Exploring The Internalization Mechanism Of Transmissible Gastroenteritis Virus By Single Particle Tracking Technique

Transmissible gastroenteritis(TGE)is a contagious intestinal infectious disease caused by transmissible gastroenteritis virus(TGEV),the main symptoms are vomiting,diarrhea and severe dehydration.The disease is most susceptible to pigs,which can cause high morbidity and mortality of piglets under two weeks of age,and cause huge economic losses to the pig industry in the world.At present,the entry mechanism of TGEV is still unclear.In this study,single particle tracing technology was used to study the process of TGEV entry,revealing the dynamic interaction between TGEV and the host cell in this process,and describing the whole dynamic process,which has certain significance for the prevention and treatment of TGE.This study is divided into the following four parts:1 Fluorescent labeling of TGEV and preparation of TGEV polyclonal antibodyIn this study,TGE Vs were successfully labeled with the lipophilic fluorescent dye DiD.TGEVs were amplified by ST cells,then viruses were collected and centrifuged at low speed to remove cell fragments,concentrated by ultracentrifugation and purified by density gradient centrifugation.Then mixed the purified viruses with the DiD dye,incubated it at room temperature,filter and remove the unbound dye.The results of confocal microscopy and fluorescence intensity statistics of individual TGEV virus particles showed that TGEV virus particles were successfully labeled,and the fluorescence intensity of each virus particle was relatively uniform.The TCID50 results showed that there was no significant difference in virus virulence between before and after TGEV labeling,indicating that this labeling method had no significant effect on virus infection.In addition,the preparation of TGEV polyclonal antibody was to immunize BALB/c mice with inactivated virus as immunogen,which was inactivated by β-propiolactone and UV.After the titer of ELISA reached 105,the serum was collected and purified.The results of SDS-PAGE and IFA showed that the purified antibody had no other protein contamination and had specific binding to TGEV.The DiD labeled TGEV and the polyclonal antibody of TGEV laid an important foundation for the following experiments.2 Study on TGEV entry via clathrin-and caveolae-mediated endocytosisClathrin-and caveolae-mediated endocytosis are two common endocytosis pathways in cells.In order to study the effect of clathrin-and caveolae-mediated endocytosis on TGEV in ST cells,ST cells were pretreated with specific inhibitors of clathrin-and caveolaemediated endocytosis as CPZ,pitstop2,nystatin and MβCD,respectively,and then infected with TGEVs for IFA and qPCR.The results showed that the treatment of inhibitors could significantly reduce the infection of TGEV,indicating that clathrin-and caveolae-mediated endocytosis had an important role in the TGEV infection.In order to further reveal the dynamic process of TGEV entry via clathrin-and caveolae-mediated endocytosis,the vectors of clathrin and caveolae as Clc-EGFP,Cav1-EGFP and Cav1-mKO2,which all fused to express fluorescent protein tags,were constructed,respectively.ST cells were transfected with Clc-EGFP and Cav1-EGFP,respectively.After 24 h,DiD-labeled TGEVs were added to the transfected cells for real-time tracking.The results showed that after the TGEV was attached on the cell membrane,TGEVs could enter the ST cells clathrin-and caveolaemediated endocytosis,and the whole process was completed within 2 min.By analyzing the dynamics of TGEV,it is found that the whole process of TGEV entry could be divided into two stages:in the first stage,TGEVs experienced anomalous diffusion at a low rate;in the latter stage,TGEVs experienced directed diffusion at a high rate.Because TGEV can enter the ST cells via clathrin-and caveolae-mediated endocytosis,in order to determine the proportion of TGEV entry into ST cells via two pathways,ST cells were transfected with Clc-EGFP and Cav1-mKO2 plasmids at the same time.After 24 h,DiD-labeled TGEVs were added for real-time tracking.The tracking experiment of TGEV entry was performed in the double transfected cells.The statistical results showed that 60.7%of TGEV could enter ST cells via clathrin-mediated endocytosis,and 39.3%could enter via caveolae-mediated endocytosis.3 Effect of actin on TGEV entryIt has been shown that actin plays an important role in the entry of some viruses.In order to study the effect of actin on the TGEV entry,ST cells were pretreated with CytoD,which is an actin inhibitor.Then ST cells were infected with TGEVs for IFA and qPCR.The results showed that the cells treated with inhibitors could significantly reduce the TGEV infection,indicating that actin plays an important role in TGEV infection.In order to reveal the dynamic process of interaction between TGEV and actin,Actin-EGFP was constructed.ST cells were transfected with Actin-EGFP,after 24 h DiD-labeled TGEVs were added for real-time tracking.The results showed that TGEV entry was dependent on actin.By analyzing the dynamics of TGEV,it is found that the whole process of TGEV entry could also be divided into two stages:in the first stage,TGEVs experienced anomalous diffusion at a low rate;in the latter stage,TGEVs experienced directed diffusion at a high rate.Similar to clathrin and caveolae,the interaction between TGEV and actin took place within 2 min before the TGEV entry,indicating that actin could be recruited to assist the TGEV entry.4 Effect of dynamin2 on TGEV entryDynamin is a protein with GTP hydrolase activity in cells,which can mediate membrane separation.It has been found that dynamin can affect the endocytosis of many viruses.In order to study the effect of dynamin on the TGEV entry,ST cells were first pretreated with dynamin specific inhibitor dynasore.Then ST cells were infected with TGEVs for IFA and qPCR.The results showed that the inhibitor could significantly inhibit the TGEV infection.In addition,in order to further verify the above results,we constructed the dynamin2 dominant negative mutant vector K44A and the shRNA targeting dynamin2.ST cells were transfected with the dominant negative mutant vector K44A and shRNA,respectively,and then infected with TGEVs for qPCR.The results showed that K44A and shRNA could significantly inhibit the infection of TGEV,indicating that dynamin2 was important for the TGEV infection.In order to study the dynamic interaction between TGEV and dynamin2 in the TGEV entry,Dyn2-EGFP was constructed.ST cells were transfected with Dyn2-EGFP,after 24 h DiD-labeled TGEVs were added for real-time tracking.The results showed that the TGEV entry depended on dynamin2.By analyzing the dynamics of TGEV,it was found that the whole process of TGEV entry could also be divided into two stages:in the first stage,TGEVs experienced anomalous diffusion at a low rate;in the latter stage,.TGEVs experienced directed diffusion at a high rate.However,unlike clathrin,caveolae and actin,the interaction between TGEV and actin took place within 2 min before the TGEV entry,indicating that dynamin2 could be recruited to mediated membrane separation in the process of TGEV entry.