we conducted a cross - sectional survey of lprss in lahore ( 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 organisation for animal health / food and agriculture organization of the united nations national reference laboratory for avian influenza in padua , italy .
location of live poultry retail shops ( x ) in 5 towns in lahore , pakistan , where avian influenza a(h9n2 ) virus isolates were identified in chickens , 20092010 .
of the 280 samples , 10 tested positive for h9n2 subtype by real - time reverse transcription pcr ( rrt - pcr ) ( 3 ) and virus isolation test ( united nations www.oie.int/fileadmin/home/eng/health_standards/tahm/2.03.04_ai.pdf ) ( table 1 ) .
each of the 10 h9n2 isolates was characterized 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 ) .
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 viruses in this study
tightly clustered within the g1 lineage along with h9n2 viruses from pakistan , india , iran , israel , saudi arabia , and bangladesh , suggesting derivation from a common ancestor : a / quail / hong kong / g1/97 ( technical appendix figure panels a , b ) .
m and np genes clustered within the g1 lineage ( technical appendix figure panels c , d ) along with an influenza ( h7n3 ) isolate from pakistan ( a / chicken / 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 eurasian lineages : these gene sequences made a separate well - supported cluster with highly similar h9n2 viruses that circulated in pakistan ( 20052008 ) , iran , india , and bangladesh ; these sequences had a high sequence identity ( > 95% ) with a / chicken / karachi / narc-100/04 ( h7n3 ) ( 2,6,7 ) ( technical appendix figure panels e , f , g ) .
the pb2 segment clustered separately with h9n2 viruses from the subcontinent and middle east ( 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 ) reassortment events from local h7n3 viruses circulating in pakistan ( nucleotide identity > 95% ) .
this suggests that intersubtypic reassortment events continuously result from mixing of aiv subtypes in domestic poultry and wild birds in pakistan ( 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 viruses and compared them to representative h9n2 lineages . when compared to the prototype g1 viruses , h9n2 viruses isolated from lprss showed that they have evolved to acquire mammalian host - specific mutations throughout the genome ( technical appendix table 1 ) .
of these mutations , certain amino acid substitutions throughout 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 uncommon elsewhere .
amino acid signature changes of human influenza viruses were also observed in internal gene segments ( table 2 ) .
each of the 10 isolates had the ql 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 substitutions : e / t a in 9 viruses and e / tv in 1 virus , and qi 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  rk 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 substitutions ( ke in 5 viruses , kg in 1 virus , and sa and wr 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 activity ( 11 ) ( technical appendix table 1 ) .
many residues in nucleoprotein and polymerase are considered 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 , vi , ta ; m2 , eg , lf ) ; in pb1 protein ( lp ) ; and in ns1 protein ( va ) of all lprs isolates and in np ( ed ) of the 2 isolates we sequenced ( technical appendix table 2 ) . in m2 protein ,
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 ek 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 .
our 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 markets ,
availability of freshly slaughtered poultry , live poultry transportation , and mixed trading of domestic animals provide a favorable environment for gene reassortment , mutation , and interspecies transmission of aivs ( 15 ) .
continuous circulation of these viruses in lprss increases the chances of their evolution into new genotypes .
close contact 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 .
continued surveillance in lprss is essential to better understand the public health risk posed by h9n2 aivs .
technical appendix . comparisons of structure and pathogenicity determinants of avian influenza isolates and phylogenetic distribution of 10 sequenced viruses from samples from chickens in live poultry retail shops in lahore , pakistan , 20092010 .
 phylogenetic analysis of influenza viruses collected during december 2009february 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 . 
The Study
Conclusions
None