The Effect Of ATF3 In The Neuron Cells Infected With Porcine Hemagglutinating Encephalomyelitis Virus

Porcine hemagglutinating encephalomyelitis(PHE)is an acute and highly contagious disease of piglets.The pathogen is porcine blood coagulating encephalomyelitis virus(Porcine hemagglutinating encephalomyelitis virus,PHEV).It is mainly infected with 1~3 week old suckling piglets,mainly vomiting failure and Ming.Obvious neurological symptoms were characteristic.Activating transcriptional activator 3(activating transcription factor 3,ATF3)is one of the members of the transcription factor ATF/CREB family.ATF3 is an adaptive response gene that is low in resting cells and increases rapidly when stimulated.Previous mRNA microarray analysis showed that ATF3 was highly expressed in mice brain and N2 a cells infected with PHEV.However,the mechanism of ATF3 overexpression and its effect on nerve cells are not clear.In this experiment,the ATF3 expression level of N2 a cells infected with PHEV at different time periods was detected.At the same time,the ATF3 expression level of N2 a cells with different titer PHEV infection was detected to analyze the relationship between the viral load and the expression level of ATF3.Secondly,the ATF3 siRNA was synthesized and the ATF3 overexpression vector was constructed,and N2 was transfected to N2 respectively.A cells,establish a stable expression of cell lines,and then inoculate PHEV,analyze the expression level of PHEV mRNA,and detect cell cycle and apoptosis,in order to explore whether ATF3 affects the replication of the virus and explore the role of ATF3 in the process of PHEV infection of the host cells in the process of infection of the neurons.In this experiment,we selected N2 a cells as models to explore the mechanism of PHEV infected neural cells.According to the content and purpose of the test,the N2 a cells were inoculated into the six hole plates for group culture.After the PHEV adsorption of N2 a cells1h,the RT-PCR and Western Blot methods were used to detect 2h,12 h,24h,and detect the nerve cells changes in the expression level of ATF3.N2 a cells transfected with siRNA and expression vector and inoculated PHEV,set 5 groups: control group,ATF3 interference with PHEV group,negative control with PEHV group,ATF3 overexpression with PEHV group and empty vector with PHEV group,3 replicates in each group,using RT-PCR technique to detect PHEV mRNA expression,explore whether ATF3 affects virus replication.The apoptosis of 12 h cells was detected by Annexin V-FITC/PI double staining,and the 12 h cell cycle was detected by PI single staining method to determine the effect of ATF3 on the apoptosis and cell cycle of PHEV infected nerve cells.The results showed that the expression level of ATF3 in normal N2 a cells was low.As the time of infection increased,the expression of PHEV mRNA and ATF3 mRNA expression increased first and then decreased,and there was a positive correlation between the two.Withthe increase of the infection time,the protein expression of ATF3 increased continuously;with the decrease of the virus titer,the expression of ATF3 was reduced.The quantity also drops.After transfection of ATF3 siRNA,the expression of ATF3 was significantly inhibited,and the expression of ATF3 increased significantly after transfection of ATF3 over expression vector.The expression level of PHEV mRNA was detected by RT-PCR,and cell apoptosis and cell cycle were detected by flow cytometry.The results showed that there was no significant difference in the expression of PHEV mRNA in the ATF3 interference group cells compared with the negative control group,and the early apoptosis was increased,and the G1 phase block was aggravated.The apoptosis rate of ATF3 overexpressing virus group was lower than that of no load group,while S phase arrest slowed down.In this study,ATF3 is positively related to the replication of the virus in the process of PHEV infection of N2 a cells.PHEV can affect the expression of ATF3,but ATF3 does not affect the replication of PHEV.ATF3 plays the role of anti apoptosis and promoting cell cycle during PHEV infection of N2 a cells.