Determining the Cellular Diversity of Hepatitis C Virus Quasispecies by Single-Cell Viral Sequencing

Abstract

Single-cell genomics is emerging as an important tool in cellular biology. We describe for the first time a system to investigate RNA virus quasispecies diversity at the cellular level utilizing hepatitis C virus (HCV) replicons. A high-fidelity nested reverse transcription (RT)-PCR assay was developed, and validation using control transcripts of known copy number indicated a detection limit of 3 copies of viral RNA/reaction. This system was used to determine the cellular diversity of subgenomic JFH-1 HCV replicons constitutively expressed in Huh7 cells. Each cell contained a unique quasispecies that was much less diverse than the quasispecies of the bulk cell population from which the single cells were derived, suggesting the occurrence of independent evolution at the cellular level. An assessment of the replicative fitness of the predominant single-cell quasispecies variants indicated a modest reduction in fitness compared to the wild type. Real-time RT-PCR methods capable of determining single-cell viral loads were developed and indicated an average of 113 copies of replicon RNA per cell, correlating with calculated RNA copy numbers in the bulk cell population. This study introduces a single-cell RNA viral-sequencing method with numerous potential applications to explore host-virus interactions during infection. HCV quasispecies diversity varied greatly between cells in vitro, suggesting different within-cell evolutionary pathways. Such divergent trajectories in vivo could have implications for the evolution and establishment of antiviral-resistant variants and host immune escape mutants.

Viral quasispecies

“…Genetic variation is generated by the accumulation of mutations during replication and their re-arrangement by genetic recombination, and genome segment reassortment in the case of segmented genomes. Mutant swarms are established within single infected cells and cell clusters (Del Portillo et al, 2011; Jung et al, 2002; McWilliam Leitch and McLauchlan, 2013; Sala et al, 1994; Sobesky et al, 2007) and in infected organisms during disease episodes, outbreaks, epidemics and pandemics [(Gire et al, 2014; Martell et al, 1992), among many other studies that have been reviewed (Domingo et al, 2012)]. Basic features of quasispecies dynamics can be studied experimentally because mutant swarms are also generated upon replication of molecular or biological viral clones in cultured cells.…”

Single-Cell Genomics for Virology

“…McWilliam, Leitch, and McLauchlan investigated HCV, a positive single-stranded RNA virus. In particular, they analyzed the viral diversity of HCV replicon quasi-species by RT-qPCR and vRNA deep-sequencing in individual cells [ 29 ]. They determined that on average, one single cell contained 113 copies of replicon RNA (ranging from 84 to 160 copies).…”

Single-Cell Analysis of Foot-and-Mouth Disease Virus

“…The gene expression profiles resulting from high-throughput single-cell RNA sequencing (scRNA-seq) can identify changes in individual cells during viral infections, revealing vital factors that can influence the life cycle of a virus. For example, Hepatitis C virus (HCV) quasispecies were detected in individual human liver Huh7 cells by scRNA-seq (McWilliam Leitch and McLauchlan, 2013); scRNA-seq also demonstrated that the thymocyte selectionassociated high mobility group box protein (TOX) was a critical regulator of CD8+ T cell persistence during chronic lymphocytic choriomeningitis virus (LCMV) infections and maintained CD8+ T cell longevity, facilitating long-term antiviral CD8+associated immunity (Yao et al, 2019). Through the examination of thousands of individual peripheral blood mononuclear cells (PBMCs), derived from six dengue patients and four healthy controls, multiple interferon (IFN)-response genes, particularly MX2 in naive B cells and CD163 in CD14+, CD16+ monocytes, were found to be upregulated in a cell-specific manner before progression to severe dengue (Zanini et al, 2018).…”