Phylogenetic analysis of influenza viruses collected during December 2009–February 2010 from chickens in live poultry retail shops in Lahore, Pakistan, showed influenza A(H9N2) lineage polymerase and nonstructural genes generate through inter- and intrasubtypic reassortments. Many amino acid signatures observed were characteristic of human isolates; hence, their circulation could enhance inter- or intrasubtypic reassortment

Aamir

Abbas

Alessandra Drago

Angélique Angot

Baigent

Cameron

Cattoli

Finkelstein

Fusaro

Giovanni Cattoli

Giulia Trovò

Hamad B. Rashid

Hulse

Ilaria Capua

Iqbal

Mamoona Chaudhry

Manzoor Hussain

Mark C. Eisler

Michael Thrusfield

Monne

Obenauer

Shanmuganatham

Sue Welburn

Tamura

Viviana Valastro

Emerging Infectious Diseases

PubMed

Phylogenetic analysis of influenza viruses collected during December 2009-February 2010 from chickens in live poultry retail shops in Lahore, Pakistan, showed influenza A(H9N2) lineage polymerase and nonstructural genes generate through inter- and intrasubtypic reassortments. Many amino acid signatures observed were characteristic of human isolates; hence, their circulation could enhance inter- or intrasubtypic reassortment.</p

Chaudhry, Mamoona

Angot, Angélique

Rashid, Hamad B

Cattoli, Giovanni

Hussain, Manzoor

Trovò, Giulia

Drago, Alessandra

Valastro, Viviana

Thrusfield, Michael

Welburn, Susan

Eisler, Mark C

Capua, Ilaria

Edinburgh Research Explorer

     Edinburgh Research Explorer                                      Reassortant Avian Influenza A(H9N2) viruses in chickens in retailpoultry shops, Pakistan, 2009-2010Citation for published version:Chaudhry, M, Angot, A, Rashid, HB, Cattoli, G, Hussain, M, Trovò, G, Drago, A, Valastro, V, Thrusfield, M,Welburn, S, Eisler, MC & Capua, I 2015, 'Reassortant Avian Influenza A(H9N2) viruses in chickens in retailpoultry shops, Pakistan, 2009-2010', Emerging Infectious Diseases, vol. 21, no. 4, pp. 673-6.https://doi.org/10.3201/eid2104.141570Digital Object Identifier (DOI):10.3201/eid2104.141570Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Publisher's PDF, also known as Version of recordPublished In:Emerging Infectious DiseasesPublisher Rights Statement:All materials published in Emerging Infectious Diseases are in the public domain and can be used withoutpermissionGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 28. Nov. 2025Mamoona Chaudhry, Angélique Angot,  Hamad B. Rashid, Giovanni Cattoli,  Manzoor Hussain, Giulia Trovò,  Alessandra Drago, Viviana Valastro,  Michael Thrusfield, Sue Welburn,  Mark C. Eisler, Ilaria CapuaPhylogenetic	 analysis	 of	 influenza	 viruses	 collected	 dur-ing	December	 2009–February	 2010	 from	 chickens	 in	 live	poultry	retail	shops	in	Lahore,	Pakistan,	showed	influenza	A(H9N2)	lineage	polymerase	and	nonstructural	genes	gen-erate	through	inter-	and	intrasubtypic	reassortments.	Many	amino	acid	signatures	observed	were	characteristic	of	hu-man	isolates;	hence,	their	circulation	could	enhance	inter-	or	intrasubtypic	reassortment.The first outbreak of illness caused by avian influenza A(H9N2) virus in Pakistan was reported in 1998; iso-lates showed a close relationship to subtype H9N2 avian influenza viruses (AIVs) circulating in Hong Kong, China during 1997 that were grouped within the G1 lineage (1). In recent years, H9N2 genes have reassorted extensively, generating novel genotypes on the Indian subcontinent. Widespread co-circulation of H9N2 with other AIVs (e.g., highly pathogenic AIVs H5N1 and H7N3) could instigate the generation of novel variant and reassorted viruses, pos-sibly with increased zoonotic potential (2).No information was available about the genetic make-up of AIVs circulating in live poultry retail shops (LPRSs) in Pakistan. This study was conducted to genetically char-acterize AIVs in LPRSs in Lahore District, Pakistan.The StudyWe conducted a cross-sectional survey of LPRSs in La-hore (Figure), which is the capital of the Punjab Province in Pakistan. In each of 280 LPRSs, we collected tracheal swab samples from 5 randomly selected chickens and pooled them into 1 composite sample, totaling 280 pooled samples. The samples were characterized at the World Or-ganisation for Animal Health/Food and Agriculture Orga-nization of the United Nations National Reference Labora-tory for Avian Influenza in Padua, Italy.Of the 280 samples, 10 tested positive for H9N2 sub-type by real-time reverse transcription PCR (rRT-PCR) (3) and virus isolation test (United Nations www.oie.int/filead-min/Home/eng/Health_standards/tahm/2.03.04_AI.pdf) (Table 1). Results were negative for H5 and H7 subtypes. Two of the 280 samples were positive for Newcastle dis-ease virus. Each of the 10 H9N2 isolates was character-ized by sequencing for 6 gene segments (hemagglutinin [HA]: neuraminidase [NA], nonstructural [NS], matrix [M], and polymerase basic 1 and 2 [PB1, PB2]); 2 isolates (A/chicken/Pakistan/10RS3039-283-11/2010 and A/chicken/Pakistan/10RS3039-289-186/2010) were sequenced for all 8 gene segments (HA, NA, NS, M, PB1, PB2, polymerase [PA], and nucleoprotein [NP]), as described (4). Sequences for these viruses were deposited into GenBank (accession nos. KF975457–KF975503, and KP223678–KP223693). The NS gene sequence of A/chicken/Pakistan/ 10RS3039-284-11/2010 could not be deposited due to poor data quality.We generated neighbor-joining phylogenetic trees for all gene segments using the distance-based method in MEGA version 5.2.2 (http://www.megasoftware.net/). We calculated bootstrap values based on 1,000 replicates of alignment (5). HA and NA genes of virus-es in this study tightly clustered within the G1 lineage along with H9N2 viruses from Pakistan, India, Iran, Is-rael, Saudi Arabia, and Bangladesh, suggesting deriva-tion from a common ancestor: A/quail/Hong Kong/G1/97 (online Technical Appendix Figure panels A, B http://wwwnc.cdc.gov/EID/article/21/4/14-1570-Techapp1.pdf). M and NP genes clustered within the G1 lineage (online Technical Appendix Figure panels C, D) along with an influenza (H7N3) isolate from Pakistan (A/chick-en/Karachi/NARC-100/2004; nucleotide identity >96% for M gene and 95% for NP gene). Two polymerase complex genes, PB1 and PA, and the NS gene did not cluster within G1 lineage or any other established Eur-asian lineages: these gene sequences made a separate well-supported cluster with highly similar H9N2 virus-es that circulated in Pakistan (2005–2008), Iran, India, and Bangladesh; these sequences had a high sequence Reassortant Avian Influenza A(H9N2)  Viruses in Chickens in Retail Poultry Shops,  Pakistan, 2009–2010	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	21,	No.	4,	April	2015	 673Author	affiliations:	University	of	Veterinary	and	Animal	Sciences,	Lahore,	Pakistan	(M.	Chaudhry,	H.B.	Rashid);	University	of	Edinburgh,	Edinburgh,	Scotland,	UK	(M.	Chaudhry,	M.	Thrusfield,	S.	Welburn);	Istituto	Zooprofilattico	Sperimentale	delle	Venezie,	Viale	dell’Università,	Legnaro,	Padua,	Italy	(A.	Angot,	G.	Cattoli,	G.	Trovò,	A.	Drago,	V.	Valastro,	I.	Capua);	Food	and	Agriculture	Organization	of	the	United	Nations,	Islamabad,	Pakistan	 (M.	Hussain);	University	of	Bristol,	Bristol,	UK	(M.C.	Eisler)DOI:	http://dx.doi.org/10.3201/eid2104.141570DISPATCHESidentity (>95%) with A/chicken/Karachi/NARC-100/04 (H7N3) (2,6,7) (online Technical Appendix Figure pan-els E, F, G). The PB2 segment clustered separately with H9N2 viruses from the subcontinent and Middle East (online Technical Appendix Figure panel H), except A/chicken/Karachi/NARC-100/04. These results indicate a separate Indian subcontinental lineage of H9N2 viruses has emerged (2).On the basis of these analyses, we could conclude that internal genes PB1, PA, and NS of these viruses originated by intersubtypic (between different HA subtypes) reassort-ment events from local H7N3 viruses circulating in Pakistan (nucleotide identity >95%). This suggests that intersubtyp-ic reassortment events continuously result from mixing of AIV subtypes in domestic poultry and wild birds in Paki-stan (8), and that PB2, M, and NP genes were acquired by intrasubtypic reassortment between H9N2 viruses of G1 lineage circulating within Pakistan. These results support speculation that the currently circulating H9N2 lineage is a reassortment of G1 lineage from Hong Kong and the highly pathogenic H7N3 virus that circulated in Pakistan and can be assigned to genetic group B (8).We aligned amino acid sequences of current virus-es and compared them to representative H9N2 lineages. When compared to the prototype G1 viruses, H9N2 vi-ruses isolated from LPRSs showed that they have evolved to acquire mammalian host-specific mutations through-out the genome (online Technical Appendix Table 1). Of these mutations, certain amino acid substitutions through-out the viral genome have become fixed (9). All LPRS isolates possessed the K-S-S-R motif at the cleavage site. The presence of lysine at position 4 was observed in H9 isolates from the Indian subcontinent (2,9), but it is un-common elsewhere. Amino acid signature changes of hu-man influenza viruses were also observed in internal gene segments (Table 2).674	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	21,	No.	4,	April	2015Figure.	Location	of	live	poultry	retail	shops	(X)	in	5	towns	in	Lahore,	Pakistan,	where	avian	influenza	A(H9N2)	virus	isolates	were	identified	in	chickens,	2009–2010.	Inset	shows	location	of	Lahore	in	Punjab	Province. Table 1.	Influenza	A(H9N2)	viruses	isolated	from	oropharyngeal	swab	samples	from	chickens	in	live	poultry	retail	shops	of	Lahore,	Pakistan,	2010 Isolates Collection	date Towns	of	Lahore	District* A/chicken/Pakistan/10RS3039-283-11/2010 Jan	23 Gulberg	Town A/chicken/Pakistan/10RS3039-284-48/2010 Jan	25 Data	Gunj	Bakhsh	Town A/chicken/Pakistan/10RS3039-285-63/2010 Jan	26 Data	Gunj	Bakhsh	Town A/chicken/Pakistan/10RS3039-286-65/2010 Jan	26 Data	Gunj	Bakhsh	Town A/chicken/Pakistan/10RS3039-287-98/2010 Jan	27 Ravi	Town A/chicken/Pakistan/10RS3039-288-102/2010 Jan	27 Ravi	Town A/chicken/Pakistan/10RS3039-289-186/2010 Feb	11 Samanabad	Town A/chicken/Pakistan/10RS3039-199-199/2010 Feb	11 Samanabad	Town A/chicken/Pakistan/10RS3039-290-230/2010 Feb	13 Allama	Iqbal	Town A/chicken/Pakistan/10RS3039-291-266/2010 Feb	14 Allama	Iqbal	Town *Lahore	District	contains 9	towns and	1	cantonment	area.    	Reassortant	Influenza	A(H9N2)	VirusesEach of the 10 isolates had the Q226L substitution (H3 numbering) in the receptor-binding site of HA, correlating to a shift in affinity from avian-type sialic acid receptor to human-type (10). We identified 3 representative substitu-tions: E/T190 A in 9 viruses and E/T190V in 1 virus, and Q227I in all 10 viruses (H3 numbering). The outcomes of these substitutions have not been investigated; further study is needed (2). Glycosylation sites at positions 551, 218, and 206 were absent in the study viruses, suggesting a frequent alteration in sequences from this region (2,6,9) and possibly signifying the selected adaptation of H9N2 to poultry (10). We did not find an R292K substitution, which is associated with resistance to the sialidase inhibitors oseltamivir and zanamivir, in the NA proteins of any of the 10 LPRS virus isolates. HB sites were also well conserved with few substi-tutions (K367E in 5 viruses, K367G in 1 virus, and S372A and W403R in all 10 viruses). An additional glycosylation site was present at position 44, which is believed to enhance virulence caused by altered antigenicity or sialidase activ-ity (11) (online Technical Appendix Table 1).Many residues in nucleoprotein and polymerase are con-sidered determinants of the host range of AIVs and increase virulence or replication in the mammalian host (2,10,12). The analyses of internal genes showed that these viruses also contained mammalian host-specific markers (2,8) that have become permanent in M protein (M1, V15I, T37A; M2, E16G, L55F); in PB1 protein (L13P); and in NS1 protein (V149A) of all LPRS isolates and in NP (E372D) of the 2 isolates we se-quenced (online Technical Appendix Table 2). In M2 pro-tein, no substitutions linked to resistant amantadine were seen. All viruses contained an uncommon K-S-E-I sequence as a PDZ ligand motif in the NS protein. Residue isoleucine at the C-terminus of PDZ ligand motif has been reported as a rare substitution (12). NS also harbored E227K mutation in the C-terminal, which has been demonstrated to modulate pathogenicity of AIVs (13) and appeared to be a rare amino acid signature, although in this study it was observed in all 10 virus isolates from chickens in LPRSs.ConclusionsOur analysis confirmed that continuous gene reassortment has occurred among influenza A(H9N2) viruses since their emergence in poultry in Pakistan. Because H9N2 viruses infect multiple species, they may donate genes to emerging human pathogens; it has been observed that H7N9 acquired internal genes from the avian H9N2 virus (14). In wet mar-kets, availability of freshly slaughtered poultry, live poul-try transportation, and mixed trading of domestic animals provide a favorable environment for gene reassortment, mutation, and interspecies transmission of AIVs (15). Con-tinuous circulation of these viruses in LPRSs increases the chances of their evolution into new genotypes. Close con-tact of humans and poultry in LPRSs with no biosecurity barriers increases the risk for emergence of novel influenza viruses with zoonotic or human pandemic potential. Con-tinued surveillance in LPRSs is essential to better under-stand the public health risk posed by H9N2 AIVs.AcknowledgmentsWe thank the staff members of the live poultry retail  shops in which the study subjects were housed for data and sample collection. The European Union project Training and technology transfer of avian influenza diagnostics and disease management skills (FLUTRAIN) provided financial support for training of MC at IZSVe, Italy. The Higher Education Commission of Pakistan provided a PhD scholarship to MC through the Overseas Schol-arship Scheme (10%- Batch-2).We acknowledge the memory of Isabel Minguez Tudela  (1955–2011), of the European Commission, who supported  this collaboration as a contribution to improving veterinary public health.Dr. Chaudhry is an academician in the Department of Epidemi-ology and Public Health, University of Veterinary and Animal Sciences, Lahore. Her research interests include epidemiology of infectious diseases of public health concern.References  1. Cameron KR, Gregory V, Banks J, Brown IH, Alexander DJ,  Hay AJ, et al. H9N2 subtype influenza A viruses in poultry in  Pakistan are closely related to the H9N2 viruses responsible for human infection in Hong Kong. Virology. 2000;278:36–41.  http://dx.doi.org/10.1006/viro.2000.0585	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	21,	No.	4,	April	2015	 675 Table 2.	Amino	acid	signature	changes	observed	in	M1,	NP,	PB1	and	NS1	proteins	of	influenza	A(H9N2) viruses	isolated	from	chickens	in	live	poultry retail	shops, Lahore,	Pakistan,	2009–2010* Protein Amino	acid position Predicted	aa Viruses	with	detected	mutation Mutation Avian Human M1 15 V I All	10	isolates	analyzed	in	this	study V15I NP 372 E D Only	2	viruses	were	analyzed (A/chicken/	Pakistan/10RS3039-283-11/2010 A/chicken/	Pakistan/10RS3039-289-186/2010) E372D PB1 13 L P All	10	isolates	analyzed	in	this	study L13P NS1 149 V A All	10	isolates	analyzed	in	this	study V149A 227 E R	or	K	(H1N1	1918) E227K *M,	matrix;	NP,	nucleoprotein;	PB,	basic	polymerase;	NS,	nonstructured.  DISPATCHES  2. Iqbal M, Yaqub T, Reddy K, McCauley JW. Novel genotypes of H9N2 influenza A viruses isolated from poultry in Pakistan containing NS genes similar to highly pathogenic H7N3 and H5N1 viruses. PLoS ONE. 2009;4:e5788. http://dx.doi.org/10.1371/ journal.pone.0005788  3. Monne I, Ormelli S, Salviato A, De Battisti C, Bettini F,  Salomoni A, et al. Development and validation of a one-step  real-time PCR assay for simultaneous detection of subtype H5,  H7, and H9 avian influenza viruses. J Clin Microbiol. 2008; 46:1769–73. http://dx.doi.org/10.1128/JCM.02204-07  4. Cattoli G, Monne I, Fusaro A, Joannis TM, Lombin LH, Aly MM, et al. Highly pathogenic avian influenza virus subtype H5N1 in Africa: a comprehensive phylogenetic analysis and molecular characterization of isolates. PLoS ONE. 2009;4:e4842  http://dx.doi.org/10.1371/journal.pone.0004842.  5. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular  Evolutionary Genetics Analysis (MEGA) software version 4.0.  Mol Biol Evol. 2007;24:1596–9. http://dx.doi.org/10.1093/ molbev/msm092  6. Aamir UB, Wernery U, Ilyushina N, Webster RG. Characterization of avian H9N2 influenza viruses from United Arab Emirates 2000 to 2003. Virology. 2007;361:45–55. http://dx.doi.org/10.1016/ j.virol.2006.10.037  7. Abbas MA, Spackman E, Swayne DE, Ahmed Z, Sarmento L,  Siddique N, et al. Sequence and phylogenetic analysis of H7N3 avian influenza viruses isolated from poultry in Pakistan 1995–2004. Virol J. 2010;7:137. http://dx.doi.org/10.1186/ 1743-422X-7-137  8. Fusaro A, Monne I, Salviato A, Valastro V, Schivo A,  Amarin NM, et al. Phylogeography and evolutionary history  of reassortant H9N2 viruses with potential human health  implications. J Virol. 2011;85:8413–21. http://dx.doi.org/ 10.1128/JVI.00219-11  9. Shanmuganatham K, Feeroz MM, Jones-Engel L, Smith GJ,  Fourment M, Walker D, et al. Antigenic and molecular char-acterization of avian influenza A(H9N2) viruses, Bangladesh. Emerg Infect Dis. 2013;19:1393–1402. http://dx.doi.org/10.3201/eid1909.130336 10. Baigent SJ, McCauley JW. Influenza type A in humans, mammals and birds: determinants of virus virulence, host-range and  interspecies transmission. Bioessays. 2003;25:657–71. http://dx.doi.org/10.1002/bies.1030311. Hulse DJ, Webster RG, Russell RJ, Perez DR. Molecular deter-minants within the surface proteins involved in the pathogenicity of H5N1 influenza viruses in chickens. J Virol. 2004;78:9954–64. http://dx.doi.org/10.1128/JVI.78.18.9954-9964.200412. Finkelstein DB, Mukatira S, Mehta PK, Obenauer JC, Su X,  Webster RG, et al. Persistent host markers in pandemic and H5N1 influenza viruses. J Virol. 2007;81:10292–9.  http://dx.doi.org/10.1128/JVI.00921-0713. Obenauer JC, Denson J, Mehta PK, Su X, Mukatira S,  Finkelstein DB, et al. Large-scale sequence analysis of avian influenza isolates. Science. 2006;311:1576–80. http://dx.doi.org/10.1126/science.112158614. Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, et al. Human infection with a novel avian-origin influenza A (H7N9) virus. N Engl J Med. 2013;368:1888–97. http://dx.doi.org/10.1056/NEJMoa130445915. Bao CJ, Cui LB, Zhou MH, Hong L, Gao GF, Wang H. Live- animal markets and influenza A (H7N9) virus infection. N Engl  J Med. 2013;368:2337–9. http://dx.doi.org/10.1056/NEJMc1306100Address for correspondence: Mamoona Chaudhry, Department of Epidemiology and Public Health, University of Veterinary and  Animal Sciences, Abdul Qadir Jilani Road, Lahore, Pakistan;  email: mamoona.chaudhry@gmail.com676	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	21,	No.	4,	April	2015February 2014:  High-Consequence PathogensIncluding:•  Poxvirus	Viability	and	Signatures	 in	Historical	Relics•  Novel	Paramyxovirus	Associated	with	 Severe	Acute	Febrile	Disease,	 South	Sudan	and	Uganda,	2012•  Subtyping	Cryptosporidium ubiquitum,	a	Zoonotic	Pathogen	Emerging	in	Humans •  Genomic	Variability	of	Monkeypox	Virus	among	Humans,	Democratic	Republic	 of	the	Congohttp://wwwnc.cdc.gov/eid/content/20/2/contents.htm

English

Reassortant Avian Influenza A(H9N2) viruses in chickens in retail poultry shops, Pakistan, 2009-2010

     Edinburgh Research Explorer                                      Reassortant Avian Influenza A(H9N2) viruses in chickens in retailpoultry shops, Pakistan, 2009-2010Citation for published version:Chaudhry, M, Angot, A, Rashid, HB, Cattoli, G, Hussain, M, Trovò, G, Drago, A, Valastro, V, Thrusfield, M,Welburn, S, Eisler, MC & Capua, I 2015, 'Reassortant Avian Influenza A(H9N2) viruses in chickens in retailpoultry shops, Pakistan, 2009-2010' Emerging Infectious Diseases, vol. 21, no. 4, pp. 673-6. DOI:10.3201/eid2104.141570Digital Object Identifier (DOI):10.3201/eid2104.141570Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Publisher's PDF, also known as Version of recordPublished In:Emerging Infectious DiseasesPublisher Rights Statement:All materials published in Emerging Infectious Diseases are in the public domain and can be used withoutpermissionGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 05. Apr. 2019Mamoona Chaudhry, Angélique Angot,  Hamad B. Rashid, Giovanni Cattoli,  Manzoor Hussain, Giulia Trovò,  Alessandra Drago, Viviana Valastro,  Michael Thrusfield, Sue Welburn,  Mark C. Eisler, Ilaria CapuaPhylogenetic	 analysis	 of	 influenza	 viruses	 collected	 dur-ing	December	 2009–February	 2010	 from	 chickens	 in	 live	poultry	retail	shops	in	Lahore,	Pakistan,	showed	influenza	A(H9N2)	lineage	polymerase	and	nonstructural	genes	gen-erate	through	inter-	and	intrasubtypic	reassortments.	Many	amino	acid	signatures	observed	were	characteristic	of	hu-man	isolates;	hence,	their	circulation	could	enhance	inter-	or	intrasubtypic	reassortment.The first outbreak of illness caused by avian influenza A(H9N2) virus in Pakistan was reported in 1998; iso-lates showed a close relationship to subtype H9N2 avian influenza viruses (AIVs) circulating in Hong Kong, China during 1997 that were grouped within the G1 lineage (1). In recent years, H9N2 genes have reassorted extensively, generating novel genotypes on the Indian subcontinent. Widespread co-circulation of H9N2 with other AIVs (e.g., highly pathogenic AIVs H5N1 and H7N3) could instigate the generation of novel variant and reassorted viruses, pos-sibly with increased zoonotic potential (2).No information was available about the genetic make-up of AIVs circulating in live poultry retail shops (LPRSs) in Pakistan. This study was conducted to genetically char-acterize AIVs in LPRSs in Lahore District, Pakistan.The StudyWe conducted a cross-sectional survey of LPRSs in La-hore (Figure), which is the capital of the Punjab Province in Pakistan. In each of 280 LPRSs, we collected tracheal swab samples from 5 randomly selected chickens and pooled them into 1 composite sample, totaling 280 pooled samples. The samples were characterized at the World Or-ganisation for Animal Health/Food and Agriculture Orga-nization of the United Nations National Reference Labora-tory for Avian Influenza in Padua, Italy.Of the 280 samples, 10 tested positive for H9N2 sub-type by real-time reverse transcription PCR (rRT-PCR) (3) and virus isolation test (United Nations www.oie.int/filead-min/Home/eng/Health_standards/tahm/2.03.04_AI.pdf) (Table 1). Results were negative for H5 and H7 subtypes. Two of the 280 samples were positive for Newcastle dis-ease virus. Each of the 10 H9N2 isolates was character-ized by sequencing for 6 gene segments (hemagglutinin [HA]: neuraminidase [NA], nonstructural [NS], matrix [M], and polymerase basic 1 and 2 [PB1, PB2]); 2 isolates (A/chicken/Pakistan/10RS3039-283-11/2010 and A/chicken/Pakistan/10RS3039-289-186/2010) were sequenced for all 8 gene segments (HA, NA, NS, M, PB1, PB2, polymerase [PA], and nucleoprotein [NP]), as described (4). Sequences for these viruses were deposited into GenBank (accession nos. KF975457–KF975503, and KP223678–KP223693). The NS gene sequence of A/chicken/Pakistan/ 10RS3039-284-11/2010 could not be deposited due to poor data quality.We generated neighbor-joining phylogenetic trees for all gene segments using the distance-based method in MEGA version 5.2.2 (http://www.megasoftware.net/). We calculated bootstrap values based on 1,000 replicates of alignment (5). HA and NA genes of virus-es in this study tightly clustered within the G1 lineage along with H9N2 viruses from Pakistan, India, Iran, Is-rael, Saudi Arabia, and Bangladesh, suggesting deriva-tion from a common ancestor: A/quail/Hong Kong/G1/97 (online Technical Appendix Figure panels A, B http://wwwnc.cdc.gov/EID/article/21/4/14-1570-Techapp1.pdf). M and NP genes clustered within the G1 lineage (online Technical Appendix Figure panels C, D) along with an influenza (H7N3) isolate from Pakistan (A/chick-en/Karachi/NARC-100/2004; nucleotide identity >96% for M gene and 95% for NP gene). Two polymerase complex genes, PB1 and PA, and the NS gene did not cluster within G1 lineage or any other established Eur-asian lineages: these gene sequences made a separate well-supported cluster with highly similar H9N2 virus-es that circulated in Pakistan (2005–2008), Iran, India, and Bangladesh; these sequences had a high sequence Reassortant Avian Influenza A(H9N2)  Viruses in Chickens in Retail Poultry Shops,  Pakistan, 2009–2010	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	21,	No.	4,	April	2015	 673Author	affiliations:	University	of	Veterinary	and	Animal	Sciences,	Lahore,	Pakistan	(M.	Chaudhry,	H.B.	Rashid);	University	of	Edinburgh,	Edinburgh,	Scotland,	UK	(M.	Chaudhry,	M.	Thrusfield,	S.	Welburn);	Istituto	Zooprofilattico	Sperimentale	delle	Venezie,	Viale	dell’Università,	Legnaro,	Padua,	Italy	(A.	Angot,	G.	Cattoli,	G.	Trovò,	A.	Drago,	V.	Valastro,	I.	Capua);	Food	and	Agriculture	Organization	of	the	United	Nations,	Islamabad,	Pakistan	 (M.	Hussain);	University	of	Bristol,	Bristol,	UK	(M.C.	Eisler)DOI:	http://dx.doi.org/10.3201/eid2104.141570DISPATCHESidentity (>95%) with A/chicken/Karachi/NARC-100/04 (H7N3) (2,6,7) (online Technical Appendix Figure pan-els E, F, G). The PB2 segment clustered separately with H9N2 viruses from the subcontinent and Middle East (online Technical Appendix Figure panel H), except A/chicken/Karachi/NARC-100/04. These results indicate a separate Indian subcontinental lineage of H9N2 viruses has emerged (2).On the basis of these analyses, we could conclude that internal genes PB1, PA, and NS of these viruses originated by intersubtypic (between different HA subtypes) reassort-ment events from local H7N3 viruses circulating in Pakistan (nucleotide identity >95%). This suggests that intersubtyp-ic reassortment events continuously result from mixing of AIV subtypes in domestic poultry and wild birds in Paki-stan (8), and that PB2, M, and NP genes were acquired by intrasubtypic reassortment between H9N2 viruses of G1 lineage circulating within Pakistan. These results support speculation that the currently circulating H9N2 lineage is a reassortment of G1 lineage from Hong Kong and the highly pathogenic H7N3 virus that circulated in Pakistan and can be assigned to genetic group B (8).We aligned amino acid sequences of current virus-es and compared them to representative H9N2 lineages. When compared to the prototype G1 viruses, H9N2 vi-ruses isolated from LPRSs showed that they have evolved to acquire mammalian host-specific mutations through-out the genome (online Technical Appendix Table 1). Of these mutations, certain amino acid substitutions through-out the viral genome have become fixed (9). All LPRS isolates possessed the K-S-S-R motif at the cleavage site. The presence of lysine at position 4 was observed in H9 isolates from the Indian subcontinent (2,9), but it is un-common elsewhere. Amino acid signature changes of hu-man influenza viruses were also observed in internal gene segments (Table 2).674	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	21,	No.	4,	April	2015Figure.	Location	of	live	poultry	retail	shops	(X)	in	5	towns	in	Lahore,	Pakistan,	where	avian	influenza	A(H9N2)	virus	isolates	were	identified	in	chickens,	2009–2010.	Inset	shows	location	of	Lahore	in	Punjab	Province. Table 1.	Influenza	A(H9N2)	viruses	isolated	from	oropharyngeal	swab	samples	from	chickens	in	live	poultry	retail	shops	of	Lahore,	Pakistan,	2010 Isolates Collection	date Towns	of	Lahore	District* A/chicken/Pakistan/10RS3039-283-11/2010 Jan	23 Gulberg	Town A/chicken/Pakistan/10RS3039-284-48/2010 Jan	25 Data	Gunj	Bakhsh	Town A/chicken/Pakistan/10RS3039-285-63/2010 Jan	26 Data	Gunj	Bakhsh	Town A/chicken/Pakistan/10RS3039-286-65/2010 Jan	26 Data	Gunj	Bakhsh	Town A/chicken/Pakistan/10RS3039-287-98/2010 Jan	27 Ravi	Town A/chicken/Pakistan/10RS3039-288-102/2010 Jan	27 Ravi	Town A/chicken/Pakistan/10RS3039-289-186/2010 Feb	11 Samanabad	Town A/chicken/Pakistan/10RS3039-199-199/2010 Feb	11 Samanabad	Town A/chicken/Pakistan/10RS3039-290-230/2010 Feb	13 Allama	Iqbal	Town A/chicken/Pakistan/10RS3039-291-266/2010 Feb	14 Allama	Iqbal	Town *Lahore	District	contains 9	towns and	1	cantonment	area.    	Reassortant	Influenza	A(H9N2)	VirusesEach of the 10 isolates had the Q226L substitution (H3 numbering) in the receptor-binding site of HA, correlating to a shift in affinity from avian-type sialic acid receptor to human-type (10). We identified 3 representative substitu-tions: E/T190 A in 9 viruses and E/T190V in 1 virus, and Q227I in all 10 viruses (H3 numbering). The outcomes of these substitutions have not been investigated; further study is needed (2). Glycosylation sites at positions 551, 218, and 206 were absent in the study viruses, suggesting a frequent alteration in sequences from this region (2,6,9) and possibly signifying the selected adaptation of H9N2 to poultry (10). We did not find an R292K substitution, which is associated with resistance to the sialidase inhibitors oseltamivir and zanamivir, in the NA proteins of any of the 10 LPRS virus isolates. HB sites were also well conserved with few substi-tutions (K367E in 5 viruses, K367G in 1 virus, and S372A and W403R in all 10 viruses). An additional glycosylation site was present at position 44, which is believed to enhance virulence caused by altered antigenicity or sialidase activ-ity (11) (online Technical Appendix Table 1).Many residues in nucleoprotein and polymerase are con-sidered determinants of the host range of AIVs and increase virulence or replication in the mammalian host (2,10,12). The analyses of internal genes showed that these viruses also contained mammalian host-specific markers (2,8) that have become permanent in M protein (M1, V15I, T37A; M2, E16G, L55F); in PB1 protein (L13P); and in NS1 protein (V149A) of all LPRS isolates and in NP (E372D) of the 2 isolates we se-quenced (online Technical Appendix Table 2). In M2 pro-tein, no substitutions linked to resistant amantadine were seen. All viruses contained an uncommon K-S-E-I sequence as a PDZ ligand motif in the NS protein. Residue isoleucine at the C-terminus of PDZ ligand motif has been reported as a rare substitution (12). NS also harbored E227K mutation in the C-terminal, which has been demonstrated to modulate pathogenicity of AIVs (13) and appeared to be a rare amino acid signature, although in this study it was observed in all 10 virus isolates from chickens in LPRSs.ConclusionsOur analysis confirmed that continuous gene reassortment has occurred among influenza A(H9N2) viruses since their emergence in poultry in Pakistan. Because H9N2 viruses infect multiple species, they may donate genes to emerging human pathogens; it has been observed that H7N9 acquired internal genes from the avian H9N2 virus (14). In wet mar-kets, availability of freshly slaughtered poultry, live poul-try transportation, and mixed trading of domestic animals provide a favorable environment for gene reassortment, mutation, and interspecies transmission of AIVs (15). Con-tinuous circulation of these viruses in LPRSs increases the chances of their evolution into new genotypes. Close con-tact of humans and poultry in LPRSs with no biosecurity barriers increases the risk for emergence of novel influenza viruses with zoonotic or human pandemic potential. Con-tinued surveillance in LPRSs is essential to better under-stand the public health risk posed by H9N2 AIVs.AcknowledgmentsWe thank the staff members of the live poultry retail  shops in which the study subjects were housed for data and sample collection. The European Union project Training and technology transfer of avian influenza diagnostics and disease management skills (FLUTRAIN) provided financial support for training of MC at IZSVe, Italy. The Higher Education Commission of Pakistan provided a PhD scholarship to MC through the Overseas Schol-arship Scheme (10%- Batch-2).We acknowledge the memory of Isabel Minguez Tudela  (1955–2011), of the European Commission, who supported  this collaboration as a contribution to improving veterinary public health.Dr. Chaudhry is an academician in the Department of Epidemi-ology and Public Health, University of Veterinary and Animal Sciences, Lahore. Her research interests include epidemiology of infectious diseases of public health concern.References  1. Cameron KR, Gregory V, Banks J, Brown IH, Alexander DJ,  Hay AJ, et al. H9N2 subtype influenza A viruses in poultry in  Pakistan are closely related to the H9N2 viruses responsible for human infection in Hong Kong. Virology. 2000;278:36–41.  http://dx.doi.org/10.1006/viro.2000.0585	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	21,	No.	4,	April	2015	 675 Table 2.	Amino	acid	signature	changes	observed	in	M1,	NP,	PB1	and	NS1	proteins	of	influenza	A(H9N2) viruses	isolated	from	chickens	in	live	poultry retail	shops, Lahore,	Pakistan,	2009–2010* Protein Amino	acid position Predicted	aa Viruses	with	detected	mutation Mutation Avian Human M1 15 V I All	10	isolates	analyzed	in	this	study V15I NP 372 E D Only	2	viruses	were	analyzed (A/chicken/	Pakistan/10RS3039-283-11/2010 A/chicken/	Pakistan/10RS3039-289-186/2010) E372D PB1 13 L P All	10	isolates	analyzed	in	this	study L13P NS1 149 V A All	10	isolates	analyzed	in	this	study V149A 227 E R	or	K	(H1N1	1918) E227K *M,	matrix;	NP,	nucleoprotein;	PB,	basic	polymerase;	NS,	nonstructured.  DISPATCHES  2. Iqbal M, Yaqub T, Reddy K, McCauley JW. Novel genotypes of H9N2 influenza A viruses isolated from poultry in Pakistan containing NS genes similar to highly pathogenic H7N3 and H5N1 viruses. PLoS ONE. 2009;4:e5788. http://dx.doi.org/10.1371/ journal.pone.0005788  3. Monne I, Ormelli S, Salviato A, De Battisti C, Bettini F,  Salomoni A, et al. Development and validation of a one-step  real-time PCR assay for simultaneous detection of subtype H5,  H7, and H9 avian influenza viruses. J Clin Microbiol. 2008; 46:1769–73. http://dx.doi.org/10.1128/JCM.02204-07  4. Cattoli G, Monne I, Fusaro A, Joannis TM, Lombin LH, Aly MM, et al. Highly pathogenic avian influenza virus subtype H5N1 in Africa: a comprehensive phylogenetic analysis and molecular characterization of isolates. PLoS ONE. 2009;4:e4842  http://dx.doi.org/10.1371/journal.pone.0004842.  5. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular  Evolutionary Genetics Analysis (MEGA) software version 4.0.  Mol Biol Evol. 2007;24:1596–9. http://dx.doi.org/10.1093/ molbev/msm092  6. Aamir UB, Wernery U, Ilyushina N, Webster RG. Characterization of avian H9N2 influenza viruses from United Arab Emirates 2000 to 2003. Virology. 2007;361:45–55. http://dx.doi.org/10.1016/ j.virol.2006.10.037  7. Abbas MA, Spackman E, Swayne DE, Ahmed Z, Sarmento L,  Siddique N, et al. Sequence and phylogenetic analysis of H7N3 avian influenza viruses isolated from poultry in Pakistan 1995–2004. Virol J. 2010;7:137. http://dx.doi.org/10.1186/ 1743-422X-7-137  8. Fusaro A, Monne I, Salviato A, Valastro V, Schivo A,  Amarin NM, et al. Phylogeography and evolutionary history  of reassortant H9N2 viruses with potential human health  implications. J Virol. 2011;85:8413–21. http://dx.doi.org/ 10.1128/JVI.00219-11  9. Shanmuganatham K, Feeroz MM, Jones-Engel L, Smith GJ,  Fourment M, Walker D, et al. Antigenic and molecular char-acterization of avian influenza A(H9N2) viruses, Bangladesh. Emerg Infect Dis. 2013;19:1393–1402. http://dx.doi.org/10.3201/eid1909.130336 10. Baigent SJ, McCauley JW. Influenza type A in humans, mammals and birds: determinants of virus virulence, host-range and  interspecies transmission. Bioessays. 2003;25:657–71. http://dx.doi.org/10.1002/bies.1030311. Hulse DJ, Webster RG, Russell RJ, Perez DR. Molecular deter-minants within the surface proteins involved in the pathogenicity of H5N1 influenza viruses in chickens. J Virol. 2004;78:9954–64. http://dx.doi.org/10.1128/JVI.78.18.9954-9964.200412. Finkelstein DB, Mukatira S, Mehta PK, Obenauer JC, Su X,  Webster RG, et al. Persistent host markers in pandemic and H5N1 influenza viruses. J Virol. 2007;81:10292–9.  http://dx.doi.org/10.1128/JVI.00921-0713. Obenauer JC, Denson J, Mehta PK, Su X, Mukatira S,  Finkelstein DB, et al. Large-scale sequence analysis of avian influenza isolates. Science. 2006;311:1576–80. http://dx.doi.org/10.1126/science.112158614. Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, et al. Human infection with a novel avian-origin influenza A (H7N9) virus. N Engl J Med. 2013;368:1888–97. http://dx.doi.org/10.1056/NEJMoa130445915. Bao CJ, Cui LB, Zhou MH, Hong L, Gao GF, Wang H. Live- animal markets and influenza A (H7N9) virus infection. N Engl  J Med. 2013;368:2337–9. http://dx.doi.org/10.1056/NEJMc1306100Address for correspondence: Mamoona Chaudhry, Department of Epidemiology and Public Health, University of Veterinary and  Animal Sciences, Abdul Qadir Jilani Road, Lahore, Pakistan;  email: mamoona.chaudhry@gmail.com676	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	21,	No.	4,	April	2015February 2014:  High-Consequence PathogensIncluding:•  Poxvirus	Viability	and	Signatures	 in	Historical	Relics•  Novel	Paramyxovirus	Associated	with	 Severe	Acute	Febrile	Disease,	 South	Sudan	and	Uganda,	2012•  Subtyping	Cryptosporidium ubiquitum,	a	Zoonotic	Pathogen	Emerging	in	Humans •  Genomic	Variability	of	Monkeypox	Virus	among	Humans,	Democratic	Republic	 of	the	Congohttp://wwwnc.cdc.gov/eid/content/20/2/contents.htm

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Reassortant Avian Influenza A(H9N2) Viruses in Chickens in Retail Poultry Shops, Pakistan, 2009–2010

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Phylogenetic analysis of influenza viruses collected during December 2009–February 2010 from chickens in live poultry retail shops in Lahore, Pakistan, showed influenza A(H9N2) lineage polymerase and nonstructural genes generate through inter- and intrasubtypic reassortments. Many amino acid signatures observed were characteristic of human isolates; hence, their circulation could enhance inter- or intrasubtypic reassortment.<br/

Rashid, Hamad B.

Welburn, Sue

Explore Bristol Research

                          Chaudhry, M., Angot, A., Rashid, H. B., Cattoli, G., Hussain, M., Trovò, G.,... Capua, I. (2015). Reassortant Avian Influenza A(H9N2) Viruses inChickens in Retail Poultry Shops, Pakistan, 2009–2010. Emerging InfectiousDiseases, 21(4), 673. [141570]. 10.3201/eid2104.141570Publisher's PDF, also known as Final Published VersionLink to published version (if available):10.3201/eid2104.141570Link to publication record in Explore Bristol ResearchPDF-documentUniversity of Bristol - Explore Bristol ResearchGeneral rightsThis document is made available in accordance with publisher policies. Please cite only the publishedversion using the reference above. Full terms of use are available:http://www.bristol.ac.uk/pure/about/ebr-terms.htmlTake down policyExplore Bristol Research is a digital archive and the intention is that deposited content should not beremoved. However, if you believe that this version of the work breaches copyright law please contactopen-access@bristol.ac.uk and include the following information in your message:• Your contact details• Bibliographic details for the item, including a URL• An outline of the nature of the complaintOn receipt of your message the Open Access Team will immediately investigate your claim, make aninitial judgement of the validity of the claim and, where appropriate, withdraw the item in questionfrom public view.Mamoona Chaudhry, Angélique Angot,  Hamad B. Rashid, Giovanni Cattoli,  Manzoor Hussain, Giulia Trovò,  Alessandra Drago, Viviana Valastro,  Michael Thrusfield, Sue Welburn,  Mark C. Eisler, Ilaria CapuaPhylogenetic	 analysis	 of	 influenza	 viruses	 collected	 dur-ing	December	 2009–February	 2010	 from	 chickens	 in	 live	poultry	retail	shops	in	Lahore,	Pakistan,	showed	influenza	A(H9N2)	lineage	polymerase	and	nonstructural	genes	gen-erate	through	inter-	and	intrasubtypic	reassortments.	Many	amino	acid	signatures	observed	were	characteristic	of	hu-man	isolates;	hence,	their	circulation	could	enhance	inter-	or	intrasubtypic	reassortment.The first outbreak of illness caused by avian influenza A(H9N2) virus in Pakistan was reported in 1998; iso-lates showed a close relationship to subtype H9N2 avian influenza viruses (AIVs) circulating in Hong Kong, China during 1997 that were grouped within the G1 lineage (1). In recent years, H9N2 genes have reassorted extensively, generating novel genotypes on the Indian subcontinent. Widespread co-circulation of H9N2 with other AIVs (e.g., highly pathogenic AIVs H5N1 and H7N3) could instigate the generation of novel variant and reassorted viruses, pos-sibly with increased zoonotic potential (2).No information was available about the genetic make-up of AIVs circulating in live poultry retail shops (LPRSs) in Pakistan. This study was conducted to genetically char-acterize AIVs in LPRSs in Lahore District, Pakistan.The StudyWe conducted a cross-sectional survey of LPRSs in La-hore (Figure), which is the capital of the Punjab Province in Pakistan. In each of 280 LPRSs, we collected tracheal swab samples from 5 randomly selected chickens and pooled them into 1 composite sample, totaling 280 pooled samples. The samples were characterized at the World Or-ganisation for Animal Health/Food and Agriculture Orga-nization of the United Nations National Reference Labora-tory for Avian Influenza in Padua, Italy.Of the 280 samples, 10 tested positive for H9N2 sub-type by real-time reverse transcription PCR (rRT-PCR) (3) and virus isolation test (United Nations www.oie.int/filead-min/Home/eng/Health_standards/tahm/2.03.04_AI.pdf) (Table 1). Results were negative for H5 and H7 subtypes. Two of the 280 samples were positive for Newcastle dis-ease virus. Each of the 10 H9N2 isolates was character-ized by sequencing for 6 gene segments (hemagglutinin [HA]: neuraminidase [NA], nonstructural [NS], matrix [M], and polymerase basic 1 and 2 [PB1, PB2]); 2 isolates (A/chicken/Pakistan/10RS3039-283-11/2010 and A/chicken/Pakistan/10RS3039-289-186/2010) were sequenced for all 8 gene segments (HA, NA, NS, M, PB1, PB2, polymerase [PA], and nucleoprotein [NP]), as described (4). Sequences for these viruses were deposited into GenBank (accession nos. KF975457–KF975503, and KP223678–KP223693). The NS gene sequence of A/chicken/Pakistan/ 10RS3039-284-11/2010 could not be deposited due to poor data quality.We generated neighbor-joining phylogenetic trees for all gene segments using the distance-based method in MEGA version 5.2.2 (http://www.megasoftware.net/). We calculated bootstrap values based on 1,000 replicates of alignment (5). HA and NA genes of virus-es in this study tightly clustered within the G1 lineage along with H9N2 viruses from Pakistan, India, Iran, Is-rael, Saudi Arabia, and Bangladesh, suggesting deriva-tion from a common ancestor: A/quail/Hong Kong/G1/97 (online Technical Appendix Figure panels A, B http://wwwnc.cdc.gov/EID/article/21/4/14-1570-Techapp1.pdf). M and NP genes clustered within the G1 lineage (online Technical Appendix Figure panels C, D) along with an influenza (H7N3) isolate from Pakistan (A/chick-en/Karachi/NARC-100/2004; nucleotide identity >96% for M gene and 95% for NP gene). Two polymerase complex genes, PB1 and PA, and the NS gene did not cluster within G1 lineage or any other established Eur-asian lineages: these gene sequences made a separate well-supported cluster with highly similar H9N2 virus-es that circulated in Pakistan (2005–2008), Iran, India, and Bangladesh; these sequences had a high sequence Reassortant Avian Influenza A(H9N2)  Viruses in Chickens in Retail Poultry Shops,  Pakistan, 2009–2010	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	21,	No.	4,	April	2015	 673Author	affiliations:	University	of	Veterinary	and	Animal	Sciences,	Lahore,	Pakistan	(M.	Chaudhry,	H.B.	Rashid);	University	of	Edinburgh,	Edinburgh,	Scotland,	UK	(M.	Chaudhry,	M.	Thrusfield,	S.	Welburn);	Istituto	Zooprofilattico	Sperimentale	delle	Venezie,	Viale	dell’Università,	Legnaro,	Padua,	Italy	(A.	Angot,	G.	Cattoli,	G.	Trovò,	A.	Drago,	V.	Valastro,	I.	Capua);	Food	and	Agriculture	Organization	of	the	United	Nations,	Islamabad,	Pakistan	 (M.	Hussain);	University	of	Bristol,	Bristol,	UK	(M.C.	Eisler)DOI:	http://dx.doi.org/10.3201/eid2104.141570DISPATCHESidentity (>95%) with A/chicken/Karachi/NARC-100/04 (H7N3) (2,6,7) (online Technical Appendix Figure pan-els E, F, G). The PB2 segment clustered separately with H9N2 viruses from the subcontinent and Middle East (online Technical Appendix Figure panel H), except A/chicken/Karachi/NARC-100/04. These results indicate a separate Indian subcontinental lineage of H9N2 viruses has emerged (2).On the basis of these analyses, we could conclude that internal genes PB1, PA, and NS of these viruses originated by intersubtypic (between different HA subtypes) reassort-ment events from local H7N3 viruses circulating in Pakistan (nucleotide identity >95%). This suggests that intersubtyp-ic reassortment events continuously result from mixing of AIV subtypes in domestic poultry and wild birds in Paki-stan (8), and that PB2, M, and NP genes were acquired by intrasubtypic reassortment between H9N2 viruses of G1 lineage circulating within Pakistan. These results support speculation that the currently circulating H9N2 lineage is a reassortment of G1 lineage from Hong Kong and the highly pathogenic H7N3 virus that circulated in Pakistan and can be assigned to genetic group B (8).We aligned amino acid sequences of current virus-es and compared them to representative H9N2 lineages. When compared to the prototype G1 viruses, H9N2 vi-ruses isolated from LPRSs showed that they have evolved to acquire mammalian host-specific mutations through-out the genome (online Technical Appendix Table 1). Of these mutations, certain amino acid substitutions through-out the viral genome have become fixed (9). All LPRS isolates possessed the K-S-S-R motif at the cleavage site. The presence of lysine at position 4 was observed in H9 isolates from the Indian subcontinent (2,9), but it is un-common elsewhere. Amino acid signature changes of hu-man influenza viruses were also observed in internal gene segments (Table 2).674	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	21,	No.	4,	April	2015Figure.	Location	of	live	poultry	retail	shops	(X)	in	5	towns	in	Lahore,	Pakistan,	where	avian	influenza	A(H9N2)	virus	isolates	were	identified	in	chickens,	2009–2010.	Inset	shows	location	of	Lahore	in	Punjab	Province. Table 1.	Influenza	A(H9N2)	viruses	isolated	from	oropharyngeal	swab	samples	from	chickens	in	live	poultry	retail	shops	of	Lahore,	Pakistan,	2010 Isolates Collection	date Towns	of	Lahore	District* A/chicken/Pakistan/10RS3039-283-11/2010 Jan	23 Gulberg	Town A/chicken/Pakistan/10RS3039-284-48/2010 Jan	25 Data	Gunj	Bakhsh	Town A/chicken/Pakistan/10RS3039-285-63/2010 Jan	26 Data	Gunj	Bakhsh	Town A/chicken/Pakistan/10RS3039-286-65/2010 Jan	26 Data	Gunj	Bakhsh	Town A/chicken/Pakistan/10RS3039-287-98/2010 Jan	27 Ravi	Town A/chicken/Pakistan/10RS3039-288-102/2010 Jan	27 Ravi	Town A/chicken/Pakistan/10RS3039-289-186/2010 Feb	11 Samanabad	Town A/chicken/Pakistan/10RS3039-199-199/2010 Feb	11 Samanabad	Town A/chicken/Pakistan/10RS3039-290-230/2010 Feb	13 Allama	Iqbal	Town A/chicken/Pakistan/10RS3039-291-266/2010 Feb	14 Allama	Iqbal	Town *Lahore	District	contains 9	towns and	1	cantonment	area.    	Reassortant	Influenza	A(H9N2)	VirusesEach of the 10 isolates had the Q226L substitution (H3 numbering) in the receptor-binding site of HA, correlating to a shift in affinity from avian-type sialic acid receptor to human-type (10). We identified 3 representative substitu-tions: E/T190 A in 9 viruses and E/T190V in 1 virus, and Q227I in all 10 viruses (H3 numbering). The outcomes of these substitutions have not been investigated; further study is needed (2). Glycosylation sites at positions 551, 218, and 206 were absent in the study viruses, suggesting a frequent alteration in sequences from this region (2,6,9) and possibly signifying the selected adaptation of H9N2 to poultry (10). We did not find an R292K substitution, which is associated with resistance to the sialidase inhibitors oseltamivir and zanamivir, in the NA proteins of any of the 10 LPRS virus isolates. HB sites were also well conserved with few substi-tutions (K367E in 5 viruses, K367G in 1 virus, and S372A and W403R in all 10 viruses). An additional glycosylation site was present at position 44, which is believed to enhance virulence caused by altered antigenicity or sialidase activ-ity (11) (online Technical Appendix Table 1).Many residues in nucleoprotein and polymerase are con-sidered determinants of the host range of AIVs and increase virulence or replication in the mammalian host (2,10,12). The analyses of internal genes showed that these viruses also contained mammalian host-specific markers (2,8) that have become permanent in M protein (M1, V15I, T37A; M2, E16G, L55F); in PB1 protein (L13P); and in NS1 protein (V149A) of all LPRS isolates and in NP (E372D) of the 2 isolates we se-quenced (online Technical Appendix Table 2). In M2 pro-tein, no substitutions linked to resistant amantadine were seen. All viruses contained an uncommon K-S-E-I sequence as a PDZ ligand motif in the NS protein. Residue isoleucine at the C-terminus of PDZ ligand motif has been reported as a rare substitution (12). NS also harbored E227K mutation in the C-terminal, which has been demonstrated to modulate pathogenicity of AIVs (13) and appeared to be a rare amino acid signature, although in this study it was observed in all 10 virus isolates from chickens in LPRSs.ConclusionsOur analysis confirmed that continuous gene reassortment has occurred among influenza A(H9N2) viruses since their emergence in poultry in Pakistan. Because H9N2 viruses infect multiple species, they may donate genes to emerging human pathogens; it has been observed that H7N9 acquired internal genes from the avian H9N2 virus (14). In wet mar-kets, availability of freshly slaughtered poultry, live poul-try transportation, and mixed trading of domestic animals provide a favorable environment for gene reassortment, mutation, and interspecies transmission of AIVs (15). Con-tinuous circulation of these viruses in LPRSs increases the chances of their evolution into new genotypes. Close con-tact of humans and poultry in LPRSs with no biosecurity barriers increases the risk for emergence of novel influenza viruses with zoonotic or human pandemic potential. Con-tinued surveillance in LPRSs is essential to better under-stand the public health risk posed by H9N2 AIVs.AcknowledgmentsWe thank the staff members of the live poultry retail  shops in which the study subjects were housed for data and sample collection. The European Union project Training and technology transfer of avian influenza diagnostics and disease management skills (FLUTRAIN) provided financial support for training of MC at IZSVe, Italy. The Higher Education Commission of Pakistan provided a PhD scholarship to MC through the Overseas Schol-arship Scheme (10%- Batch-2).We acknowledge the memory of Isabel Minguez Tudela  (1955–2011), of the European Commission, who supported  this collaboration as a contribution to improving veterinary public health.Dr. Chaudhry is an academician in the Department of Epidemi-ology and Public Health, University of Veterinary and Animal Sciences, Lahore. Her research interests include epidemiology of infectious diseases of public health concern.References  1. Cameron KR, Gregory V, Banks J, Brown IH, Alexander DJ,  Hay AJ, et al. H9N2 subtype influenza A viruses in poultry in  Pakistan are closely related to the H9N2 viruses responsible for human infection in Hong Kong. Virology. 2000;278:36–41.  http://dx.doi.org/10.1006/viro.2000.0585	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	21,	No.	4,	April	2015	 675 Table 2.	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N Engl  J Med. 2013;368:2337–9. http://dx.doi.org/10.1056/NEJMc1306100Address for correspondence: Mamoona Chaudhry, Department of Epidemiology and Public Health, University of Veterinary and  Animal Sciences, Abdul Qadir Jilani Road, Lahore, Pakistan;  email: mamoona.chaudhry@gmail.com676	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	21,	No.	4,	April	2015February 2014:  High-Consequence PathogensIncluding:•  Poxvirus	Viability	and	Signatures	 in	Historical	Relics•  Novel	Paramyxovirus	Associated	with	 Severe	Acute	Febrile	Disease,	 South	Sudan	and	Uganda,	2012•  Subtyping	Cryptosporidium ubiquitum,	a	Zoonotic	Pathogen	Emerging	in	Humans •  Genomic	Variability	of	Monkeypox	Virus	among	Humans,	Democratic	Republic	 of	the	Congohttp://wwwnc.cdc.gov/eid/content/20/2/contents.htm

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Reassortant Avian Influenza A(H9N2) viruses in chickens in retail poultry shops, Pakistan, 2009-2010

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