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

Abstract

Virus replication displays a large cell-to-cell heterogeneity; yet, not all sources of this variability are known. Here, we study the effect of defective interfering (DI) particle (DIP) co-infection on cell-to-cell variability in influenza A virus (IAV) replication. DIPs contain a large internal deletion in one of their eight viral RNAs (vRNA) and are, thus, defective in virus replication. Moreover, they interfere with virus replication. Using single-cell isolation and reverse transcription polymerase chain reaction, we uncovered a large between-cell heterogeneity in the DI vRNA content of infected cells, which was confirmed for DI mRNAs by single-cell RNA sequencing. A high load of intracellular DI vRNAs and DI mRNAs was found in low-productive cells, indicating their contribution to the large cell-to-cell variability in virus release. Furthermore, we show that the magnitude of host cell mRNA expression (some factors may inhibit virus replication), but not the ribosome content, may further affect the strength of single-cell virus replication. Finally, we show that the load of viral mRNAs (facilitating viral protein production) and the DI mRNA content are, independently from one another, connected with single-cell virus production. Together, these insights advance single-cell virology research toward the elucidation of the complex multi-parametric origin of the large cell-to-cell heterogeneity in virus infections.

Cell culture-based production of defective interfering influenza A virus particles in perfusion mode using an alternating tangential flow filtration system

“…The total amount of IAV particles was quantified using a hemagglutination assay and was expressed as log 10 hemagglutinin units/100 μL (log 10 HA units/100 μL) (Kalbfuss et al 2008). PFU were determined by a plaque assay, conducted as described previously (Hein et al 2021a; Hein et al 2021b; Kupke et al 2020). Here, for the quantification of released infectious virus particles in the interference assay, parental adherent MDCK cells were used, only allowing propagation of STVs.…”

Antiviral Activity of Influenza A Virus Defective Interfering Particles against SARS-CoV-2 Replication In Vitro through Stimulation of Innate Immunity

“…Influenza A virus (IAV) defective interfering particles (DIPs) were previously proposed for antiviral treatment against IAV infections [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ], but also for pan-specific treatments of other respiratory viral diseases [ 23 , 24 ]. IAV DIPs typically carry a large internal deletion in their genome, rendering them defective in virus replication [ 25 , 26 , 27 ].…”

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].…”