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 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.

登革热病毒（DENV）感染可能导致严重的并发症。然而，对严重性的分子相关性的理解是有限的，部分是由于难以定义体内含有DENV RNA的外周血单核细胞（PBMC）。因此，目前尚无可预测发展为严重登革热（SD）的生物标志物。大量的转录组学数据难以解释，因为血液由多种细胞类型组成，这些细胞类型可能对感染的反应不同。在这里，我们应用了包含病毒的单细胞RNA-seq方法（viscRNA-Seq）来分析成千上万的单个PBMC的转录组，这些单个PBMC在疾病过程中源自六个登革热患者和四个健康对照，并描述了具有不同特征的白细胞亚型病毒RNA（vRNA）。多种IFN反应基因⁺ CD16 ⁺单核细胞在发展为SD之前以细胞特异性方式上调。两名进展为SD的患者血液中大多数含vRNA的细胞是表达CD69和CXCR4受体及各种抗病毒基因的幼稚IgM B细胞，其次是单核细胞。旁观者，不含vRNA的B细胞也显示出免疫激活作用，两名患者的IgG1成浆细胞表现出克隆性扩增。最后，DENV基因组序列的组装揭示了意想不到的位点的多样性。这项研究提出了人类天然登革热感染的多方面分子阐明，对任何组织和病毒感染都有影响，并提出了预测SD的候选生物标志物。