Single-Cell Analysis Of The Impact Of Host Cell Heterogeneity On Infection With Foot-and-Mouth Disease Virus

Cellular heterogeneity within a population of-cells is apparent even when the cells possess the same genotype or are derived from the same clone,as evidenced by morphological differences in single-celled organisms,cell polarity differentiation in higher organisms,phenotypic differences in in vitro cell culture,and differences in growth and the cell cycle.Cellular heterogeneity occurs in mixed cell populations exhibiting different functional phenotypes that exist in a dynamic balance and undergo phenotypic transformation among different states.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,which affects virus infection.Therefore,studies on mechanism of cell heterogeneity formation and transformation have important scientific significance and application value in revealing the mechanism of promoting or controlling host cell heterogeneity formation by viral infections,and exploring the occurrence and development of viral infections.In this study,the wild type and the persistently infected foot-and-mouth disease virus(FMDV)strain were purified by plaque purification.We demonstrated that the plaque-forming ability of the persistently infected virus was weaker than that of the wild-type FMDV through plaque assay.Then the genome open reading frame(ORF)sequences of the two viruses were sequenced.The result of sequence alignment showed that 34 amino acid mutations occurred in the persistently infected FMDV.The mutation rate of VP1 protein was the highest,accounting for 32.35%of the total mutation rate.The mutations occurred in persistently infected virus may have a potential role in maintaining the persistent FMDV infections.Next,we sorted single cells using fluorescence-activated cell sorting(FACS)and quantified viral RNA copy numbers using single-cell RT-qPCR to determine inter-cell replication differences.The method has the characteristics of simple operation,rapidity,good stability,and high sensitivity,which could directly quantitative detection of viral RNA content in single cell,get the original data of virus replication in single cell and objectively reflect the differences among single cells.The results of single cell analysis revealed marked variability in the positive-and negative-strand viral RNA levels in FMDV-infected cells,ranging from below the detection limit to millions.And such variability was not dependent on the virus titer,which might be related to host cell heterogeneity.In order to verify the relationship between FMDV infection and—host cell heterogeneity,we investigated the effects of host cell heterogeneity on FMDV infection(acute infection and persistent infection)and replication from three aspects:cell size,number of inclusions and cell cycle status.We evaluated viral proteins,RNA,and infectious particles from heterogeneous cells and found that larger cells and cells with a greater number of inclusions generated more viral RNA copies,viral protein,and a higher proportion of infectious cells.Furthermore,we investigated the single-cell FMDV RNA content and the percentage of 3D-positive cells and found variation in the distribution of FMDV RNA in different cell-cycle phases.Cells in the G2/M phase contained more viral RNAs and a higher percentage of 3 D-positive cells than cells in other phases.We also found that larger cells and cells with a greater number of inclusions generated more viral RNA copies than did smaller cells and those with fewer inclusions in persistently FMDV infection,which was consistent with the results of acute infection.These results suggested that cell heterogeneity can both affect the acute and persistent FMDV infection and replication.Finally,we explored the mechanism that cellular heterogeneity influenced on viral infection.We demonstrated that heterogeneity in cell size and inclusion number affects the adsorption of FMDV due to differences in the levels of FMDV integrin receptors expression.We used drugs to arrest the cell cycle in different phases and found that the viral titer was 10-to 100-fold higher in cells in G2/M phase than those in other cell cycle phases.The results suggested that cells arrested in the G2/M phase had increased replication efficiency and favored progeny virus production.Additionally,the levels of FMDV integrin receptors in G2/M phase cells were higher than those in other cell cycle phases.We speculated that the G2/M phase may affect virus infection by affecting virus adsorption.We also found correlations between heterogeneity in cell size,number of inclusions,and cell-cycle status,which all affect the infection and replication of FMDV.This study propose that direct correlations exist between FMDV infection and host cell heterogeneity and that control of host cell heterogeneity factors is conducive to viral infection and replication,offering new ideas for studies of the correlation between FMDV infection mechanisms and host cells.