Zika virus (ZIKV), a mosquito-transmitted flavivirus responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, reemerged in the last decade causing serious human diseases, including microcephaly, congenital malformations, and Guillain-Barré syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to the enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently lacking. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic; it is absent in many of the African isolates but present in all isolates from the recent outbreaks. In the present study, we investigated the roles of this sequence motif and glycosylation of the E protein in the pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or in which the N-linked glycosylation site is mutated by single-amino-acid substitution are highly attenuated and nonlethal. The mutant viruses replicate poorly in the brains of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion.

IMPORTANCE The recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases, including microcephaly and Guillain-Barré syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein, whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for the ability to cause mortality in a mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein is an important factor contributing to ZIKV pathogenicity.

Zika病毒（ZIKV）是一种蚊子传播的黄病毒，在非洲和亚洲引起零星的轻度和高热疾病的爆发，在过去十年中再次出现，引起了严重的人类疾病，包括小头畸形，先天性畸形和格林-巴利综合征。尽管基因组和系统发育分析表明，遗传进化可能导致ZIKV的毒力增强，但目前尚缺乏支持特定遗传变化在毒力中的作用的实验证据。一个序列基序VNDT是多态性的，它在包膜（E）蛋白中包含一个N-连接的糖基化位点；在许多非洲分离株中都没有这种病毒，但在最近爆发的所有分离物中都存在这种病毒。在本研究中，我们调查了该序列基序和E蛋白的糖基化在ZIKV致病性中的作用。我们首先在新型线性载体中构建了ZIKV的稳定全长cDNA克隆，从中可回收感染性病毒。从感染性克隆产生的重组ZIKV含有VNDT基序，具有很高的致病性，并在小鼠模型中引起致死性。相反，从中缺失VNDT基序或其中N-连接的糖基化位点通过单氨基酸取代突变的重组病毒高度减毒并且是非致命性的。皮下接种时，突变病毒在受感染小鼠的大脑中复制能力很差，但在颅内接种后，复制病毒能够很好地复制。我们的发现提供了第一个证据，证明E蛋白的N联糖基化是ZIKV毒力和神经侵袭的重要决定因素。从感染性克隆产生的重组ZIKV含有VNDT基序，具有很高的致病性，并在小鼠模型中引起致死性。相反，从中删除VNDT基序或通过单氨基酸取代使N-连接的糖基化位点突变的重组病毒高度减毒并且是非致命性的。皮下接种时，突变病毒在受感染小鼠的大脑中复制能力很差，但在颅内接种后，复制病毒能够很好地复制。我们的发现提供了第一个证据，证明E蛋白的N联糖基化是ZIKV毒力和神经侵袭的重要决定因素。从感染性克隆产生的重组ZIKV含有VNDT基序，具有很高的致病性，并在小鼠模型中引起致死性。相反，从中缺失VNDT基序或其中N-连接的糖基化位点通过单氨基酸取代突变的重组病毒高度减毒并且是非致命性的。皮下接种时，突变病毒在受感染小鼠的大脑中复制能力很差，但在颅内接种后，复制病毒能够很好地复制。我们的发现提供了第一个证据，证明E蛋白的N联糖基化是ZIKV毒力和神经侵袭的重要决定因素。缺失VNDT基序或通过单氨基酸取代使N-连接的糖基化位点突变的重组病毒高度减毒且非致死性。皮下接种时，突变病毒在受感染小鼠的大脑中复制能力很差，但在颅内接种后，复制病毒能够很好地复制。我们的发现提供了第一个证据，证明E蛋白的N联糖基化是ZIKV毒力和神经侵袭的重要决定因素。缺失VNDT基序或通过单氨基酸取代使N-连接的糖基化位点突变的重组病毒高度减毒且非致死性。皮下接种时，突变病毒在受感染小鼠的大脑中复制能力很差，但在颅内接种后，复制病毒能够很好地复制。我们的发现提供了第一个证据，证明E蛋白的N联糖基化是ZIKV毒力和神经侵袭的重要决定因素。

重要性美洲最近出现的寨卡病毒（ZIKV）引起了全球范围内的重大公共卫生问题。该病毒似乎已获得明显的致病性，导致严重的人类疾病，包括小头畸形和格林-巴利综合征。尽管遗传变化已证明可促进病毒传播，但目前尚不了解导致致病性ZIKV出现的因素。最近爆​​发的所有分离株在病毒包膜（E）蛋白中均包含一个N-连接的糖基化位点，而非洲谱系病毒的许多分离株都缺少该位点。为了阐明糖基化在ZIKV致病性中的功能重要性，产生了来自感染性克隆的重组ZIKV，这些感染性克隆在E蛋白上带有或不带有聚糖。缺乏聚糖的ZIKV在小鼠模型中导致死亡的能力大大减弱，并因神经侵袭而严重受损。我们的研究表明，E蛋白的糖基化是导致ZIKV致病性的重要因素。