West Nile Virus-Inclusive Single-Cell RNA Sequencing Reveals Heterogeneity in the Type I Interferon Response within Single Cells

Abstract

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

Single-Cell Analysis Uncovers a Vast Diversity in Intracellular Viral Defective Interfering RNA Content Affecting the Large Cell-to-Cell Heterogeneity in Influenza A Virus Replication

“…Thus, the measurements were affected by a high degree of technical noise, preventing the accurate identification of host cell transcripts that are specific for high-or low-yield cells. Note that in other studies, host cell mRNAs of virus-infected single cells could be reliably identified [37,39,40,43,44]. We suppose that the following technical and experimental differences of our study are responsible for the noisy data.…”

“…The origin of the large cell-to-cell heterogeneity in virus infections is still not fully understood [29][30][31][32][34][35][36][37][38][39][40]43,44]. We conducted single-cell analysis of IAV replication and reveal an association of DIP co-infections and single-cell progeny virus yields.…”

“…It was speculated that specific host cell determinants are responsible for a supposed suppression of virus replication. Such determinants may be the innate immune response [36][37][38][39], and other antiviral host cell factors that were previously identified by scRNA-seq [37,39,40,43,44].…”

“…In order to identify additional factors that can contribute to the large cell-to-cell heterogeneity in IAV replication, we next performed whole transcriptome analysis by scRNA-seq using Illuminabased next-generation sequencing (NGS) technologies. Although similar studies have been conducted previously [37,43,44] including IAV-infected cells [39,40], only the viral mRNA level was used as a read-out to evaluate virus replication. In contrast, we also quantified the extracellular In order to identify additional factors that can contribute to the large cell-to-cell heterogeneity in IAV replication, we next performed whole transcriptome analysis by scRNA-seq using Illumina-based next-generation sequencing (NGS) technologies.…”

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 or 384 well plate containing the necessary reagents for cDNA synthesis and mRNA barcoding 22 .…”