病毒在不同的細胞中繼代培養會產生不同的突變點，這些突變點可能會造成病毒細胞趨性與毒力的改變。本篇論文主要是針對感染性選殖株衍生之登革病毒第四型DENV-4 2A病毒株在人類胚胎肺臟纖維母細胞 (MRC-5 cells) 繼代培養後產生位於套膜蛋白domainⅢ C loop位置的適應性突變點Glu345-Lys (E345-K)進行研究 (Liu et al., PLoS ONE, 2008;e1810) 。另一個位於套膜蛋白domain III BC loop的突變點Glu327Gly (E327G)，是在恆河猴胚胎肺臟細胞 (FRhL cells) 繼代培養後產生的適應性突變點，研究指出Glu327Gly這個適應性突變會造成病毒的感染力以及抗原性降低 (Anez et al., J Virol. 2009;83(20): 10384-94)。將兩個單點突變 Glu345-Lys和Glu327Gly利用標靶突變的技術分別構築於兩種登革疫苗候選株DENV-4 2A與DEN-4 2AΔ30的感染性選殖株骨架上，DENV-4 2AΔ30是DENV-4 2A在3’ 非轉譯區有30個核苷酸缺失突變的衍生病毒株。以反向遺傳法做出帶有單點突變的病毒，接著將突變的病毒分別在MRC-5與非洲綠猴腎細胞 (Vero cells)兩種細胞株中繼代培養三代。突變點Glu345Lys在Vero細胞中繼代培養的過程中會有回復成Glu345的現象出現，而突變點Glu327Gly則可以穩定地存在；Glu345Lys突變點在MRC-5細胞中的繼代培養，能穩定地存在。適應性突變點造成病毒感染力的改變或許可以由兩種突變病毒的病毒蝕斑明顯變小獲得證明；新生小鼠的神經毒力實驗可以觀察到兩種帶有適應性突變點的病毒DENV-4 2A Glu345Lys與DENV-4 2A Glu327Gly所造成的毒力較野生型病毒DEN-2 NGS C病毒株及其病毒母株DEN-4 2A弱。Glu345Lys與Glu327Gly在分子模型的預測中會造成套膜蛋白表面的電性由原本的負電性變成正電性，而套膜蛋白表面電性的改變，可能會影響病毒與宿主細胞受體間結合的能力，實驗結果證明DENV-4 2A Glu345Lys與DENV-4 2A Glu327Gly病毒對於Heparin的結合能力都明顯高於其病毒母株DENV-4 2A。本篇論文對於適應性點突變的研究，可以提供登革病毒減毒活毒疫苗開發重要的資訊。

Passage of virus in different cell lines is possible to produce host-cell specific mutations, which may affect cell tropism and virus virulence. We have previously reported that a dengue type 4 virus vaccine candidate passaged in human fetal lung fibroblast (MRC-5) cells rapidly acquired a Glu345-Lys substitution in the envelope protein domain III (Liu et al., PLoS ONE, 2008;e1810). It was also reported that another Glu327Gly substitution of the same vaccine candidate was selected after three passages in fetal rhesus lung (FRhL) cells with reduced infectivity and immunogenicity in rhesus monkeys (Anez et al., J Virol. 2009;83(20): 10384-94). Therefore, single point mutation at Glu327Gly and Glu345Lys, respectively, was constructed in two infectious cDNA clones of the dengue type 4 virus vaccine candidates, DEN-4 2A and its derived 3’ NCR deletion mutant DEN-4 2AΔ30. Using PCR-mediated site-directed mutagenesis method, the infectious cDNA clone-derived Glu345-Lys mutants of DEN-4 2A and DEN-4 2AΔ30 were passaged in MRC-5 cells for three consecutive times. Similarly, the infectious cDNA clone-derived Glu327Gly DEN-4 2A and DEN-4 2AΔ30 were three time-passaged in Vero cells. Single point mutation of Glu345-Lys was found to revert to Glu345 when the virus was passaged in Vero cells. The Glu345-Lys substitution predicted using molecular modeling show the increase of more positive charges on the surface of E protein than the Glu327Gly substitution. The virulence of these recombinant mutant viruses were analyzed in newborn ICR mice. The neurovirulence of these mutated viruses was significantly less than the DEN-2 NGC strain and their parent strain virus. Virulence attenuation inducing by adaptation mutation implicated important information to the development of live-attenuated dengue vaccine.

Anez G, Men R, Eckels KH, Lai CJ. 2009. Passage of dengue virus type 4 vaccine candidates in fetal rhesus lung cells selects heparin-sensitive variants that result in loss of infectivity and immunogenicity in rhesus macaques. J Virol 83: 10384-10394.
Basu A, Chaturvedi UC. 2008. Vascular endothelium: the battlefield of dengue viruses. FEMS Immunol Med Microbiol 53: 287-299.
Blaney JE, Jr., Johnson DH, Firestone CY, Hanson CT, Murphy BR, Whitehead SS. 2001. Chemical mutagenesis of dengue virus type 4 yields mutant viruses which are temperature sensitive in vero cells or human liver cells and attenuated in mice. J Virol 75: 9731-9740.
Blaney JE, Jr., Manipon GG, Firestone CY, Johnson DH, Hanson CT, Murphy BR, Whitehead SS. 2003. Mutations which enhance the replication of dengue virus type 4 and an antigenic chimeric dengue virus type 2/4 vaccine candidate in Vero cells. Vaccine 21: 4317-4327.
Bordignon J, Strottmann DM, Mosimann AL, Probst CM, Stella V, Noronha L, Zanata SM, Dos Santos CN. 2007. Dengue neurovirulence in mice: identification of molecular signatures in the E and NS3 helicase domains. J Med Virol 79: 1506-1517.
Chen Y, Maguire T, Hileman RE, Fromm JR, Esko JD, Linhardt RJ, Marks RM. 1997. Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate. Nat Med 3: 866-871.
Dorrance WR, Frankel JW, Gordon I, Patterson PR, Schlesinger RW, Winter JW. 1956. Clinical and serologic response of man to immunization with attenuated dengue and yellow fever viruses. J Immunol 77: 352-364.
Fujita N, Oda K, Yasui Y, Hotta S. 1969. Research on dengue in tissue culture. IV. Serologic responses of human beings to combined inoculations of attenuated, tissue-cultured type I dengue virus and yellow fever vaccine. Kobe J Med Sci 15: 163-180.
Gomez SY, Ocazionez RE. 2008. [Yellow fever virus 17D neutralising antibodies in vaccinated Colombian people and unvaccinated ones having immunity against dengue]. Rev Salud Publica (Bogota) 10: 796-807.
Grachev VP, Khapchaev I. 2008. [Use of continuous human and animal cell lines for the production of viral vaccines]. Zh Mikrobiol Epidemiol Immunobiol: 82-90.
Gubler DJ. 1998. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 11: 480-496.
Guy B, Guirakhoo F, Barban V, Higgs S, Monath TP, Lang J. 2010. Preclinical and clinical development of YFV 17D-based chimeric vaccines against dengue, West Nile and Japanese encephalitis viruses. Vaccine 28: 632-649.
Halstead SB. 1989. Antibody, macrophages, dengue virus infection, shock, and hemorrhage: a pathogenetic cascade. Rev Infect Dis 11 Suppl 4: S830-839.
—. 2006. Dengue in the Americas and Southeast Asia: do they differ? Rev Panam Salud Publica 20: 407-415.
Hanley KA, Manlucu LR, Manipon GG, Hanson CT, Whitehead SS, Murphy BR, Blaney JE, Jr. 2004. Introduction of mutations into the non-structural genes or 3' untranslated region of an attenuated dengue virus type 4 vaccine candidate further decreases replication in rhesus monkeys while retaining protective immunity. Vaccine 22: 3440-3448.
Heinz FX, Allison SL. 2003. Flavivirus structure and membrane fusion. Adv Virus Res 59: 63-97.
Hotta S. 1952. Experimental studies on dengue. I. Isolation, identification and modification of the virus. J Infect Dis 90: 1-9.
Huang CY, Butrapet S, Moss KJ, Childers T, Erb SM, Calvert AE, Silengo SJ, Kinney RM, Blair CD, Roehrig JT. 2010. The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion. Virology 396: 305-315.
Jessie K, Fong MY, Devi S, Lam SK, Wong KT. 2004. Localization of dengue virus in naturally infected human tissues, by immunohistochemistry and in situ hybridization. J Infect Dis 189: 1411-1418.
Lee E, Lobigs M. 2002. Mechanism of virulence attenuation of glycosaminoglycan-binding variants of Japanese encephalitis virus and Murray Valley encephalitis virus. J Virol 76: 4901-4911.
Lee E, Hall RA, Lobigs M. 2004. Common E protein determinants for attenuation of glycosaminoglycan-binding variants of Japanese encephalitis and West Nile viruses. J Virol 78: 8271-8280.
Lee E, Wright PJ, Davidson A, Lobigs M. 2006. Virulence attenuation of Dengue virus due to augmented glycosaminoglycan-binding affinity and restriction in extraneural dissemination. J Gen Virol 87: 2791-2801.
Liu CC, Lee SC, Butler M, Wu SC. 2008. High genetic stability of dengue virus propagated in MRC-5 cells as compared to the virus propagated in vero cells. PLoS One 3: e1810.
Mandl CW, Kroschewski H, Allison SL, Kofler R, Holzmann H, Meixner T, Heinz FX. 2001. Adaptation of tick-borne encephalitis virus to BHK-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo. J Virol 75: 5627-5637.
McArthur JH, Durbin AP, Marron JA, Wanionek KA, Thumar B, Pierro DJ, Schmidt AC, Blaney JE, Jr., Murphy BR, Whitehead SS. 2008. Phase I clinical evaluation of rDEN4Delta30-200,201: a live attenuated dengue 4 vaccine candidate designed for decreased hepatotoxicity. Am J Trop Med Hyg 79: 678-684.
Men R, Bray M, Clark D, Chanock RM, Lai CJ. 1996. Dengue type 4 virus mutants containing deletions in the 3' noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys. J Virol 70: 3930-3937.
Miagostovich MP, dos Santos FB, Fumian TM, Guimaraes FR, da Costa EV, Tavares FN, Coelho JO, Nogueira RM. 2006. Complete genetic characterization of a Brazilian dengue virus type 3 strain isolated from a fatal outcome. Mem Inst Oswaldo Cruz 101: 307-313.
Nickells J, Cannella M, Droll DA, Liang Y, Wold WS, Chambers TJ. 2008. Neuroadapted yellow fever virus strain 17D: a charged locus in domain III of the E protein governs heparin binding activity and neuroinvasiveness in the SCID mouse model. J Virol 82: 12510-12519.
Nybakken GE, Oliphant T, Johnson S, Burke S, Diamond MS, Fremont DH. 2005. Structural basis of West Nile virus neutralization by a therapeutic antibody. Nature 437: 764-769.
Ramos C, Sanchez G, Pando RH, Baquera J, Hernandez D, Mota J, Ramos J, Flores A, Llausas E. 1998. Dengue virus in the brain of a fatal case of hemorrhagic dengue fever. J Neurovirol 4: 465-468.
Rosenzweig EC, Babione RW, Wisseman CL, Jr. 1963. Immunological studies with group B arthropod-borne viruses. IV. Persistence of yellow fever antibodies following vaccination with 17D strain yellow fever vaccine. Am J Trop Med Hyg 12: 230-235.
Sabin AB. 1952. Research on dengue during World War II. Am J Trop Med Hyg 1: 30-50.
Sabin AB, Schlesinger RW. 1945. Production of Immunity to Dengue with Virus Modified by Propagation in Mice. Science 101: 640-642.
Webster DP, Farrar J, Rowland-Jones S. 2009. Progress towards a dengue vaccine. Lancet Infect Dis 9: 678-687.
Wu SJ, et al. 2000. Human skin Langerhans cells are targets of dengue virus infection. Nat Med 6: 816-820.