Sequence analysis for VP4 of enterovirus 71 isolated in Beijing during 2007 to 2008

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1 Journal of Microbes and Infection, March 2009, Vol. 4, No VP4 1, 2, 2, 2, 1, 2, 2, 2, 1, 2 1., ; 2., : ( EV71), EV71( 1, 2 ) EV71( 3, 2 ), RNA, - ( RT-PCR) VP4,, GenBank EV71 VP4, 8 EV71 VP4 207 bp, 69, ( Mr) EV71 VP4 94% 100%, GenBank EV71 VP4 82% 100%, EV71 VP4 VP ( : 7, 54 ; 7, 54 ), 8 EV71 VP4 EV71 VP4 100% 8 EV71 VP4 3 VP4 ( BrCr MS NCKU9822), 8 ( BJ97 BJ110B BJ110Y BJ4243) ( BJ25 BJ47 BJ65 BJ67) VP4, VP4, 8 EV71 C4, EV71 VP4, EV71 VP4, 2 EV71 C4 : 71 ; VP4 ; ; Sequence analysis for VP4 of enterovirus 71 isolated in Beijing during 2007 to 2008 LI Yu-Yun 1, 2, ZHU Ru-Nan 2, QIAN Yuan 2, 1, DENG Jie 2, SUN Yu 2, WANG Fang 2, ZHAO Lin- Qing 1, 2 1. Graduate School of Peking Union Medical College, Beijing , China; 2. Laboratory of Virology, Capital Institute of Pediatrics, Beijing , China Abstract: To understand the relationship between variation of enterovirus 71 ( EV 71) and its virulence, the sequence of the VP4 gene of EV71 virus was analyzed. Eight strains of EV71 isolated from clinical samples collected from infants and children with hand, foot and mouth disease in the Children s Hospital Affiliated to Capital Institute of Pediatrics in Beijing during 2007 to 2008 were used for comparison. Full length VP4 genes from these eight EV71 strains were amplified by reverse-transcriptase PCR ( RT-PCR) and sequenced after the amplicons were cloned into pucm-t. The sequences were compared with VP4 genes from EV71 in GenBank. Sequence analysis and type identification were performed by bioinformatics ( DNAStar). The full length of VP4 gene has 207 bp coding for a protein of 69 amino acids with estimated molecular weight of The nucleotide homology of VP4 genes among the eight strains were 94% 100% and homology of the deduced amino acids were 100%. The nucleotide sequence homology between these eight strains isolated in Beijing and those isolated in Fuyang, Shenzhen and Taiwan was : ( 2008BAI70B00) :, net Corresponding author: QIAN Yuan, yqianbjc@ 263. net

2 Journal of Microbes and Infection, March 2009, Vol. 4, No higher than those isolated elsewhere. The homology of deduced amino acid sequences encoded by VP4 between these eight isolates in Beijing and those in GenBank was 100% except one strain isolated from India. The amino acids at the 7th and 54th position of VP4 of the strain from India were different from others. There were many differences between the nucleotide sequences of VP4 from the eight Beijing strains and those from severe cases ( BrCr, MS and NCKU9822), as reported in the literature, whereas only a few nucleotide differences were observed between severe cases ( BJ97, BJ110B, BJ110Y and BJ4243) and mild cases ( BJ25, BJ47, BJ65 and BJ67) among these eight Beijing strains. Phylogenetic analysis of the VP4 sequences from these eight strains indicates that the EV71 viruses that circulated in Beijing during 2007 to 2008 should be classified as subtype C4. The VP4 genes from the isolates in Beijing from 2007 and 2008 were highly conserved. There was no consistent divergence in the sequences of VP4 between strains isolated from severe cases and those from mild cases, suggesting that the virulence of the EV71 virus does not seem to be related to the sequence of VP4. Key words: Enterovirus 71; VP4 gene; Hand, foot and mouth disease; Beijing, 2008,, 71 ( enterovirus 71, EV71) EV71,,, [ 1] EV71,, [ 2 ], EV71,,,?? 2 EV71 VP /,, 1. 2 Vero,,, Trizol ( Invitrogen ) RNA, ( ) M-MLV ( Invitrogen ) cdna, EV71 VP1 [ 3 ] PCR, 8, BJ25 BJ47 BJ4243 BJ65 BJ67 BJ97 BJ110B BJ110Y; BJ25 BJ47 BJ , 2008 BJ97 BJ110B, BJ25 BJ47 BJ110Y BJ4243, BJ65 BJ67 BJ97 BJ110B BJ110Y BJ4243 (, ), 1. 3 VP4 Kung [ 4 ] VP4 : 5 -CTACTTTGGGTGTCCGTGTT-3 ; : 5 -GGG( A) AACTTCCAG( A) TACCAT ( G) CC-3, 655 bp, VP4 200 bp VP4 VP4 248 bp Taq ( TOYOBO ) cdna VP4, 94 3 min, s, s, s, 35 pucm-t( ) T4 DNA 16, DH5,, 37, ( ) 1. 4 VP4 EV71VP4 GenBank, ( BJ25 BJ47 BJ65 BJ67 BJ4243 BJ97 BJ110B BJ110Y), EU EU EU EU EU EU EU EU862484, GenBank EV71 VP4 ( EU EU EU AY EU DQ DQ EU DQ AJ AB DQ AB239756

3 Journal of Microbes and Infection, March 2009, Vol. 4, No. 1 EU U22521 AY179602,,,,,,,,,,, ) DNAStar EditSeq MegAlign, 3 EV71 BrCr( U22521) MS ( U22522) [ 5 ] NCKU9822( AF136379) [ 6 ] EV71 GenBank EV71 VP , 1, 5 EV71( 7. 0%, 5 / 71), 4 EV71( 5. 7% ), 1 EV71 ( % ) , 11, 117 EV71( 59. 7%, 117 /196), 112 EV71 ( 60. 5%, 112 /185), 5 ( 45. 5%, 5 /11) ( BJ4243) 2 ( BJ25 BJ47), ( BJ97 BJ110B BJ110Y) 2 ( BJ65 BJ67) EV71 8 EV71 VP4 207 bp, 69, 94% 100%,, GenBank EV71 VP4 82% 100%, EV71 VP4 VP4 VP4 100%, 1 EV71( R13223-IND-01, GenBank AY179602) [ 7 ] VP4 2, 7 54, 7 54, 8 EV71 GenBank EV71 1, 1 8 EV71 VP4 GenBank EV71 VP4 ( % ) Tab 1. Nucleotide identities among VP4 of EV71 isolated in Beijing and those in GenBank( % ) Country/ District GenBank Number EU EU EU EU EU EU EU EU Beijing EU Beijing EU Beijing EU Beijing EU Beijing EU Beijing EU Beijing EU Beijing EU Fuyang EU Fuyang EU Fuyang EU Shenzhen AY Taiwan EU Korea DQ Malaysia DQ Australia EU Singapore DQ UK AJ Turkey AB Norway DQ Japan AB Viet Nam EU USA U Indian AY

4 Journal of Microbes and Infection, March 2009, Vol. 4, No EV71 GenBank 3 VP4 1 8 EV71 VP4 3 EV71( BrCr MS NCKU9822) VP4 8 EV71 VP4,, VP4 3 EV71 VP VP4 2, EV71 C4, 8 EV71 C4

5 Journal of Microbes and Infection, March 2009, Vol. 4, No. 1 The GenBank access numbers of BrCr, MS and NCKU9822 are U22521, U22522 and AF136379, respectively. 1 8 VP4 GenBank 3 VP4 Fig 1. Comparison of VP4 among 8 strains of EV71 isolated in Beijing and 3 strains of EV71 from severe cases in GenBank Referring Lin s method [ 8]. The strains of GenBank number of U22521, AY817061, AY123957, AY125984, AY125993, AY125986, AF119795, DQ341355, D and AY were selected as subtype A, B1, B2, B3, B4, C1, C2, C3, C4 and D. 2 VP4 Fig 2. Phylogenetic analysis based on VP4 3 EV VP1 ( ) VP2( ) VP3( ) VP4( ),,, 60, VP1 VP2 VP3, VP4, VP1, VP4, VP4, EV71 EV71, RNA ( Picornaradae) 1974 Schmidt, , EV71, ( 1975) ( 1978) ( 1986) ( 2000 ) ( ) EV71 A B C, A BrCr, B C 4 : B1 B4 C1 C4 Deshpande EV71 D [ 7 ]

6 Journal of Microbes and Infection, March 2009, Vol. 4, No. 1 21, EV71 CA16, EV71, [ 2 ], EV71,,, [ 2],,, 8 EV71 VP4,,, 1, 8 EV71 EV71 VP4, 1 8 EV71 VP4 3 EV71( BrCr MS NCKU9822) VP4, 8 EV71 VP4, BJ65 BJ110Y VP4,, 2, EV71 VP4 100%, VP4, [ 1] Chang LY, Huang LM, Gau SS, et al. Neurodevelopment and cognition in children after enterovirus 71 infection [ J]. N Engl J Med, 2007, 356( 12) : [ 2] Lin TY, Twu SJ, Ho MS, et al. Enterovirus 71 outbreaks, Taiwan: Occurrence and recognition[ J]. Emerg Infect Dis, 2003, 9( 3) : [ 3],,,. 71 A 16 [ J]., 2007, 28( 10) : [ 4] Kung CM, King CC, Lee CN, et al. Differences in replication capacity between enterovirus 71 isolates obtained from patients with encephalitis and those obtained from patients with herpangina in Taiwan[ J]. J Med Virol, 2007, 79( 1) : [ 5] Brown BA, Pallansch MA. Complete nucleotide sequence of enterovirus 71 is distinct from poliovirus[ J]. Virus Res, 1995, 39( 2-3) : [ 6] Yan JJ, Wang JR, Liu CC, et al. An outbreak of enterovirus 71 infection in Taiwan 1998: A comprehensive pathological, virological, and molecular study on a case of fulminant encephalitis[ J]. J Clin Virol, 2000, 17( 1) : [ 7] Deshpande JM, Nadkarni SS, Francis PP. Enterovirus 71 isolated from a case of acute flaccid paralysis in India represents a new genotype[ J]. Curr Sci, 2003, 84( 10) : [ 8] Lin KH, Hwang KP, Ke GM, et al. Evolution of EV71 genogroup in Taiwan from 1998 to 2005: An emerging of subgenogroup C4 of EV71 [ J]. J Med Virol, 2006, 78 ( 2) : ( : )