腸病毒A71型屬於小RNA病毒家族中的腸病毒屬，臨床上經常造成小兒的手足口症和疱疹性咽峽炎等；有些感染者會引起腦炎等神經性併發症，甚至死亡。為了防止腸病毒A71型的感染，疫苗研發的重要性日漸上升。密碼子使用度偏倚描述在每一個胺基酸之下對應到的每一種同義密碼子之使用度是不同的，若將常用的密碼子置換為不常用的密碼子，便稱為密碼子去優化。先前研究指出，密碼子去優化可以成功的降低病毒蛋白質生成的速率，進而影響病毒的複製，甚至是毒性。我們利用基因序列合成的技術製造的密碼子去優化殼蛋白VP1序列，建立了攜帶此序列之四株腸病毒A71型。由實驗結果我們發現部分密碼子去優化不會使腸病毒A71型的複製速率下降，但是降低了腸病毒A71型的在細胞形成溶斑的大小。由密碼子去優化的四株病毒免疫小鼠過後的抗血清，針對四株病毒交叉進行中和試驗後的結果發現，四種抗血清針對四種病毒都有中和的能力，產生的中和抗體效價都在4倍的差異以內，顯示密碼子去優化保留了相同的氨基酸序列的確不影響病毒的抗原特性。然而由於病毒受到偌大的生長壓力，因此基因穩定性降低，出現許多位點的突變。為了克服此情形，我們又建立了帶有高保真性聚合酶點突變之密碼子去優化的腸病毒A71型，並且發現這樣的確使突變出現的數量降低。除此之外帶有高保真性聚合酶點突變之密碼子去優化的腸病毒A71型的生長速率較低。並且在B6小鼠身上也看到高保真性密碼子去優化的腸病毒A71型的毒性較低，相較於原病毒株使得老鼠的體重增加變慢並且有一腳癱瘓之情形出現，施打高保真性密碼子去優化的腸病毒A71型的小鼠體重增加的速率與施打培養液的組別相似，成長過程也未出現臨床表徵，顯示高保真性密碼子去優化的腸病毒A71型之毒性較低。以原病毒株和高保真性的密碼子去優化腸病毒A71型同時免疫BALB/c小鼠也看見抗體量均有上升。綜合以上實驗結果，我們認為密碼子去優化結合高保真性聚合酶之點突變，可以降低腸病毒A71型的毒性並維持抗原性及基因穩定性，有潛力作為疫苗候選病毒株。

Enterovirus A71 (EV-A71) is the major pathogen which causes hand-foot-and-mouth disease (HFMD), and it caused large outbreaks in Taiwan in 1998, 2004-5, 2008 and 2012. To prevent EV-A71 infection, it is necessary and urgent to develop EV-A71 vaccine against EV-A71. In this study, we tried to develop a new synthetic EV-A71 vaccine based on codon deoptimization (CD). Codon usage bias is defined that the frequency of each synonymous codon usage is different. CD means the exchange of the optimal codon to deoptimal codon. Previous studies showed that codon deoptimization decreases the protein expression level, which further affects replication and virulence of virus. We analyzed the codon usage of EV-A71 and constructed four EV-A71 which contained codon deoptimized VP1 capsid protein. The replication rate of CD and wild-type virus was analyzed, and we found that partial CD didn’t affect replication rate, but the plaque size formed by the partial CD viruses was smaller than parental strain. The antigenicity was not affected due to there’s no change of amino acid sequence. The antisera raised by CD viruses showed similar neutralizing antibody titers against CD and wild-type EV-A71. Each antiserum showed neutralizing antibody titers with less than four-fold difference against each virus. However, the genome stability showed that there were some mutations appeared during passages of the CD viruses. Thus, we further constructed VP1-CD EV-A71 containing high-fidelity (CD-HF) determinants of G64R and L123F in 3D polymerase and found that mutation rate of CD-HF EV-A71 was decreased. The CD-HF viruses showed decreased replication rate compared with parental strain 4643C4VP in RD cells. In addition, B6 mice infected with the CD-HF viruses showed higher body weight than parental strain 4643C4VP and no appearance of paralysis and clinical score, indicating less virulence of CD-HF viruses than parental strain. The BALB/c mice infected with parental strain and CD-HF viruses all showed antibody titers increase after 14 days post-immunization via intraperitoneal route. Furthermore, mouse antisera raised by CD-HF viruses showed similar neutralizing antibody titer against CD-HF and wild-type EV-A71. In conclusion, we found that VP1 codon deoptimization combined with high-fidelity determinants 3D-G64R/L123F decreased EV-A71 infectivity and mutation rates but retained their antigenicity, which may be good candidates for next generation EV-A71 vaccine candidate.

1. Adams, M.J., R.C. Hendrickson, D.M. Dempsey, and E.J. Lefkowitz, Tracking the changes in virus taxonomy. Arch Virol, 2015. 160(5): p. 1375-83.
2. Whitton, J.L., C.T. Cornell, and R. Feuer, Host and virus determinants of picornavirus pathogenesis and tropism. Nat Rev Microbiol, 2005. 3(10): p. 765-76.
3. Tsao, K.C., C.G. Huang, Y.L. Huang, F.C. Chen, P.N. Huang, Y.C. Huang, T.Y. Lin, S.R. Shih, and S.C. Chang, Epidemiologic features and virus isolation of enteroviruses in Northern Taiwan during 2000-2008. J Virol Methods, 2010. 165(2): p. 330-2.
4. Tee, K.K., T.T.Y. Lam, Y.F. Chan, J.M. Bible, A. Kamarulzaman, C.Y.W. Tong, Y. Takebe, and O.G. Pybus, Evolutionary Genetics of Human Enterovirus 71: Origin, Population Dynamics, Natural Selection, and Seasonal Periodicity of the VP1 Gene. J Virol, 2010. 84(7): p. 3339-50.
5. Zhang, Y., X. Tan, A. Cui, N. Mao, S. Xu, Z. Zhu, J. Zhou, J. Shi, Y. Zhao, X. Wang, X. Huang, S. Zhu, Y. Zhang, W. Tang, H. Ling, and W. Xu, Complete Genome Analysis of the C4 Subgenotype Strains of Enterovirus 71: Predominant Recombination C4 Viruses Persistently Circulating in China for 14 Years. PLoS One, 2013. 8(2).
6. van der Sanden, S., M. Koopmans, G. Uslu, and H. van der Avoort, Epidemiology of enterovirus 71 in the Netherlands, 1963 to 2008. J Clin Microbiol, 2009. 47(9): p. 2826-33.
7. Yip, C.C., S.K. Lau, B. Zhou, M.X. Zhang, H.W. Tsoi, K.H. Chan, X.C. Chen, P.C. Woo, and K.Y. Yuen, Emergence of enterovirus 71 "double-recombinant" strains belonging to a novel genotype D originating from southern China: first evidence for combination of intratypic and intertypic recombination events in EV71. Arch Virol, 2010. 155(9): p. 1413-24.
8. van der Sanden, S.M.G., G. Koen, H. van Eijk, S.M. Koekkoek, M.D. de Jong, and K.C. Wolthers, Prediction of Protection against Asian Enterovirus 71 Outbreak Strains by Cross-neutralizing Capacity of Serum from Dutch Donors, The Netherlands. Emerg Infect Dis, 2016. 22(9): p. 1562-9.
9. Bessaud, M., R. Razafindratsimandresy, A. Nougairède, M.-L. Joffret, J.M. Deshpande, A. Dubot-Pérès, J.-M. Héraud, X. de Lamballerie, F. Delpeyroux, and J.-L. Bailly, Molecular Comparison and Evolutionary Analyses of VP1 Nucleotide Sequences of New African Human Enterovirus 71 Isolates Reveal a Wide Genetic Diversity. PLOS ONE, 2014. 9(3): p. e90624.
10. Rossmann, M.G., E. Arnold, J.W. Erickson, E.A. Frankenberger, J.P. Griffith, H.J. Hecht, J.E. Johnson, G. Kamer, M. Luo, A.G. Mosser, and et al., Structure of a human common cold virus and functional relationship to other picornaviruses. Nature, 1985. 317(6033): p. 145-53.
11. Ma, H.C., Y. Liu, C. Wang, M. Strauss, N. Rehage, Y.H. Chen, N. Altan-Bonnet, J. Hogle, E. Wimmer, S. Mueller, A.V. Paul, and P. Jiang, An interaction between glutathione and the capsid is required for the morphogenesis of C-cluster enteroviruses. PLoS Pathog, 2014. 10(4): p. e1004052.
12. Yi, E.J., Y.J. Shin, J.H. Kim, T.G. Kim, and S.Y. Chang, Enterovirus 71 infection and vaccines. Clin Exp Vaccine Res, 2017. 6(1): p. 4-14.
13. Lin, J.-Y., T.-C. Chen, K.-F. Weng, S.-C. Chang, L.-L. Chen, and S.-R. Shih, Viral and host proteins involved in picornavirus life cycle. Journal of Biomedical Science, 2009. 16(1): p. 103.
14. Toyoda, H., D. Franco, K. Fujita, A.V. Paul, and E. Wimmer, Replication of poliovirus requires binding of the poly(rC) binding protein to the cloverleaf as well as to the adjacent C-rich spacer sequence between the cloverleaf and the internal ribosomal entry site. J Virol, 2007. 81(18): p. 10017-28.
15. Hogle, J.M., M. Chow, and D.J. Filman, Three-dimensional structure of poliovirus at 2.9 A resolution. Science, 1985. 229(4720): p. 1358-65.
16. Foo, D.G., S. Alonso, M.C. Phoon, N.P. Ramachandran, V.T. Chow, and C.L. Poh, Identification of neutralizing linear epitopes from the VP1 capsid protein of Enterovirus 71 using synthetic peptides. Virus Res, 2007. 125(1): p. 61-8.
17. Toyoda, H., M.J. Nicklin, M.G. Murray, C.W. Anderson, J.J. Dunn, F.W. Studier, and E. Wimmer, A second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein. Cell, 1986. 45.
18. Byrd, M.P., M. Zamora, and R.E. Lloyd, Translation of eukaryotic translation initiation factor 4GI (eIF4GI) proceeds from multiple mRNAs containing a novel cap-dependent internal ribosome entry site (IRES) that is active during poliovirus infection. J Biol Chem, 2005. 280(19): p. 18610-22.
19. Aldabe, R., A. Barco, and L. Carrasco, Membrane permeabilization by poliovirus proteins 2B and 2BC. J Biol Chem, 1996. 271(38): p. 23134-7.
20. van Kuppeveld, F.J., J.M. Galama, J. Zoll, P.J.v.a.n.d.e.n. Hurk, and W.J. Melchers, Coxsackie B3 virus protein 2B contains cationic amphipathic helix that is required for viral RNA replication. J Virol, 1996. 70.
21. Ulferts, R., L. van der Linden, H.J. Thibaut, K.H. Lanke, P. Leyssen, B. Coutard, A.M. De Palma, B. Canard, J. Neyts, and F.J. van Kuppeveld, Selective serotonin reuptake inhibitor fluoxetine inhibits replication of human enteroviruses B and D by targeting viral protein 2C. Antimicrob Agents Chemother, 2013. 57(4): p. 1952-6.
22. Lai, J.K.F., I.C. Sam, and Y.F. Chan, The Autophagic Machinery in Enterovirus Infection. Viruses, 2016. 8(2).
23. Lai, J., I.-C. Sam, P. Verlhac, J. Baguet, E.-L. Eskelinen, M. Faure, and Y. Chan, 2BC Non-Structural Protein of Enterovirus A71 Interacts with SNARE Proteins to Trigger Autolysosome Formation. Viruses, 2017. 9(7): p. 169.
24. Teoule, F., C. Brisac, I. Pelletier, P.O. Vidalain, S. Jegouic, C. Mirabelli, M. Bessaud, N. Combelas, A. Autret, F. Tangy, F. Delpeyroux, and B. Blondel, The Golgi protein ACBD3, an interactor for poliovirus protein 3A, modulates poliovirus replication. J Virol, 2013. 87(20): p. 11031-46.
25. Dodd, D.A., T.H. Giddings, Jr., and K. Kirkegaard, Poliovirus 3A protein limits interleukin-6 (IL-6), IL-8, and beta interferon secretion during viral infection. J Virol, 2001. 75(17): p. 8158-65.
26. Dorner, A.J., P.G. Rothberg, and E. Wimmer, The fate of VPg during in vitro translation of poliovirus RNA. FEBS Lett, 1981. 132.
27. Lama, J., M.A. Sanz, and P.L. Rodriguez, A role for 3AB protein in poliovirus genome replication. J Biol Chem, 1995. 270(24): p. 14430-8.
28. Nayak, A., I.G. Goodfellow, and G.J. Belsham, Factors required for the Uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3Dpol) in vitro. J Virol, 2005. 79(12): p. 7698-706.
29. Gamarnik, A.V. and R. Andino, Interactions of viral protein 3CD and poly(rC) binding protein with the 5' untranslated region of the poliovirus genome. J Virol, 2000. 74(5): p. 2219-26.
30. Jore, J., B. De Geus, R.J. Jackson, P.H. Pouwels, and B.E. Enger-Valk, Poliovirus protein 3CD is the active protease for processing of the precursor protein P1 in vitro. J Gen Virol, 1988. 69.
31. Clark, M.E., P.M. Lieberman, A.J. Berk, and A. Dasgupta, Direct cleavage of human TATA-binding protein by poliovirus protease 3C in vivo and in vitro. Mol Cell Biol, 1993. 13(2): p. 1232-7.
32. Kundu, P., S. Raychaudhuri, W. Tsai, and A. Dasgupta, Shutoff of RNA polymerase II transcription by poliovirus involves 3C protease-mediated cleavage of the TATA-binding protein at an alternative site: incomplete shutoff of transcription interferes with efficient viral replication. J Virol, 2005. 79(15): p. 9702-13.
33. Clark, M.E., T. Hammerle, E. Wimmer, and A. Dasgupta, Poliovirus proteinase 3C converts an active form of transcription factor IIIC to an inactive form: a mechanism for inhibition of host cell polymerase III transcription by poliovirus. Embo J, 1991. 10.
34. Plotch, S.J. and O. Palant, Poliovirus protein 3AB forms a complex with and stimulates the activity of the viral RNA polymerase, 3Dpol. J Virol, 1995. 69(11): p. 7169-79.
35. Takegami, T., R.J. Kuhn, C.W. Anderson, and E. Wimmer, Membrane-dependent uridylylation of the genome-linked protein VPg of poliovirus. Proc Natl Acad Sci USA, 1983. 80.
36. Li, L., Y. He, H. Yang, J. Zhu, X. Xu, J. Dong, Y. Zhu, and Q. Jin, Genetic characteristics of human enterovirus 71 and coxsackievirus A16 circulating from 1999 to 2004 in Shenzhen, People's Republic of China. J Clin Microbiol, 2005. 43(8): p. 3835-9.
37. Ooi, M.H., S.C. Wong, P. Lewthwaite, M.J. Cardosa, and T. Solomon, Clinical features, diagnosis, and management of enterovirus 71. Lancet Neurol, 2010. 9(11): p. 1097-105.
38. Schmidt, N.J., E.H. Lennette, and H.H. Ho, An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis, 1974. 129(3): p. 304-9.
39. Blomberg, J., E. Lycke, K. Ahlfors, T. Johnsson, S. Wolontis, and G. von Zeipel, Letter: New enterovirus type associated with epidemic of aseptic meningitis and-or hand, foot, and mouth disease. Lancet, 1974. 2(7872): p. 112.
40. Deibel, R., L.L. Gross, and D.N. Collins, Isolation of a new enterovirus (38506). Proc Soc Exp Biol Med, 1975. 148(1): p. 203-7.
41. Chonmaitree, T., M.A. Menegus, E.M. Schervish-Swierkosz, and E. Schwalenstocker, Enterovirus 71 infection: report of an outbreak with two cases of paralysis and a review of the literature. Pediatrics, 1981. 67(4): p. 489-93.
42. Hayward, J.C., S.M. Gillespie, K.M. Kaplan, R. Packer, M. Pallansch, S. Plotkin, and L.B. Schonberger, Outbreak of poliomyelitis-like paralysis associated with enterovirus 71. Pediatr Infect Dis J, 1989. 8(9): p. 611-6.
43. Brown, B.A., D.R. Kilpatrick, M.S. Oberste, and M.A. Pallansch, Serotype-specific identification of enterovirus 71 by PCR. J Clin Virol, 2000. 16(2): p. 107-12.
44. Fujimoto, T., S. Yoshida, T. Munemura, K. Taniguchi, M. Shinohara, O. Nishio, M. Chikahira, and N. Okabe, Detection and quantification of enterovirus 71 genome from cerebrospinal fluid of an encephalitis patient by PCR applications. Jpn J Infect Dis, 2008. 61(6): p. 497-9.
45. Komatsu, H., Y. Shimizu, Y. Takeuchi, H. Ishiko, and H. Takada, Outbreak of severe neurologic involvement associated with Enterovirus 71 infection. Pediatr Neurol, 1999. 20(1): p. 17-23.
46. Chan, L.G., U.D. Parashar, M.S. Lye, F.G. Ong, S.R. Zaki, J.P. Alexander, K.K. Ho, L.L. Han, M.A. Pallansch, A.B. Suleiman, M. Jegathesan, and L.J. Anderson, 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, 2000. 31(3): p. 678-83.
47. Chan, K.P., K.T. Goh, C.Y. Chong, E.S. Teo, G. Lau, and A.E. Ling, Epidemic hand, foot and mouth disease caused by human enterovirus 71, Singapore. Emerg Infect Dis, 2003. 9(1): p. 78-85.
48. Jee, Y.M., D.S. Cheon, K. Kim, J.H. Cho, Y.S. Chung, J. Lee, S.H. Lee, K.S. Park, J.H. Lee, E.C. Kim, H.J. Chung, D.S. Kim, J.D. Yoon, and H.W. Cho, Genetic analysis of the VP1 region of human enterovirus 71 strains isolated in Korea during 2000. Arch Virol, 2003. 148(9): p. 1735-46.
49. Zhang, Y., X.J. Tan, H.Y. Wang, D.M. Yan, S.L. Zhu, D.Y. Wang, F. Ji, X.J. Wang, Y.J. Gao, L. Chen, H.Q. An, D.X. Li, S.W. Wang, A.Q. Xu, Z.J. Wang, and W.B. Xu, An outbreak of hand, foot, and mouth disease associated with subgenotype C4 of human enterovirus 71 in Shandong, China. J Clin Virol, 2009. 44(4): p. 262-7.
50. Ho, M., E.R. Chen, K.H. Hsu, S.J. Twu, K.T. Chen, S.F. Tsai, J.R. Wang, and S.R. Shih, An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group. N Engl J Med, 1999. 341(13): p. 929-35.
51. Huang, S.W., Y.W. Hsu, D.J. Smith, D. Kiang, H.P. Tsai, K.H. Lin, S.M. Wang, C.C. Liu, I.J. Su, and J.R. Wang, Reemergence of enterovirus 71 in 2008 in taiwan: dynamics of genetic and antigenic evolution from 1998 to 2008. J Clin Microbiol, 2009. 47(11): p. 3653-62.
52. Lin, K.H., K.P. Hwang, G.M. Ke, C.F. Wang, L.Y. Ke, Y.T. Hsu, Y.C. Tung, P.Y. Chu, B.H. Chen, H.L. Chen, C.L. Kao, J.R. Wang, H.L. Eng, S.Y. Wang, L.C. Hsu, and H.Y. Chen, Evolution of EV71 genogroup in Taiwan from 1998 to 2005: an emerging of subgenogroup C4 of EV71. J Med Virol, 2006. 78(2): p. 254-62.
53. Chang, L.Y., Enterovirus 71 in Taiwan. Pediatr Neonatol, 2008. 49(4): p. 103-12.
54. Huang, Y.P., T.L. Lin, C.Y. Kuo, M.W. Lin, C.Y. Yao, H.W. Liao, L.C. Hsu, C.F. Yang, J.Y. Yang, P.J. Chen, and H.S. Wu, The circulation of subgenogroups B5 and C5 of enterovirus 71 in Taiwan from 2006 to 2007. Virus Res, 2008. 137(2): p. 206-12.
55. Yeh, M.T., S.W. Wang, C.K. Yu, K.H. Lin, H.Y. Lei, I.J. Su, and J.R. Wang, A single nucleotide in stem loop II of 5'-untranslated region contributes to virulence of enterovirus 71 in mice. PLoS One, 2011. 6(11): p. e27082.
56. Kung, Y.H., S.W. Huang, P.H. Kuo, D. Kiang, M.S. Ho, C.C. Liu, C.K. Yu, I.J. Su, and J.R. Wang, Introduction of a strong temperature-sensitive phenotype into enterovirus 71 by altering an amino acid of virus 3D polymerase. Virology, 2010. 396(1): p. 1-9.
57. Huang, S.W., Y.F. Wang, C.K. Yu, I.J. Su, and J.R. Wang, Mutations in VP2 and VP1 capsid proteins increase infectivity and mouse lethality of enterovirus 71 by virus binding and RNA accumulation enhancement. Virology, 2012. 422(1): p. 132-43.
58. Liu, Y., C. Fu, S. Wu, X. Chen, Y. Shi, B. Zhou, L. Zhang, F. Zhang, Z. Wang, Y. Zhang, C. Fan, S. Han, J. Yin, B. Peng, W. Liu, and X. He, A novel finding for enterovirus virulence from the capsid protein VP1 of EV71 circulating in mainland China. Virus Genes, 2014. 48(2): p. 260-72.
59. Li, P., Y. Yue, N. Song, B. Li, H. Meng, G. Yang, Z. Li, L. An, and L. Qin, Genome analysis of enterovirus 71 strains differing in mouse pathogenicity. Virus Genes, 2016. 52(2): p. 161-71.
60. Wang, S.M., H.Y. Lei, K.J. Huang, J.M. Wu, J.R. Wang, C.K. Yu, I.J. Su, and C.C. Liu, Pathogenesis of enterovirus 71 brainstem encephalitis in pediatric patients: roles of cytokines and cellular immune activation in patients with pulmonary edema. J Infect Dis, 2003. 188(4): p. 564-70.
61. Lin, T.Y., S.H. Hsia, Y.C. Huang, C.T. Wu, and L.Y. Chang, Proinflammatory cytokine reactions in enterovirus 71 infections of the central nervous system. Clin Infect Dis, 2003. 36(3): p. 269-74.
62. Rimmelzwaan, G.F., M. Baars, P. de Lijster, R.A.M. Fouchier, and A. Osterhaus, Inhibition of Influenza Virus Replication by Nitric Oxide. J Virol, 1999. 73(10): p. 8880-3.
63. Kawanishi, M., Nitric oxide inhibits Epstein-Barr virus DNA replication and activation of latent EBV. Intervirology, 1995. 38(3-4): p. 206-13.
64. Saura, M., C. Zaragoza, A. McMillan, R.A. Quick, C. Hohenadl, J.M. Lowenstein, and C.J. Lowenstein, An Antiviral Mechanism of Nitric Oxide. Immunity, 1999. 10(1): p. 21-28.
65. López-Guerrero, J.A. and L. Carrasco, Effect of Nitric Oxide on Poliovirus Infection of Two Human Cell Lines. J Virol, 1998. 72(3): p. 2538-40.
66. Sanders, S.P., E.S. Siekierski, S.M. Richards, J.D. Porter, F. Imani, and D. Proud, Rhinovirus infection induces expression of type 2 nitric oxide synthase in human respiratory epithelial cells in vitro and in vivo. J Allergy Clin Immunol, 2001. 107(2): p. 235-43.
67. Sanders, S.P., E.S. Siekierski, J.D. Porter, S.M. Richards, and D. Proud, Nitric Oxide Inhibits Rhinovirus-Induced Cytokine Production and Viral Replication in a Human Respiratory Epithelial Cell Line. J Virol, 1998. 72(2): p. 934-42.
68. Li, M.-L., T.-A. Hsu, T.-C. Chen, S.-C. Chang, J.-C. Lee, C.-C. Chen, V. Stollar, and S.-R. Shih, The 3C Protease Activity of Enterovirus 71 Induces Human Neural Cell Apoptosis. Virology, 2002. 293(2): p. 386-395.
69. Lin, J.Y., G. Brewer, and M.L. Li, HuR and Ago2 Bind the Internal Ribosome Entry Site of Enterovirus 71 and Promote Virus Translation and Replication. PLoS One, 2015. 10(10).
70. McCullough, K.C., F. De Simone, E. Brocchi, L. Capucci, J.R. Crowther, and U. Kihm, Protective immune response against foot-and-mouth disease. J Virol, 1992. 66(4): p. 1835-40.
71. Mizuta, K., Y. Aoki, A. Suto, K. Ootani, N. Katsushima, T. Itagaki, A. Ohmi, M. Okamoto, H. Nishimura, Y. Matsuzaki, S. Hongo, K. Sugawara, H. Shimizu, and T. Ahiko, Cross-antigenicity among EV71 strains from different genogroups isolated in Yamagata, Japan, between 1990 and 2007. Vaccine, 2009. 27(24): p. 3153-8.
72. Hayden, F.G., D.T. Herrington, T.L. Coats, K. Kim, E.C. Cooper, S.A. Villano, S. Liu, S. Hudson, D.C. Pevear, M. Collett, and M. McKinlay, Efficacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of 2 double-blind, randomized, placebo-controlled trials. Clin Infect Dis, 2003. 36(12): p. 1523-32.
73. Wang, S.M., C.C. Liu, H.W. Tseng, J.R. Wang, C.C. Huang, Y.J. Chen, Y.J. Yang, S.J. Lin, and T.F. Yeh, Clinical spectrum of enterovirus 71 infection in children in southern Taiwan, with an emphasis on neurological complications. Clin Infect Dis, 1999. 29(1): p. 184-90.
74. Chi, C.Y., T.H. Khanh, P.K. Thoa le, F.C. Tseng, S.M. Wang, Q. Thinh le, C.C. Lin, H.C. Wu, J.R. Wang, N.T. Hung, T.C. Thuong, C.M. Chang, I.J. Su, and C.C. Liu, Milrinone therapy for enterovirus 71-induced pulmonary edema and/or neurogenic shock in children: a randomized controlled trial. Crit Care Med, 2013. 41(7): p. 1754-60.
75. Chou, A.H., C.C. Liu, J.Y. Chang, R. Jiang, Y.C. Hsieh, A. Tsao, C.L. Wu, J.L. Huang, C.P. Fung, S.M. Hsieh, Y.F. Wang, J.R. Wang, M.H. Hu, J.R. Chiang, I.J. Su, and P.C. Chong, Formalin-inactivated EV71 vaccine candidate induced cross-neutralizing antibody against subgenotypes B1, B4, B5 and C4A in adult volunteers. PLoS One, 2013. 8(11): p. e79783.
76. Zhu, F., W. Xu, J. Xia, Z. Liang, Y. Liu, X. Zhang, X. Tan, L. Wang, Q. Mao, J. Wu, Y. Hu, T. Ji, L. Song, Q. Liang, B. Zhang, Q. Gao, J. Li, S. Wang, Y. Hu, S. Gu, J. Zhang, G. Yao, J. Gu, X. Wang, Y. Zhou, C. Chen, M. Zhang, M. Cao, J. Wang, H. Wang, and N. Wang, Efficacy, safety, and immunogenicity of an enterovirus 71 vaccine in China. N Engl J Med, 2014. 370(9): p. 818-28.
77. Li, R., L. Liu, Z. Mo, X. Wang, J. Xia, Z. Liang, Y. Zhang, Y. Li, Q. Mao, J. Wang, L. Jiang, C. Dong, Y. Che, T. Huang, Z. Jiang, Z. Xie, L. Wang, Y. Liao, Y. Liang, Y. Nong, J. Liu, H. Zhao, R. Na, L. Guo, J. Pu, E. Yang, L. Sun, P. Cui, H. Shi, J. Wang, and Q. Li, An inactivated enterovirus 71 vaccine in healthy children. N Engl J Med, 2014. 370(9): p. 829-37.
78. Zhu, F.-C., F.-Y. Meng, J.-X. Li, X.-L. Li, Q.-Y. Mao, H. Tao, Y.-T. Zhang, X. Yao, K. Chu, Q.-H. Chen, Y.-M. Hu, X. Wu, P. Liu, L.-Y. Zhu, F. Gao, H. Jin, Y.-J. Chen, Y.-Y. Dong, Y.-C. Liang, N.-M. Shi, H.-M. Ge, L. Liu, S.-G. Chen, X. Ai, Z.-Y. Zhang, Y.-G. Ji, F.-J. Luo, X.-Q. Chen, Y. Zhang, L.-W. Zhu, Z.-L. Liang, and X.-L. Shen, Efficacy, safety, and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in China: a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial. The Lancet, 2013. 381(9882): p. 2024-2032.
79. Arita, M., N. Nagata, N. Iwata, Y. Ami, Y. Suzaki, K. Mizuta, T. Iwasaki, T. Sata, T. Wakita, and H. Shimizu, An attenuated strain of enterovirus 71 belonging to genotype a showed a broad spectrum of antigenicity with attenuated neurovirulence in cynomolgus monkeys. J Virol, 2007. 81(17): p. 9386-95.
80. Zhao, H., H.-Y. Li, J.-F. Han, Y.-Q. Deng, S.-Y. Zhu, X.-F. Li, H.-Q. Yang, Y.-X. Li, Y. Zhang, E.D. Qin, R. Chen, and C.-F. Qin, Novel recombinant chimeric virus-like particle is immunogenic and protective against both enterovirus 71 and coxsackievirus A16 in mice. 2015. 5: p. 7878.
81. Premanand, B., T.K. Kiener, T. Meng, Y.R. Tan, Q. Jia, V.T. Chow, and J. Kwang, Induction of protective immune responses against EV71 in mice by baculovirus encoding a novel expression cassette for capsid protein VP1. Antiviral Res, 2012. 95(3): p. 311-5.
82. Kim, Y.I., J.H. Song, B.E. Kwon, H.N. Kim, M.D. Seo, K. Park, S. Lee, S.G. Yeo, M.N. Kweon, H.J. Ko, and S.Y. Chang, Pros and cons of VP1-specific maternal IgG for the protection of Enterovirus 71 infection. Vaccine, 2015. 33(48): p. 6604-10.
83. Wu, C.N., Y.C. Lin, C. Fann, N.S. Liao, S.R. Shih, and M.S. Ho, Protection against lethal enterovirus 71 infection in newborn mice by passive immunization with subunit VP1 vaccines and inactivated virus. Vaccine, 2001. 20(5-6): p. 895-904.
84. Crick, F.H., Codon--anticodon pairing: the wobble hypothesis. J Mol Biol, 1966. 19(2): p. 548-55.
85. Hershberg, R. and D.A. Petrov, Selection on codon bias. Annu Rev Genet, 2008. 42: p. 287-99.
86. Kunec, D. and N. Osterrieder, Codon Pair Bias Is a Direct Consequence of Dinucleotide Bias. Cell Reports, 2016. 14(1): p. 55-67.
87. Martinez, M.A., A. Jordan-Paiz, S. Franco, and M. Nevot, Synonymous Virus Genome Recoding as a Tool to Impact Viral Fitness. Trends Microbiol, 2016. 24(2): p. 134-47.
88. Shen, S.H., C.B. Stauft, O. Gorbatsevych, Y. Song, C.B. Ward, A. Yurovsky, S. Mueller, B. Futcher, and E. Wimmer, Large-scale recoding of an arbovirus genome to rebalance its insect versus mammalian preference. Proc Natl Acad Sci U S A, 2015. 112(15): p. 4749-54.
89. Coleman, J.R., D. Papamichail, S. Skiena, B. Futcher, E. Wimmer, and S. Mueller, Virus attenuation by genome-scale changes in codon pair bias. Science, 2008. 320(5884): p. 1784-7.
90. Le Nouen, C., T. McCarty, M. Brown, M.L. Smith, R. Lleras, M.A. Dolan, M. Mehedi, L. Yang, C. Luongo, B. Liang, S. Munir, J.M. DiNapoli, S. Mueller, E. Wimmer, P.L. Collins, and U.J. Buchholz, Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure. Proc Natl Acad Sci U S A, 2017. 114(3): p. E386-e395.
91. Mueller, S., J.R. Coleman, D. Papamichail, C.B. Ward, A. Nimnual, B. Futcher, S. Skiena, and E. Wimmer, Live attenuated influenza virus vaccines by computer-aided rational design. Nat Biotech, 2010. 28(7): p. 723-726.
92. Le Nouen, C., L.G. Brock, C. Luongo, T. McCarty, L. Yang, M. Mehedi, E. Wimmer, S. Mueller, P.L. Collins, U.J. Buchholz, and J.M. DiNapoli, Attenuation of human respiratory syncytial virus by genome-scale codon-pair deoptimization. Proc Natl Acad Sci U S A, 2014. 111(36): p. 13169-74.
93. Tulloch, F., N.J. Atkinson, D.J. Evans, M.D. Ryan, and P. Simmonds, RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies. eLife, 2014. 3: p. e04531.
94. Simmonds, P., F. Tulloch, D.J. Evans, and M.D. Ryan, Attenuation of dengue (and other RNA viruses) with codon pair recoding can be explained by increased CpG/UpA dinucleotide frequencies. Proc Natl Acad Sci U S A, 2015. 112(28): p. E3633-4.
95. Atkinson, N.J., J. Witteveldt, D.J. Evans, and P. Simmonds, The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication. Nucleic Acids Res, 2014. 42(7): p. 4527-45.
96. Yu, C.H., Y. Dang, Z. Zhou, C. Wu, F. Zhao, M.S. Sachs, and Y. Liu, Codon Usage Influences the Local Rate of Translation Elongation to Regulate Co-translational Protein Folding. Mol Cell, 2015. 59(5): p. 744-54.
97. Nogales, A., S.F. Baker, E. Ortiz-Riano, S. Dewhurst, D.J. Topham, and L. Martinez-Sobrido, Influenza A virus attenuation by codon deoptimization of the NS gene for vaccine development. J Virol, 2014. 88(18): p. 10525-40.
98. Cheng, B.Y., E. Ortiz-Riano, A. Nogales, J.C. de la Torre, and L. Martinez-Sobrido, Development of live-attenuated arenavirus vaccines based on codon deoptimization. J Virol, 2015. 89(7): p. 3523-33.
99. Cheng, B.Y., A. Nogales, J.C. de la Torre, and L. Martinez-Sobrido, Development of live-attenuated arenavirus vaccines based on codon deoptimization of the viral glycoprotein. Virology, 2017. 501: p. 35-46.
100. Mueller, S., D. Papamichail, J.R. Coleman, S. Skiena, and E. Wimmer, Reduction of the rate of poliovirus protein synthesis through large-scale codon deoptimization causes attenuation of viral virulence by lowering specific infectivity. J Virol, 2006. 80(19): p. 9687-96.
101. Zhang, J., M. Wang, W. Liu, J. Zhou, H. Chen, L. Ma, Y. Ding, Y. Gu, and Y. Liu, Analysis of codon usage and nucleotide composition bias in polioviruses. Virol J, 2011. 8: p. 146.
102. Abil, Z., X. Xiong, and H. Zhao, Synthetic Biology for Therapeutic Applications. Mol Pharm, 2015. 12(2): p. 322-31.
103. Cello, J., A.V. Paul, and E. Wimmer, Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. Science, 2002. 297(5583): p. 1016-8.
104. Verity, E.E., S. Camuglia, C.T. Agius, C. Ong, R. Shaw, I. Barr, D. Middleton, and S. Rockman, Rapid generation of pandemic influenza virus vaccine candidate strains using synthetic DNA. Influenza Other Respir Viruses, 2012. 6(2): p. 101-9.
105. Campagnola, G., S. McDonald, S. Beaucourt, M. Vignuzzi, and O.B. Peersen, Structure-Function Relationships Underlying the Replication Fidelity of Viral RNA-Dependent RNA Polymerases. J Virol, 2015. 89(1): p. 275-86.
106. Domingo, E. and J.J. Holland, RNA virus mutations and fitness for survival. Annu Rev Microbiol, 1997. 51: p. 151-78.
107. Woodman, A., J.J. Arnold, C.E. Cameron, and D.J. Evans, Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination. Nucleic Acids Res, 2016. 44(14): p. 6883-95.
108. Boehr, D.D., J.J. Arnold, I.M. Moustafa, and C.E. Cameron, Structure, Dynamics, and Fidelity of RNA-Dependent RNA Polymerases, in Nucleic Acid Polymerases, K.S. Murakami and M.A. Trakselis, Editors. 2014, Springer Berlin Heidelberg: Berlin, Heidelberg. p. 309-333.
109. Freistadt, M.S., J.A. Vaccaro, and K.E. Eberle, Biochemical characterization of the fidelity of poliovirus RNA-dependent RNA polymerase. Virology Journal, 2007. 4(1): p. 44.
110. Castro, C., J.J. Arnold, and C.E. Cameron, Incorporation fidelity of the viral RNA-dependent RNA polymerase: a kinetic, thermodynamic and structural perspective. Virus Res, 2005. 107(2): p. 141-9.
111. McDonald, S., A. Block, S. Beaucourt, G. Moratorio, M. Vignuzzi, and O.B. Peersen, Design of a Genetically Stable High Fidelity Coxsackievirus B3 Polymerase That Attenuates Virus Growth in Vivo. J Biol Chem, 2016. 291(27): p. 13999-4011.
112. Arias, A., J.J. Arnold, M. Sierra, E.D. Smidansky, E. Domingo, and C.E. Cameron, Determinants of RNA-dependent RNA polymerase (in)fidelity revealed by kinetic analysis of the polymerase encoded by a foot-and-mouth disease virus mutant with reduced sensitivity to ribavirin. J Virol, 2008. 82(24): p. 12346-55.
113. Lauring, A.S. and R. Andino, Quasispecies theory and the behavior of RNA viruses. PLoS Pathog, 2010. 6(7): p. e1001005.
114. Coffin, J.M., HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy. Science, 1995. 267(5197): p. 483-9.
115. Meng, T. and J. Kwang, Attenuation of human enterovirus 71 high-replication-fidelity variants in AG129 mice. J Virol, 2014. 88(10): p. 5803-15.
116. Vignuzzi, M., E. Wendt, and R. Andino, Engineering attenuated virus vaccines by controlling replication fidelity. Nat Med, 2008. 14(2): p. 154-61.
117. Van Slyke, G.A., J.J. Arnold, A.J. Lugo, S.B. Griesemer, I.M. Moustafa, L.D. Kramer, C.E. Cameron, and A.T. Ciota, Sequence-Specific Fidelity Alterations Associated with West Nile Virus Attenuation in Mosquitoes. PLOS Pathogens, 2015. 11(6): p. e1005009.
118. Naito, T., K. Mori, H. Ushirogawa, N. Takizawa, E. Nobusawa, T. Odagiri, M. Tashiro, R.L. Ohniwa, K. Nagata, and M. Saito, Generation of a Genetically Stable High-Fidelity Influenza Vaccine Strain. J Virol, 2017. 91(6).
119. Sadeghipour, S., E.J. Bek, and P.C. McMinn, Ribavirin-resistant mutants of human enterovirus 71 express a high replication fidelity phenotype during growth in cell culture. J Virol, 2013. 87(3): p. 1759-69.
120. Tan, C.W., I.C. Sam, V.S. Lee, H.V. Wong, and Y.F. Chan, VP1 residues around the five-fold axis of enterovirus A71 mediate heparan sulfate interaction. Virology, 2017. 501: p. 79-87.
121. Tan, C.W., C.L. Poh, I.C. Sam, and Y.F. Chan, Enterovirus 71 uses cell surface heparan sulfate glycosaminoglycan as an attachment receptor. J Virol, 2013. 87(1): p. 611-20.
122. Zhou, M., T. Wang, J. Fu, G. Xiao, and Y. Liu, Nonoptimal codon usage influences protein structure in intrinsically disordered regions. Mol Microbiol, 2015. 97(5): p. 974-87.
123. Rocha, E.P.C., Codon usage bias from tRNA's point of view: Redundancy, specialization, and efficient decoding for translation optimization. Genome Res, 2004. 14(11): p. 2279-86.
124. Quax, T.E., N.J. Claassens, D. Soll, and J. van der Oost, Codon Bias as a Means to Fine-Tune Gene Expression. Mol Cell, 2015. 59(2): p. 149-61.
125. Pfeiffer, J. and K. Kirkegaard, A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity. PNAS, 2003. 100.
126. Arita, M., Y. Ami, T. Wakita, and H. Shimizu, Cooperative effect of the attenuation determinants derived from poliovirus sabin 1 strain is essential for attenuation of enterovirus 71 in the NOD/SCID mouse infection model. J Virol, 2008. 82(4): p. 1787-97.
127. Lin, Y.-C., C.-N. Wu, S.-R. Shih, and M.-S. Ho, Characterization of a vero cell-adapted virulent strain of enterovirus 71 suitable for use as a vaccine candidate. Vaccine, 2002. 20(19): p. 2485-2493.
128. Lee, M.S., High-growth enterovirus 71 strains and vaccines. 2016, Google Patents.
129. Burns, C.C., J. Shaw, R. Campagnoli, J. Jorba, A. Vincent, J. Quay, and O. Kew, Modulation of poliovirus replicative fitness in HeLa cells by deoptimization of synonymous codon usage in the capsid region. J Virol, 2006. 80(7): p. 3259-72.
130. Stobart, C.C., C.A. Rostad, Z. Ke, R.S. Dillard, C.M. Hampton, J.D. Strauss, H. Yi, A.L. Hotard, J. Meng, R.J. Pickles, K. Sakamoto, S. Lee, M.G. Currier, S.M. Moin, B.S. Graham, M.S. Boukhvalova, B.E. Gilbert, J.C. Blanco, P.A. Piedra, E.R. Wright, and M.L. Moore, A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation. Nat Commun, 2016. 7: p. 13916.
131. Rostad, C.A., C.C. Stobart, B.E. Gilbert, R.J. Pickles, A.L. Hotard, J. Meng, J.C. Blanco, S.M. Moin, B.S. Graham, P.A. Piedra, and M.L. Moore, A Recombinant Respiratory Syncytial Virus Vaccine Candidate Attenuated by a Low-Fusion F Protein Is Immunogenic and Protective against Challenge in Cotton Rats. J Virol, 2016. 90(16): p. 7508-18.
132. Pfeiffer, J.K. and K. Kirkegaard, Increased Fidelity Reduces Poliovirus Fitness and Virulence under Selective Pressure in Mice. PLoS Pathog, 2005. 1(2).
133. Weeks, S.A., C.A. Lee, Y. Zhao, E.D. Smidansky, A. August, J.J. Arnold, and C.E. Cameron, A Polymerase Mechanism-based Strategy for Viral Attenuation and Vaccine Development. J Biol Chem, 2012. 287(38): p. 31618-22.