Infection Of Different Biotypes Of BVDV Induces Cell Cycle Arrest In MDBK

Bovine viral diarrhea virus（BVDV）is the causative agent of bovine viral diarrhea（BVD）and belongs to the genus border disease virus（BDV）and classical swine fever virus（CSFV）.BVDV infection bring respiratory,digestive,reproductive and immune system diseases,which can cause significant economic losses to the cattle industry.The cell cycle is a process of cellular replication which,once initiated,can proceed in an orderly manner.Studies have shown that virus infection creates a favourable environment for virus proliferation by regulating changes in the cell cycle.In this study,MDBK cells were infected with different biotypic BVDV strains to explore the effects of cytopathogenic and non-cytopathogenic strains on cell cycle arrest.1.BVDV infection induces cell cycle arrest in MDBK cellsBVDV TC strain and NADL strain were used to infect MDBK cells,respectively.NADL strain infection can produce lesions of cytopathic type（CP）,while TC strain infection showed no lesions to non-cytopathic type（NCP）.After IFA,it was observed that the cytoplasm infected by the two strains showed specific green fluorescence,indicating that both strains of viruses could proliferate in MDBK cells.The CCK-8 test determined the test infectious dose to be 1MOI.In order to understand the relationship between the infection process and the cell cycle,MDBK cells were infected with BVDV NADL strain and TC strain respectively,and the cells were collected at 12 h,24 h,36 h and 48 h.After PI staining,the distribution of cell cycle phases was detected by flow cytometry.The distribution of different phases of the cycle,TC strain（NCP）infection induced MDBK cells to arrest in S phase,NADL strain（CP）infection induced cells to arrest in G0/G1 phase.Synchronized processing results are synchronized and asynchronous.2.BVDV NADL and TC strains cause cell cycle arrest via p53 inductionTo explore the effect of BVDV infection of different biotypes on cell cycle arrest.The total RNA and protein of infected cells were extracted respectively,and the G0/G1 phase and G1/S phase associated cell cycle proteins Cyclin D1,Cyclin E2 and cell cycle protein-dependent kinases CDK2,CDK4,CDK6,p53 pathway ATM,p53,p21,p-p53（Ser-15）were detected by real-time fluorescence quantitative PCR and Western Blot and GAPDH as internal reference.The results showed that the mRNA expression levels of CDK4,CDK6,Cyclin D1,Cyclin E2,CDK2,ATM,p53,and p21 genes were elevated after infection of TC strain（NCP）,and Cyclin E2 mRNA expression was significantly reduced;the protein expression levels of CDK2,CDK4,Cyclin D1 and p21 were reduced after infection,and CDK6,Cyclin E2,p53 and p-p53（Ser-15）protein expression levels were increased.mRNA expression levels of other genes were the same as those of the TC strain after infection with the NADL strain（CP）,except for no difference in ATM mRNA expression levels;in terms of protein expression levels,only CDK4 protein expression was increased.To clarify the relationship between BVDV-induced cell cycle arrest and the p53 pathway,cells were treated with p53 inhibitor and the cell cycle was detected by flow cytometry.It was found that the inhibitor treatment reduced the blockage of S phase by TC strain（NCP）and G0/G1 phase by NADL strain（CP）.Cyclin p53 and p-p53（ser-15）were detected by Western blot,and the proteins were barely expressed after inhibitor treatment.3.Effect of BVDV-induced cell cycle block on viral replicationTo investigate the effect of cell cycle block on BVDV replication.Cells synchronized in G0/G1,G1/S and G2/M phases of the cell cycle were treated with serum starvation,thymidine and nocodazole,respectively,and desynchronized cells were used as controls.The 1 MOI virus was used to infect synchronized and non-synchronized cells respectively.48 h after infection,mRNA levels of BVDV 5’-UTR were measured by real-time fluorescence quantitative PCR,viral TCID₅₀ was measured by IPMA,and cell cycle temporal distribution was detected by flow cytometry.Results It was found that G0/G1 phase promotes the replication of NADL strain（NCP）and inhibits the replication of TC strain（NCP）;G1/S phase promotes the replication of TC strain（NCP）and NADL strain（CP）;G2/M phase promotes TC strain（NCP）replication,has inhibitory effect on NADL strain（CP）replication.In summary,this study investigated the effect of BVDV infection of different biotypes on MDBK cell cycle arrest.It is clear that TC strain（NCP）infection induces MDBK cells to block in S phase and NADL strain（CP）infection induces cells to block in G0/G1 phase;different biotypes of BVDV can cause cell cycle block through the p53 pathway.The G0/G1 phase promoted NADL strain（CP）replication,and the G1/S phase promoted TC strain（NCP）and NADL strain（CP）replication,the G2/M phase promoted TC（NCP）strain replication,confirming that cell cycle arrest facilitates its proliferation in cells.