Interactions Among Expressed MicroRNAs And MRNAs In The Early Stages Of Fowl Adenovirus Aerotype 4-infected Leghorn Male Hepatocellular Cells

Hydropericardiμm-hepatitis syndrome(HHS)is caused by some strains of fowl adenovirus serotype 4(FAd V-4).HHS,first observed in Pakistan Angara in 1987,therefore,it has also been called “Angara Disease”.Since 2015,it has posed a serious threat to the broiler industry in most parts of China,causes significant losses to the poultry industry.FAd V-4 is highly pathogenic and shows a high affinity for hepatic,endothelial and lymphatic cells.Upon histological examination,the most consistent findings in the liver are small multifocal areas of necrosis and infiltration of mononuclear cells,including basophilic intranuclear inclusion bodies in hepatocytes surrounded by a clear halo or filling the entire nucleus.However,the mechanism of FAd V-4 entry was not well understood.We choosed Leghorn male hepatocellular cells(LMH cells)as target to analyzed the mechanism of FAd V-4 entry and got the following results:1.Isolation and identification of a local strains in FAd V-4 region,and preparation of anti hexon and penton polyclonal antibodies.FAd V-4 was isolated from a liver sample from a broiler chicken during a recent HHS outbreak.The results of genome evolution analysis suggested that the isolate belongs to the FAd V-4 epidemic strain group,and has the closest genetic relationship with the strain isolated in most of China in recent years.The FAd V-4 isolate is highly pathogenic and can cause typical tissue lesions and cell lesions.The recombinant hexon and penton proteins were expressed by prokaryotic expression system.The proteins were collected to immunize rabbits.The serum of the rabbits was harvested and purified.The purified polyclonal antibodies were specific,the purity of antibodies was higher than 85%,and the titers was higher than 512 K.2.The analysis of mi RNA-seq in LMH cells during FAd V-4 entry.In total,784 differentially expressed mi RNAs(DESs)were identified in LMHs during FAd V-4 infection.Among these genes,520,312,and 246 DESs were identified at 30 min,60 min,and 120 min,respectively.Target gene prediction of differential mi RNAs was conducted.The results showed that these mi RNAs are correlated with gene targets found in pathways that involve virus entry.The GO and KEGG pathway analysis of the differentially expressed mi RNAs revealed that the target genes were involved in many signalling pathways,including endocytosis,binding,receptor activity,metabolic process,and biological regulation.3.The analysis of m RNA-seq in LMH cells during FAd V-4 entry.In total,724 differentially expressed m RNAs(DEGs)were identified in LMHs during FAd V-4 infection.Among these genes,90,259,and 625 DEGs were identified at 30 min,60 min,and 120 min,respectively.The GO and KEGG pathway analysis revealed that the DEGs were involved in many signalling pathways,including binding,receptor activity,metabolic process,and biological regulation,and the DEGs were mostly related to the Toll like signalling pathway,TNF signalling pathway,and MAPK signalling pathway.4.A conjoint analysis of mi RNA-seq and m RNA-seq in LMH cells during FAd V-4 entry,and functional gene screening.A conjoint analysis of mi RNA-seq and m RNA-seq was utilized with LMH cells at 30 min,60 min,and 120 min after FAd V-4 infection.The conjoint analysis of the obtained data identified 856 negative mi RNAs-m RNAs pairs at the three time points.Among these mi RNAs-m RNAs pairs,41,154,and 661 were differentially expressed at 30 min,60 min,and 120 min,respectively.The GO and KEGG pathway analysis revealed that the mi RNAs-m RNAs pairs were mostly related to pathways in Toll like signalling pathway,TNF signalling pathway,and MAPK signalling pathway.5.To validate the correlations between the mi RNAs and m RNAs,the relationship between gga-mi R-128-2-5p and its target OBSL1 was confirmed using a dual-luciferase reporter system and a real time quantitative polymerase chain reaction(RT-q PCR)assay.To investigate the biological function of gga-mi R-128-2-5p in FAd V-4 entry,FAd V-4 proliferation in LMHs treated with gga-mi R-128-2-5p mimics and inhibitors was detected.Virus copy number analysis indicated that gga-mi R-128-2-5p mimics-treated and OBSL1 si RNA-treated cells showed inhibited FAd V-4 entry.Similarly,CLSM revealed that gga-mi R-128-2-5p mimics and OBSL1 si RNA treatment inhibit the expression of green fluorescent signals specific to the FAd V-4 in the cell.Therefore,FAd V-4 entry was inhibited in gga-mi R-128-2-5p mimics and OBSL1 si RNA treated cells.Thus,gga-mi R-128-2-5p regulated the expression of OBSL1,which plays a negative role in FAd V-4 entry.In sμmmary,a FAd V-4 strain was isolated,obtained anti hexon and penton polyclonal antibodies.The mi RNAs and m RNAs expression profiles of LMH cells during FAd V-4 entry were obtained.The conjoint analysis of the obtained data identified 856 negative mi RNAs-m RNAs pairs.Further experiments confirmed that gga-mi R-128-2-5p against FAd V-4 entry by down-regulating its target OBSL1 during virus invasion.Our work suggests that unveiling differential mi RNAs-m RNAs coexpression properties help to understand the molecular mechanisms underlying the pathogenesis of FAd V-4.