West Nile virus-inclusive single-cell RNA sequencing reveals heterogeneity in the type I interferon response within single cells

Abstract

232 words; Importance: 122 words; and Text: 4,850 words. 22 ABSTRACT 23West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus of global importance. 24Neuroinvasive WNV infection results in encephalitis and can lead to prolonged 25 neurological impairment or death. Type I interferon (IFN-I) is crucial for promoting 26 antiviral defenses through the induction of antiviral effectors, which function to restrict 27 viral replication and spread. However, our understanding of the antiviral response to 28 WNV infection is mostly derived from analysis of bulk cell populations. It is becoming 29 increasingly apparent that substantial heterogeneity in cellular processes exists among 30 individual cells, even within a seemingly homogenous cell population. Here, we present 31 WNV-inclusive single-cell RNA sequencing (scRNA-seq), an approach to examine the 32 transcriptional variation and viral RNA burden across single cells. We observed that 33 only a few cells within the bulk population displayed robust transcription of IFN-β mRNA, 34 and this did not appear to depend on viral RNA abundance within the same cell. 35 Furthermore, we observed considerable transcriptional heterogeneity in the IFN-I 36 response, with genes displaying high unimodal and bimodal expression patterns. 37 Broadly, IFN-stimulated genes negatively correlated with viral RNA abundance, 38 corresponding with a precipitous decline in expression in cells with high viral RNA levels. 39 Altogether, we demonstrated the feasibility and utility of WNV-inclusive scRNA-seq as a 40 high-throughput technique for single-cell transcriptomics and WNV RNA detection. This 41 approach can be implemented in other models to provide insights into the cellular 42 features of protective immunity and identify novel therapeutic targets. 43 44 45 IMPORTANCE 46 West Nile virus (WNV) is a clinically relevant pathogen responsible for recurrent 47 epidemics of neuroinvasive disease. Type I interferon is essential for promoting an 48 antiviral response against WNV infection; however, it is unclear how heterogeneity in 49 the antiviral response at the single-cell level impacts viral control. Specifically, 50 conventional approaches lack the ability to distinguish differences across cells with 51 varying viral abundance. The significance of our research is to demonstrate a new 52 technique for studying WNV infection at the single-cell level. We discovered extensive 53 variation in antiviral gene expression and viral abundance across cells. This protocol 54 can be applied to primary cells or in vivo models to better understand the underlying 55 cellular heterogeneity following WNV infection for the development of targeted 56 therapeutic strategies. 57 58 59 60 61

Analysis of cell-associated DENV RNA by oligo(dT) primed 5’ capture scRNAseq

“…Recent advances in single cell RNA sequencing (scRNAseq) technology have revolutionized the field of cellular biology, providing insight into the heterogeneity of cellular transcription in an unbiased yet high-resolution fashion [16]. scRNAseq has also been leveraged to quantify the cellular tropism of several RNA viruses including influenza [17, 18], West Nile [19], Zika [20], and DENV [20, 21]. The majority of these published reports utilized a variant of the Smart-seq2 scRNAseq technology, wherein individual cells are deposited into separate wells in a 96 well plate containing the necessary reagents for cDNA synthesis and mRNA barcoding [22].…”

Viral and host heterogeneity and their effects on the viral life cycle

“…These cultures comprise ciliated, non ciliated, goblet and basal cells, thus recapitulating the complex multicellular envi ronment of the lung 5 . Single cell RNA sequencing (scRNA seq) [6][7][8][9][10][11][12][13][14][15][16][17] techniques reveal cell to cell variabil ity in viral infections and provide important insights into the heterogeneity of transcriptional responses and viral growth. Live cell imaging and advances in single particle tracking have also increased our understanding of heterogeneous viral infections of individual cells 18 .…”

The Application of Single-Cell RNA Sequencing in Vaccinology

“…Several groups have recently used dual scRNA-seq to profile virally infected cells and draw insights from transcriptome information [38][39][40][41][42]. O'Neal et al revealed the feasibility and value of West Nile Virus-(WNV-) inclusive scRNA-seq as a method for single-cell transcriptomics and WNV RNA detection [38]. There was extreme heterogeneity in viral quantity and antiviral gene expression among in vitro infected cells.…”