Analysis Of Differentially Expressed MiRNAs In Liver Tissue Of Duck Enteritis Virus (DEV)-infected Ducks

Duck enteritis virus(DEV),also known as duck plague virus(DPV),is a member of the Alphaherpesvirinae genus Mardivirus of the family Herpesviridae,which can cause Avian to be an acute contact septicemic infectious disease characterized by vascular injury,hemorrhage of body cavity,inflammation of digestive tract and degeneration of parenchymal organs.one of the important pathogens.Micro RNA(mi RNA)is a class of highly conserved non-coding single-stranded RNA molecules with regulatory functions in eukaryotes,with a length of about 18-25 nucleotides,which can regulate the expression of target gene m RNAs by interacting with them,plays an important role in cell proliferation,tissue differentiation and virus replication.Existing data studies have shown that overexpression of DEV-mi RNA-D22-5P can inhibit DEV proliferation through its targeted binding to DEV-ICP4 gene m RNA,suggesting that mi RNAs play an important role in DEV proliferation.In this study,the mi RNA expression profile of DEV-infected duck liver was constructed,the differentially expressed mi RNAs were screened and their effects on DEV proliferation were analyzed,in order to provide new clues and basis for elucidating the pathogenesis of DEV.The main research contents are as follows:1.Analysis of differentially expressed mi RNAs in DEV-infected duck liver tissue(1)DEV artificial infection duck model was established.Thirty clinically healthy ducks were selected and randomly divided into an experimental group(20 ducks,inoculated with0.2 m L of DEV virus solution in leg muscles)and a control group(10 ducks,inoculated with0.2 m L of normal saline).(66 h),during the period of obvious symptoms(90 h)and at the dying stage(114 h),the test ducks were dissected to observe the gross tissue lesions and PCR detection of viral nucleic acid.There was no abnormality in the detection,etc.The ducks in the experimental group first showed rough coat,depression and loss of appetite,and then there were symptoms such as eyelid edema,dyspnea,yellow-green or brown blood-stained loose stools;necropsy showed damaged blood vessels,intestinal bleeding and liver dysfunction.necrosis spots,etc.;the duck liver tissue of the three time periods can amplify DEV-specific DNA fragments(133 bp in size),indicating that this study successfully established a DEV artificial infection duck model.(2)The mi RNA expression profile of DEV-infected duck liver was established.The liver tissue samples of the ducks in the above experimental group were collected at 66 h,90 h and114 h.Through mi RNA extraction and high-quality screening,mi RNA sequence alignment and differential expression analysis,and KEGG and GO database analysis,the results showed that DEV infection was performed for 66 h.There were 227 differentially expressed mi RNAs in duck liver at the time of infection,mainly enriched in protein digestion and absorption,ECM-receptor interaction and synaptic vesicle cycling,etc.;at 90 h of infection,there were225 differentially expressed mi RNAs,mainly enriched in relaxin signaling pathway,Wnt signaling pathway,and exocytosis,etc.There were 231 differentially expressed mi RNAs at114 h of infection,mainly enriched in protein digestion and absorption,relaxing signaling pathway,and focal adhesions.The immune pathways involved in the differentially expressed mi RNAs in the livers of DEV-infected ducks were mainly JAK-STST signaling pathway,of which the most significant differences were mi R-97 and mi R-111.The above results indicated that this study successfully established the mi RNA expression profile of DEV-infected duck liver.2.Validation of relationship between differentially expressed mi R-97 and mi R-111 and SOCS3 gene targetsIn order to explore the target relationship between mi R-97 and mi R-111 and the host cell SOCS3 gene,in this experiment,after constructing mi R-97 and mi R-111 mimics vectors,they were combined with the constructed SOCS3-3’UTR vector(including WT type and MUT type,respectively).co-transfected 293 t cells,and the dual-luciferase reporter gene technology was used to verify the target relationship.The results showed that the fluorescence activity of mi R-97+SOCS3-3’UTR-WT group was significantly lower than that of mi RNA NC+SOCS3-3’ The UTR-WT group(P<0.01)was significantly lower than the mi R-97+SOCS3-3’UTR-MUT group(P<0.05).Meanwhile,the fluorescence activity of the cells in the mi R-111+SOCS3-3’UTR-WT group was significantly lower than that in the mi RNANC+SOCS3-3’UTR-WT group and the mi R-111+SOCS3-3’UTR-MUT group(P<0.01),indicating that both mi R-97 and mi R-111 can target and bind to SOCS3-3’ UTR.3.Effects of mi R-97 and mi R-111 on SOCS3 expression and DEV replicationIn order to verify the effects of mi R-97 and mi R-111 on SOCS3 gene expression and DEV replication,the mi R-97 and mi R-111 mimics vectors constructed above were used in this experiment to transfect DEV-infected DEF cells,respectively,and cells were collected at different time periods.Samples,the expression levels of SOCS3 gene and DEV-NP gene after overexpression of mi R-97 and mi R-111 were analyzed by fluorescence quantitative PCR and ELISA.Both were extremely significantly decreased(P<0.01),while the relative expression of DEV-NP gene decreased(P<0.05),indicating that mi R-97 and mi R-111 may regulate DEV replication by inhibiting the expression of SOCS3 gene.To sum up,this study successfully established a duck DEV artificial infection model and a mi RNA expression profile of DEV-infected duck liver.There are many differentially expressed mi RNAs in DEV-infected duck liver,and most of them are related to signal pathways such as virus proliferation and immune response.Among them,mi R-97 and mi R-111 may regulate DEV proliferation by inhibiting the expression of SOCS3 gene,providing new ideas for elucidating the pathogenesis of DEV.