the influenza viruses include three genera , a , b , and c within the family orthomyxoviridae . avian influenza viruses ( aivs ) , all of which are contained in the genus influenza virus a , are an economically important cause of disease in fowl and occasionally affect humans , pigs , and horses [ 1 , 2 ] . each genus of the virus is further subdivided into serotypes based on the surface proteins , consisting of 16 different hemagglutinin ( h ) and 9 neuraminidase ( n ) subtypes . while only a limited number of h and n subtypes are circulating in humans and other mammalian species , all the h and n subtypes are found in avian species [ 3 , 4 ] . the genome of influenza a viruses consists of 8 unique segments of single - stranded negative sense rna . the viral rna segments encode 10 recognized gene products , pb1 , pb2 , and pa polymerases , h , np , n , m1 , m2 , ns1 , and ns2 proteins . the h6 subtype is one of the most commonly recognized subtypes in domestic ducks in southern china [ 6 , 7 ] and in migratory birds in north america and in europe [ 810 ] . h6 viruses have caused several outbreaks in commercial poultry worldwide that resulted in decreased egg production and increased mortality [ 1113 ] . during the hong kong h5n1 incident in 1997 , an h6n1 avian influenza virus , teal / hong kong / w312/97 ( w312 ) , was isolated from birds in a live poultry market . genetic characterization of this virus revealed that except for the h gene , the remaining 7-gene segments were closely related to those of highly pathogenic avian influenza h5n1 viruses ( hpaivs ) found in both poultry and humans . the present study reports on the outbreak of avian influenza ( ai ) caused by an h6n1 subtype of aiv isolated during january 2009 in kibbutz gvulot , in the southern part of israel . the present isolate , a / turkey / israel/09 ( h6n1 ) , is the third h6 aiv obtained in israel since the year 2000 . therefore , circulation of these viruses was concurrent with that of the highly prevalent h9n2 aiv in israel [ 15 , 16 ] . while the former h6 aiv isolates , a / duck / israel/289/01 ( h6n2 ) and a / mallard / israel/320/01 ( h6n2 ) , were isolated from water birds , the present isolate was obtained from domestic turkey poults . 2 ) located in one farm , kibbutz gvulot , exhibited an increased daily mortality of approximately 1% over a two - day period . the clinical signs were swollen infraorbital sinuses , nasal discharge , respiratory rales , rattles , and lethargy . birds in the affected poultry house had developed aiv antibodies , as detected by elisa and aiv h6 haemagglutination inhibition ( hi ) tests . the elisa test was performed with the flockchek avian influenza antibody test kit ( idexx , usa ) according to the manufacturer 's instructions , and the hi test employed a panel of reference antisera to h5 , h6 , h7 , and h9 . blood samples were tested for antibodies by the hi test using 4 ( ha ) units of the h6 subtype antigen . turkey poults from the six houses were distributed to 4 secondary farms , mishmeret , ramon , ein zurim , and evron . tracheal and cloacal swabs of clinically - affected turkeys were assayed for the presence of aiv by pcr and virus isolation in 11-day - old embryonated eggs . the presence of aiv was initially detected in the allantoic fluids ( af ) of dead eggs after 2 - 3 days of incubation , by the hemagglutination ( ha ) assay . testing of ha - positive af was performed by reverse transcriptase polymerase chain reaction ( rt - pcr ) . in parallel , rt - pcr was performed on rna extracted directly from tracheal and cloacal swabs . viral rna was extracted directly from the tracheal and cloacal swabs and from af using the qiaamp viral rna mini kit ( qiagen , valencia , ca ) according to the manufacturer 's instructions . firstly , aiv was detected using a primer pair specific for the m gene of influenza a virus , mf ( 5-ttctaaccgaggtyraaacgt-3 ) and mr ( 5-ctgggcacggtgagcgt-3 ) to amplify a 200  bp product ( panshin a. , kimron veterinary institute , bet dagan , israel , unpublished ) . rt - pcr was performed using a one step rt - pcr kit , ( qiagen ltd . ) . a 25  l mixture containing 5  l of buffer , 1  l of dntp mix , 1  l of each primer , 1  l of one step enzyme mix , 13  l nuclease - free water , and 3  l rna template was used in the assay as follows : 30  min at 50c , 10  min at 95c , 30 cycles at 94c for 30  sec , then 30  sec at 54c , 30  sec at 72c , 5  min at 72c , and 15  min at 4c using the dna engine thermal cycler ( bio rad , waltham , ma , usa ) . secondly , molecular subtyping was performed with a set of specific primers for genes of h subtypes h1h4 , h6 , h9 , and h10   and h5 , h7 , and n subtypes n1   n2 , n3 , n7 , n9 ( panshin a. , kimron veterinary institute , bet dagan , israel , unpublished ) . the primers used to amplify a 623  bp segment of the n2 gene were n2f ( 5-ytgyacagtrgtaatgacbgatggr-3 ) and n2r ( 5-cratrctryttgadgtccaccabac-3 ) ; the primers to used to to amplify a 470  bp product of the n3 gene were n3f ( 5-aayagrccwtggrtgagrat-3 ) and n3r ( 5-cccgatccaggttcattgt-3 ) . the primers used to amplify a 509  bp segment of the n7 gene were n7f ( 5-ttggrtggtcragyacaagctgcc-3 ) and n7r ( 5-accaggrctyccagtdatwggattg-3 ) , the primers used to amplify a 520  bp segment of n9 gene were n9f ( 5-caaccaatgcaagccaaacaata-3 ) and n9r ( 5-tttcgggcccatgtgttgatttc-3 ) . the amplification conditions for h and n genes were similar to those of the m gene . the pcr products were purified with the megaquick - spin pcr & agarose gel extraction system ( intron biotechnology , inc , gyeonggi - do , korea ) according to the manufacturer 's instructions . sequencing reactions were performed by the weizmann institute of science , rehovot , israel , using a 3700 dna analyzer ( perkin - elmer , applied biosystems , foster city , ca . , u.s.a . ) and analysed by capillary electrophoresis . the bioedit package , version 7.3 , and dnastar system software were used for sequence analysis and alignment . nucleotide sequences of all gene segments and available sequence data from genbank were used to generate phylogenetic trees . the intravenous pathogenicity index ( ivpi ) of aiv isolates was determined as described in the oie manual of standards for diagnostic tests and vaccines . the test employs a standard volume of virus which is inoculated intravenously into spf chickens . the ivpi test yields an index of virulence valued from 0 to 3 , which is calculated according to illness severity and the viability period following inoculation . the mishmeret farm received poults from the original poultry houses nos . 1 and 4 and developed a low level of hi antibodies ( geometric mean titer ( gmt ) = 1.8 ) ; the evron farm received poults from the original poultry houses nos . 5 , and 6 and did not develop hi antibodies ( gmt = 0 ) ; the ramon farm received poults from the original poultry houses nos . 1 , 4 , 5 and 6 , and did not develop hi antibodies ( gmt = 0 ) ; the ein zurim farm received poults from the original poultry house no . 2 , that was infected with the a / turkey / israel/09 ( h6n1 ) isolate , and from house no . , birds on the ein zurim farm developed hi antibodies to h6 and showed clinical signs ( gmt = 6.3 ) . to identify the aiv h and n gene , specificity amplifications and hi assays the virus was isolated and characterized by classical methods ( oie ) and genome was detected by pcr . a / turkey / israel/09 ( h6n1 ) was characterized as low pathogenic avian influenza virus ( lpai ) with an ivpi of 0.00 . infection in the chickens used in the ivpi assay was confirmed by the hi test . the amino acid sequence of the h gene cleavage site was examined to support the virulence determination of the a / turkey / israel/09/(h6n1 ) isolate . the amino acid sequence at the cleavage site was pqietr*glf , indicating that the isolate was of low pathogenicity , since it did not contain multiple basic amino acids , characteristic of hpaivs . most lpaivs have a single arginine at the cleavage site , whereas hpaivs usually exhibit a multibasic amino acid motif ( r and k ) flanking the cleavage site [ 22 , 23 ] . the genome of influenza a viruses consists of eight unique segments of single - stranded rna , which are of negative polarity ( i.e. , complementary to the mrna sense ) . the 8 gene segments of the a / turkey / israel/09 ( h6n1 ) isolate were sequenced . the nucleotide sequences obtained in the present study were compared to those of aivs from the genebank ( figures 18 ) . figure 1 shows the phylogenetic tree of the complete h gene , subtype h6 , of representative recent aiv h6 isolates reported worldwide , including the previous two israeli h6 isolates , which were not reported to the genebank . it seems that the h gene of the present isolate , a / turkey / israel/09 ( h6n1 ) , differed from the h genes of the two previous israeli isolates , as they belonged to different clades , although the differences in the nucleotide content were about 3.7 - 3.8% . it is notable that the h6 aiv , that shaped the clade to which the present isolate belonged , originated from waterbirds . figure 2 shows the phylogenetic tree of the n gene of n1 subtypes , based on the analysis of nucleotides 204 to 1149 . the analysis includes representative aiv n1 genes compared on the respective gene fragment . while the compared sequences differed up to 31% , the present isolate , a / turkey / israel/09 ( h6n1 ) , was closest to the n1 from a / mute swan / aktau/06 ( h5n1 ) and a / swan / mangystau/06 ( h5n1 ) . it is notable to mention that the hong kong avian a / teal / hong kong / w312/97 ( h6n1 ) and the human influenza virus a / hong kong/156/97 ( h5n1 ) showed a very high nucleotide homology in the 6 aiv internal genes , and especially in the n1 gene sequence [ 14 , 24 ] . that similarity might indicate a common precursor and that the present virus could become a potential source of novel pathogenic aiv strains . figures 3 , 4 , 5 , 6 , 7 , and 8 show the phylogenetic comparison of the a / turkey / israel/09 ( h6n1 ) isolate complete internal genes , ns , m , np , pa , pb1 and pb2 , respectively , to genes of representative aivs . the main feature revealed from these phylogenetic trees was the close similarity between the former two h6n2 aiv isolates , a / duck / israel/289/01 and a / mallard / israel/320/01 , as compared to the phylogenetic divergence of the present h6n1 isolate , a / turkey / israel/09 . the phylogenetic distances between the 2009 h6n1 isolate and the two previous h6n2 isolates were calculated . while the two previously described h6 isolates , a / duck / israel/289/01 ( h6n2 ) and a / mallard / israel/320/01 ( h6n2 ) , were identical in their ns , m , np , pa , pb1 , and pb2 genes , the a / turkey / israel/09 ( h6n1 ) isolate differed from the former two isolates by 3% , 2.3% , 6% , 2.5% , 3.6% , and 5% , respectively . in conclusion , the present paper describes a third h6 aiv isolate ( h6n1 ) in israel , which unlike the two previous h6n2 isolates that were obtained from ducks and mallard , was now detected in turkeys . this h6n1 lpaiv was shown to possess a low - pathogenic amino acid sequence at the cleavage site and had a low ivpi . a / turkey / israel/09 ( h6n1 ) was not highly transmissible among commercial flocks . that phenomenon was supported by two observations ; ( a ) the absence of clinically - affected cases in adjacent poultry houses and ( b ) the detection of only three cases of aiv h6 in israel .
an avian influenza virus ( aiv ) , a / turkey / israel/09 subtype h6n1 , was isolated from turkey poults exhibiting typical pathology associated with aiv infection . 
 the virus was characterized by rt - pcr using aiv subtype - specific primers and by the haemagglutination inhibition test using aiv subtype - specific antisera . 
 the virus has an intravenous pathogenicity index of 0 and possessed a nucleotide sequence at the cleavage site of the hemagglutinin gene , pqietr*glf , associated with avian influenza viruses of low pathogenicity . unlike the two previous h6n2 isolates originating from domestic ducks and mallard , the a / turkey / israel/09 ( h6n1 ) was isolated from turkeys . 
 the gene sequences of the a / turkey / israel/09 ( h6n1 ) virus show divergence from the former israeli h6 isolates .