Evolution of Enterovirus 71

1. Evolution of Enterovirus 71 Jen-Ren Wang, Ph. D. (王貞仁) • Dept. of Medical Laboratory Science and Biotechnology, National Cheng Kung University • NHRI Tainan Virology Laboratory for Diagnosis and Research • Taiwan CDC Virology Contract Laboratory

2. Outline • Enterovirus surveillance in Taiwan • Molecular analysis of EV71 • Virulent Gene Analysis of Enterovirus 71 and Disease Association

3. Taiwan CDC and Virology Contract Laboratories Taipei Veterans General Hospital Chang Gung Memorial Hospital, Linkou Tri-Service General Hospital Taiwan University Hospital China Medical University Hospital Buddhist Tzu Chi General Hospital Chang Gung Memorial Hospital, Kaoshiung Cheng Kung University Hospital Kaoshiung Medical University Hospital ◎ CDC, Taiwan Taichung Veterans General Hospital Chunghua Christian Hospital Kaoshiung Veterans General Hospital

4. Enterovirus positive cases, 2000-2005 Taiwan (Virology contract Labs)

5. Enteroviruses isolated in contract labs-I 2000 (n=1310)2001 (n=2114) 2002 (n=1839) Rank Serotype % Serotype % Serotype % 1 Untyped 44.4 Untyped 43.8 Untyped 45.9 2 EV 71 18.2CA 16 15.5CA 16 16.2 3 CA 16 17.1EV 71 15.0 E 6 10.2 4 CB 3 4.9E 30 12.1CB 5 9.4 5 E 9 4.6E 6 5.4 EV 71 7.9 6 CB 4 4.0 P 3.8CA 24 3.3 7 E 4 1.3 CB 4 1.6 CB 2 2.3 8 P 0.8 CA 24 0.8 P 1.6 9 CA 4 0.7 CA 9 0.5 E 9 0.8 10 E 3 0.7 CB 5 0.5 E 4 0.5 11 CB 1 0.6 CA 10 0.3 CB 4 0.5 12 CA 24 0.5 CA 6 0.1 CA 10 0.4 13 CA 9 0.3CB 1 0.1 CA 4 0.3 14 CA 2 0.3 CB 3 0.1 E 11 0.2 15 most frequent serotypes 98.4 99.6 99.5

6. Enteroviruses isolated in contract labs-II 2003 (n=2066) 2004 (n=2389) 2005 (n=2031) Rank Serotype % Serotype % Serotype % 1 CA 16 35.8 CA 4 23.8 CB 3 30.6 2 Untyped17.7CA 10 22.7CA 16 25.5 3 EV 71 13.0CB 4 16.5EV 71 13.9 4 E 9 7.5Untyped 9.8CA 6 6.6 5 E 11 6.2EV 71 8.5 CA 5 5.9 6 CA 2 4.7 CA 6 2.6Untyped 5.3 7 CA 6 3.9 CA 9 2.6 CA 2 3.8 8 CA 5 3.2 CB 3 2.2 E 9 3.3 9 CB 2 2.0 CA 5 2.0 CB 2 1.4 10 CB 5 1.3 CA 2 1.8 CA 10 1.0 11 P 0.9 CA 16 1.7 P 0.9 12 CA 11 0.6 CB 2 1.2 CA 4 0.6 13 E 30 0.6E 25 1.0 CA 9 0.5 14 CA 10 0.5 E 11 0.9 CB 1 0.2 15 most frequent serotypes 97.9 97.3 99.5

7. Clinical presentations of EV71 • Infection of EV71 can cause hand-foot-and-mouth disease (HFMD), herpangina, aseptic meningitis, acute flaccid paralysis (AFP), brainstem encephalitis, and pulmonary edema. • Several death-associated outbreaks of EV71 infections occurred in Taiwan from 1998 to 2005.

8. Enterovirus 71(EV71) 1975, Bulgaria 1978, Hungary 1997, Japan 1969, California 1998, Taiwan 1998, Singapore 1997, Malaysia 1999, Australia Ho, M, et al. Engl J Med. 1999.

9. EV71 in West Pacific region • Large EV71 epidemics were also reported in Malaysia, Japan, Singapore, Australia, China, Korea, India.

10. Isolations of CA16 and EV71

11. Outline • Enterovirus surveillance in Taiwan • Molecular analysis of EV71 • Virulent Gene Analysis of Enterovirus 71 and Disease Association

12. 1986 1998 1999 2000 2001 2002 2003 2004 2005 CA16 A BrCr 7423-MS-87 72 237-TW-86 Phylogenetic analysis of EV71 isolates in Taiwan from 1986 and 1998-2005 based on VP1 region Both genotypes were able to cause mild and severe diseases. 242-TW-86 99 99 N2838-TW-03 9-97-SHA89 99 5145-TW-98 236-TW-86 97 99 N3793-TW-01 99 N0991-TW-01 S0320-TW-02 81 B N2443-TW-02 N3496-TW-02 5666-SIN-002209 99 91 5865-SIN-000009 99 N0781-TW-01 N1643-TW-00 H0148-TW-00 80 S0875-TW-99 S0942-TW-99 S0943-TW-99 99 M0167-TW-00 N0716-TW-00 M0201-TW-00 99 S18191-SAR-02 02363-Thai-02 93 1814-JAP-01 1585-JAP-01 99 99 1695-JAP-01 2086-TW-98 99 5746-TW-98 H0001-TW-98 H0358-TW-98 NCKU9822 6092-TW-98 99 H0139-TW-98 4643-TW-98 1245a-TW-98 4-SHZH-01 C 2779-JAP-02 N3340-TW-02 SHZH03 99 40-SHZH-02 03-SHZH-01 08-SHZH-01 99 62-SHZH-02 N2735-TW-04 S0584-TW-04 N2121-TW-05 99 N2472-TW-04 N1862-TW-05 N2490-TW-04 N1614-TW-04 N0003-TW-05 N3031-TW-04 N2411-TW-04 0.1 0.1

13. Phylogenetic tree for VP1 region of EV71 strain isolated in Yamagata, Japan, between 1998 and 2003 and reference strains. Mizuta et al, J Clin Micro 43: 6171, 2005

14. Phylogenetic analysis of EV71 based on VP4 gene sequence. Lin et al, J Med Virol 78: 254, 2006

15. Appearance of intratypic recombination of EV71 • Recombination is a well-known phenomenon for enteroviruses. • The actual extent of recombination within circulating EV71 is not known.

16. Genetic analysis of EV71

17. 1986 1998 1999 2000 2001 2002 2003 2004 2005 CA16 A BrCr 7423-MS-87 72 237-TW-86 Phylogenetic analysis of EV71 isolates in Taiwan from 1986 and 1998-2005 based on VP1 region 242-TW-86 99 99 N2838-TW-03 9-97-SHA89 99 5145-TW-98 236-TW-86 97 99 N3793-TW-01 99 N0991-TW-01 S0320-TW-02 81 B N2443-TW-02 N3496-TW-02 5666-SIN-002209 99 91 5865-SIN-000009 99 N0781-TW-01 N1643-TW-00 H0148-TW-00 80 S0875-TW-99 S0942-TW-99 S0943-TW-99 99 M0167-TW-00 N0716-TW-00 M0201-TW-00 99 S18191-SAR-02 02363-Thai-02 93 1814-JAP-01 1585-JAP-01 99 99 1695-JAP-01 2086-TW-98 99 5746-TW-98 H0001-TW-98 H0358-TW-98 NCKU9822 6092-TW-98 99 H0139-TW-98 4643-TW-98 1245a-TW-98 4-SHZH-01 C 2779-JAP-02 N3340-TW-02 SHZH03 99 40-SHZH-02 03-SHZH-01 08-SHZH-01 99 62-SHZH-02 N2735-TW-04 S0584-TW-04 N2121-TW-05 99 N2472-TW-04 N1862-TW-05 N2490-TW-04 N1614-TW-04 N0003-TW-05 N3031-TW-04 N2411-TW-04 0.1 0.1

18. CA16 A BrCr NCKU9822 6092-TW-98 2086-TW-98 5746-TW-98 H0001-TW-98 H0139-TW-98 H0358-TW-98 4643-TW-98 1245a-TW-98 SHZH03 99 C N3340-TW-02 N0003-TW-05 N1862-TW-05 N2735-TW-04 S0584-TW-04 N2490-TW-04 N3031-TW-04 N2411-TW-04 N2472-TW-04 N1614-TW-04 N2121-TW-05 237-TW-86 99 242-TW-86 236-TW-86 7423-MS-87 N2838-TW-03 5145-TW-98 93 9-97-SHA89 99 N2443-TW-02 99 N0991-TW-01 N3793-TW-01 B S0320-TW-02 N3496-TW02 89 5666-SIN-002209 5865-SIN-000009 98 N1643-TW-00 N0781-TW-01 91 M0201-TW-00 N0716-TW-00 M0167-TW-00 S0875-TW-99 H0148-TW-00 S0942-TW-99 S0943-TW-99 0.1 Phylogenetic analysis of EV71 isolates in Taiwan from 1986 and 1998-2005 based on 3D region 1986 1998 1999 2000 2001 2002 2003 2004 2005

19. CA16 A BrCr 2086-TW-98 99 H0358-TW-98 H0139-TW-98 4643-TW-98 C NCKU9822 1245a-TW-98 6092-TW-98 5746-TW-98 H0001-TW-98 N3340-TW-02 83 81 SHZH03 99 N0003-TW-05 N2121-TW-05 99 N1862-TW-05 B S0584-TW-04 1986 N2735-TW-04 N2472-TW-04 1998 N2490-TW-04 1999 N2411-TW-04 99 2000 N3031-TW-04 N1614-TW-04 2001 7423-MS-87 242-TW-86 2002 99 236-TW-86 2003 237-TW-86 2004 9-97-SHA89 91 5145-TW-98 2005 N2838-TW-03 N0991-TW-01 N3793-TW-01 92 99 S0320-TW-02 B N3496-TW-02 N2443-TW-02 N0781-TW-01 N1643-TW-00 5666-SIN-002209 5865-SIN-000009 M0201-TW-00 N0716-TW-00 S0942-TW-99 S0943-TW-99 S0875-TW-99 H0148-TW-00 M0167-TW-00 0.1 Phylogenetic analysis of EV71 isolates in Taiwan from 1986 and 1998-2005 based on 2B region

20. Year T otal EV71 EV71 analyzed Genotype of EV71 tested based on five regions isolated in this study B C Genotype of Recombinant no. (%) no. (%) no (%) 5' - UTR VP1 VP4 - VP2 2B 3D 1986 unknown 7 7 (100.0) 0 0 B B B B B 1998 132 10 2 (20.0) 0 B B B B B 8 (80.0) C C C C C 1999 13 4 4 (100.0) 0 0 B B B B B 2000 171 15 15 (100.0) 0 0 B B B B B 2001 116 5 5 (10 0.0) 0 0 B B B B B 2002 25 8 7 (100.0) 0 B B B B B 1 (12.5) C C C B C 2003 10 4 4 (100.0) 0 0 B B B B B 2004 32 12 0 0 12 (100.0) C C C B C 2005 34 8 0 1 (12.5) C C C C C 7 (87.5) C C C B C Recombination of EV71 from 1986 and 1998-2005

21. 3B 5′UTR 3′UTR VP2 VP3 VP4 2A 2C 3A 3C 3D 2B VP4 Simplot compared N3340/TW/02 with EV71 reference strains

22. 3B 5′UTR 3′UTR VP2 VP3 VP4 2A 2C 3A 3C 3D 2B VP4 Bootscan compared N3340/TW/02 with EV71 reference strains

23. Summary of EV71 recombination study • Intratypic recombination of EV71 among nature circulating isolates was found to be initiated in 2002 and became predominant in 2004 and 2005. • Recombinant points were located at the 3'-termini of 2A and 3C regions, suggesting that nonstructural genes recombined to produce new EV71 variants.

24. Implications of EV71 study • The results indicated that standard typing may produce misleading results or even sequencing of multiple partial regions of EV71 will not always be sufficient. • It remains to be determined what role of the emergence of this new EV71 recombinant plays (e.g. clinical significance, vaccine selection).