Exploring The Infection Mechanism Of Mink Enteritis Virus By Quantum Dots-Based Single Virus Tracking

Mink enteritis virus(MEV)is a single stranded DNA virus with strong environmental resistance,which has caused huge economic losses to the global mink breeding industry.At present,the mechanism of MEV infection is not clear,which makes its prevention and control extremely difficult.It’s important to clarify the infection mechanism of virus for formulating effective anti-virus strategy.Single virus tracking technology based on quantum dots has become a practical method to explore the molecular mechanism of virus infection because it can realize virus imaging in living cells.This study selected the labeling strategy of Biotin-SA system,built a single virus tracking platform based on quantum dots,visualized the process of MEV infecting CRFK cells,analyzed the mechanism of MEV infecting host cells,and provided a basis for the research and development of anti-MEV drugs.The main results of this paper are shown as follows:1.Establishment of MEV tracking system based on QDsUsing Biotin-SA system to complete the labeling of MEV by QDs,and a MEV single virus tracking platform based on quantum dots was built.The purified MEV virus were reacted with biotin to obtain biotinylated virus,and then incubated with SA-QDs to obtain QDs-MEV.The virus was characterized by electron microscope before or after modification,the result showed that the modified MEV was combined with QDs.The virulence of the virus was detected by TCID50,the result showed that the modified virus still maintained normal biological activity.Dual channel imaging of Delta Vision was performed on CRFK cells labeled with Di O and QDs-MEV.The red fluorescence signal of QDs-MEV could enter the cytoplasmic region wrapped with Di O green fluorescence,QDs-MEV could normally infect CRFK cells.QDs-MEV could be used for dynamic tracking to complete virus imaging research.2.Exploring the endosomes depended transport mechanism of MEVThe interaction between MEV and endosome system was explored by MEV single-virus visualization system.Rab5 protein(early endosome marker)or Rab7 protein(late endosome marker)in CRFK cells were labeled by transient transfection of plasmid,and QDs-MEV was added for dual channel imaging.The result showed that red fluorescence signal of MEV entered the cell,Rab5 green fluorescence signal approached the virus rapidly until MEV was captured by early endosomes,and the fluorescence signals colocated and moved continuously.Rab7 green fluorescence signal was colocated with MEV red fluorescence,then two signals separated,the virus escaped from the late endosome.These results suggested that the transport of MEV in host cells depend on endosome system.CRFK cells were treated with endosome acidification inhibitors,and the amount of virus was detected by general methods to explore the effect of low-p H environment on MEV infection.The results of q PCR and Western blot showed that the relative expression of virus VP2 gene and protein decreased significantly after the addition of inhibitors,and the virus infection was inhibited obviously,indicating that the infection of MEV was dependent on the acidic environment of endosomes.3.Exploring the cytoskeleton depended transport mechanism of MEVThe interaction between MEV transport and cytoskeleton was explored by MEV singlevirus visualization system.CRFK microfilaments or microtubules were labeled by transient transfection,and QDs-MEV was added for dual channel imaging.The imaging results showed that the MEV red fluorescence signal did not colocate with the microfilament,but colocated with the microtubule,and made a rapid directional movement along the microtubules.It was supported by general research methods.CRFK cells were treated with microfilament or microtubule inhibitors and the amount of virus was detected.The results of q PCR and Western blot showed that after adding microfilament inhibitor,the virus infection did not affected.After adding microtubule inhibitor,the relative expression of MEV VP2 gene and protein decreased significantly,and the virus infection was inhibited obviously.The above results showed that the transport of MEV depends on microtubule.In conclusion,the MEV single-virus tracking system was constructed based on QDs,and was used to explored the relationship between MEV infection and acidic endosomes,cytoskeleton.MEV enter the host cell through endocytosis,it depends on endosomes to transport along microtubule.The acidic environment of endosome is necessary for MEV to start effective infection.This study provides strong visual evidence for improving the infection mechanism of MEV,and is expected to provide theoretical basis for the development of antiMEV drugs.