extraction of viral rna , rt - pcr and nucleotide sequencing : the isolation and characterization of apmv / shimane/67 have been previously described 
 .
viral genomic rna was extracted from infected allantoic fluid using qiaamp viral rna mini kit ( qiagen , tokyo , 
 japan ) , according to the manufacturer s instructions manual .
the cdna of the apmv / shimane67 genome was synthesized using primescript reverse transcriptase ( takara 
 bio , otsu , japan ) and amplified using sapphireamp fast pcr master mix ( takara bio ) and pairs of oligonucleotide primers .
after purification from agarose gel using qiaquick gel extraction kit ( qiagen ) , pcr products were sequenced using 
 bigdye terminater v3.1 sequencing kit ( applied biosystems , foster city , ca , u.s.a . ) and analyzed using the 3130 xl genetic analyzer ( applied biosystems ) .
determination of 3- and 5-ends of the viral genome sequence : the 3-end of the viral genome ( le region ) sequence of apmv / shimane67 was 
 determined by a method previously described . to determine the 5-end of the viral genome ( tr region ) , cdna from the 
 5-end of the genome
was amplified using smart pcr cdna synthesis kit ( clontech , palo alto , ca , u.s.a . ) and virus - specific primers , according to the 
 manufacturer s instructions manual .
analysis of nucleotide and deduced amino acid sequences : the molecular weight ( mw ) and isoelectric point ( pi ) of protein were calculated by 
 compute pi / mw tool ( http://web.expasy.org/compute_pi/ ) .
the transmembrane region of hn protein was predicted by the 
 sosui system ( http://harrier.nagahama-i-bio.ac.jp/sosui/sosui_submit.html ) .
the alignment of nt and deduced amino acid 
 ( aa ) sequences and calculation of evolutionary distance in nt substitutions per site were conducted using the clustal x program .
phylogenetic trees were generated by neighbor - joining method   with 1,000 bootstraps using clustal x 
 program and then visualized with njplot .
the nucleotide sequence data reported in this study have been deposited in 
 the ddbj database under the accession number lc041132 .
whole genome sequences of other apmvs for comparison with apmv / shimane67 were from the following sources 
 ( abbreviation and accession number ) : apmv-1/goose / alaska/415/91 ( apmv-1/415 , ab524405 ) ; apmv-1 strain lasota ( apmv-1/lasota , af077761 ) ; apmv-1 strain sf02 
 ( apmv-1/sf02 , af473851 ) ; apmv-2/chicken / california / yucaipa/56 ( apmv-2/yukaipa , eu338414 ) ; apmv-2/finch / northern ireland / bangor/73 ( apmv-2/bangor , hm159995 ) ; 
 apmv-3/parakeet / netherland/449/75 ( apmv-3/nld , eu403085 ) ; apmv-3/turkey / wisconsin/68 ( apmv-3/wi , eu782025 ) ; apmv-4/duck / hong kong / d3/75 ( apmv-4/hk / d3 , fj177514 ) ; 
 apmv-4/kr / yj/06 ( apmv-4/kr / yj , eu877976 ) ; apmv-5/budgerigar / kunitachi/74 ( apmv-5/kunitachi , gu206351 ) ; apmv-6/duck / hong kong/18/199/77 ( apmv-6/hk / d199 , eu622637 ) ; 
 apmv-6/duck / italy/4524 - 2/07 ( apmv-6/ita/4524 - 2 , gq406232 ) ; apmv-7/dove / tennessee/4/75 ( apmv-7/tn , fj231524 ) ; apmv-8/goose / delaware/1053/76 ( apmv-8/de , fj215863 ) ; 
 apmv-8/pintail / wakuya/20/78 ( apmv-8/wakuya , fj215864 ) ; apmv-9/duck / new york/22/78 ( apmv-9/ny , eu910942 ) ; apmv-10/penguin / falkland islands/324/2007 ( apmv-10/flk , 
 hm147142 ) ; apmv-11/common snipe / france/100212/2010 ( apmv-11/fra , jq886184 ) ; and apmv-12/wigeon / italy/3920 - 1/2005 ( apmv-12/ita/3920 - 1 , kc333050 ) .
genomic features of apmv / shimane67 : the genome characteristics of apmv / shimane67 and some other apmvs are summarized in table 1table 1.comparison of nucleotide and amino acid length between apmv / shimane67 , apmv-1 , -9 and-12geneapmv / shimane67apmv-1/lasotaapmv-1/415apmv-1/sf02apmv-9/nyapmv-12/ita/3920 - 1full genome ( nt)16,14615,18615,19815,19215,43815,132leader ( nt)555555555555trailer ( nt)7761141141144760n3-noncoding ( nt)606666666666orf ( nt)1,4821,4701,4701,4701,4701,4825-noncoding ( nt)179210210217192162total ( nt)1,7211,7461,7461,7531,7281,710amino acid493489489489489493n - p intergenic ( nt)14221197p3-noncoding ( nt)9583838311395orf ( nt)1,1941,1881,2001,1881,2601,2185-noncoding ( nt)223180180180248190total ( nt)1,5121,4511,4631,4511,6211,503amino acid397395399395419405p - m intergenic ( nt)111163m3-noncoding ( nt)343434343434orf ( nt)1,1011,0951,0951,0951,0951,0955-noncoding ( nt)200112112112161151total ( nt)1,3351,2411,2411,2411,2901,280amino acid366364364364364364m - f intergenic ( nt)21113011f 3-noncoding ( nt)454646465552orf ( nt)1,6381,6621,6621,6621,6561,6415-noncoding ( nt)1758484846788total ( nt)1,8581,7921,7921,7921,7781,781amino acid545553553553551546f - hn intergenic ( nt)143131312261hn3-noncoding ( nt)929191919791orf ( nt)1,8331,7341,8511,7161,7401,8455-noncoding ( nt)14517759195293136total ( nt)2,0702,0022,0012,0022,1302,072amino acid610577616571579614hn - l intergenic ( nt)25474847042l3-noncoding ( nt)131111111111orf ( nt)6,6006,6156,6156,6156,6336,6095-noncoding ( nt)15077777769106total ( nt)6,7636,7036,7036,7036,7136,727amino acid2,2012,2042,2042,2042,2102,202a ) the nucleotide sequence of apmv / shimane67 f gene was reported by yamamoto et al . .. the genome of apmv / shimane67 comprised 16,146 nt that was slightly au - rich ( a 25.9% , c 22.1% , g 20.5% and u 31.5% ) .
the genome of apmv / shimane67 
 contained six viral protein genes in the order 3-n - p - m - f - hn - l-5 , which was identical to other apmvs , except for apmv-6 .
the sh protein gene existing in the 
 apmv-6 genome was not present in the apmv / shimane67 genome .
the full genome sequence of apmv / shimane67 had the highest nt identity with apmv-12/ita/3920 - 1 
 ( 62.2% ) , intermediate nt identities with apmv-1 and -9 ( 53.7%55.0% ) , and lower nt identities with apmv-2 , -3 , -4 , -5 , -6 , -7 , -8 , -10 and -11 ( 41.9%44.8% ) 
 ( table 2table 2.nucleotide ( nt ) and deduced amino acid ( aa ) sequence identities between apmv / shimane67 and other apmvs ( % ) virusfull genomenp vmhnlntaantaaaantaantaantaaapmv-1/lasota55.058.657.953.842.338.556.753.356.955.156.654.4apmv-1/41555.059.058.353.640.534.956.553.956.155.356.755.6apmv-1/sf0254.658.456.452.342.837.656.352.856.155.157.055.5apmv-2/yucaipa44.847.939.839.624.225.243.127.844.434.146.337.5apmv-2/bangor44.647.039.340.122.924.443.827.843.334.546.432.0apmv-3/nld41.948.538.741.023.720.540.225.243.837.243.633.2apmv-3/wi41.947.738.640.024.219.341.125.545.436.943.833.4apmv-4/hk / d342.348.837.040.521.724.139.424.544.236.543.432.5apmv-4/kr / yj42.348.437.039.722.525.140.824.444.036.343.132.6apmv-5/kunitachi43.548.736.940.922.925.243.429.943.433.846.437.5apmv-6/hk / d19942.949.140.641.322.426.743.629.644.331.947.538.4apmv-6/ita/4524 - 243.449.040.242.922.726.143.630.442.930.747.638.5apmv-7/tn44.849.139.138.720.026.744.230.345.237.947.540.3apmv-8/de44.847.839.240.624.927.242.429.543.834.247.139.1apmv-8/wakuya44.747.739.440.625.226.742.028.743.433.847.339.1apmv-9/ny53.758.656.051.240.432.155.350.854.554.756.053.4apmv-10/flk44.647.940.439.023.323.342.028.143.435.246.438.0apmv-11/fra43.048.140.240.123.726.638.925.845.537.447.040.1apmv/12/ita/3920 - 162.267.674.461.451.345.265.173.661.360.563.165.5 ) .
a ) the nucleotide sequence of apmv / shimane67 f gene was reported by yamamoto et al . .
the le sequence of apmv / shimane67 was 55 nt in length , which was the same as that of other apmvs .
in contrast , the tr sequence of apmv / shimane67 was 776 nt in 
 length , which was the longest in the avulavirus genus .
marcos et al .   reported that the first 18 nt of 
 the le and tr regions and thrice - repeated motif ( 3-nnnngc-5 ) at 7390 nt of the apmv-1 genomic and antigenomic rna functioned as promoters of viral genome 
 replication .
fourteen nt of the le and tr sequences , 
 with the exception of the 9th nt from the 3- and 5-terminal of the genome , were complementary in the apmv / shimane67 genome ( fig .
alignment of the ( b ) 3-leader and ( c ) 5-trailer regions of the sequences from apmv / shimane67 and other apmvs .
identical nucleotides with 
 apmv / shimane67 and gaps are shown by dots and dashes , respectively .
twelve nt of the 3 le and 11 nt of the 5 tr of the apmv / shimane67 genome were relatively conserved with those of other apmvs and were completely 
 matched with apmv-1 ( fig .
the three times repeated motifs , 3-gguggc-5 , 
 3-acaagc-5 and 3-ucaggc-5 , and 3-auuucc-5 , 3-uccagc-5 and 
 3-uucagc-5 were found in 7390 nt from the 3-terminus of the genome and antigenome of apmv / shimane67 , respectively .
the gs signal of 
 apmv / shimane67 was well preserved , and its consensus sequence was  acgggcagaa 
2.(a ) the gene - start , gene - end and intergenic sequences of apmv / shimane67 .
( b ) alignment of the consensus gene - start and gene - end sequences from 
 apmv / shimane67 and other apmvs .
in contrast , the preservation of ge signal sequences of apmv / shimane67 was relatively low .
the consensus sequence of the apmv / shimane67 ge signal was 
 ttaaga56 , whereas that of the m gene diverged at positions 1 ( a ) , 2 ( a ) , 3 ( t ) and 5 ( t ) .
the hn gene had one nt difference at the fifth position 
 ( t ) ; whereas the l gene also contained two nt differences at the second ( a ) and third ( g ) positions .
apmv / shimane67 gs and ge sequences had similarities with 
 those of apmv-1 , -9 and -12 ( fig .
p - m , m - f , f - hn and hn - l junctions was 14 nt , one nt , two nt , 14 nt and 25 nt , 
 respectively .
the last nt at igs of apmv / shimane67 was t at all times , and this could act with the gs sequences to initiate mrna transcription .
alignment of the ( b ) 3-leader and ( c ) 5-trailer regions of the sequences from apmv / shimane67 and other apmvs .
identical nucleotides with 
 apmv / shimane67 and gaps are shown by dots and dashes , respectively .
( a ) the gene - start , gene - end and intergenic sequences of apmv / shimane67 .
( b ) alignment of the consensus gene - start and gene - end sequences from 
 apmv / shimane67 and other apmvs .
n gene and n protein : the n gene of apmv / shimane67 was 1,721 nt in length and contained an open reading frame ( orf ) that encoded 493 aa , with mw 
 of 54,045 da and pi of 5.29 .
the nt sequence of apmv / shimane67 n gene orf was 67.6% identical with apmv-12/ita/3920 - 1 , 58.4%59.0% identical with apmv-1 and -9 , 
 and 47.0%49.1% identical with the rest of the apmvs .
the predicted aa sequence of apmv / shimane67 n gene demonstrated 74.4% , 56.0%58.3% and 36.9%40.6% 
 identities with apmv-12/ita/3920 - 1 , apmv-1 and -9 and the rest of the apmvs , respectively ( table 2 ) .
there are three 
 highly conserved regions [ region 1 , qxw(i , v)xxxk(a , c)xt ; region 2 , fxxt(i , l)(r , k)(g , a)(l , i , v)xt ; and region 3 , fxxxxypxxsamg ] ( where x represents any amino 
 acid and  represents an aromatic amino acid and , either of the residues in parentheses can be present at that position ) in the central domain of the n protein of 
 the subfamily paramyxovirinae . among these regions ,
region 3 is particularly important , because this 
 region is thought to be involved in the n 
the n protein of apmv / shimane67 contained aa 
 sequences similar to these motifs : 
 qiwvtlakamt , 
 ffltlkygint and 
 fapaeyslmysfsmg ( fig . 

the motifs are shown at the upper lines ( x and  represent any amino acid and 
 aromatic amino acid , respectively ) .
numbers indicate the amino acid 
 positions of the apmv / shimane67 n protein . ) .
the 7th ( p ) and 13th ( a ) aa of region 3 motif were replaced with s and s , respectively .
the aa sequence of region 2 was 
 completely identical among apmv / shimane67 , apmv-1 and apmv-12 .
the motifs are shown at the upper lines ( x and  represent any amino acid and 
 aromatic amino acid , respectively ) .
p gene and p / v proteins : the p gene of apmv / shimane67 genome was 1,512 nt long .
the p gene of avulaviruses encodes two proteins , p and v [ 35 , 38 ] .
the p protein is expressed from mrna that is directly transcribed from the 
 genomic rna .
the nt identities of apmv / shimane67 p protein orf were 61.4% with apmv-12/ita/3920 - 1 , 51.2%53.8% with apmv-1 and -9 , and 38.7%42.9% with the other 
 remaining apmvs ( table 2 ) .
the v proteins is produced through mrna that contains insertion of one non - template g 
 residues at the rna editing site .
the nt at positions 436444 of the p gene orf , 5-aaaaaaggg-3 ( mrna sense ) , was predicted as the rna editing site of 
 apmv / shimane67 ( fig .
4.alignment of ( a ) nucleotide sequences of rna editing sites ( mrna sense ) and ( b ) c - terminal regions of v protein .
numbers 
 indicate the nucleotide or amino acid positions of the ( a ) p gene orf and ( b ) v protein of apmv / shimane67 , respectively . ) .
this nt sequence was completely identical with that of apmv-5/kunitachi , apmv-6/hk / d199 , apmv-7/tn , apmv-9/ny and apmv-12/ita/3920 - 1 .
the deduced aa 
 lengths of p and v proteins were 397 aa ( mw 41,992 da , pi 6.22 ) and 241 aa ( mw 25,929 da , pi 4.80 ) , respectively .
these two proteins shared the n - terminal 148 aa . 
 the aa identities in p and v proteins between apmv / shimane67 and other apmvs were comparatively low , ranging from 20.0% ( apmv-7/tn ) to 51.3% ( apmv-12/ita/3920 - 1 ) 
 and 19.3% ( apmv-3/wi ) to 45.2% ( apmv-12/ita/3920 - 1 ) , respectively ( table 2 ) .
seven cysteine residues ( c , 
 c , c , c , c , c and c ) and the hrre and 
 wcnp motifs were conserved at the c - terminus of apmv / shimane67 v protein as well as in other apmvs ( fig .
4b ) . alignment of ( a ) nucleotide sequences of rna editing sites ( mrna sense ) and ( b ) c - terminal regions of v protein .
numbers 
 indicate the nucleotide or amino acid positions of the ( a ) p gene orf and ( b ) v protein of apmv / shimane67 , respectively .
m gene and m protein : the m gene of apmv / shimane67 contained 1,335 nt and encoded a protein with 366 aa with mw of 39,902 da and pi of 9.63 .
the 
 orf and predicted aa sequence of apmv / shimane67 m gene had identities in 65.1% and 73.6% with apmv-12/ita/3920 - 1 , 55.3%56.7% and 50.8%53.9% with apmv-1 and -9 , 
 and 38.9%44.2% and 24.4%30.4% with the remaining apmvs , respectively ( table 2 ) .
there are two functional aa 
 sequences one is nuclear localization signal ( nls ) , and the other is the late domain in the m protein of apmv-1 [ 7 , 10 ] .
nls comprised a bipartite clustering of basic amino acids ( e.g. , 
 rkgkkvtfdklekkirs of apmv-1/lasota m 
 protein ) . in the apmv / shimane67 m protein , the putative bipartite nls motif 
 ( kgnkisvdklelkirr ) 
 was found at positions 248263 aa ( fig . 5afig .
5.alignment of ( a ) putative bipartite nuclear localization signal and ( b ) late domain of apmv m protein .
numbers indicate the amino acid positions of 
 the apmv / shimane67 m protein . ) .
the fpiv late domain that contributes to efficient viral release and replication was at positions 2326 aa of the apmv-1 m protein .
furthermore , a comparable sequence motif in other apmv m proteins was reported [ 37 , 44 , 45 ] .
a similar aa sequence motif ( fpvv ) was found at positions 2326 aa in the m 
 protein of apmv / shimane67 ( fig .
5b ) . alignment of ( a ) putative bipartite nuclear localization signal and ( b ) late domain of apmv m protein .
hn gene and hn protein : the hn gene for apmv / shimane67 comprised 2,070 nt with one orf that encoded a protein with 610 aa with mw of 67,492 da 
 and pi of 5.42 .
the nt and deduced aa of apmv / shimane67 hn gene had the highest identities with apmv-12/ita/3920 - 1 at 61.3% and 60.5% , followed by that with 
 apmv-1 and -9 at 54.5%56.9% and 54.7%55.3% , and other apmvs at 42.9%45.5% and 30.7%37.9% , respectively ( table 2 ) . 

the transmembrane region of apmv / shimane67 hn protein was predicted at positions 2547 by 
 the sosui system .
five n - glycosylation motifs ( n - x - s / t ) were found at positions 119 , 341 , 392 , 481 and 604 aa of 
 apmv / shimane67 hn protein ( table 3table 3.comparison of amino acid position and sequences of functional domains of the hn protein between apmv / shimane67 and other apmvsvirusamino acid positions of potential n - linked glycosylation sitesialic acid - binding motifsialic acid - binding and neuraminidase activity apmv / shimane67119 , 341 , 392 , 481 , 604nrkscsr , i , d , k , e , y , y , 
 e , r , r , y , e   apmv-1/lasota119 , 341 , 433 , 481 , 538 ................. apmv-2/yucaipa119 , 278 , 345 , 392 , 481 ................. apmv-3/nld33 , 53 , 58 , 115 , 309 , 322 , 380 , 493 , 494 .................
apmv-4/hk / d311 , 57 , 142 , 322 , 380 , 392 , 443 ................. apmv-5/kunitachi60 , 119 , 148 , 278 , 346 , 392 ................. apmv-6/hk / d199119 , 278 , 346 , 377 , 392 , 438 , 483 ................. apmv-7/tn60 , 119 , 145 , 278 , 343 , 377 , 392 , 484 , 513 , 564 ................. apmv-8/de119 , 278 , 392 , 507 ................. apmv-9/ny119 , 147 , 228 , 341 , 348 , 433 , 481 ................. apmv-10/flk119 , 147 , 278 , 352 , 432 , ................. apmv-11/fra147 , 150 , 266 , 278 , 345 , 392 , 431 , 479 , 484 ................. apmv-12/ita/3920 - 1119 , 147 , 341 , 348 , 594 ................. a ) numbering of amino acid residues corresponds to apmv / shimane67 hn protein sequnece .
b ) the identical amino acids between apmv / shimane67 and other apmvs 
 are shown by dot . ) .
two of these , at positions 119 and 392 aa , were relatively conserved among avulaviruses , because the hn proteins of apmv-1 , -2 , -5 , -6 , -7 , -8 , 
 -9 , -10 and -12 , and apmv-2 , -4 , -5 , -6 , -7 , -8 and -11 also contained the n - glycosylation motif at positions corresponding to the 119 and 392 aa , respectively . 

the sialic acid - binding motif nrkscs preserved among hn proteins of paramyxoviruses   was identified at positions 
 234239 of the hn protein of apmv / shimane67 ( table 3 ) .
twelve aa ( r , i , d , 
 k , e , y , y , e , r , r , y and e ) 
 engaged in sialic acid - binding and neuraminidase ( na ) activity of apmv-1 [ 8 , 9 , 14 ] were also completely conserved in the hn protein of apmv / shimane67 ( table 3 ) . 
 moreover , the hn protein of other apmvs perfectly possessed these aa . the aa sequence alignment of hn protein from apmvs demonstrated that the hn protein of 
 apmv / shimane67 , apmv-9/mallard / italy/5709/2007 and apmv-12/ita/3920 - 1 contained aa sequences corresponding to the 45 aa extension , which was found in the 
 c - terminal end of hn protein of lentogenic apmv-1 , such as strains d26 , ulster and 415 ( fig .
6.alignment of c - terminal 45 extension of apmv-1 hn protein and the corresponding regions of apmv / shimane67 , apmv-9 and 12 .
the genbank accession of apmv-1/d26 , ulster and apmv-9/ita/5709 is m19432 , m19478 and gu068587 , respectively . ) .
the removal of c - terminal 42 aa extension by proteolytic cleavage converts into the biologically active form of lentogenic apmv-1 hn protein [ 27 , 28 ] .
recently , yuan et al .   demonstrated that the c - terminal 45 aa extension of lentogenic apmv-1 hn protein autoinhibited receptor binding and catalytic activities by blocking 
 the na - active and second sialic binding sites .
in addition , the intermolecular disulfide bond formed by the cysteine residue at 596 aa in the c - terminal extension 
 was critical for the expression of autoinhibition of hn activities .
although there was no cysteine residue and aa identity in the c - terminal extension region of 
 apmv / shimane67 , the three extreme c - terminal residues ( swp ) , which masked the na active site in the apmv-1 hn protein , were identical to apmv-1 .
a ) numbering of amino acid residues corresponds to apmv / shimane67 hn protein sequnece .
b ) the identical amino acids between apmv / shimane67 and other apmvs 
 are shown by dot .
alignment of c - terminal 45 extension of apmv-1 hn protein and the corresponding regions of apmv / shimane67 , apmv-9 and 12 .
the genbank accession of apmv-1/d26 , ulster and apmv-9/ita/5709 is m19432 , m19478 and gu068587 , respectively .
l gene and l protein : the apmv / shimane67 l gene was 6,763 nt long and encoded a single orf , giving a deduced protein of 2,199 aa ( mw 248,209 da 
 and pi 6.86 ) .
the orf and aa sequences of apmv / shimane67 l gene were 63.1% and 65.5% identical with apmv-12/ita/3920 - 1 , and 56.0%57.0% and 53.4%55.6% identical 
 with apmv-1 and -9 , respectively .
in contrast , fewer identities ( 43.1%47.6% in nt and 32.0%40.3% in aa sequences ) were observed between apmv / shimane67 and 
 apmv-2 to -8 , apmv-10 and apmv-11 ( table 2 ) .
the l protein of non - segmented negative - strand rna viruses contains six 
 conserved aa domains ( i - vi ) . among these domains ,
domain iii corresponding to at positions 635826 aa of 
 apmv / shimane67 was relatively highly conserved among avulaviruses ( fig . 7afig .
7.alignment of ( a ) the conserved domain iii of l protein of non - segmented negative strand rna viruses and ( b ) the putative atp - binding site of the l protein . 
 the qgdnq and k - x18 - 21g - x - g - x - g motifs are underscored .
numbers indicate the amino 
 acid positions of the apmv / shimane67 l protein . ) .
the qgdnq sequence , identified in the motif c of domain iii as the putative active site for nucleotide polymerization , was found on the apmv / shimane67 l protein at positions 747751 aa . the putative atp - binding site comprising k - x1821-g - x - g - x - g was found 
 at positions 17561782 aa of domain iv of apmv / shimane67 l protein ( fig .
7b ) [ 12 , 
 32 ] . alignment of ( a ) the conserved domain iii of l protein of non - segmented negative strand rna viruses and ( b ) the putative atp - binding site of the l protein . 
 the qgdnq and k - x18 - 21g - x - g - x - g motifs are underscored .
phylogenetic analysis : to understand genetic relationships , phylogenetic trees were constructed based on the nt sequences of full length genome 
 and the n , p , m , hn and l genes of apmv / shimane67 and other viruses from the five members of the family paramyxovirinae 
 ( avulavirus , henipavirus , morbillivirus , respirovirus and rubulavirus ) 
 ( fig . 8fig .
the numbers at the 
 branches represent bootstrap values from 1,000 replicates . the number of nucleotide substitutions per site ( scale bar ) is shown . and supplementary figs .
essentially , all trees demonstrated similar grouping patterns , 
 except for apmv-7 and -11 , and clearly divided according to the five classifications of paramyxoviruses .
apmv / shimane67 was classified as a member of the genus avulavirus , but distinct from other 
 apmvs .
the apmv / shimane67 fell into the group that comprised apmv-1 , -9 and -12 and had the closest relationship with apmv-12/ita/3920 - 1 .
in the present study , we determined the nt sequences of n , p , m , hn and l genes , le , tr and igs of apmv / shimane67 . together with our previous study of the f gene 
 sequencing , whole genome sequencing of apmv / shimane67 was completed .
the genome of apmv / shimane67 basically had following common features with other apmvs : agreement with the  rule of six  ; gene order ( 3-n - p - m - f - hn - l-5 ) and 
 existence of gs and ge signal , and igs ; complementation of 3- and 5-terminus sequence ; existence of three times repeated motif ; existence of the putative rna 
 editing site of p gene .
in addition , the coding proteins of apmv / shimane67 genome contained following conserved aa sequence motifs with other apmvs : n protein 
 self - assembly motif ; cysteine - rich region and hrre and wcnp motifs of v protein ; putative bipartite nls motif and the late domain of m protein ; amino acids 
 constituting the sialic acid binding site of hn protein ; putative active site for nucleotide polymerization and atp - binding site of l protein .
these nt and aa 
 characteristics of apmv / shimane67 can contribute to the efficient replication and transcription of viral genome , and viral growth .
by contrast , the genome of apmv / shimane67 had differences with other apmvs in terms of some details , such as the nt length of whole genome , six genes ( n , p , m , 
 f , hn and l ) and igs , and predicted aa length of six genes , which were almost unique in apmv / shimane67 ( table 1 ) . the 
 genome size of apmv / shimane67 was 16,146 nt long .
this genome size is different from any other known apmvs genome and was the fifth longest among apmvs . with the 
 exception of apmv / shimane67 , there are four apmvs , which have genome longer than 16,000 nt : apmv-3 with 16,18216,272 nt ; apmv-5 with 17,262 nt ; apmv-6 with 
 16,17416,236 nt ; and apmv-11 with 17,412 nt [ 3 , 6 , 17 , 18 , 37 , 42 , 45 ] . the relatively large genome size of apmv / shimane67 is attributable to its long tr sequence ( 776 nt ) . the tr sequence 
 longer than 700 nt was also found in apmv-3/nld genome . however , the significance of these long tr sequence is 
 unclear , and a further study is needed to clarify this issue .
the phylogenetic trees indicated that apmv / shimane67 had the closest relationship with 
 apmv-12/ita/3920 - 1 .
when comparing full genome sequences , the evolutionary distance between apmv / shimane67 and apmv-12/ita/3920 - 1 was 0.357 nt substitutions per 
 site .
this distance was longer than those observed within serotypes , such as the distance between apmv-2/yucaipa and apmv-2/bangor at 0.291 , apmv-3/nld and 
 apmv-3/wi at 0.292 , and apmv-6/twn / y1 and apmv-6/ita/4524 - 2 at 0.265 .
these strains were antigenically and genetically divided into subgroups in each serotype 
 [ 2 , 18 , 39 , 45 ] .
thus , the apmv / shimane67 and apmv-12/ita/3920 - 1 had greater degrees of genetic diversity than that found within the subgroups of apmv-2 , -3 or -6 . 
 although there are no defined genetic criteria to differentiate the typing of apmvs , recent studies attempted and demonstrated that classification based on 
 genetic analysis was correlated with conventional serotyping [ 3 , 22 , 35 , 41 ] .
our previous serological analysis demonstrated that apmv / shimane67 was distinct 
 from apmv-1 , -2 , -3 , -4 , -6 and -7 .
moreover , sequence analysis of f gene of apmv / shimane67 indicated that 
 apmv / shimane67 was genetically diverse from other apmv serotypes .
thus , the results obtained in this study emphasized the possibility that apmv / shimane67 would be 
 a novel apmv type .
quite recently , karamendin et al .   reported the whole genome sequence of a novel 
 apmv isolated from a white fronted goose in northern kazakhstan ( genbank accession number ku64513 ) .
the genome length of apmv / shimane67 is 150 nt longer than that 
 of kazakhstan apmv , while genome nt identity of these strains was very high ( approximately 96% ) .
the deduced aa sequence identities of the n , p , m , f , hn and l 
 proteins between apmv / shimane67 and kazakhstan apmv are 99.6% , 99.2% , 98.9% , 99.6% , 99.1% and 99.6% , respectively .
although there is no serological evidence for 
 the close relationship between apmv / shimane67 and kazakhstan apmv , the genomic similarities of apmv / shimane67 and kazakhstan apmv suggest that these two strains 
 would be classified as the same serotype , apmv-13 .
 the nucleotide sequences of nucleocapsid protein ( n ) ; phosphoprotein ( p ) ; matrix protein ( m ) ; hemagglutinin - neuraminidase ( hn ) ; and large polymerase protein 
 ( l ) genes , 3-end leader , 5-end trailer and intergenic regions of the avian paramyxovirus ( apmv ) strain goose / shimane/67/2000 ( apmv / shimane67 ) were determined . 
 
 together with previously reported data on fusion protein ( f ) gene sequence [ 46 ] , the determination of the genome sequence 
 of apmv / shimane67 has been completed in this study . 
 the genome of apmv / shimane67 comprised 16,146 nucleotides in length and contains six genes in the order of 
 3-n - p - m - f - hn - l-5. the features of the apmv / shimane67 genome ( e.g. , nucleotide length of whole genome and each of the six genes , and predicted 
 amino acid length of each of the six genes ) were distinct from those of other apmv serotypes . 
 phylogenetic analysis indicated that although apmv / shimane67 was 
 grouped with apmv-1 , -9 and -12 , the evolutionary distance between apmv / shimane67 and these viruses was longer than that observed between intra - serotype 
 viruses . 
 these results show that the genome sequence of apmv / shimane67 contains specific characteristics and is distinguishable from other types of apmv . 