nasopharyngeal and cloacal swab samples were collected from sick and dead birds found on farms near ibadan , nigeria , and injected into 10-day - old embryonating chicken eggs . retrospective analysis of isolates obtained in or near january 2006 identified influenza virus ( h5n1 ) in 3 samples . in addition , 4 newcastle disease virus isolates were obtained , which highlights the cocirculation of newcastle disease virus and influenza viruses ( h5n1 ) in nigerian poultry and emphasizes the need for specific virologic testing to distinguish the clinically similar poultry diseases caused by these 2 pathogens . the 3 avian influenza ( h5 ) isolates were designated influenza a / chicken / nigeria/228 - 5/2005 , a / chicken / nigeria/228 - 6/2006 , and a / chicken / nigeria/228 - 10/2006 . nucleotide sequences of the coding regions of all 8 segments of the 3 viruses demonstrated that all isolates possessed a multiple basic amino acid at the hemagglutinin ( ha ) cleavage site with the sequence pqgerrrkkr . sequences at this site were identical to those of highly pathogenic avian influenza ( hpai ) subtype h5n1 viruses from europe , russia , asia , and from recent isolates from the lagos state of nigeria ( 2 ) ; it lacks a single basic residue when compared with ha from strains from southeastern people s republic of china , vietnam , cambodia ( pqrerrrkkrg ) , and thailand ( pqrekrrkkrg ) ( 3 ) . other notable features of the sequences were the absence of the h274y genetic change associated with high - level resistance to oseltamivir in influenza neuraminidase 1 ( n1 ) viruses ( 4 ) . the nonstructural ( ns ) 1 open reading frame encodes a 5-aa deletion at positions 8084 , as has been observed since 2001 in subtype h5n1 isolates from poultry . one of the isolates ( a / chicken / nigeria/228 - 10/2006 ) also has a c - terminal amino - acid extension , which is predicted to affect the function of the pdz - ligand domain otherwise present at the c terminus of the ns1 protein ( 5,6 ) . this sequence change did not detectably affect the ability of ns1 to block interferon induction when expressed transiently in 293 t cells ( data not shown ) . the polymerase basic 2 protein ( pb2 ) of these viruses possesses a lysine residue at position 627 , an amino acid previously implicated in mammalian adaptation of subtype h5n1 viruses ( 79 ) . phylogenetic analysis based on the ha sequence and on complete genome sequences of hpai ( h5n1 ) strains grouped the 3 new isolates from nigeria with other isolates from europe , the middle east , and lagos state of nigeria . according to recent classification by the h5n1 evolution working group , the viruses belong to clade 2.2.2 ( previously referred to as the european - middle eastern - african clade 1 ) ( 10 ) . the technical appendix , shows the phylogenetic trees generated on the basis of the sequences of ha , na , nucleocapsid protein ( np ) , and ns segments . on the basis of phylogenetic analyses of all 8 segments , no evidence of reassortment was observed among the newly sequenced hpai isolates from nigeria . although prior reports ( 2,3,11 ) have suggested 3 independent introductions of hpai viruses into nigeria , our analysis of viruses in this study and in genbank identified only 2 clades ( clades 2.2.2 and 2.2.3 ) among the nigeria isolates , suggesting 2 unique introductions into nigeria . the virulence of the hpai virus isolated in poultry was assessed by intravenous injection of a 1:10 dilution of allantoic fluid into groups of 4-week - old spf white leghorn chickens , 8 per group , as previously described ( 12 ) . according to results of this assay , all 3 isolates were highly pathogenic ( table ) ; mean times to death were 1.0 , 1.3 , and 1.4 days , similar to times previously reported for other european - asian lineage hpai ( h5n1 ) viruses ( 13 ) . * virus isolated from tissues and swabs sample on 1 and 2 days postinoculation ( dpi ) . limit of detection < 10 eid50/g of tissue or < 10 eid50/ml sample . to determine infectivity and pathogenicity , using a simulated natural respiratory route of exposure , we intranasally inoculated ten 4-week - old white leghorn chickens with strain a / chicken / nigeria/228 - 5/2005 ( 10 mean embryo infectious doses ) . two chickens that were not inoculated and were maintained as negative controls were euthanized ( 100 mg sodium pentobarbital / kg body weight ) at 0 days postinoculation ( dpi ) . at 1 dpi , 2 of the injected birds were euthanized . on 2 dpi , postmortem examinations were performed on 2 birds that had died , and samples were collected for virus isolation and for histopathologic and immunohistochemical examination of a variety of tissues . for immunohistochemical examination , a monoclonal antibody against influenza a nucleoprotein was used as previously described ( 14 ) . virus isolation and titration were conducted with brain , heart , lung , kidney , breast muscle , oropharyngeal , and cloacal samples ; embryonating chicken eggs were used ( 12 ) . after intranasal inoculation with a / chicken / nigeria/228 - 5/2005 , all birds died or were euthanized for severe illness within 3 dpi ( mean time to death 2.125 days ) . at 1 dpi , both intranasally inoculated chickens sampled were infected , as evidenced by low virus titers in oropharyngeal swabs ; however , only 1 chicken had systemic infection with moderate titers of virus in tissues ( table ) . histologically , both chickens lacked lesions , but the spleen of 1 chicken had a few avian influenza virus  positive histiocytes . the intranasally inoculated chickens that died and were necropsied on 2 dpi had exhibited mild to moderate listlessness , a hunched posture , and partial closure of eyelids a few hours before death . gross lesions included scattered petechia in epicardial fat within the coronary groove ( figure , panel a ) , a few petechia in mucosa of the ventriculi ( gizzards ) , slight increase in pericardial fluid , and swollen kidneys . swabs from respiratory and alimentary tracts and the tissues had high titers of the virus ( table ) . histologically , tissues with lesions coincided with sites of virus replication and indicated severe systemic infection . the most severe lesions were severe interstitial pneumonia with edema ( figure , panel b ) , moderate to severe myocyte degeneration and necrosis in the heart , moderate nonsuppurative encephalitis , moderate necrotizing rhinitis , moderate lymphohistiocytic depletion and apoptosis in the spleen , and mild to moderate degeneration and necrosis of pancreatic acinar epithelium ( figure , panel c ) . scattered lesions of necrosis and inflammation were seen in liver , cloacal bursa , thymus , proventriculus , ventriculus , and pancreatic islets . influenza a virus was localized to necrotic cells , which most frequently included blood vessel endothelium throughout the body ( figure , panel d ) , cardiac myocytes ( figure , panel e ) , pulmonary histiocytes and heterophils ( figure , panel d ) , neurons and glial cells in the brain , splenic histiocytes and cellular debris , and renal tubular epithelium . virus was less frequently identified in kupffer cells and hepatocytes ; histiocytes in lamina propria of alimentary tract , thymus , and cloacal bursa ; pancreatic acinar and islet epithelium ( figure , panel f ) ; proventricular epithelium ; and bone marrow myeloid cells . experimental studies in chickens inoculated intranasally with highly pathogenic influenza a / chicken / nigeria/228 - 5/2005 ( h5n1 ) and sampled 2 days postinoculation . a ) photograph ( scale bar = 1 cm ) of a gross lesion , showing petechia in epicardial fat in the coronary groove . b , c ) photomicrographs ( scale bars = 50 m ) of tissue sections stained with hematoxylin and eosin , showing severe histiocytic and heterophilic interstitial pneumonia with moderately interlobular edema ( panel b ) and pancreatic acinar epithelium ( panel c ) . d  f ) photomicrographs ( scale bars = 50 m ) of tissue sections stained immunohistochemically to demonstrate avian influenza virus nucleoprotein , showing avian influenza virus antigen in pulmonary and blood vessel endothelial cells and histiocytes ( panel d ) , nuclei and cytoplasm of cardiac myocytes ( panel e ) , and pancreatic acinar epithelium and islet cells ( panel f ) . the 3 early hpai ( h5n1 ) isolates from nigeria , which belonged to clade 2.2.2 , produced in chickens a systemic infection characterized by virus replication and associated necrotic and inflammatory lesions in critical internal organs such as the heart , brain , and lungs . a prominent vascular tropism of the virus was evidenced by widespread replication in blood vessel endothelium throughout the body and is typical of other hpai viruses ( h5n1 ) of the asian lineage .
three highly pathogenic avian influenza subtype h5n1 and 4 newcastle disease viruses were isolated from sick or dead chickens in southwestern nigeria . 
 sequencing and phylogenetic analysis placed them within h5n1 subclade 2.2.2 . 
 intravenous and intranasal pathogenicity tests produced systemic disease with vascular endothelial cell tropism in chickens .