Analysis of cell-associated DENV RNA by oligo(dT) primed 5' capture scRNAseq.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
03 06 2020
03 06 2020
Historique:
received:
20
02
2020
accepted:
12
05
2020
entrez:
5
6
2020
pubmed:
5
6
2020
medline:
18
12
2020
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 32493997
doi: 10.1038/s41598-020-65939-5
pii: 10.1038/s41598-020-65939-5
pmc: PMC7270085
doi:
Substances chimiques
DNA Primers
0
DNA, Complementary
0
Oligodeoxyribonucleotides
0
RNA, Viral
0
oligo (dT)
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
9047Subventions
Organisme : NIAID NIH HHS
ID : P01 AI034533
Pays : United States
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