Virus-inclusive single cell RNA sequencing reveals molecular signature predictive of progression to severe dengue infection
題目：通過(guò)包含病毒的單細(xì)胞測(cè)序揭露重癥登革熱發(fā)展過(guò)程的分子信號(hào)
作者及單位：
Fabio Zanini, Makeda Robinson, Derek Croote, Malaya Kumar Sahoo, Ana Maria Sanz, Eliana Ortiz-Lasso, Ludwig Luis Albornoz, Fernando Rosso Suarez, Jose G Montoya, Benjamin A Pinsky, Stephen Quake, Shirit Einav
Stephen Quake帜消、Shirit Einav
Stanford University;
發(fā)表期刊及時(shí)間：
PNAS December 26, 2018 115 (52) E12363-E12369; published ahead of print December 7, 2018
摘要：
Dengue virus (DENV) infection can result in severe complications. However, the understanding of the molecular correlates of severity is limited, partly due to difficulties in defining the peripheral blood mononuclear cells (PBMCs) that contain DENV RNA in vivo. Accordingly, there are currently no biomarkers predictive of progression to severe dengue (SD). Bulk transcriptomics data are difficult to interpret because blood consists of multiple cell types that may react differently to infection. Here, we applied virus-inclusive single-cell RNA-seq approach (viscRNA-Seq) to profile ==transcriptomes of thousands of single PBMCs==（注意此處是數(shù)千個(gè)單核細(xì)胞，而不是數(shù)千個(gè)轉(zhuǎn)錄組） derived early in the course of disease from six dengue patients and four healthy controls and to characterize distinct leukocyte subtypes that harbor viral RNA (vRNA). Multiple IFN response genes, particularly MX2 in naive B cells and CD163 in CD14+ CD16+ monocytes, were up-regulated in a cell-specific manner before progression to SD. The majority of vRNA-containing cells in the blood of two patients who progressed to SD were naive IgM B cells expressing the CD69 and CXCR4 receptors and various antiviral genes, followed by monocytes. Bystander, non-vRNA–containing B cells also demonstrated immune activation, and IgG1 plasmablasts from two patients exhibited clonal expansions. Lastly, assembly of the DENV genome sequence revealed diversity at unexpected sites. This study presents a multifaceted molecular elucidation of natural dengue infection in humans with implications for any tissue and viral infection and proposes candidate biomarkers for prediction of SD.
登革熱病毒(DENV)感染可導(dǎo)致嚴(yán)重并發(fā)癥浓体。 然而泡挺，對(duì)嚴(yán)重程度的分子相關(guān)性的認(rèn)識(shí)是有 限的，部分原因是難以定義體內(nèi)含有 DENV RNA 的外周血單核細(xì)胞(PBMCs)命浴。 因此娄猫，目前沒(méi) 有可預(yù)測(cè)進(jìn)展為嚴(yán)重登革熱(SD)生物標(biāo)志物。 因?yàn)檠河啥喾N細(xì)胞類型組成生闲，它們對(duì)感染的 反應(yīng)可能不同媳溺，所以大量轉(zhuǎn)錄組數(shù)據(jù)很難解釋。在本研究中碍讯，我們采用病毒包容性單細(xì)胞 RNA-seq 方法(viscRNA-Seq)悬蔽，對(duì) 6 名登革熱患者和 4 名健康對(duì)照患者在疾病早期獲得的數(shù) 千個(gè)單個(gè) PBMCs 轉(zhuǎn)錄組進(jìn)行了分析，并對(duì)攜帶病毒 RNA (vRNA)的白細(xì)胞亞型進(jìn)行了表征捉兴。 多種 IFN 應(yīng)答基因蝎困，尤其是原 B 細(xì)胞中的 MX2 和 CD14+ CD16+單核細(xì)胞中的 CD163，在 進(jìn)展為 SD 前均以細(xì)胞特異性方式上調(diào)轴术。 在兩位進(jìn)展到 SD 的病人的血液中含 vRNA 的細(xì)胞 的主體是未分化的 IgM B 細(xì)胞难衰，表達(dá)了 CD69 和 CXCR4 受 體和多樣的抗病毒基因，單核細(xì) 胞也是這樣逗栽。 同時(shí)盖袭，不含 vRNA 的 B 細(xì)胞也表現(xiàn)出免疫激活，兩例患者 IgG1 質(zhì)粒表現(xiàn)出克隆 擴(kuò)增彼宠。 最后鳄虱， DENV 基因組序列的組裝在意想不到的位點(diǎn)顯示出多樣性。 本研究提出了一個(gè) 多層面的人類自然登革熱感染的分子闡明凭峡，對(duì)任何組織和病毒感染都有意義拙已，并提出了預(yù)測(cè) SD 的候選生物標(biāo)志物。
圖表選析：
Fig. 1. FACS-assisted viscRNA-Seq workflow on PBMCs from DENV-infected and healthy control human subjects.
圖1. 對(duì)感染登革熱的病人和健康的對(duì)照個(gè)體取外周血單核細(xì)胞后摧冀， 進(jìn) 行熒光激活細(xì)胞分選術(shù)的含病毒單細(xì)胞 RNA-seq 的工作流程
(Aand B) Blood samples were collected from human subjects enrolled in the Colombia cohort (healthy, dengue, and SD).
(A and B)從哥倫比亞群體中收集的血液樣本（包含正常個(gè)體倍踪， 登革熱感染體和惡化的登革熱感染個(gè)體）
(C) PBMCs were isolated via Ficoll centrifugation and stained with three antibody panels to distinguish various cell types: T, B, NK, DC, and monocytes.
（C） 通過(guò)聚蔗糖離心分離外周血單核細(xì)胞， 并用三個(gè)抗體板染色 以區(qū)分各種細(xì)胞類型： T 細(xì)胞索昂、 B 細(xì)胞建车、 自殺型細(xì)胞、 樹(shù)狀細(xì)胞和單 核細(xì)胞
(D) Single cells from each ==aliquot== were sorted and processed by viscRNA-Seq to simultaneously quantify single-cell virus abundance and host transcriptome changes.
(D)對(duì)來(lái)自每個(gè)等分的單個(gè)細(xì)胞進(jìn)行分類并用含病毒單細(xì)胞 seq 處理椒惨， 以同時(shí)定量單細(xì)胞病毒豐度和宿主轉(zhuǎn)錄組變化缤至。
(E) The information provided for each single cell includes: cell type, immune activation, infection state, and virus population genomics. N.A., nonapplicable.
(E)為每個(gè)單細(xì)胞提供的信息包括： 細(xì)胞類型、 免疫激活康谆、 感染狀 態(tài)和病毒群體基因組领斥。 N.A 代表不適用嫉到。
==aliquot==：等分的，指的是每個(gè)病人的起始細(xì)胞上樣量一致
Fig. 2. Overview of the types of PBMCs surveyed. (A) Two-dimensional representation of the cells color coded by the expression level of cell type-specific marker genes or the abundance of virus reads within the cell (>30 virus reads per million reads in samples from two SD patients, p1-026-1 and p1-036-1). (B) Number of cells analyzed for each cell type from each subject, see also SI Appendix, Table S6. (C) tSNE visualizations within T, NK, B cells, and monocytes, highlighting some broad cell subpopulations. The colored lines were drawn manually following inspection of the marker genes for visualization only.
圖 2：調(diào)查的PBMC類型概述月洛。（A） 按照細(xì)胞特異標(biāo)志基因或細(xì)胞中病毒 reads豐度（兩 個(gè)重癥登革熱患者樣本中每百萬(wàn) reads出現(xiàn)超過(guò) 30 個(gè)病毒reads） 給細(xì)胞上色何恶， 并且用二維 圖的形式展示出來(lái)。（B） 每個(gè)實(shí)驗(yàn)者每種類型細(xì)胞的數(shù)量膊存。（C） 對(duì) T 細(xì)胞导而， NK 細(xì)胞， B 細(xì) 胞和單核細(xì)胞進(jìn)行 t-SNE可視化分析隔崎， 高亮出一些邊緣細(xì)胞亞種群今艺。 為了可視化， 這些上色 的線是依照標(biāo)志基因的檢查結(jié)果手工繪制的
Fig. 3. Differential expression across disease severity and cell types shows hallmarks of progression to SD. (A) Genes that are overexpressed in subjects before progressing to SD versus all other subjects across cell types and subtypes. Color (white to red) indicates the average log-fold change; size of the dot indicates lower Pvalue in a distribution statistical comparison (two sample Kolmogorov–Smirnov). (B) Many inflammatory genes such as IFITM1 are expressed ubiquitously during both mild and SD infection. (C) Other genes such as IFIT3 are specifically expressed before the development of SD in various types of lymphocytes. (D) A number of genes show double specificity for both SD and a single cell type, for instance CD163 in monocytes. (E) Averaging across cells within specific cell types and subtypes indicates promising candidate predictors of SD as assessed by ROC curves at increasing discriminatory thresholds for gene expression versus disease severity. The numbers after the gene name indicate log-twofold changes of average expression in patients progressing to SD versus other dengue patients, indicating an overexpression of these genes by a 100-fold or more in our cohort. D, dengue; H, healthy subject.
圖 3 所示爵卒。不同疾病嚴(yán)重程度和細(xì)胞類型的差異表達(dá)表現(xiàn)出 SD 進(jìn)展的特征虚缎。 (A) 與細(xì)胞類 型和亞型的所有其他研究對(duì)象相比，在發(fā)展為 SD 之前在研究對(duì)象中過(guò)度表達(dá)的基因钓株。 顏色 (從白色到紅色)表示對(duì)數(shù)的平均變化;點(diǎn)的大小表明分布統(tǒng)計(jì)比較中 P 值較低(兩個(gè)樣本 Kolmogorov-Smirnov)实牡。 (B)許多炎癥基因，如 IFTMI轴合，在輕度感染和 SD 感染過(guò)程中均普遍表 達(dá)创坞， (C)其他基因，如 IFT3受葛，在 sD 發(fā)生前在各種類型的淋巴細(xì)胞中均有特異性表達(dá)题涨。 (D)許多 基因?qū)?SD 和單細(xì)胞類型都具有雙重特異性，例如單核細(xì)胞中的 CD163总滩。 (E) 在特定細(xì)胞類 型和亞型內(nèi)的細(xì)胞間取平均值表明纲堵，通過(guò) ROC 曲線評(píng)估，在提高基因表達(dá)與疾病嚴(yán)重程度 的區(qū)別閾值方面闰渔，有希望成為 SD 的候選預(yù)測(cè)因子席函。 該基因名稱后面的數(shù)字表明，與其他登 革熱患者相比冈涧，進(jìn)展為 SD 的患者平均表達(dá)量發(fā)生了對(duì)數(shù)倍的變化茂附，表明這些基因在我們這一組中過(guò)表達(dá)了 100 倍或更多。 D,登革熱組;H,健康組督弓。
討論：
We recently developed viscRNA-Seq, a scalable approach to quantify host and nonpolyadenylated vRNAs from the same cell (21). In the current study, we apply viscRNA-Seq to in vivo samples and show that it can be used to effectively profile the landscape of host transcripts and vRNA in thousands of single immune cells during natural dengue infection of human subjects. The human samples studied here posed additional challenges, beyond those presented in cultured cells. First, the exact sequence of the viral strain infecting the patients was unknown. We therefore designed an oligonucleotide for virus capture in a conserved region of the viral genome (21). Second, the target cell types of DENV in vivo are incompletely characterized, mandating assembly of antibody panels for FACS that maximize the probability of capturing the vRNA-containing cell population(s) (Fig. 1C). Third, cell viability and integrity of the RNA after freezing, shipping, and thawing the PBMC samples was much lower than in cultured cells, especially for certain cell types. Although we could not recover all types of blood cells (e.g., granulocytes, see SI Appendix, Fig. S3) we increased the throughput and PCR preamplification to ensure a sufficient number of high-quality cells from many cell types. These modifications have successfully addressed the above challenges, as indicated by our findings. Because the viscRNA-seq approach can be extended to any virus of interest and is compatible with surface markers for FACS, we expect it to be readily extensible to dissociated solid tissues, for instance to characterize viral reservoirs of various viral infections. A similar approach was recently applied to influenza infection in mouse lungs (37) and to in vitro Zika virus infection of neuronal stem cells (22).
我們最近開(kāi)發(fā)了Viscrna-seq营曼，一種從同一細(xì)胞(21)中量化宿主和非多聚腺苷化vrnas的可伸縮方法。在 目前的研究中咽筋，我們將viscrna-seq應(yīng)用于體內(nèi)樣品溶推，并表明它可以有效地描述人體自然感染過(guò)程中數(shù)千 個(gè)單個(gè)免疫細(xì)胞中宿主轉(zhuǎn)錄本和vrna的情況徊件。這里研究的人體樣本除了培養(yǎng)的細(xì)胞外奸攻，還帶來(lái)了額外的 挑戰(zhàn)蒜危。 首先，感染患者的病毒株的確切序列尚不清楚睹耐。因此辐赞，我們?cè)O(shè)計(jì)了一種在病毒基因組(21) 保守區(qū)域捕獲病毒的寡核苷酸。 第二硝训， DENV在體內(nèi)的目標(biāo)細(xì)胞類型不完全地被描述响委，要求為 FACS組裝抗體面板，以最大化捕獲含有vrna的細(xì)胞群的概率(圖)窖梁。 1C)赘风。第三， rna在冷凍纵刘、運(yùn) 輸和解凍后的細(xì)胞活力和完整性明顯低于培養(yǎng)的細(xì)胞邀窃，特別是某些細(xì)胞類型。雖然我們不能恢復(fù) 所有類型的血細(xì)胞(如粒細(xì)胞假哎，見(jiàn)SI附錄瞬捕，圖)。 S3)我們?cè)黾恿送掏铝亢蚉CR預(yù)擴(kuò)增舵抹，以確保足夠 數(shù)量的高質(zhì)量細(xì)胞從許多細(xì)胞類型肪虎。如我們的調(diào)查結(jié)果所示，這些修改成功地解決了上述挑戰(zhàn)惧蛹。因?yàn)?Viscrna-seq方法可以擴(kuò)展到任何感興趣的病毒扇救，并且可以與FACS的表面標(biāo)記物兼容，我們期望它可以 很容易地?cái)U(kuò)展到分離的固體組織赊淑，例如描述各種病毒感染的病毒庫(kù)爵政。最近，一種類似的方法被應(yīng)用于小 鼠肺部的流感感染(37)和神經(jīng)干細(xì)胞的體外Zika病毒感染(22)
翻譯小組：
陳凱星陶缺、王俊豪钾挟、鄧峻瑋、倪豪辰饱岸、陳志榮掺出、鄭凌伶
