茲卡病毒屬於黃病毒科的一員，茲卡病毒首次在1947年烏干達的rhesus monkey中被發現，從那時起，茲卡病毒已經在非洲和亞洲從人類和蚊子中分離出不同品系的茲卡病毒，茲卡病毒是一種由Aedes mosquitoes所傳播的病毒，受到感染後會導致茲卡熱。在近幾年的研究中發現，茲卡病毒的感染和嬰兒小頭畸形以及格林-巴利症候群有關。茲卡病毒和其他黃病毒有許多相似之處，例如：登革病毒，黃熱病毒，日本腦炎病毒和西尼羅河病毒。雖然已有有效疫苗治療上述黃病毒，但是目前對於茲卡病毒並沒有有效的疫苗或治療方法，所以研究茲卡病毒是重要的。類病毒顆粒之套膜蛋白跟病毒一樣會有酸性所會引發的套膜蛋白排列重組及進入細胞之膜融和反應，類病毒顆粒不具感染力，因此具安全性，可以大量生產及可以被操控，因此類病毒顆粒被認為是用於病毒疫苗很有潛力的方式。
在我的研究中，我們與中興大學趙黛瑜教授合作，大量生產茲卡類病毒（Zika VLP）並利用負染色電子顯微鏡技術，拍攝茲卡類病毒的影像，發現純化出的顆粒有大小分布，然後我們利用免疫金標定來標定茲卡類病毒，發現被金標定的茲卡類病毒大小介於在30至50 nm間。接著，我們使用低溫電子顯微鏡(cryo-electron microscope, cryo-EM)的技術，拍攝茲卡類病毒的影像，透過二維影像分類圖，發現50 nm顆粒的二維影像密度圖較其他大小粒子具較結實及清晰的構造，代表相較其他顆粒更具對稱性的特徵。因此我們判斷50nm粒子是我們的類病毒顆粒，我們透過EMAN2重組其結構。透過三維結構，我們發現所解析出來的結構大小和目前在2016年發表的茲卡病毒結構相似，但我們的類病毒為一個空心的結構，此外，我們將以發表的茲卡病毒enveloped glycoprotein (E protein) 排列在我們的類病毒顆粒表面，發現E protein 能夠良好的平鋪在在結構表層，並以T=3排列，我們期待將茲卡類病毒結構解析度提高，高解析度結構將顯示出可能的抗原決定位極可能的標定，這些位點將可以提供在研發茲卡病毒的藥物或疫苗上提供更多資訊。

Zika virus is a member of the virus family Flaviviridae. Zika virus was first isolated from rhesus monkey in Uganda in 1947. Since that time, Zika virus has been isolated from human and mosquito in both Africa and Asia. It was spreaded by Aedes mosquitoes and caused Zika fever. In recent years, some studies showed that Zika virus is related to microcephaly and Guillain Barré syndrome. Zika virus is related to the dengue virus, yellow fever virus, Japanese encephalitis virus and West Nile virus. Although there are vaccines for treat those flavivirus, there is no effective vaccine or treatment for Zika virus therefore it is important to study Zika virus. VLP containing envelope proteins have been demonstrated to be a potential vaccine candidate, since their ordered E structures are similar to those on the virion surface and also undergo low-pH-induced rearrangements and membrane fusion similar to viral particle. More importantly, the advantages of VLP include no infection, high production, and can be manipulated.
Here, we produced Zika virus-like particle (Zika VLP). We checked the Zika VLP by negative stain electron microscope (EM). We found that there was size distribution under EM inspection. To identify the Zika VLP, we performed the immunogold labeling. We found the size of the Zika VLP which was recognized by nanogold were the particles with the size of 30nm-50nm in diameter. Next, we used cryo-electron microscope (cryo-EM) and 3D reconstruction approach to reconstruct the structure of Zika VLP. By 2D classification analyses, we found the structure of 50 nm particles have more solid structure than other particles meaning that 50nm particles have features of icosahedral symmetry; therefore 50nm particles were suffering from the following 3D reconstruction process. Cryo-EM structure of ZIKV VLP was similar to the ZIKV virion structure published in 2016 except that VLP had a hollow structure. Furthermore, fitting the enveloped glycoprotein (E protein) of ZIKV into our density map showed that the E protein could arrange well on the surface and had T=3 arrangement. In the future we will continue improving resolution, the high resolution structure will reveal the possible epitopes and potential targets which will be the hints for antiviral candidates and effective vaccine development.

1. Dick, G.W., S.F. Kitchen, and A.J. Haddow, Zika virus. I. Isolations and serological specificity. Trans R Soc Trop Med Hyg, 1952. 46(5): p. 509-20.
2. Macnamara, F.N., Zika virus: a report on three cases of human infection during an epidemic of jaundice in Nigeria. Trans R Soc Trop Med Hyg, 1954. 48(2): p. 139-45.
3. Duffy, M.R., et al., Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med, 2009. 360(24): p. 2536-43.
4. Zanluca, C., et al., First report of autochthonous transmission of Zika virus in Brazil. Mem Inst Oswaldo Cruz, 2015. 110(4): p. 569-72.
5. Althouse, B.M., et al., Potential for Zika Virus to Establish a Sylvatic Transmission Cycle in the Americas. PLoS Negl Trop Dis, 2016. 10(12): p. e0005055.
6. Basarab, M., et al., Zika virus. BMJ, 2016. 352: p. i1049.
7. Kuno, G., et al., Phylogeny of the genus Flavivirus. J Virol, 1998. 72(1): p. 73-83.
8. Cao-Lormeau, V.M., et al., Guillain-Barre Syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet, 2016. 387(10027): p. 1531-1539.
9. Schuler-Faccini, L., et al., Possible Association Between Zika Virus Infection and Microcephaly - Brazil, 2015. MMWR Morb Mortal Wkly Rep, 2016. 65(3): p. 59-62.
10. Miner, J.J. and M.S. Diamond, Zika virus pathogenesis and tissue tropism. Cell Host Microbe, 2017. 21(2): p. 134-42.
11. Hamel, R., et al., Biology of Zika Virus Infection in Human Skin Cells. J Virol, 2015. 89(17): p. 8880-96.
12. Saiz, J.C., et al., Zika Virus: the Latest Newcomer. Front Microbiol, 2016. 7: p. 496.
13. Roby, J.A., et al., Post-translational regulation and modifications of flavivirus structural proteins. J Gen Virol, 2015. 96(Pt 7): p. 1551-69.
14. Sirohi, D., et al., The 3.8 A resolution cryo-EM structure of Zika virus. Science, 2016. 352(6284): p. 467-70.
15. Kuhn, R.J., et al., Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell, 2002. 108(5): p. 717-25.
16. Hasan, S.S., et al., A human antibody against Zika virus crosslinks the E protein to prevent infection. Nat Commun, 2017. 8: p. 14722.
17. Perera, R. and R.J. Kuhn, Structural proteomics of dengue virus. Curr Opin Microbiol, 2008. 11(4): p. 369-77.
18. Yu, I.M., et al., Structure of the immature dengue virus at low pH primes proteolytic maturation. Science, 2008. 319(5871): p. 1834-7.
19. Zhang, Y., et al., Structures of immature flavivirus particles. EMBO J, 2003. 22(11): p. 2604-13.
20. Dai, L., et al., Structures of the Zika Virus Envelope Protein and Its Complex with a Flavivirus Broadly Protective Antibody. Cell Host & Microbe, 2016. 19(5): p. 696-704.
21. Bressanelli, S., et al., Structure of a flavivirus envelope glycoprotein in its low‐pH‐induced membrane fusion conformation. The EMBO Journal, 2004. 23(4): p. 728-738.
22. Navarro‐Sanchez, E., et al., Dendritic‐cell‐specific ICAM3‐grabbing non‐integrin is essential for the productive infection of human dendritic cells by mosquito‐cell‐derived dengue viruses. EMBO reports, 2003. 4(7): p. 723-728.
23. Tassaneetrithep, B., et al., DC-SIGN (CD209) Mediates Dengue Virus Infection of Human Dendritic Cells. The Journal of Experimental Medicine, 2003. 197(7): p. 823-829.
24. Richter, M.K.S., et al., Immature Dengue Virus Is Infectious in Human Immature Dendritic Cells via Interaction with the Receptor Molecule DC-SIGN. PLOS ONE, 2014. 9(6): p. e98785.
25. Pokidysheva, E., et al., Cryo-EM Reconstruction of Dengue Virus in Complex with the Carbohydrate Recognition Domain of DC-SIGN. Cell, 2006. 124(3): p. 485-493.
26. Beckett, C.G., et al., Evaluation of a prototype dengue-1 DNA vaccine in a Phase 1 clinical trial. Vaccine, 2011. 29(5): p. 960-8.
27. Ferraro, B., et al., Clinical applications of DNA vaccines: current progress. Clin Infect Dis, 2011. 53(3): p. 296-302.
28. Martin, J.E., et al., A West Nile virus DNA vaccine induces neutralizing antibody in healthy adults during a phase 1 clinical trial. J Infect Dis, 2007. 196(12): p. 1732-40.
29. Larocca, R.A., et al., Vaccine protection against Zika virus from Brazil. Nature, 2016. 536(7617): p. 474-8.
30. Abbink, P., et al., Protective efficacy of multiple vaccine platforms against Zika virus challenge in rhesus monkeys. Science, 2016. 353(6304): p. 1129-32.
31. Muthumani, K., et al., In vivo protection against ZIKV infection and pathogenesis through passive antibody transfer and active immunisation with a prMEnv DNA vaccine. NPJ Vaccines, 2016. 1: p. 16021.
32. Schlake, T., et al., Developing mRNA-vaccine technologies. RNA Biol, 2012. 9(11): p. 1319-30.
33. Richner, J.M., et al., Modified mRNA Vaccines Protect against Zika Virus Infection. Cell, 2017. 168(6): p. 1114-1125 e10.
34. Pardi, N., et al., Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination. Nature, 2017. 543(7644): p. 248-251.
35. Monath, T.P., et al., An inactivated cell-culture vaccine against yellow fever. N Engl J Med, 2011. 364(14): p. 1326-33.
36. Heinz, F.X. and K. Stiasny, Flaviviruses and flavivirus vaccines. Vaccine, 2012. 30(29): p. 4301-6.
37. Monath, T.P., et al., Inactivated yellow fever 17D vaccine: development and nonclinical safety, immunogenicity and protective activity. Vaccine, 2010. 28(22): p. 3827-40.
38. Tauber, E., et al., Safety and immunogenicity of a Vero-cell-derived, inactivated Japanese encephalitis vaccine: a non-inferiority, phase III, randomised controlled trial. Lancet, 2007. 370(9602): p. 1847-53.
39. Durbin, A.P., et al., A 12-Month-Interval Dosing Study in Adults Indicates That a Single Dose of the National Institute of Allergy and Infectious Diseases Tetravalent Dengue Vaccine Induces a Robust Neutralizing Antibody Response. J Infect Dis, 2016. 214(6): p. 832-5.
40. Barrett, A.D. and D.E. Teuwen, Yellow fever vaccine - how does it work and why do rare cases of serious adverse events take place? Curr Opin Immunol, 2009. 21(3): p. 308-13.
41. Priyamvada, L., et al., Humoral cross-reactivity between Zika and dengue viruses: implications for protection and pathology. Emerging Microbes &Amp; Infections, 2017. 6: p. e33.
42. Dejnirattisai, W., et al., Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus. Nat Immunol, 2016. 17(9): p. 1102-8.
43. Charles, A.S. and R.C. Christofferson, Utility of a Dengue-Derived Monoclonal Antibody to Enhance Zika Infection In Vitro. PLoS Curr, 2016. 8.
44. Paul, L.M., et al., Dengue virus antibodies enhance Zika virus infection. Clin Transl Immunology, 2016. 5(12): p. e117.
45. Stettler, K., et al., Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection. Science, 2016. 353(6301): p. 823-6.
46. Kawiecki, A.B. and R.C. Christofferson, Zika Virus-Induced Antibody Response Enhances Dengue Virus Serotype 2 Replication In Vitro. J Infect Dis, 2016. 214(9): p. 1357-1360.
47. Parra, B., et al., Guillain-Barre Syndrome Associated with Zika Virus Infection in Colombia. N Engl J Med, 2016. 375(16): p. 1513-1523.
48. Sur, D.K., D.H. Wallis, and T.X. O'Connell, Vaccinations in pregnancy. Am Fam Physician, 2003. 68(2): p. 299-304.
49. Bozzo, P., A. Narducci, and A. Einarson, Vaccination during pregnancy. Can Fam Physician, 2011. 57(5): p. 555-7.
50. Munoz, F.M., Infant Protection Against Influenza Through Maternal Immunization: A Call for More Immunogenic Vaccines. JAMA Pediatr, 2016. 170(9): p. 832-3.
51. Zinkernagel, R.M., On the Role of Dendritic Cells Versus Other Cells in Inducing Protective CD8+ T Cell Responses. Front Immunol, 2014. 5.
52. Beyer, T., et al., Bacterial carriers and virus-like-particles as antigen delivery devices: role of dendritic cells in antigen presentation. Curr Drug Targets Infect Disord, 2001. 1(3): p. 287-302.
53. Wang, J.W. and R.B. Roden, Virus-like particles for the prevention of human papillomavirus-associated malignancies. Expert Rev Vaccines, 2013. 12(2): p. 129-41.
54. Roldao, A., et al., Virus-like particles in vaccine development. Expert Rev Vaccines, 2010. 9(10): p. 1149-76.
55. Chroboczek, J., I. Szurgot, and E. Szolajska, Virus-like particles as vaccine. Acta Biochim Pol, 2014. 61(3): p. 531-9.
56. Allison, S.L., et al., Synthesis and secretion of recombinant tick-borne encephalitis virus protein E in soluble and particulate form. J Virol, 1995. 69(9): p. 5816-20.
57. Burghardt, R.C. and R. Droleskey, Transmission electron microscopy. Curr Protoc Microbiol, 2006. Chapter 2: p. Unit 2B 1.
58. Bai, X.C., G. McMullan, and S.H. Scheres, How cryo-EM is revolutionizing structural biology. Trends Biochem Sci, 2015. 40(1): p. 49-57.
59. De Carlo, S. and J.R. Harris, Negative staining and cryo-negative staining of macromolecules and viruses for TEM. Micron, 2011. 42(2): p. 117-31.
60. Fabig, G., et al., Labeling of ultrathin resin sections for correlative light and electron microscopy. Methods Cell Biol, 2012. 111: p. 75-93.
61. Penczek, P., M. Radermacher, and J. Frank, Three-dimensional reconstruction of single particles embedded in ice. Ultramicroscopy, 1992. 40(1): p. 33-53.
62. Elmlund, D. and H. Elmlund, Cryogenic electron microscopy and single-particle analysis. Annu Rev Biochem, 2015. 84: p. 499-517.
63. Kostyuchenko, V.A., et al., Structure of the thermally stable Zika virus. Nature, 2016. 533(7603): p. 425-8.
64. Boigard, H., et al., Zika virus-like particle (VLP) based vaccine. PLoS Negl Trop Dis, 2017. 11(5).
65. Espinosa, D., et al., Passive Transfer of Immune Sera Induced by a Zika Virus-Like Particle Vaccine Protects AG129 Mice Against Lethal Zika Virus Challenge. EBioMedicine, 2018. 27: p. 61-70.
66. Kostyuchenko, V.A., et al., Immature and Mature Dengue Serotype 1 Virus Structures Provide Insight into the Maturation Process. J Virol, 2013. 87(13): p. 7700-7.
67. Allison, S.L., et al., Two distinct size classes of immature and mature subviral particles from tick-borne encephalitis virus. J Virol, 2003. 77(21): p. 11357-66.