[PubMed] [CrossRef] [Google Scholar] 24

[PubMed] [CrossRef] [Google Scholar] 24. Collectively, these results further our understanding of RO-5963 the evolution of a virus in a single host cell. IMPORTANCE It is important to understand how host cell heterogeneity affects viral contamination and replication. Using single-cell analysis, we found that viral genome replication levels exhibited dramatic variability in foot-and-mouth disease virus (FMDV)-infected cells. We also found a strong correlation between heterogeneity in cell size, inclusion number, and cell cycle status and that all of these characteristics affect the contamination and replication of FMDV. Moreover, we found that host cell heterogeneity influenced the viral adsorption as differences in the levels of FMDV integrin receptors’ expression. This study provided new ideas for the studies of correlation between FMDV contamination mechanisms and host cells. cell culture, and differences in growth and the cell cycle (1,C3). Intrinsic factors, such as random mutations during transcription and translation or cell switching controlled by genotype and epigenetics, or external factors, such as adaptive transformation caused by environmental changes, can induce cellular heterogeneity (4, 5). Cellular heterogeneity occurs in mixed cell populations exhibiting different functional phenotypes that exist in a dynamic balance and undergo phenotypic transformation among different says (6). The RO-5963 switch between functional phenotypes directly regulates the conversation of cells with viruses. It has been suggested that fluctuations in viral protein expression result in the generation of small subpopulations of latent cells during human immunodeficiency virus (HIV) replication. The presence of these heterogeneous cell subpopulations hinders drug efficacy, contributing to long-term viral transmission and persistent contamination (7). Moreover, persistent hepatitis C virus (HCV) and HIV infections significantly reduce the number of cells in the G1 and S phases but increase the number of G2/M phase cells (8, 9). Differences in cellular characteristics, such as size and cell cycle, also result in significant differences in the number of viral progeny in vesicular stomatitis virus (VSV)-infected cells (10, 11). Early studies showed that host cells produce at least six different phenotypes during the course of RO-5963 persistent contamination with foot-and-mouth disease virus (FMDV) and that these altered phenotypes Tnfrsf1b were caused by inheritable cell modifications that were selected during virus persistence (12). Similarly, we found that FMDV-infected BHK-2l RO-5963 cells exhibit morphological heterogeneities that are different from those of normal BHK-2l cells (13, 14). Thus, studying the mechanisms of cellular heterogeneity and their role in viral contamination could impact the development of antiviral strategies. However, studies around the occurrence, development, and completion of the viral contamination cycle have been confined to whole populations of infected cells, yielding only the average response of the cellular population, and few studies have focused on a single infected cell. Although all host cells can be infected simultaneously, viral replication kinetics are different in each cell due to cellular heterogeneity (15, 16), which is usually attributed to a variety of factors, such as cell size, inclusion, and cell cycle heterogeneity in normal host cells (17,C19). FMDV, a positive-strand RNA virus in the family (20), causes acute and persistent infections in host cells and cloven-hoofed animals (21,C23). Cells coexist with virus without obvious cytopathic effects (CPE) and produce infectious virions during serial passage of BHK-21 cells persistently infected with FMDV (14, 24). We sorted single cells using fluorescence-activated cell sorting (FACS) and decided viral RNA copy numbers using single-cell reverse transcriptase quantitative PCR (RT-qPCR) to determine intercell replication differences. The results revealed marked variability in the positive- and negative-strand viral RNA levels in FMDV-infected cells, ranging from below the detection limit to millions. We next investigated the effects of host cell heterogeneity, including cell size, number of inclusions, and cell cycle status, on FMDV contamination (acute and persistent) and replication. We evaluated viral proteins, RNA, and infectious particles from heterogeneous cells and found that the viral outcome depends on cell size and number of inclusions. Furthermore, we exhibited that heterogeneity in cell size and inclusion number also affects the adsorption of FMDV by altering the expression of FMDV integrin receptors. Cells in the G2/M phase were more amenable to viral contamination. Finally, we found correlations between heterogeneity in cell size, number of inclusions, and cell cycle status, which all affect the contamination and replication.

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