Long non-coding RNAs (lncRNAs) are involved in numerous biological processes and are pivotal mediators of the immune response, yet little is known about their properties at the single-cell level. Here, we generate a multi-tissue bulk RNAseq dataset from Ebola virus (EBOV) infected and not-infected rhesus macaques and identified 3979 novel lncRNAs. To profile lncRNA expression dynamics in immune circulating single-cells during EBOV infection, we design a metric, Upsilon, to estimate cell-type specificity. Our analysis reveals that lncRNAs are expressed in fewer cells than protein-coding genes, but they are not expressed at lower levels nor are they more cell-type specific when expressed in the same number of cells. In addition, we observe that lncRNAs exhibit similar changes in expression patterns to those of protein-coding genes during EBOV infection, and are often co-expressed with known immune regulators. A few lncRNAs change expression specifically upon EBOV entry in the cell. This study sheds light on the differential features of lncRNAs and protein-coding genes and paves the way for future single-cell lncRNA studies.

Long non-coding RNAs (lncRNAs) are pivotal mediators of systemic immune response to viral infection, yet most studies concerning their expression and functions upon immune stimulation are limited to in vitro bulk cell populations. This strongly constrains our understanding of how lncRNA expression varies at single-cell resolution, and how their cell-type specific immune regulatory roles may differ compared to protein-coding genes. Here, we perform the first in-depth characterization of lncRNA expression variation at single-cell resolution during Ebola virus (EBOV) infection in vivo. Using bulk RNA-sequencing from 119 samples and 12 tissue types, we significantly expand the current macaque lncRNA annotation. We then profile lncRNA expression variation in immune circulating single-cells during EBOV infection and find that lncRNAs' expression in fewer cells is a major differentiating factor from their protein-coding gene counterparts. Upon EBOV infection, lncRNAs present dynamic and mostly cell-type specific changes in their expression profiles especially in monocytes, the main cell type targeted by EBOV. Such changes are associated with gene regulatory modules related to important innate immune responses such as interferon response and purine metabolism. Within infected cells, several lncRNAs have positively and negatively correlated expression with viral load, suggesting that expression of some of these lncRNAs might be directly hijacked by EBOV to attack host cells. This study provides novel insights into the roles that lncRNAs play in the host response to acute viral infection and paves the way for future lncRNA studies at single-cell resolution.