Analysis of cell-associated DENV RNA by oligo(dT) primed 5\u2019 capture scRNAseq

Mark A Sanborn,Damon Ellison,Tao Li,Hayden Siegfried,Stefan Fernandez,Irina Maljkovic Berry,Heather Friberg,Kaitlin Victor,Christian Fung,Richard G Jarman,Jeffrey R Currier,Alan L Rothman,Anon Srikiatkhachorn,Adam T Waickman

doi:10.1038/s41598-020-65939-5

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.

Highlights

Dengue is one of the most widespread vector-borne viral diseases in the tropical and sub-tropical regions of the world
The majority of these published reports utilized a variant of the Smart-seq[2] 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]
The addition of a dengue virus (DENV)-specific cDNA synthesis primer did not appreciably increase either the total amount of DENV RNA captured by 5′ capture scRNAseq analysis or the fraction of cells flagged as containing DENV RNA

Summary

Introduction

Dengue is one of the most widespread vector-borne viral diseases in the tropical and sub-tropical regions of the world. We verified that 5′ capture scRNAseq can be utilized to detect/quantify cell-associated DENV RNA in PBMC from an individual experiencing a natural secondary DENV-1 infection This analysis recapitulated previous reports of DENV-infected B cells and identified cell-associated viral variants that were not present in contemporaneous serum samples. These results demonstrate for the first time that DENV can be detected and quantified in physiologically relevant conditions using oligo(dT) primed 5′ capture scRNAseq, and provides insight into sequence variability of the DENV genome contained within infected cells

Methods

Results

Conclusion