近日，中國科學院武漢病毒研究所羅敏華課題組在寨卡病毒（ZIKV）致神經損傷機制的研究方面取得新進展，發現寨卡病毒感染神經幹細胞時顯著下調其重要標誌分子雙皮質素（doublecortin， DCX），為寨卡病毒感染影響腦皮層結構和大腦神經發育提出新的潛在機制。本工作在線發表於Frontiers in Micorbiology。

寨卡病毒（Zika virus，ZIKV），是黃病毒科中之黃病毒屬，經由埃及斑蚊傳播，該病毒最早在1947年於烏干達的寨卡森林中的獼猴體內分離出來，因而得名。近兩年，寨卡病毒爆發於中南美洲和東南亞地區，並導致該地區小頭畸形（microcephaly）和其他出生缺陷率顯著升高，引起全球關注。已有報道顯示人神經幹細胞（neural progenitor cell， NPC）能夠被寨卡病毒感染，提示其導致小頭畸形可能是通過影響神經幹細胞的細胞命運而導致的，而其分子機制尚未解析。

該研究通過對寨卡病毒感染人原代NPC進行蛋白質組學分析，發現細胞內影響神經祖細胞增殖、分化、遷移的重要標誌分子的蛋白水平都有所改變.而其中雙皮質素（DCX）在NPC的分化、遷移中起著重要作用。結果顯示，寨卡病毒感染NPC時，能同時下調DCX的蛋白質水平及mRNA水平。而在寨卡病毒感染胎鼠腦模型中，DCX的蛋白水平也有明顯下調，且伴隨著胎鼠體重、胎腦重和胎腦大小的異常及腦皮質結構的破壞。通過進一步篩查寨卡病毒蛋白，並使其NPC中逐一表達，結果發現NS4A和NS5與DCX蛋白水平和mRNA水平的下調存在相關性。這些數據說明寨卡病毒對DCX的調節是發生於感染之後，而病毒蛋白NS4A，NS5參與DCX的調控。

該研究工作揭示了寨卡病毒與NPC細胞命運及胎腦發育異常之間的聯繫，指出了關鍵分子DCX在皮層感染病理結果發揮的重要作用。同時，篩選到病毒蛋白NS4A，NS5下調關鍵分子DCX，進一步揭示了寨卡病毒影響胎腦發育的分子機制。後續將繼續確認是病毒蛋白與DCX 相互作用的機制。

武漢病毒所神經病毒學科組博士後姜旋為第一作者，羅敏華研究員和廣州婦女兒童醫療中心唐亞平教授為共同通訊作者，該工作獲得國家自然科學基金、國家重點實驗室和廣州婦女兒童醫療中心的合作與支持。

2015年6月伊始，寨卡病毒（Zika Virus，ZIKV）在美洲大規模流行，導致大批嬰兒腦發育不全。截至目前，針對ZIKV的感染，尚沒有獲得授權的疫苗上市，也沒有特異性的抗病毒治療措施。近期，中國科學院武漢病毒研究所王漢中研究員團隊成功利用合成工程技術研製出新型ZIKV弱毒疫苗。

合成減毒病毒工程技術（synthetic attenuated virus engineering，SAVE），又稱為」密碼對去優化技術」，在不改變氨基酸種類及儘可能不影響RNA空間結構的情況下，提高病毒基因組中罕見的密碼對所佔的比例，從而降低病毒的複製翻譯效率，使病毒致病性減弱。該技術製備弱毒疫苗具有周期短，安全，以及免疫原性強等特點。

研究人員利用反向遺傳學操作技術成功拯救出三株致弱的寨卡病毒(Min E, Min NS1和Min E+NS1)。其中Min E+NS1的基因組中引入了2568個同義突變，單次免疫後就可以刺激小鼠產生高滴度中和抗體，誘導產生清除性的免疫，獲得完全的攻毒保護，並且可以阻止ZIKV通過母體垂直傳播給子代。由於基因組中含有成百上千的同義突變，回復突變的風險極低。

該研究證明利用密碼對去優化技術可以將ZIKV高效地致弱，MinE+NS1具有潛力成為一種安全的疫苗候選，預防ZIKV的感染。研究成果在病毒學權威雜誌Journal ofVirology上在線發表，武漢病毒所2014級博士生李朋輝為論文第一作者，王漢中研究員與鄭振華青年研究員為論文通訊作者。該研究得到了國家重點研發計劃，國家自然科學基金和中國科學院青年創新促進會的支持。

Proteomic Analysis of Zika Virus Infected Primary Human Fetal Neural Progenitors Suggests a Role for Doublecortin in the Pathological Consequences of Infection in the Cortex.

Abstract

Zika virus (ZIKV) infection is associated with severe neurological defects in fetuses and newborns, such as microcephaly. However, the underlying mechanisms remain to be elucidated. In this study, proteomic analysis on ZIKV-infected primary human fetal neural progenitor cells (NPCs) revealed that virus infection altered levels of cellular proteins involved in NPC proliferation, differentiation and migration. The transcriptional levels of some of the altered targets were also confirmed by qRT-PCR. Among the altered proteins, doublecortin (DCX) plays an important role in NPC differentiation and migration. Results showed that ZIKV infection downregulated DCX, at both mRNA and protein levels, as early as 1 day post infection (1 dpi), and lasted throughout the virus replication cycle (4 days). The downregulation of DCX was also observed in a ZIKV-infected fetal mouse brain model, which displayed decreased body weight, brain size and weight, as well as defective cortex structure. By screening the ten viral proteins of ZIKV, we found that both the expression of NS4A and NS5 were correlated with the downregulation of both mRNA and protein levels of DCX in NPCs. These data suggest that DCX is modulated following infection of the brain by ZIKV. How these observed changes of DCX expression translate in the pathological consequences of ZIKV infection and if other cellular proteins are equally involved remains to be investigated.

Zika Virus Attenuation by Codon Pair Deoptimization Induces Sterilizing Immunity in Mouse Models.

Abstract

Zika virus (ZIKV) infection during the large epidemics in the Americas is related to congenital abnormities or fetal demise. To date, there is no vaccine, antiviral drug, or other modality available to prevent or treat Zika virus infection. Here we designed novel live attenuated ZIKV vaccine candidates using a codon pair deoptimization strategy. Three codon pair-deoptimized ZIKVs (Min E, Min NS1, and Min E+NS1) were de novo synthesized, and recovered by reverse genetics, containing large amounts of underrepresented codon pairs in E gene and/or NS1 gene. Amino acid sequence was 100% unchanged. The codon pair-deoptimized variants had decreased replication fitness in Vero cells (Min NS1 ? Min E > Min E+NS1), replicated more efficiently in insect cells than in mammalian cells, and demonstrated diminished virulence in a mouse model. In particular, Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer, and a single immunization achieved complete protection against lethal challenge and vertical ZIKV transmission during pregnancy. More importantly, due to the numerous synonymous substitutions in the codon pair-deoptimized strains, reversion to wild-type virulence through gradual nucleotide sequence mutations is unlikely. Our results collectively demonstrate that ZIKV can be effectively attenuated by codon pair deoptimization, highlighting the potential of Min E+NS1 as a safe vaccine candidate to prevent ZIKV infections.IMPORTANCE Due to unprecedented epidemics of Zika virus (ZIKV) across the Americas and the unexpected clinical symptoms including Guillain-Barré syndrome, microcephaly and other birth defects in human, there is an urgent need for ZIKV vaccine development. Here, we provided the first attenuated versions of ZIKV with two important genes (E and/or NS1) that were subjected to codon pair deoptimization. Compared to parental ZIKV, the codon pair-deoptimized ZIKVs were mammalian-attenuated, and preferred insect to mammalian Cells. Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer, and achieved complete protection against lethal challenge and vertical virus transmission during pregnancy. More importantly, the massive synonymous mutational approach made it impossible to revert to wild-type virulence. Our results have proven the feasibility of codon pair deoptimization as a strategy to develop live-attenuated vaccine candidates against flavivirues like ZIKV, Japanese encephalitis virus and West Nile virus.

來源：中科院武漢病毒所

本期編輯：Tony

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