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

Abstract

Dengue is one of the most widespread vector-borne viral diseases in the world. However, the size, heterogeneity, and temporal dynamics of the cell-associated viral reservoir during acute dengue virus (DENV) infection remains unclear. In this study, we analyzed cells infected in vitro with DENV and PBMC from an individual experiencing a natural DENV infection utilizing 5’ capture single cell RNA sequencing (scRNAseq). Both positive- and negative-sense DENV RNA was detected in reactions containing either an oligo(dT) primer alone, or in reactions supplemented with a DENV-specific primer. The addition of a DENV-specific primer did not increase the total amount of DENV RNA captured or the fraction of cells identified as containing DENV RNA. However, inclusion of a DENV-specific cDNA primer did increase the viral genome coverage immediately 5’ to the primer binding site. Furthermore, while the majority of intracellular DENV sequence captured in this analysis mapped to the 5’ end of the viral genome, distinct patterns of enhanced coverage within the DENV polyprotein coding region were observed. The 5’ capture scRNAseq analysis of PBMC not only recapitulated previously published reports by detecting virally infected memory and naïve B cells, but also identified cell-associated genomic variants not observed in contemporaneous serum samples. These results demonstrate that oligo(dT) primed 5’ capture scRNAseq can detect DENV RNA and quantify virus-infected cells in physiologically relevant conditions, and provides insight into viral sequence variability within infected cells.IMPORTANCEDengue is one of the most widespread vector-borne viral diseases in the world. However, it is still unclear which cells harbor virus during DENV infection, and how viral reservoirs in serum and infected cells are related. These results demonstrate for the first time that intracellular DENV RNA can be identified and infected cells quantified by 5’ capture scRNAseq. This strategy provides a significantly greater throughput and higher sensitivity than previously published methods and has the potential to provide additional information on the genomic heterogeneity of intracellular DENV RNA.

Global and cell type-specific immunological hallmarks of severe dengue progression

“…These cells are also susceptible to DENV infection in vitro 3, 20 . Here, we not only show that B cells harbor the majority of positive-strand vRNA in children’s blood, as we and others have reported in adult patients 12, 24 , but also measure active replication in myeloid and B cells via three orthogonal experimental methods: negative-strand vRNA detection in patient cells, E protein expression measurement via flow cytometry, and viral spread detection upon co-culture of with permissive cells. Evidence for infectious DENV production was previously demonstrated only in healthy donor-derived B cells in vitro infected at a high multiplicity of infection (MOI=20) 25 , but not in patient-derived B cells.…”

Dangerous Liaisons: Gammaherpesvirus Subversion of the Immunoglobulin Repertoire

“…Simultaneous sequencing of the genome and transcriptome of the host and pathogen will also be critical for understanding virus latency, autoimmunity, and viral oncogenesis. Recently, these methods were used with success to study memory B cells generated in response to influenza virus [ 250 ] as well as the transcription profile of Dengue virus [ 251 ]. Single-cell genomics and transcriptomics is revealing novel functions of tumor infiltrating immune cells [ 252 ], recently in EBV-associated carcinoma [ 253 ].…”

A single-cell atlas of theCulex tarsalismidgut during West Nile virus infection

“…We utilized a scRNA-seq approach previously demonstrated to be flavivirus RNA inclusive, which allowed us to detect WNV viral RNA (vRNA) in addition to host transcripts (21). Through this approach we identified distinct midgut populations corresponding with midgut cell types previously described in Drosophila and Aedes aegypti midguts -enterocyte (nutrient absorption cells), enteroendocrine (secretory cells), cardia (peritrophic matrix secreting cells), intestinal stem cell/enteroblast (undifferentiated progenitor cells), proliferating intestinal stem cell/enteroblast, visceral muscle cells, and hemocyte (immune cells) -and characterized the infection and replication dynamics of WNV within each population (5,6,14,16,17).…”