during october 2009october 2010 , a total of 448 respiratory specimens were obtained from 448 patients ( 258 male patients and 190 female patients ) with rtis and fevers in a virus surveillance system in osaka , japan ( 9 ) . the mean  sd age of the patients was 41.4  53.7 months ( range < 1404 months ) , and 351 ( 78.3% ) were < 5 years of age .
procedures for viral nucleic acid extraction and cdna synthesis have been reported ( 9 ) .
pcr for detecting hrv and enterovirus was conducted by using evp4 and ol68 - 1 primers , which detected hrv and human enterovirus , respectively , in amplicons of 530 and 650 bp , respectively ( 7 ) .
results showed 178 positive specimens ( 140 for hrv , 16 for human enterovirus , 7 for hrv and human enterovirus , and 15 for an unexpected amplicon of 600 bp ) . to identify the 600-bp amplicon , we sequenced viral protein 4 ( vp4 ) and vp1 genes .
blast analysis ( www.ncbi.nlm.nih.gov/ ) showed that these isolates had high identity with ev68 vp4 ( 98.5%99.5% with the pav25426868 strain [ genbank accession no
. hm370293 ] ) and vp1 ( 96.7%97.5% with the md021 strain [ genbank accession no
phylogenetic analysis using vp1 sequences ( 14 of 15 osaka strains were sequenced ) demonstrated that osaka strains were clustered in 1 group and differed from previously reported strains ( figure 2 ) .
monthly distribution of enterovirus 68 ( ev68 ) in osaka , japan , october 2009october 2010 , bars indicate no .
phylogenetic tree of enterovirus 68 viral protein 1 gene sequences constructed by using a 927-nt sequence corresponding to nt sequence 23553281 in strain 3799 , osaka , japan , october 2009october 2010 .
genbank accession numbers for strains used in this analysis were fermon , ay426531 ; 3799 , ef107098 ; jpoc10200 , ab601872 ; jpoc10290 , ab601882 ; jpoc10373 , ab601873 ; jpoc10378 , ab601883 ; jpoc10396 , ab601884 ; jpoc10402 , ab601874 ; jpoc10404 , ab601885 ; jpoc10412 , ab601875 ; jpoc10441 , ab601876 ; jpoc10445 , ab601877 ; jpoc10471 , ab601878 ; jpoc10573 , ab601879 ; jpoc10616 , ab601880 ; jpoc10618 , ab601881 ; md021 , ay426491 ; tx021 , ay426495 ; mo00 , ay426493 ; wi00 , ay426494 ; mn98 , ay426497 ; tx99 , ay426498 ; md99 , ay426499 ; tx03 , ay426500 ; mn89 , ay426489 ; ny93 , ay426490 ; ev70 , d00820 ; and ev94 , dq916376
nucleotide and amino acid identities among 4 osaka ev68 ( jpoc10 - 290 , 378 , 396 , and 404 strains ; nt 5017265 corresponding to the 3799 strain ) , fermon , and 3799 strains were determined . to determine sequences , we synthesized cdna by using specific primers and amplified 4 segments ( nt 1601153 , 5433391 , 31324032 , and 37477333 corresponding to the 3799 strain ) .
the fermon and 3799 strains are the only ev68 strains for which complete genome sequences are available .
the 3799 strain was isolated from a 6-year - old girl with pneumonia in 1998 ( h. norder , pers .
the sequenced region coded partial 5 untranslated regions ( utrs ) and all structural and nonstructural viral proteins ( vp43d ) .
identities between strains were calculated by using bioedit version 7.09 ( www.mbio.ncsu.edu/bioedit/bioedit.html ) ( table 2 ) . *
osaka strains , jpoc10 - 290 , jpoc10 - 378 , jpoc10 - 396 , and jpoc10 - 404 ; vp , viral protein . among osaka strains ,
nucleotide and amino acid sequences were highly conserved ( nt identity 96.5%100% and aa identity 98.6%100% ) .
in contrast , osaka strains had lower similarities with the fermon strain ( nt identity 83.5%91.7% and aa identity 90.6%100% ) than with the 3799 strain ( nt identity 91.9%98.4% and aa identity 95.4%100% ) . when we compared individual viral proteins in osaka strains with those in the fermon strain , no gene except for vp4 showed > 90% nt sequence identity ; gene 2b showed the lowest identity ( 83.5%85.8% ) .
regarding amino acids , < 95% identity was observed in vp1 , vp2 , and vp3 in the fermon strain , and no genes with < 95% aa identity were found in the 3799 strain in contrast with osaka strains .
moreover , no integration or deletion of nucleotides was observed in vp43d sequences among osaka , fermon , and 3799 strains . to clarify why ev68 osaka strain genomes were smaller than those of other enteroviruses and the ev68 fermon strain ( 7 ) , we aligned the partial 5 utr sequences ( nt 541820 corresponding to the fermon strain ) of 4 osaka , fermon , and 3799 strains ( figure a1 ) .
results showed that the osaka and 3799 strains had deletions at nt 681704 and 717727 in contrast with the fermon strain .
moreover , a 1-nt deletion in osaka strains was identified at nt 641 in contrast with the fermon and 3799 strains .
because 14 patients with ev68 were detected during 20062009 ( 8) , detection of 15 patients with ev68 during a 4-month period suggests an ev68 epidemic in the summer of 2010 in japan .
phylogenetic analysis with vp1 sequences showed that osaka strains differed genetically from previously reported strains . for precise analysis of osaka , fermon , and 3799 strains ,
results showed that osaka strains more closely resembled the 3799 strain than the fermon strain .
alignment of partial 5 utr sequences showed that osaka and 3799 strains had deletions in 2 regions in contrast with the fermon strain , and the amplicon was shorter than expected .
the 5 utr of enterovirus contains an internal ribosome entry site ( 10 ) that is associated with translational efficiency and virulence of the enterovirus ( 11,12 ) .
deleted regions of osaka strains appear to be in the flanking region between the internal ribosome entry site and an open reading frame ( 1 ) .
detection of ev68 in numerous patients was reported in france during 2008 ( 4 ) and italy during 20082009 ( 13 ) . because this deletion was found in the 3799 strain in 1998 , recent detection of ev68 in japan
reported that serum samples from 281 pregnant women in finland in 1983 , 1993 , and 2002 had high titers of neutralizing antibody against ev68 ( 14 ) .
all ev68-positive patients in this study were < 5 years of age and had lower respiratory tract inflammation .
seroepidemiologic studies in finland showed that most adults might have been previously infected with ev68 and therefore might have neutralizing antibodies ( 14 ) . increased detection of ev68 , especially in infants and children
this result indicates that ev68 can replicate in blood and may damage the central nervous system .
ev68 was detected in cerebrospinal fluid of a young adult patient with acute flaccid paralysis ( 5 ) .
epidemiologic data for ev68 are lacking , and little information is available regarding virologic characteristics .
if one considers results of phylogenetic analyses and nucleotide and amino acid identities , evolutionary changes might have occurred in ev68 .
our results show the potential role of ev68 infection in infants and children with rtis .
 enterovirus 68 strains were detected in 14 specimens from children with respiratory tract infections and 1 specimen from a child with febrile convulsions during 2010 in osaka , japan . 
 these strains had deletions in the 5 untranslated region and were genetically different from reported strains . 
 this virus is associated with respiratory tract infections in japan . 