Isolation And Identification Of Cell-derived Exosomes From Bovine Viral Diarrhea Virus Infection And Their Role In Virus Infection

Bovine Viral Diarrhea Virus(BVDV)is the pathogen that causes bovine viral diarrhea mucosal disease,which is a huge harm to the cattle industry in the world and even in my country.The pathogenic mechanism of BVDV has not yet been fully elucidated.In recent years,studies have shown that viruses can also be infected through exosomes,providing new ideas for the study of the pathogenic mechanism of BVDV.Exosomes are the smallest extracellular vesicles(EVs),with a diameter of 30 nm ～ 150 nm.They were the first to be widely used in the study of cancer markers.This study examines the effect of BVDV infection on the release of exosomes from MDBK cells.The effect of exosomes on virus infection has been preliminary studied.This study established a method for the isolation and purification of exosomes derived from BVDV infected cells,using PEG6000 to sediment the exosomes in the virus culture supernatant,ultracentrifugation and CD9 immunomagnetic beads to purify the exosomes.The exosomes were identified by protein concentration determination,electron microscopy,NTA detection,SDS-PAGE and Western-blot methods.After purification,the exosomes showed a typical cup-shaped vesicle structure with a particle size between 100-150 nm.After 72 h of BVDV infection,the release of exosomes was higher.CD9,a surface marker of exosomes,can be detected.In order to study the role of BVDV in the release of exosomes,CD9,a surface marker of exosomes,was used as an indicator for detection of exosomes.Fluorescence quantitative PCR was used to detect the effects of different infectious doses,different infection times,and different viral components on the release of exosomes.The mRNA expression level of genes in the pathway related to secretion production and release.The infection time is the same.As the virus infection dose increases,the exosomal CD9 gene mRNA expression level increases,and is dose-dependent;when the virus infection dose is the same,the exosomal CD9 gene mRNA level increases with the infection The increase of time increases.Poly(I:C)is an analog of double-stranded RNA,which simulates virus infection.The exosomal CD9 gene mRNA level of poly(I:C)group and live virus group increased significantly,and the exosomal CD9 gene mRNA level of inactivated BVDV group No change,BVDV infection can release exosomes through Alix-related signaling pathways.BVDV infection can promote the release of exosomes.For this reason,we conducted a preliminary study on the role of exosomes in BVDV replication.After adding exosomes to MDBK cells,the exosomes produced by BVDV infection have a certain degree of infectivity,and can also inhibit the expression of antiviral cytokines;the exosome inhibitor GW4869 inhibits the release of exosomes and reduces the BVDV Infectious.The addition of exosomes promoted the proliferation of BVDV in cells.The results show that BVDV exosomes are infective in vitro and contribute to virus infection.In summary,this study isolated and identified BVDV-infected cell-derived exosomes,and confirmed that while BVDV infection promotes the release of exosomes from cells,the exosomes released also promote the proliferation of viruses in cells.The research on the pathogenic mechanism of BVDV provides new ideas and a theoretical basis for the prevention and treatment of BVD.