antigen and antiserum will be needed to confirm their antigenic diversity. In the NA protein, 20 amino acid deletions at the stalk region (with acid positions 49 to 68) were observed in all Bangladeshi isolates as compared to H5N1 AIVs isolated from wild birds prior to 2003. One isolate (A/chicken/ Bangladesh/1151-11/2010) had deletion of 25 amino acids at the same region (positions 48 to 72). This deletion in the NA protein is considered a hallmark of adaptation of H5N1 viruses from wild birds to domestic poultry. Only three conserved glycosylation sites (88NSS, 146NGT/NRT and 235 NGS/NVS) were predicted in the NA of Bangladeshi isolates. The glycosylation site 353NGS disappeared in one isolate (A/chicken/Bangladesh/7140-6/2012) with the amino acid substitution N235D. On the other hand, one isolate (A/chicken/Bangladesh/7140-1/20121) acquired a new potential glycosylation site (35NHS) with the amino acid substitution S35N. None of the Bangladeshi isolates exhibited any mutations in the NA that are considered to be associated with Oseltamivir resistance (E119V, H275Y, R293K or N295S; N1 ATG numbering).

Analysis of the deduced amino acid sequences of the M2 gene revealed that three Bangladeshi isolates (A/chicken/ Bangladesh/1151-9/2010, A/chicken/Bangladesh/12VIR7140-5/2012 and A/chicken/Bangladesh/10410/2011) acquired amantadine resistance with the amino acid substitution S31N as suggested by Abed et al. (2005) and Govorkova et al. (2013). The NS1 protein of all Bangladeshi isolates had deletion of five amino acids (positions 80 to 84). This deletion has been suggested to increase the virulence of H5N1 viruses (Long et al., 2008). At the carboxy terminus of NS1, all HA clade 2.2 isolates had ESKV while all HA clade 2.3.2.1 viruses possessed ESEV, both of which could serve as a PDZ domain binding motif. Avian and human-type signature amino acids. Deduced amino acid sequences of all eight genome segments were searched for the presence of residues that are considered a signature of pandemic human influenza viruses (Chen et al., 2006). The findings are presented in Table 4. All HA clade 2.2 isolates but not clade 2.3.2.1 isolates had 627K in the PB2. On the other hand, all clade 2.3.2.1 isolates had 404S in the PA protein. Five clade 2.3.2.1 isolates acquired an

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M. E. Haque et al.

Figure 4. Mutation hotspots in HA, as observed in the present study, shown in a three-dimensional model of avian influenza virus HA (single chain). Positions of the residues prone to mutation are indicated with closed circles and H5 numbering (H3 numbering in parentheses). Details of the mutations can be seen in Table 3.

additional human influenza virus-like residue in the PA protein (28L, 57Q, 100A or 409S). The Bangladeshi isolates are still avian type in nature, although some of the isolates have acquired a few human influenza virus-like residues. However, animal experimentation with reverse genetics strains having defined mutations will be needed to establish the significance of these mutations.

to monitor the evolution of circulating avian influenza viruses in Bangladesh, because such information is very important in assessing the pathogenic, zoonotic and pandemic potential of the viruses and selecting appropriate vaccine strains.

Supplementary Data Conclusion

Supplementary data for this article can be accessed online.

The newly introduced clade 2.3.2.1 H5N1 viruses have apparently replaced previously circulating clade 2.2 viruses in Bangladesh. This needs to be confirmed by extensive and systematic surveillance. There has been an event of reassortment between H5N1 virus of clade 2.3.2.1 and H9N2 virus of Eurasian lineage (“Quail/HK/G1/97-like” sublineage) in Bangladesh, where H5N1 virus has acquired the PB1 gene of an H9N2 virus. Point mutations are accumulating in Bangladeshi isolates with potential modification of receptor binding site and antigenic sites. Extensive and continuous molecular epidemiological studies are necessary

References Abed, Y., Goyette, N. & Boivin, G. (2005). Generation and characterization of recombinant influenza A (H1N1) viruses harboring amantadine resistance mutations. Antimicrobial Agents and Chemotherapy, 49, 556–559. Ahmed, S.S., Ersbøll, A.K., Biswas, P.K., Christensen, J.P. & Toft, N. (2011). Spatio-temporal magnitude and direction of highly pathogenic avian influenza (H5N1) outbreaks in Bangladesh. PLoS One, 6, e24324. Ahmed, S.S., Themudo, G.E., Christensen, J.P., Biswas, P.K., Giasuddin, M., Samad, M.A., Toft, N. & Ersbøll, A.K. (2012). Molecular

Table 4. Presence of specific amino acid residues in Bangladeshi H5N1 HPAI virus isolates, which are considered signatory to human pandemic influenza viruses.

Residue usually observed in Protein PB2 PA

Amino acid position

Avian strain

Human strain

Bangladeshi isolates having human-like residues

627 28 57 100

E P R V

K L Q A

404 409

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All clade 2.2 isolates from Bangladesh A/chicken/Bangladesh/12VIR-7140-15/2012 A/chicken/Bangladesh/12VIR-7140-7/2012 A/chicken/Bangladesh/12VIR-7140-13/2012 A/chicken/ Bangladesh/12VIR-7140-16/2012 All clade 2.3.2.1 isolates from Bangladesh A/chicken/Bangladesh/12VIR-7140-14/2012

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