Study On The Role Of Exosomes In Mediating BVDV Cell-to-Cell Transmission

Bovine viral diarrhea（BVD）,also known as bovine mucosal disease,is an acute or persistent and chronic infectious disease of cattle caused by bovine viral diarrhea mucosal virus（BVDV）of pestivirus of Flaviviridae family.BVDV can be transmitted in cattle at all ages,especially in young cattle.Exosomes are extracellular vesicles with a particle size of30-150 nm originated from cell inclusions,which can regulate the biological process of cells that ingest these exosomes through their loaded proteins,nucleic acids,lipids and other active substances.Recent studies have shown that exosome mediated virus plays an important role in intercellular transmission and infection.The purpose of this study is to reveal the role and mechanism of exosomes induced by BVDV infection in virus transmission.The research contents and results are as follows:1.Characteristics of BVDV infected cell-derived exosomes.After BVDV（NADL strain）infected bovine kidney cells（MDBK）,the cell supernatant was collected,and the exosomes were precipitated and purified by differential centrifugation,ultracentrifugation and sucrose gradient centrifugation.The purity of the extracted exosomes was identified by transmission electron microscope and Western blot.The results showed that typical"saucer like"vesicle like exosomes could be observed under transmission electron microscope;The secretory marker proteins CD63,CD81 and TSG101 were detected by Western blot,RT-PCR was used to detect the whole genome sequence of BVDV in the exosomes of BVDV infected cells.Based on liquid chromatography combined with mass spectrometry,it was found that the exosomes were loaded with virus structural protein E^rnsand non-structural protein NS3;2.Effect of BVDV infection on secretion level:the effect of BVDV infection on secretion level was detected by Western blot and flow cytometry.The results showed that the expression levels of exosome marker molecules CD63 and CD81 in the supernatant of MDBK cells infected with BVDV（CP and NCP）were significantly higher than those in the uninfected group;In order to clarify the role of viral protein in the increase of secretion level induced by BVDV infection,MBDK cell line stably expressing structural proteins C,E₁,E₂,E^rnsand non-structural protein N^proof BVDV NADL strain was constructed to further clarify that BVDV E₂protein and N^proprotein play an important role in inducing secretion level.3.Pathway of exosomes into absorbing cells:MDBK derived exosomes were labeled with lipid fluorescent dye PKH-26,and the pathway of exosomes into absorbing cells was detected.MDBK cells were treated with caveolin blocker（MβCD,Nystatin）,clathrin blocker（CPZ）,macropinocytosis blocker（EIPA）,dynosore inhibitor and related interfering RNA,the entry pathway of MDBK derived exosomes was detected by flow cytometry and laser confocal technology.The results showed that the pathway of exosomes entering the absorbing cells was jointly regulated by caveolin and clathrin mediated endocytosis pathway,PI3K involved macropinocytosis pathway and dynein dependent entry pathway.4.The role of exosomes in mediating BVDV intercellular transmission:after infecting MDBK cells with BVDV CP and NCP respectively,the exosomes were extracted from the culture supernatant.After the exosomes were co-incubated with absorbing cells,the proliferation level of BVDV in the incubated cells was detected;The virus infected cell-derived exosomes were further incubated with antiviral serum,and then co incubated with absorbing cells to detect the neutralization effect of exosome mediated virus cell-cell transmission and escape specific antiserum.After incubation for 24h,48h and 72h,the virus infection and proliferation were detected by cellular CPE（cytopathic effect）and real-time fluorescence quantitative PCR.The results showed that BVDV could be detected in the cells after incubation of BVDV infected cell-derived exosomes and absorbing cells for 24 hours,48 hours and 72 hours,and its proliferation level was dependent on incubation time.In addition,the control group of free virus infection can be effectively neutralized and inhibited by specific antiserum,while the exosomes from MDBK cells infected with BVDV CP and NCP can escape the neutralization of antiserum and mediate the virus to produce proliferative infection in the absorbing cells.In order to further study the effect of exosome inhibitor GW4869 on exosome mediated BVDV transmission,the exosomes were extracted from the supernatant after BVDV（CP type and NCP type）was infected with MDBK cells respectively.After co-incubation with absorbing cells for 72 hours,the virus proliferation level in absorbing cells was detected.The results of TCID₅₀showed that the virus titer secreted by the absorbing cells into the supernatant was inhibited by drugs.The cells were further pretreated with different concentrations of GW4869（exosome inhibitor）and inoculated with BVDV（CP type and NCP type）for 24 hours.After 48 hours,the exosomes of the infected group were extracted and incubated with the absorbing cells for 72 hours.The results of fluorescence quantitative PCR showed that the virus replication level in the absorbing cells decreased with the increase of GW4869 concentration.In conclusion,BVDV infection induced an increase in the release level of MDBK derived exosomes,which contained the whole genome sequence of the virus and BVDV NS3 and E^rnsproteins;BVDV infected cell-derived exosomes can enter MDBK cells through caveolin,clathrin,macropinocytosis and dynein mediated endocytosis,and virus infection and proliferation occur in the absorbing cells.Exosome release level affects exosome mediated BVDV intercellular infection.BVDV E₂and N^proproteins play an important role in inducing exosome secretion level.This study provides a certain basis for further exploring the participation of exosomes in the transmission and proliferation of BVDV between cells,and provides a new idea for further exploring the pathogenic mechanism of BVDV.