Single-cell analysis reveals the relevance of foot-and-mouth disease virus persistence to emopamil-binding protein gene expression in host cells

Abstract

Foot-and-mouth disease virus (FMDV) infects host cells in either an acute or persistent manner. In this study, we examined the relevance of the establishment of FMDV persistence to the expression of the emopamil-binding protein (EBP) gene in 231 individual persistently infected baby hamster kidney (BHK-21) cells after passages 28, 38, and 68 (PI28, PI38, and PI68). At PI28, the stage at which persistent infection of FDMV becomes unstable, the percentage of cells carrying FMDV was 66.7%, while 80.2% of cells were EBP positive. Additionally, in 55.6% of the EBP-positive cells at PI28, EBP expression was upregulated approximately 149.9% compared to uninfected BHK-21 cells. This was the highest expression level among all cell passages measured. Interestingly, in a parallel experiment, the average EBP expression level in the whole cell population at PI28 was only slightly higher (108.2%) than that in uninfected BHK-21 cells. At PI38, 98.7% of the cells were positive for FMDV 3D (an RNA-dependent RNA polymerase enzyme gene), and its maximum expression level observed at this passage. The expression level of EBP in 78.2% of the total cells, however, was reduced significantly. At PI68, 95.8% of the cells were 3D positive, and the expression of both the EBP and 3D genes were at the lowest levels of all the passages. Our studies using single cells yielded data that are otherwise inaccessible a using whole cell population. These results suggest that the establishment of persistent infection by FMDV is a dynamic process that results from the continuous adaptation and coevolution of viruses and cells to reach an equilibrium.

The Carrier Conundrum; A Review of Recent Advances and Persistent Gaps Regarding the Carrier State of Foot-and-Mouth Disease Virus

“…Other investigations have studied gene expression profiles in vitro using persistently infected cultured cells, either derived from FMDV-susceptible cell lines [101][102][103] or the primary culture of bovine pharyngeal epithelial cells [104,105]. The in vitro approach is particularly attractive as it eliminates the ethical concerns of animal experimentation, is less resource demanding and may therefore overcome limitations associated with small group sizes and high within-group variance.…”

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

“…The establishment of a persistent cell culture can provide a system for elucidating the mechanisms underlying viral persistence. On the basis of this system, a relationship between FMDV infections and the host emopamil-binding protein (EBP) gene was identified, through the global transcriptional analysis of single cells isolated from a model of persistent FMDV infection (Fang et al, 2017). In the study, 231 individual persistently infected BHK-21 cells were isolated by micromanipulation after passages 28, 38, and 68 (PI28, PI38, and PI68, representing early, middle, and late infection, respectively), and were used to investigate variations in the expression levels of the FMDV virus 3D gene and the host EBP gene by sc-qRT-PCR.…”

“…In a cell population, FMDV RNA levels fluctuate in different cells because of cell heterogeneity (Huang et al, 2009;Li et al, 2009;Fang et al, 2017;Xin et al, 2018). Cells have many characteristics, but which of these influences FMDV replication?…”

Single-Cell Sequencing Yields Insights in the Evolution of Foot-and-Mouth Disease Virus Persistent Infection

“…MAPK/p38 and MAPK/ERK-related signaling pathways were active in the late phase of acute infection, and factors associated with viral replication are differentially expressed in persistent infection cells, such that persistent infection cells achieve their resistance to viral replication by downregulating the expression of MAPK14 and Hspb1 in the MAPK signaling pathway (Li et al, 2020). Emopamil Binding Protein (EBP) protein was shown to promote FMDV replication, and EBP expression was upregulated in acutely infected BHK-21 cells, whereas the opposite was true in persistently infected cells (Fang et al, 2017). Our experiments have also verified that Cav1 was required for FMDV-Op proliferation during persistent infection, and that downregulation of Ccnd1 was one of the causes of wild-type FMDV resistance in BHK-Op cells (Han et al, 2021).…”