The Study Of The Effect Of Pseudorabies Virus On PI3K/Akt Signaling Pathway In Trigeminal Ganglion Cells

Pseudorabies virus(PRV),belongs to varicella virus of?-herpesvirinae of Herpesviridae,is a double stranded DNA virus,PRV can cause pigs respiratory neurological symptoms and reproductive disorders.Pigs are the main susceptible animals of PRV and one of the most important sources of PRV infection.Lethal to other mammals and wild animals.In recent years the prevalent PRV variant strains have caused huge economic losses to China's pig breeding industry.As one of the representatives of herpesvirus,PRV also has the characteristics of herpesvirus neurotropism and latent infection.The mortality rate of piglets infected with PRV can be as high as 100%.However,adult pigs usually have no obvious but mild clinical symptoms after infection,and most of the cases are transient,after rehabilitation,PRV will not be completely eliminated in the body,and it often lurks in the trigeminal ganglion(TG)of adult pigs.The infected pigs will carry the virus for life and will continually emiss virus to the outside world,which cause other pigs in the same group to be infected.Control and elimination of PRV latent infectionin pigs is the key point of PRV research.There are few studies on the molecular mechanism of PRV latent infection in TG at home and abroad.Research shows that the PI3K/Akt signaling pathway plays an important role in the maintenance and activation of herpes virus latent infection.Studies have shown that PI3K/Akt signaling pathway plays an important role in the maintenance and activation of herpes virus latent infection.However,there is no report on the effect of PRV infection on the PI3K/Akt signaling pathway and the maintenance and activation of latent infection in primary TG cells.In order to explore this scientific problem,this experiment used PRV to infect mice and prepare primary mouse TG cells to explore the effect of PRV on PI3K/Akt signaling pathway and the molecular mechanism of PI3K/Akt signaling pathway in vivo and in vitro,to provide clues for understanding the mechanism of PI3K/Akt signaling pathway in PRV infected TG and its cell latent infection and activation.The relevant research contents of this expertiment are as follows:1.Effect of PRV infection on PI3K/Akt signaling pathway in trigeminal ganglion of miceIn this study,mice were used as the model of PRV infection in vivo.The virulent strain of PRV Guizhou DY(Gen Bank:JX417716)isolated and identified previously in the laboratory was challenged in vivo by nasal drip.The experiment was divided into two groups:low dose group with 10 000 TCID₅₀and high dose group with 50000 TCID₅₀.The clinical symptoms were observed at 12 h,24 h and 48 h after infection,and some mice were killed to remove TG.The mice infected with high dose PRV showed clinical symptoms such as excitement,pruritus,messy hair,scratching the face and ears,and then bleeding and ulceration,and died 48 h after infection;the mice infected with low-dose PRV had no obvious clinical symptoms and did not die within 7 days after infection.PCR showed that PRV nucleic acid was detected in TG of mice infected with PRV,indicating that PRV infected mice were successful.Relative quantitative Q-PCR and WB were used to detect the expression of PI3K and Akt mRNA in TG and the protein expression of PI3K/Akt signaling pathway in PRV infected mice.The showed that compared with the control group,the expression of PI3K and Akt mRNA in each infection group was significantly down regulated(P<0.05),PI3K mRNA was down regulated more significantly with the increase of viral infection(P<0.05),but expression of Akt mRNA may not be related to the amount of virus infection(P>0.05),and the down-regulation of PI3K and Akt mRNA expression in each group may not be related to the infection time(P>0.05).WB results showed that the protein expression of PI3K,Akt and p-Akt had no significant change after PRV infection,and the difference was not statistically significant(P>0.05),but the expression of PI3K/Akt signaling pathway related proteins was increased in high infection group,which showed a time-dependent induction mode(P<0.05).High infection PRV may activate PI3K/Akt signaling pathway.2.Effect of PRV infection on PI3K/Akt signaling pathway in primary TG cellsAccording to the previous research in the laboratory and related literature,the primary culture mode of mouse TG cells was optimized firstly.The main optimization methods were to increase the inoculation density of TG cells,the second adherent method,change the culture medium and polylysine plate.The results showed that after increasing the cell density and twice adherent culture,not only did not promote cell adhesion,but inhibited cell adhesion,and the cell density did not increase but decreased;DMEM/F12 was replaced with DMEM(high glucose),the density of TG cells did not increase,but the cell status was better;use Poly-D-lysine to culture TG cells can effectively promote the adhesion and growth of TG cells,the cell density has been greatly improved,but the growth and development speed of cells has not been significantly improved.These results indicate that the method of D-type polylysine plate is feasible for primary culture of TG cells.Transmission electron microscope was used to observe whether PRV infected TG cells and proliferated in them.The results showed that PRV could infect TG cells.Use1 MOI PRV infected TG cells,the mRNA expression of PI3K and Akt and the protein expression were detected.The results showed that in the early stage of PRV infection,the expression levels of PI3K and Akt mRNA were significantly up-regulated,the expression of PI3K mRNA peaked at about 30 min and Akt mRNA peaked at 15 min,then decreased rapidly.In general,the expression of PI3K and Akt mRNA in TG cells infected with PRV was first promoted and then inhibited.In protein expression,after PRV infected TG cells,the expression of PI3K protein was significantly inhibited in the early stage of infection(P<0.01),the expression of Akt protein was first promoted and then inhibited(P<0.01),which was consistent with the expression of mRNA.PRV could induce the phosphorylation of Akt in TG cells at the early stage of infection,and the phosphorylation of Akt was down regulated and disappeared at 3?6 h after infection.At the same time,we observed that PRV infected TG cells could induce Akt phosphorylation in the early stage by IFA.These results suggest that PRV infected TG cells can activate PI3K/Akt signaling pathway.3.Effect of PI3K/Akt signaling pathway on apoptosis of PRV infected TG cellsTo confirm herpesvirus can use PI3K/Akt signaling pathway to regulate its latent infection and activation in cells.TG cells are one of the main target cells of PRV infection.Whether PRV regulates its latent infection in TG cells through PI3K/Akt signaling pathway is still unclear.In this study,PI3K inhibitor LY294002 was used to block the PI3K/Akt signaling pathway in TG cells,and the blocking effect was verified by WB.The results showed that 10?mol LY294002 could effectively inhibit the phosphorylation of Akt induced by PRV infection in TG cells,and activation of PI3K/Akt pathway in TG cells was induced in a PI3K dependent manner,indicating that LY294002 can effectively block the PI3K/Akt pathway in TG cells.Transmission electron microscope was used to observe whether PRV infected TG cells and proliferated in them.The results showed that immature virus particles were found in the nucleus of TG cells without CPE and TG cells with CPE.Meanwhile,cytoplasmic cytopathic characteristics such as vacuole,autophagy and mitochondrial swelling were observed,which indicated that PRV could infect TG cells.In order to explore whether PI3K/Akt signaling pathway inhibits PRV induced apoptosis of TG cells,Annexin V-FITC in situ cell staining was used to observe the apoptosis under microscope.For exclude the effect of fat solubility on TG cell apoptosis,the experiment was divided into six groups:blank control group,LY294002 control group,PRV 3 h and 6 h infection groups,PRV+LY294002 3 h and PRV+LY294002 6 h infection groups.The results showed that there were no or few red and green fluorescence staining in control group,LY294002 group and PRV infection group,but with the extension of PRV infection time,a small number of TG cells showed apoptosis;The number of apoptotic cells in PRV+LY294002 group was significantly higher than that in PRV group,and the number of apoptotic cells increased with the extension of infection time.The results of flow cytometry showed that blocking PI3K/Akt signaling pathway significantly increased the number of apoptosis induced by PRV infection in TG cells,indicating PI3K/Akt signaling pathway plays an important role in inhibiting the apoptosis of TG cells induced by PRV infection.