Abstract

Background

The molecular mechanisms underpinning the neurologic and congenital pathologies caused by Zika virus (ZIKV) infection remain poorly understood. It is also unclear why congenital ZIKV disease was not reported prior to the recent epidemics in French Polynesia and the Americas, despite evidence that Zika virus has actively circulated in parts of Africa and Asia since 1947 and 1966, respectively.

Methods

Due to advances in the stem cell-based technologies, we can now model ZIKV infections of the central nervous system in human stem cell-derived neural progenitor cells and cerebral organoids, which recapitulate complex 3-dimensional neural architecture. We apply Seq-Well — a simple, portable platform for massively parallel single-cell RNA sequencing — to characterize these neural models infected with ZIKV. We detect and quantify host mRNA transcripts and viral RNA with single-cell resolution, thereby defining transcriptional features of both uninfected and infected cells.

Results

Although flavivirus RNAs lack a poly(A) tail, we present evidence that viral RNAs are specifically primed for reverse transcription at internal runs of adenosines, and that sequencing reads cover the entire non-polyadenylated viral genome. In neural progenitor cells, single cell sequencing reveals that while uninfected bystander cells strongly upregulate interferon pathway genes, these pathways are largely suppressed in cells infected with ZIKV within the same culture dish. Single cell sequencing identifies multiple cell types in our cerebral organoids including neural progenitor cells, intermediate progenitor cells, and neurons of varied maturity. Using this model, we find that neurons, not typically considered targets of ZIKV in the developing brain, contain high copy numbers of ZIKV genomes. It remains uncertain whether neurons are directly infected, or if infected neural progenitor cells differentiate into neurons, carrying virus with them. Notably, the neuronal bystander cell population shows limited interferon gene pathway upregulation compared to neural progenitors.

Conclusions

Overall, our work provides insight into the pathogenesis of ZIKV associated microcephaly, identifies potential new tropisms of ZIKV in the human brain, and suggests that both virus replication and host response mechanisms underlie the neuropathology of ZIKV infection.

中文翻译：

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#33：人类干细胞源性神经祖细胞和脑类器官中寨卡病毒感染的单细胞 RNA 测序分析

摘要

背景

支持由寨卡病毒 (ZIKV) 感染引起的神经系统和先天性疾病的分子机制仍然知之甚少。尽管有证据表明寨卡病毒自 1947 年和 1966 年以来一直在非洲和亚洲的部分地区活跃，但为什么在最近的法属波利尼西亚和美洲流行之前没有报告先天性 ZIKV 疾病，这一点也不清楚。

方法

由于基于干细胞的技术的进步，我们现在可以模拟人类干细胞衍生的神经祖细胞和大脑类器官中中枢神经系统的 ZIKV 感染，这些细胞重现了复杂的 3 维神经结构。我们应用 Seq-Well——一个用于大规模并行单细胞 RNA 测序的简单、便携的平台——来表征这些感染 ZIKV 的神经模型。我们以单细胞分辨率检测和量化宿主 mRNA 转录物和病毒 RNA，从而定义未感染和感染细胞的转录特征。

结果

尽管黄病毒 RNA 缺乏 poly(A) 尾，但我们提供的证据表明，病毒 RNA 在腺苷的内部运行中专门用于逆转录，并且测序读数涵盖整个非聚腺苷酸化病毒基因组。在神经祖细胞中，单细胞测序显示，虽然未感染的旁观者细胞强烈上调干扰素通路基因，但在同一培养皿中感染 ZIKV 的细胞中，这些通路在很大程度上受到抑制。单细胞测序可识别我们大脑类器官中的多种细胞类型，包括神经祖细胞、中间祖细胞和不同成熟度的神经元。使用该模型，我们发现神经元（通常不被视为发育中大脑中 ZIKV 的目标）包含高拷贝数的 ZIKV 基因组。尚不确定神经元是否被直接感染，或者受感染的神经祖细胞是否分化为神经元，并携带病毒。值得注意的是，与神经祖细胞相比，神经元旁观者细胞群显示出有限的干扰素基因通路上调。

结论

总体而言，我们的工作提供了对 ZIKV 相关小头畸形发病机制的深入了解，确定了 ZIKV 在人脑中的潜在新趋向性，并表明病毒复制和宿主反应机制是 ZIKV 感染的神经病理学的基础。