Analysis Of Lentivirus-mediated Occludin Overexpression To Regulate The Replication Of Bovine Viral Diarrhea Virus In Vivo

Bovine viral diarrhea/mucosal disease(BVD/MD),as a contact infectious disease,after infecting domestic and wild animals,it causes a series of clinical symptoms such as high fever,diarrhea,mucosal congestion,decreased milk production of mothers and animals,abortion and stillbirth.Occludin(OCLN)is a tight junction-related protein(tight junction,TJ)responsible for maintaining the barrier regulation function of cell osmotic pressure and the barrier function of helping the formation of cell polarity.A large number of research reports,OCLN can play a role in connecting the target cell receptor when the Hepatitis C Virus(HCV)enters the cell,thereby initiating the replication of the virus in the cell.BVDV and HCV belong to the Flaviviridae,and the mechanism of action between OCLN and BVDV has not been reported.As a virus of the same family,does OCLN play a key role in the process of BVDV infection?In the early stage,our research team has used CRISPR/Cas9 gene knockout and RNAi knockdown technologies to knock out/lower the expression of OCLN in BVDV target cells MDBK.It was found that OCLN knockout/low will severely affect the replication of BVDV in host cells.This study was passed Lentivirus-mediated OCLN overexpression in BALB/c mice Using BVDV to infect mice,to study whether OCLN has an effect on BVDV replication in vivo,to explore the relationship between OCLN and BVDV,Provide a theoretical basis for the prevention and control of BVD/MD and Anti-BVDV.The specific content is as follows:Objective:To study whether tight junction protein(Occludin,OCLN)affects the replication of bovine viral diarrhea virus(BVDV)in BALB/c mice.Methods:Construct lentiviral expression vectors,and combine the OCLN-GFP vector with helper plasmids(p SPAX2 and p MD2.G)Co-transfected into HEK-293T cells(select the GFP plasmid transfection group as the control group),Observe whether there is a lot of green fluorescence in the transfected cells through a fluorescence microscope at 48 h After the green fluorescent protein is expressed in large quantities,collect the OCLN-GFP lentivirus suspension and determine its titer;The 1-month-old BALB/c mice were randomly divided into five groups:A(0 d),B(4 d),C(8 d),D(10 d),and E(15 d).Each group was injected with lentiviral suspension of OCLN-GFP(experimental group)and GFP(control group)with a titer of 5×10⁷IU respectively,and injected twice with an interval of 48 h.96 h after the second injection,BVDV was injected into the tail vein with a titer of 1.68×10⁵ TCID₅₀ per animal.At 0,4,8,10,and 15 days after the BVDV challenge,the mice were dissected and tissues from different organs were collected,total RNA and total protein were extracted,and the concentration was determined.Use Real-time fluorescent quantitative PCR(q RT-PCR)technology to detect OCLN m RNA expression in different tissues and BVDV viral load in different tissues for different days;Western blot technology was used to detect the expression of OCLN protein in different tissues;the remaining tissues were made into tissue sections of different days to observe the pathological changes.Results:Observing the large-scale expression of green fluorescent protein under a fluorescence microscope and determining the lentivirus titer to meet the standards of injected animal virus,which proved the successful packaging of OCLN-GFP and GFP lentivirus;96 h after lentivirus infection of BALB/c mice,Compared with the GFP group,OCLN-GFP showed a significant increase in real-time fluorescence quantitative detection of OCLN m RNA content and Western blot detection of protein expression levels in each tissue;Compared with GFP,the viral load of each tissue detected by q RT-PCR in the OCLN-GFP group increased significantly from 4 days after challenge;The pathological section results showed that 4 days after the BVDV challenge,the lungs and liver organs of the mice showed obvious lesions at the earliest,while the lesions of other organs were not obvious.8 days after the challenge,liver and lung lesions became more severe,and other organs showed obvious lesions with aggravation;Compared with the GFP group,the tissue lesions in the BVDV mice were more severe in the OCLN-GFP group than in the GFP group.The results of this study indicate that lentivirus-mediated OCLN overexpression can significantly increase the replication of BVDV in BALB/c mice.Compared with the GFP group,the BVDV-infected OCLN-GFP group caused serious changes in liver,spleen,lung and other organ diseases.Provide an important theoretical basis for elucidating the mechanism of OCLN-mediated BVDV replication.