High-Throughput Single-Cell Kinetics of Virus Infections in the Presence of Defective Interfering Particles

Abstract

T he infection of a cell by a virus produces a mixture of viable and noninfectious progeny particles (1-3). A common class of noninfectious particles has defective genomes, often carrying deletions in essential genes that disable their ability to productively infect cells. However, in coinfections with viable or helper virus, the genomes of these defective particles compete with the viral replication machinery and packaging processes, interfering with infectious virus production in vitro (4, 5), and often reducing virulence in vivo (6, 7). These so-called defective interfering particles (DIPs) have for many decades been observed in laboratory cultures of virtually every class of DNA and RNA virus (4,8). More recently, DIPs have been isolated and characterized from patients infected with influenza virus (9), individuals infected with dengue virus (10, 11), and birds infected with West Nile virus (12). Moreover, sequencing of patient and natural isolates has contributed to a growing list of diverse viral genomes that carry deletions in essential genes or regulatory sequences, including hepatitis C virus (HCV) (13), polyomavirus BK (14), hepatitis B virus (15), human papillomavirus type 16 (16), and baculovirus (17). Notably, for hepatitis C virus, trans-complementation studies showed that defective HCV genomes could be encapsidated and released from cells as infectious virus-like particles (13)

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

“…Previously, single-cell virus replication under the influence of DIP co-infections has been studied in vesicular stomatitis virus (VSV)-infected cells [60,61] and IAV-infected cells [39], however, the latter study focused on the effect of DIP co-infections on the host cell response. In particular, an association between defective viral genomes and the stimulation of the innate immune response was observed [39].…”

Single-Cell Virology: On-Chip Investigation of Viral Infection Dynamics

“…In relation to viral infection, the noisy nature of gene expression would include between-cell differences in the expression of receptors for viral entry, pathogen recognition receptors, and any of the many, many host factors contributing to establishment of a productive viral infection. Therefore, it is not a surprise that the few studies that have been performed to date evaluating virus infection at the single-cell level have succeeded in observing the noise in viral infection (Akpinar et al, 2016; Heldt et al, 2015; Schulte and Andino, 2014; Thompson and Yin, 2010; Warrick et al, 2016). The single-cell platform reported here permits acquisition of complete time courses of infection for hundreds of cells even at low multiplicity of infection.…”

“…Indeed, studying viral infections on the single-cell level should enhance our understanding of viral mutant phenotypes, virus-host interactions, and/or antiviral therapeutic mechanisms. Previous studies of viral infection on the single-cell level have been limited in the number of cells monitored, restricted to high multiplicities of infection, unable to distinguish primary from secondary infections, and/or unable to monitor a complete time course of infection (Akpinar et al, 2016; Heldt et al, 2015; Schulte and Andino, 2014; Thompson and Yin, 2010; Warrick et al, 2016). …”

Extreme heterogeneity of influenza virus infection in single cells

“…Over 70 years ago, Max Delbruck showed that there was a ~100-fold range in the number of progeny virions produced per cell by clonal bacteria infected with clonal bacteriophage ( Delbruck, 1945 ). Subsequent work has shown similar heterogeneity during infection with other viruses ( Zhu et al, 2009 ; Schulte and Andino, 2014 ; Combe et al, 2015 ; Akpinar et al, 2016 ), including Influenza virus ( Heldt et al, 2015 ).…”