viruses and cells : apmv / shimane67 was isolated from goose fecal samples 
 collected in shimane prefecture , the wintering location for wild waterfowl , such as ducks , 
 geese and swans . electron microscopy : purification of apmv / shimane67 was conducted by 
 differential centrifugation and two successive sedimentations through a 10 to 50% sucrose 
 gradient as described previously . the purified 
 virus particles stained with 2% phosphotungstic acid ( ph 6.8 ) were observed under the 
 jem-100cx electron microscope ( jeol , tokyo , japan ) at 80 kv . hemagglutination ( ha ) and hemagglutination inhibition ( hi ) test : ha and hi 
 tests were performed using established procedures as described previously . for hi test , chicken hyperimmune sera against 
 apmv / shimane67 , ndv / goose / alaska/415/91 ( ndv / ak/415 ) , apmv-2/chicken / california / yucaipa/56 
 ( apmv-2/yucaipa ) , apmv-3/turkey / wisconsin/68 ( apmv-3/wi ) , apmv-4/duck / mississippi/320/75 
 ( apmv-4/ms ) , apmv-6/duck / hong kong / d199/77 ( apmv-6/hk / d199 ) and apmv-7/dove / tennessee/4/75 
 ( apmv-7/tn ) were prepared as described previously . propagation of apmv / shimane67 in cultured cells : madin - darby canine kidney 
 ( mdck ) , madin - darby bovine kidney ( mdbk ) , african green monkey kidney ( vero ) and baby 
 hamster kidney ( bhk21 ) cells were grown in eagle s minimal essential medium ( e - mem ) 
 containing 10% fetal bovine serum ( fbs ) . human embryonic kidney ( 293 ) and chicken embryo 
 fibroblast ( df-1 ) cells were grown in dulbecco s modified eagle s medium ( d - mem ) containing 
 10% fbs . cell monolayers prepared in 6-well plates were infected with 10 50% egg 
 infectious dose ( eid50 ) of apmv / shimane67 . after adsorption for 1 hr at 37c , 
 cells were washed three times with phosphate buffered saline ( pbs , ph7.2 ) and were incubated 
 at 37c in e - mem or d - mem containing 0.3% bovine serum albumin and 0.5 g 
 of tpck - treated trypsin ( sigma - aldrich , st . ten - fold diluted supernatant ( 100 
 l ) was inoculated into the allantoic cavities of five 10-day - old 
 embryonated chicken eggs and were incubated at 37c for 3 days unless death of the embryo 
 was detected . after the inoculated eggs were chilled at 4c , their allantoic fluids were 
 tested for hemagglutination activity . pathogenicity test : the mean death time ( mdt ) at the minimum lethal dose 
 for chicken embryos and the intracerebral pathogenicity index ( icpi ) in 1-day - old chickens 
 were measured in order to assess the virulence of this virus , as described previously . nucleotide sequencing and phylogenetic analysis : viral rnas were extracted 
 from purified apmv / shimane67 using the qiaamp viral rna mini kit ( qiagen , tokyo , japan ) , and 
 were transcribed to cdna using f10 primer and rav-2 reverse transcriptase ( takara bio , otsu , 
 japan ) in accordance with the manufacturer s instructions . f10 primer was designed to bind 
 with the gene - stop signal region of the matrix gene and gene - start signal of the f gene of 
 ndv / ak/415 . pcr was carried out using pwo dna 
 polymerase ( roche , mannheim , germany ) and a cycling sequence of 94c for 2 min , followed by 
 35 cycles of 94c for 30 sec , 55c for 30 sec and 72c for 2 min , which was then followed by 
 a final extension at 72c for 5 min . pcr products were purified using a qiaquick gel 
 extraction kit ( qiagen ) and were sequenced using the thermo sequenase cy5.5 dye terminator 
 cycle sequencing kit ( amersham pharmacia biotech , piscataway , nj , u.s.a . ) and a gene rapid 
 dna sequencer ( amersham pharmacia biotech ) . nucleotide sequences of pcr and sequencing 
 primers are shown in table 1table 1.oligonucleotide primers used in this studyprimernucleotide sequence ( 5 to 3)for pcrf10attagaaaaaacacgggtagaafr1215caacgacagatgacctgttgf270gaaacattatcccgcattctaachnr39gaatgagcaatttgataaccccagfor sequencingf10attagaaaaaacacgggtagaafr258gcaatgattgtttggcacaatcf270gaaacattatcccgcattctaacf588taacccttgaatccaggctaggf905gttcaaccttactagaaacattagcf1090catgtctcaatggaaatctaagtgafr1215caacgacagatgacctgttgfr1545gtgattgcaattattaagcataccactgf1689actcccccatccagccacatfr1746acctacatgttggagtgccahnr39gaatgagcaatttgataaccccag . the nucleotide and deduced amino acid sequences of apmv / shimane67 f gene were 
 compared to those of published apmvs by clustal x . phylogenetic trees were generated using the neighbor - joining ( nj ) method with 
 1,000 bootstraps . electron microscopy : electron microscopy of apmv / shimane67 showed typical 
 characteristics of a paramyxovirus . virions were spherical or pleomorphic , enveloped 
 particles ( 250300 nm in diameter ) , and virion surfaces were covered with spiked projections 
 of about 810 nm in length ( fig . 1.negative contrast electron micrograph of apmv / shimane67 virus showing a partially 
 disrupted particle with nucleocapsid emerging . negative contrast electron micrograph of apmv / shimane67 virus showing a partially 
 disrupted particle with nucleocapsid emerging . hi test : in order to investigate the antigenic relationships between 
 apmv / shimane67 and other serotypes of apmv , hi tests were carried out ( table 2table 2.antigenic analysis of apmv / shimane67 by hi tests with antisera against 
 representative apmv strainsantigenantiserum toapmv / shimane67ndv / ak/415apmv-2/yucaipaapmv-3/wiapmv-4/miapmv-6/hkapmv-7/tnapmv / shimane6712801,280<40<40640<40320ndv / ak/415<405,120apmv-2/yucaipa<40160apmv-3/wi<40160apmv-4/mi<405,120apmv-6/hk<40640apmv-7/tn<402,560a ) hi titer represents the reciprocal of the serum dilution inhibiting the activity 
 of 4 hemagglutinating units of virus antigen . as apmv-5 has no ha activity , and apmv-8 to 12 were not available in our 
 laboratory , hi tests against these 6 apmvs were not performed . antiserum against 
 apmv / shimane67 showed the highest hi titer ( 1:1,280 ) with the homologous antigen , but little 
 or no hi activity against other apmv serotypes . reactivity of antisera against ndv / ak/415 , 
 apmv-2/yucaipa , apmv-3/wisconsin , apmv-4/mississippi , apmv-6/hong kong and apmv-7/tennessee 
 on hi test was highest with each homologous antigen , while these antisera showed lower 
 activity against apmv / shimane67 ( 1:<40  1:1,280 ) . a ) hi titer represents the reciprocal of the serum dilution inhibiting the activity 
 of 4 hemagglutinating units of virus antigen . growth in cell culture and pathogenicity of apmv / shimane67 : growth of 
 apmv / shimane67 in avian and mammalian origin cell lines ( df-1 , mdck , mdbk , bhk , vero and 293 
 cells ) was measured . in the presence of trypsin , regardless of cell lines , apmv / shimane67 
 efficiently grew and reached approximately 10 eid50/0.1 
 ml at 72 hr post - infection . syncytium formation , a typical characteristic 
 of paramyxovirus cytopathic effects , was clearly observed , particularly in mdbk cells ( fig . 2.mdbk cells infected with apmv / shimane67 . ) . in df-1 cells without trypsin , titers of apmv / shimane67 reached about 
 10 eid50/0.1 ml at 72 hr post - infection . these 
 results indicate that external proteases are necessary for efficient cleavage of the f 
 protein . the mdt and icpi of apmv / shimane67 were 120 hr < and 0.0 , respectively . nucleotide sequencing and phylogenetic analysis : a total of 1,878 
 nucleotides of the genome sequence of apmv / shimane67 were determined . the putative 
 initiation codon , which better matched the kozak consensus sequence ( ( g / a ) nnatgg ) , was the third atg from 5-end of the f10 primer 
 binding site ( data not shown ) . it contained a long open reading frame ( orf ) composed of 
 1,638 nucleotides and encoded 545 amino acids . the nucleotide sequence of this orf showed 
 similarities with the f genes of ndvs and apmv-12/wigeon / italy/3920_1/2005 ( apmv-12/ita ) by 
 discontiguous megablast search , indicating that 
 this orf was the f gene of apmv / shimane67 . the nucleotide and deduced amino acid identities 
 between the f gene of apmv / shimane67 and other apmv serotypes ranged from 42.9% with 
 apmv-3/nld to 62.7% with apmv-12/ita , and from 28.9% with apmv-3/nld to 67.3% with 
 apmv-12/ita , respectively ( table 3table 3.nucleotide ( upper right ) and deduced amino acid ( lower left ) identities ( % ) among 
 the fusion protein gene of avian paramyxovirusesvirusshimane 67ndvapmv-2apmv-3apmv-4apmv-5apmv-6apmv-7apmv-8apmv-9apmv-10apmv-11apmv-12ak/ 415lasotasf02yucaipabangornldwihk / d3kr / yjkunitachitwn / y1ita/ 4524 - 2tnde/1053wakuyany/22ita/5709flk/324fra/ 100212ita/ 3920_1apmv / shimane67 - 53.656.154.345.644.642.943.043.743.346.244.645.343.546.646.853.353.446.343.862.7ndv / ak/41552.8 - 71.871.546.446.843.341.042.842.054.444.645.044.747.047.458.558.445.643.155.1ndv / lasota53.787.3 - 84.447.546.642.341.941.641.446.844.445.043.546.746.958.260.346.142.155.5ndv / sf0252.684.888.6 - 47.548.042.542.242.741.745.344.944.444.647.347.358.458.745.543.254.8apmv-2/yucaipa37.340.440.640.4 - 69.840.741.142.841.850.250.951.847.260.560.946.347.259.248.546.7apmv-2/bangor36.540.741.341.379.1 - 41.041.542.441.850.451.152.348.360.560.046.146.559.850.046.1apmv-3/nld28.931.231.230.730.831.5 - 66.847.847.442.342.742.441.343.443.240.740.140.842.043.2apmv-3/wi29.430.030.230.030.030.370.1 - 46.847.143.242.342.642.541.641.741.441.241.544.341.6apmv-4/hk / d332.331.031.331.033.634.032.033.5 - 92.343.542.943.540.844.244.141.942.443.341.940.9apmv-4/kr / yj32.130.831.330.633.634.232.233.197.7 - 42.543.144.040.644.143.741.142.442.441.041.3apmv-5/kunitachi39.340.141.041.446.545.731.030.933.933.5 - 56.557.047.950.550.744.945.052.146.944.6apmv-6/twn / y136.936.837.837.848.849.431.629.833.633.053.7 - 72.647.349.349.445.144.952.348.945.6apmv-6/ita/4524 - 237.337.137.437.649.248.631.229.533.833.654.185.7 - 47.050.450.044.84552.249.245.1apmv-7/tn35.738.13938.838.138.828.530.130.129.338.037.036.9 - 49.249.042.843.748.853.044.4apmv-8/de/105336.440.141.141.163.763.731.830.635.935.945.548.247.338.3 - 97.147.347.659.548.246.8apmv-8/wakuya36.240.141.141.163.763.731.830.635.935.745.548.247.338.199.4 - 47.548.059.548.446.4apmv-9/ny/2248.456.455.956.437.839.128.228.328.428.636.537.436.635.637.937.9 - 89.245.542.854.3apmv-9/ita/570950.158.158.658.437.839.629.329.228.628.837.837.937.536.639.439.492.7 - 46.542.655.2apmv-10/flk/32437.839.539.939.561.760.329.028.132.432.246.348.148.038.261.461.438.939.1 - 48.046.5apmv-11/fra/10021231.632.633.933.942.441.831.033.032.532.239.939.741.736.040.440.432.733.339.4 - 43.9apmv-12/ita/3920_167.355.154.653.838.137.329.528.532.131.937.437.737.936.536.136.152.353.237.832.7- ) . the deduced amino acid sequence of the putative cleavage site of the 
 apmv / shimane f gene was qvrenr / lvg ( fig . 3fig . ndv ( lentogenic ) ; strain 
 lasota , ndv ( velogenic ) ; strain herts 33 , apmv-4/hk / d3 ; apmv-4/duck / hong kong / d3/1975 , 
 apmv-5/kunitachi ; apmv-5/budgerigar/ japan / kunitachi/1974 , apmv-6/twn / y1 ; 
 apmv-6/duck / taiwan / y1/1998 , apmv-8/de ; apmv-8/goose / delaware/1053/1976 , apmv-9/ny ; 
 duck / new york/22/1978 , apmv-10/flk/324 ; apmv10/penguin / falkland islands/324/2007 , 
 apmv-11/fra/100212 ; apmv-11/common_snipe / france/100212/2010 . this resembled the motif , a pair of single basic residues , of the lentogenic ndv . 
 the phylogenetic tree of the f gene constructed with apmvs and members of subfamily 
 paramyxovirinae revealed that apmv / shimane67 is a member of genus 
 avulavirus and is grouped with ndv , apmv-9 and apmv-12 , while distinct 
 from these apmv serotypes ( fig . the phylogenetic tree was generated using the 
 neighbor - joining algorithm with 1,000 bootstrap replicates in clustal x. ) . ndv ( lentogenic ) ; strain 
 lasota , ndv ( velogenic ) ; strain herts 33 , apmv-4/hk / d3 ; apmv-4/duck / hong kong / d3/1975 , 
 apmv-5/kunitachi ; apmv-5/budgerigar/ japan / kunitachi/1974 , apmv-6/twn / y1 ; 
 apmv-6/duck / taiwan / y1/1998 , apmv-8/de ; apmv-8/goose / delaware/1053/1976 , apmv-9/ny ; 
 duck / new york/22/1978 , apmv-10/flk/324 ; apmv10/penguin / falkland islands/324/2007 , 
 apmv-11/fra/100212 ; apmv-11/common_snipe / france/100212/2010 . the phylogenetic tree was generated using the 
 neighbor - joining algorithm with 1,000 bootstrap replicates in clustal x. the typing of apmvs has mainly been conducted by hi test . in addition , because the nucleotide sequence data from various apmvs have been 
 collected in dna databases , genetic analysis has also been attempted to classify new apmv 
 isolates , and the results obtained have demonstrated the effectiveness of this approach 
 [ 4 , 12 , 19 ] . on hi test , the anti - apmv / shimane67 serum reacted 
 strongly ( hi titer of 1:1,280 ) with homologous virus and did not react with the other apmvs 
 ( hi titer of 1 : < 40 ) . in addition , sera against other apmvs ( ndv , apmv-2 , 3 , 4 , 6 and 7 ) 
 showed weaker reactivity with apmv / shimane67 than with homologous viruses ( table 2 ) . these results indicate that apmv / shimane67 
 is serologically distinct from ndv , apmv-2 , 3 , 4 , 6 and 7 . anti - ndv , apmv-4 and 7 sera 
 showed some cross reactivity ( hi titer of 1:3201:1,280 ) with apmv / shimane67 . it is possible 
 that this cross reactivity is caused by high titers of hyperimmune sera ( hi titer of more 
 than 1:2,560 against homologous viruses ) . the sera used in this study were prepared by 
 multiple immunization of apmv antigen . miller et al .   also reported cross reactivity on hi test of apmvs using hyperimmune 
 serum . analysis of the f gene of apmv / shimane67 also revealed the relationship of this virus with 
 other apmvs . the orf of the apmv / shimane67 f gene was 1,638 nucleotides long and encoded 545 
 amino acids . although these nucleotide and amino acid lengths for the f gene were the same 
 as those of apmv-6/duck / italy/4524 - 2/07 , the 
 identities of nucleotide and amino acid sequences between apmv / shimane67 and 
 apmv-6/duck / italy/4524 - 2/07 were low ( 45.3% and 37.3% , respectively ) ( table 3 ) . the amino acid sequence at the f protein cleavage site 
 of apmv / shimane67 was deduced as being qvrenr / lvg , which resembles the motif of lentogenic 
 ndv ( fig . this motif was observed in the f 
 proteins of apmv / shimane and ndv , apmv-9 and 12 , and interestingly , these four apmvs 
 containing conserved amino acid sequence motifs at the f protein cleavage site also formed 
 one phylogenetic cluster ( fig . the 
 phylogenetic tree showed that apmv / shimane67 is a member of genus 
 avulavirus , is distinct from other apmvs and showed close relationship 
 with apmv / wigeon / italy/3920 - 1/2005 ( apmv-12/ita/3920 - 1 ) . apmv-2 , 3 and 6 viruses have 
 antigenic differences constituting the antigenic subgroup within each serotype [ 3 , 9 , 18 ] . similar situations in phylogenetic analysis were 
 also observed in the nucleotide and amino acid identities of the f gene ( table 3 ) . apmv / shimane67 exhibited the highest 
 nucleotide and amino acid identities ( 62.7% and 67.3% , respectively ) with apmv-12 . these 
 identities were lower than those observed between the subgroups , apmv-2/yucaipa and 
 apmv-2/bangor ( 69.8% in nucleotide and 79.1% in amino acid sequence ) , and apmv-3/nld and 
 apmv-3/wi ( 66.8% in nucleotide and 70.1% in amino acid sequence ) ( table 3 ) . these observations confirm a distant relationship 
 corresponding to apmv serotype between apmv / shimane67 and apmv-12/ita/3920 - 1 . the pathogenicity of apmv / shimane67 to chickens was evaluated by mdt and icpi tests . mdt 
 ( 120 hr < ) and icpi ( 0.0 ) of apmv / shimane67 were applicable to the lentogenic ndv and 
 revealed that the virus had no virulence in chickens . the amino acid sequence at the f 
 protein cleavage site is the major determinant of ndv pathogenicity to chickens . as 
 described above , the putative cleavage site of the apmv / shimane f protein is qvrenr / lvg , 
 which resembles lentogenic ndv . the 
 introduction of an amino acid sequence of velogenic ndv at the f protein cleavage site by 
 reverse genetics did not increase the virulence of apmv-2 , 4 and 7 in chickens [ 7 , 17 , 22 ] . however , it has been reported that lentogenic ndv 
 isolated from wild geese changed to velogenic virus by amino acid substitutions in the f 
 protein cleavage site during serial passage in chickens . whether the virulent variant of apmv / shimane67 will emerge with multiple 
 passaging of apmv / shimane67 in chickens is of interest . recently , whole genome sequences from various apmv serotypes have been reported , and the 
 classification of apmvs using those whole genome sequence data has been proposed [ 4 , 12 , 14 , 19 ] . in this 
 study , only the f gene of apmv / shimane67 was identified ; however , data obtained have 
 suggested that apmv / shimane67 is a novel apmv serotype , apmv-13 . to emphasize this idea , the 
 full - length genome sequence of apmv / shimane67 needs to be determined .
an avian paramyxovirus ( apmv ) isolated from goose feces ( apmv / shimane67 ) was 
 biologically , serologically and genetically characterized . 
 apmv / shimane67 showed typical 
 paramyxovirus morphology on electron microscopy . 
 on hemagglutination inhibition test , 
 antiserum against apmv / shimane67 revealed low reactivity with other apmv serotypes and 
 vice versa . 
 the fusion ( f ) protein gene of apmv / shimane67 contained 
 1,638 nucleotides in a single open reading frame encoding a protein of 545 amino acids . 
 the cleavage site of f protein contained a pair of single basic amino 
 acid ( vrenr / l ) . 
 the nucleotide and deduced amino acid sequences of the f gene 
 of apmv / shimane67 had relatively low identities ( 42.962.7% and 28.967.3% , respectively ) 
 with those of other apmvs . 
 phylogenetic analysis showed that apmv / shimane67 was related to 
 ndv , apmv-9 and apmv-12 , but was distinct from those apmv serotypes . 
 these results suggest 
 that apmv / shimane67 is a new apmv serotype , apmv-13 .