Construction and characterization of recombinant flaviviruses bearing insertions between E and NSI genes
Construction and characterization of recombinant flaviviruses bearing insertions between E and NSI genes
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DOI：
10.1186/1743-422x-4-115
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发表时间：
2007-10-30
期刊：
影响因子：
4.8
通讯作者：
Galler, Ricardo
中科院分区：
文献类型：
--
作者：
Bonaldo, Myrna C.;Mello, Samanta M.;Galler, Ricardo
背景：黄热病病毒是黄病毒属的成员，是一种节肢动物传播的病原体，可引起人类严重疾病。减毒黄热病17 D病毒株已用于人类疫苗接种70年，并具有开发新的减毒活疫苗所需的几个特征。我们在这里描述了一种方法来构建一个可行的，免疫原性重组黄热病17 D病毒表达的绿色荧光蛋白变体（EGFP）。该方法考虑了E和NSI基因间区侧翼的功能基序和氨基酸序列保守性的存在，以复制并将它们融合到外源基因，从而允许病毒多蛋白前体的正确加工。YF 17 D EGFP重组病毒在Vero细胞中生长，并在感染后96小时达到约6.45 +/-0.4 log 10 PFU/mL的峰值滴度。免疫沉淀和共聚焦激光扫描显微镜证实了EGFP的表达，它保留在内质网中，而不是从感染的细胞分泌。与ER隔室的关联不干扰YF组装，因为重组病毒完全有能力复制并离开细胞。该病毒在Vero细胞中连续传代至第10代时遗传稳定。重组病毒能够引起中和抗体反应的YF和抗体的增强型绿色荧光蛋白，证明了由ELISA测试。结论：该系统为进一步研究YF 17 D/DEN 4嵌合重组病毒奠定了基础，为进一步开发YF 17 D减毒活疫苗奠定了基础此外，将外源基因插入黄病毒基因组中还可以允许对黄病毒细胞和组织嗜性以及与黄病毒感染相关的细胞过程进行体内研究。
Background: The yellow fever virus, a member of the genus Flavivirus, is an arthropod-borne pathogen causing severe disease in humans. The attenuated yellow fever 17D virus strain has been used for human vaccination for 70 years and has several characteristics that are desirable for the development of new, live attenuated vaccines. We described here a methodology to construct a viable, and immunogenic recombinant yellow fever 17D virus expressing a green fluorescent protein variant (EGFP). This approach took into account the presence of functional motifs and amino acid sequence conservation flanking the E and NSI intergenic region to duplicate and fuse them to the exogenous gene and thereby allow the correct processing of the viral polyprotein precursor.Results: YF 17D EGFP recombinant virus was grew in Vero cells and reached a peak titer of approximately 6.45 +/- 0.4 log10 PFU/mL at 96 hours post- infection. Immunoprecipitation and confocal laser scanning microscopy demonstrated the expression of the EGFP, which was retained in the endoplasmic reticulum and not secreted from infected cells. The association with the ER compartment did not interfere with YF assembly, since the recombinant virus was fully competent to replicate and exit the cell. This virus was genetically stable up to the tenth serial passage in Vero cells. The recombinant virus was capable to elicit a neutralizing antibody response to YF and antibodies to EGFP as evidenced by an ELISA test. The applicability of this cloning strategy to clone gene foreign sequences in other flavivirus genomes was demonstrated by the construction of a chimeric recombinant YF 17D/DEN4 virus.Conclusion: This system is likely to be useful for a broader live attenuated YF 17D virus-based vaccine development for human diseases. Moreover, insertion of foreign genes into the flavivirus genome may also allow in vivo studies on flavivirus cell and tissue tropism as well as cellular processes related to flavivirus infection.