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1 DOH94-DC-1019 参 1

2 参 2

3 ABSTRACT Research purposes: Enterovirus 71 (EV71) and Coxsackievirus B (CVB) infections can lead to devastating clinical outcomes in children, with increasing number of severe cases worldwide lately. It would be important to study the molecular evolution of these viruses to implement the related measurements for prevention. Research Methods: The genetic and antigenic variability of EV71 strains isolated in Taiwan from 1998 to 2004 was evaluated by partial nucleotide sequencing of the VP1 gene and neutralization assay. We also analyzed the nucleic acid sequences of CVB3 isolated from 2004 and 2005 in Taiwan area. Main findings: Phylogenetic analyses showed that most EV71 from the 1998 epidemic belonged to the sub-genogroup C2, while small portion of them were the sub-genogroups B1 and B4. The following five years between , EV71 strains classified into the sub-genogroup B4 became most prevalent, with a conversion of the predominant sub-genogroup to the C4 in Antibodies raised in rabbits or collected from infected patients were able to neutralize EV71 virus stocks to high efficacies regardless of the sub-genogroup of the challenging virus. While there was high sequence homology for the CVB3 isolates from the year 3

4 of 2004 and 2005, they exhibited significant diversity from those isolated in Conclusions and Suggestions: The presence of phylogenetically distinct yet antigenically similar populations of EV71 in Taiwan is of concern for herd immunity and vaccine development. Analysis of sequences of the Taiwan CVB3 strains provided the molecular evolution, benefiting development of the approaches for control of the virus. Keywords: Enterovirus 71; Coxsackievirus B3; Molecular evolution; Phylogenetic tree; neutralizing antibody (acute flaccid paralysis) 4

5 RDHEp-2Vero (cytopathic effectcpe) ( IFA ) (Chemicon) 5

6 RNA (RT) 酶 (PCR) (virus stock) TRIZOLGibco, BRL isopropanol RNA 50 µl 5 µl 75 mm KCl50 mm Tris-HCl3 mm MgCl 2 10 mm DTT0.5 mm dntp RNasin 38 U/ul50 pmole (antisense primer) #162 #159: ACY ATG AAA YTG TGC AAG G #162: CCR GTA GGK GTR CAC GCR AC 23,24 VP1 cdnacdna 50 mm KCl10 mm Tris-HCl 1.5 mm MgCl 2 0.1Triton-X 100 dntp 5 units Taq polymerasepromega Inc, WI, USA 94 denature % agarose QIA quick-gel extraction kit (Qiagen) (RT) 酶 RT-PCR Sanger dideoxy cycle sequencing reaction (PE Applied Biosystems) cycle sequencing reaction RT-PCR 6

7 ( Phylogenetic tree ) Molecular Evolutionary Genetics Analysis (MEGA) version 2.1 Neighbor-joining Kimura 2 parameter (Bootstrap) 1, ~ µl PFU 37CO 2 0.5ml2% (Noble agar) (titer) 5CC TCID 7

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14 VP1 苷 ; (herd immunity) 14

15 1. Racaniello, V. R. (2001). Picornaviridae: the viruses and their replication. In Fields Virology, fourth ed., vol. 1, pp Edited by Knipe, D.M. & Howley, P.M. Philadelphia: Lippincott Williams 2. Brown BA, Pallansch MA. Complete nucleotide sequence of enterovirus 71 is distinct from poliovirus. Virus Res. Dec 1995;39(2-3): AbuBakar S, Chee HY, Al-Kobaisi MF, Xiaoshan J, Chua KB, Lam SK. Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia. Virus Res. May 1999;61(1): Fujimoto T, Chikahira M, Yoshida S, et al. Outbreak of central nervous system disease associated with hand, foot, and mouth disease in Japan during the summer of 2000: detection and molecular epidemiology of enterovirus 71. Microbiol Immunol. 2002;46(9): Ho M, Chen ER, Hsu KH, et al. An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group. N Engl J Med. Sep ;341(13): Lin TY, Twu SJ, Ho MS, Chang LY, Lee CY. Enterovirus 71 outbreaks, Taiwan: occurrence and recognition. Emerg Infect Dis. Mar 2003;9(3): McMinn P, Stratov I, Nagarajan L, Davis S. Neurological manifestations of enterovirus 71 infection in children during an 15

16 outbreak of hand, foot, and mouth disease in Western Australia. Clin Infect Dis. Jan ;32(2): McMinn P, Lindsay K, Perera D, Chan HM, Chan KP, Cardosa MJ. Phylogenetic analysis of enterovirus 71 strains isolated during linked epidemics in Malaysia, Singapore, and Western Australia. J Virol. Aug 2001;75(16): Chan LG, Parashar UD, Lye MS, et al. Deaths of children during an outbreak of hand, foot, and mouth disease in sarawak, malaysia: clinical and pathological characteristics of the disease. For the Outbreak Study Group. Clin Infect Dis. Sep 2000;31(3): Gilbert GL, Dickson KE, Waters MJ, Kennett ML, Land SA, Sneddon M. Outbreak of enterovirus 71 infection in Victoria, Australia, with a high incidence of neurologic involvement. Pediatr Infect Dis J. Jul 1988;7(7): Hayward JC, Gillespie SM, Kaplan KM, et al. Outbreak of poliomyelitis-like paralysis associated with enterovirus 71. Pediatr Infect Dis J. Sep 1989;8(9): Kehle J, Roth B, Metzger C, Pfitzner A, Enders G. Molecular characterization of an Enterovirus 71 causing neurological disease in Germany. J Neurovirol. Feb 2003;9(1): Lum LC, Wong KT, Lam SK, et al. Fatal enterovirus 71 encephalomyelitis. J Pediatr. Dec 1998;133(6): Wang SM, Liu CC, Tseng HW, et al. Clinical spectrum of enterovirus 71 infection in children in southern Taiwan, with an emphasis on neurological complications. Clin Infect Dis. Jul 1999;29(1):

17 15. Nolan MA, Craig ME, Lahra MM, et al. Survival after pulmonary edema due to enterovirus 71 encephalitis. Neurology. May ;60(10): Wang SM, Lei HY, Huang KJ, et al. Pathogenesis of enterovirus 71 brainstem encephalitis in pediatric patients: roles of cytokines and cellular immune activation in patients with pulmonary edema. J Infect Dis. Aug ;188(4): Chang LY, Lin TY, Hsu KH, et al. Clinical features and risk factors of pulmonary oedema after enterovirus-71-related hand, foot, and mouth disease. Lancet. Nov ;354(9191): Wang JR, Tuan YC, Tsai HP, Yan JJ, Liu CC, Su IJ. Change of major genotype of enterovirus 71 in outbreaks of hand-foot-and-mouth disease in Taiwan between 1998 and J Clin Microbiol. Jan 2002;40(1): Shih SR, Li YS, Chiou CC, et al. Expression of capsid [correction of caspid] protein VP1 for use as antigen for the diagnosis of enterovirus 71 infection. J Med Virol. Jun 2000;61(2): Wu CN, Lin YC, Fann C, Liao NS, Shih SR, Ho MS. Protection against lethal enterovirus 71 infection in newborn mice by passive immunization with subunit VP1 vaccines and inactivated virus. Vaccine. Dec ;20(5-6): Brown BA, Oberste MS, Alexander JP, Jr., Kennett ML, Pallansch MA. Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to J Virol. Dec 1999;73(12): Brown BA, Kilpatrick DR, Oberste MS, Pallansch MA. 17

18 Serotype-specific identification of enterovirus 71 by PCR. J Clin Virol. Apr 2000;16(2): Oberste MS, Maher K, Kilpatrick DR, Flemister MR, Brown BA, Pallansch MA. Typing of human enteroviruses by partial sequencing of VP1. J Clin Microbiol. May 1999;37(5): Oberste MS, Maher K, Kilpatrick DR, Pallansch MA. Molecular evolution of the human enteroviruses: correlation of serotype with VP1 sequence and application to picornavirus classification. J Virol. Mar 1999;73(3): Cardosa MJ, Perera D, Brown BA, et al. Molecular epidemiology of human enterovirus 71 strains and recent outbreaks in the Asia-Pacific region: comparative analysis of the VP1 and VP4 genes. Emerg Infect Dis. Apr 2003;9(4): Shimizu H, Utama A, Onnimala N, et al. Molecular epidemiology of enterovirus 71 infection in the Western Pacific Region. Pediatr Int. Apr 2004;46(2):

19 1A B4 B3 Genogroup B B2 B1 C1 C3 C2 Genogroup C C4 Genotype A CA

20 1B MAA00B MAA99B M0200TW 00 M0177TW B N5101TW MAA97B MAA97B MAA00B4 S1971-SAR-03MAB4 S19741-SAR-03MAB MAA97B MAA97B MAA97B MAA97B3 B MAA97B3 EV71MS 8102-W A87-B PA87-B W A87-B OH82-B2 B OR83-B ND79-B NY77-B NM78-B TN80-B VNR3 TW A US 74-B 1 B NY72-B A US 74-B A US 74-B 1 BrCr-USA70-A 20

21 1C NCKU9822 TW TW TX97-C OK97-C2 2381MA 2641-AUS95-C AUS95-C MAA97-C MAA97-C NM94-C CAN91-C VA95-C CT94-C CT94-C1 001-KOR-00-C3 KOR-EV71-07-C3 013-KOR-00-C3 KOR-EV71-06-C3 KOR-EV71-05-C3 H25-CHN-00-C4 SHH02-17CHN R1 HK1159 F1-CH-00-C CHN-CQ SHZH03-China BrCr-USA70-A C C1 99 C3 88 C4

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25 VP1 EV71 subgenotypes Comparison %Homology range Mean % homology Within B Within B Within C Within C B2 B B2 C B2 C B4 C B4 C C2 C B1 A B4 A C2 A C4 A B1 B B1 B B1 C B1 C B2 B B4 B C2 B C4 B B2 C B4 C C2 C C4 C B2 C B4 C C2 C C4 C

26 EV71 subgenotypes Comparison %Homology range Mean % homology Within B Within B Within C Within C B2 B B2 C B2 C B4 C B4 C C2 C B1 A B4 A C2 A C4 A B1 B B1 B B1 C B1 C B2 B B4 B C2 B C4 B B2 C B4 C C2 C C4 C B2 C B4 C C2 C C4 C

27 a b Sera Virus B C c EV71B B1 > B2 > B3 >1024 >1024 B4 > B5 > B6 > B7 >1024 >1024 d EV71C C1 > C2 > C3 > C4 > C5 > C6 > # # B5 <8 <8 疱 <8 <8 16 <8 <8 a ) ( b ( ) c B d C 27

福利部疾病管制署,2013;Lin, Wang, Tung, & Chen, 2009) 腸病毒的型別繁多, 廣泛的分布於全球, 在室溫中存活穩定, 適合在濕熱的環境下生存, 常發生在夏季及早秋, 而在熱

福利部疾病管制署,2013;Lin, Wang, Tung, & Chen, 2009) 腸病毒的型別繁多, 廣泛的分布於全球, 在室溫中存活穩定, 適合在濕熱的環境下生存, 常發生在夏季及早秋, 而在熱預防並不難 - 談腸病毒的照護與預防台北醫學大學公共衛生系趙映婷基隆長庚醫院護理部陳淑賢基隆長庚醫院小兒科蔡明翰前言腸病毒這個病名我們並不陌生, 但是我們到底對腸病毒的了解有多少? 很多人常常把腸病毒感冒流感病毒