Multi-omics Study Of Early African Swine Fever Virus Infection

The African swine fever virus(ASFV)is highly contagious and has a substantial mortality rate,significantly endangering the global pig farming industry.Currently,there are no commercial vaccines or effective treatments available.Studies indicate that the cell nucleus plays a crucial role in ASFV replication,with early infection causing alterations in host nuclear structure and epigenetic landscape.This implies that ASFV’s early infection process involves intricate regulatory mechanisms occurring at multiple levels.In this study,we employed multi-omics techniques to analyze ASFV-infected porcine alveolar macrophages(PAMs)at multiple dimensions during the early stages of infection.Using DLO-Hi-C technology developed by our research group,we mapped chromatin interactions in host cells during early ASFV infection and analyzed changes in host chromatin threedimensional conformation across multiple scales.Combining RNA-seq and RNAP Ⅱ ChIPseq data allowed us to characterize variations in host gene expression patterns as well as assess how early-stage viral infections impact RNAP Ⅱ utilization within the host genome.Further analysis revealed potential interrelationships between alterations in host chromatin architecture,virus-host chromatin interactions,and gene expression regulation.The primary conclusions drawn from this study include:(1)DNA-FISH results indicate that during the early stages of ASFV infection(4 hpi),the ASFV genome was detected within nucleus of PAM cells,confirming that ASFV infection has a nuclear stage;(2)DLO-Hi-C data reveal the changes in host chromatin interaction and chromatin three-dimensional structure,our results reveal that early ASFV infection cause changes in the host chromatin interaction frequency and chromatin states more often transitioning from active to repressed.In this study,we identified 3,210 TADs and 11,151 Loops pre-infection and 3,195 TADs and 16,957 Loops post-infection.Among these variations,there was a alteration in 36.8%(1,289)of TAD boundaries along with the loss or reorganization of1,905 Loops;(3)By employing transcriptome sequencing(RNA-seq)technology,we analyzed the differential gene expression characteristics of PAM cells during early ASFV infection.Among the 243 identified differentially expressed genes(DEGs),there were 129 upregulated and 114 downregulated genes.These genes were primarily enriched in antiviral response,immune defense,chemokine activity,interferon activation,and receptorassociated signaling pathways;(4)Utilizing chromatin immunoprecipitation sequencing(ChIP-seq)technology,we examined the distribution of RNA polymerase Ⅱ(RNAP Ⅱ)binding sites on host chromatin.The study reveals that at 4 hpi,both the number of RNAP Ⅱ binding sites and signal intensity at transcription start sites(TSS)in PAM cell chromatin are significantly diminished,while no RNAP Ⅱ binding sites were detected on the ASFV viral genome.This indicates that while ASFV replication and gene expression may not be dependent or directly reliant upon host RNAP Ⅱ,early stages of ASFV infection do suppress its interaction with host chromatin.Western blot results demonstrate that this inhibitory effect is not a consequence of protein degradation.;(5)Multi-omics integrative analysis found that the regulation of host gene expression levels during early ASFV infection may be associated with alterations in host chromatin Compartments and Loops,while no direct link was found between the suppression of RNAP Ⅱ binding to host chromatin and changes in host chromatin conformation.This suggests that changes in host chromatin three-dimensional structure may be a potential regulatory mechanism for early ASFV infection,while RNAP Ⅱ has limited influence on chromatin three-dimensional structure;(6)Virus-host chromatin interaction analysis revealed that during early ASFV infection,there was extensive chromatin interaction between viruses and PAM cells.686virus-host interaction hotspots(virus-host interaction hotspot,VHIH)identified,with significantly changed expression levels of genes inside VHIH regions,suggesting potential association between them.In addition,motif analysis revealed that sequences within VHIH areas contain an abundance of b HLH family transcription factor motifs.This further implies that ASFV might modulate host gene expression through its interactions with host chromatin,and those b HLH transcription factors may play important role.In summary,this study found that early ASFV infection not only causes changes in host chromatin interaction and chromatin three-dimensional structure but also inhibits host genome utilization of RNAP Ⅱ.ASFV may regulate host gene expression levels by affecting host chromatin three-dimensional structure or directly interacting with host chromatin.Early infection mainly affects host antiviral immunity,chemokines and receptor-related genes.This study focuses on chromatin interaction and changes in chromatin three-dimensional conformation from multiple dimensions during early ASFV infection,providing new insights into the study of ASFV infection mechanisms and has important theoretical and practical significance.