| Muscovy duck reovirus infection is an immunosuppressive disease caused by Muscovy duck reovirus(MDRV),resulting in the death of a large number of muscovy ducklings,the stagnant growth of the resistant muscovy ducking,Which had caused huge economic losses to the duck breeding industry.Both MDRV and chicken reovirus(CRV)belong to avian orthoreovirus(ARV),but there is a certain difference between their encoded protein.It has been reported that CRV was represented by strain S1133 invading cells through both caveolin-1 and dynein-2-dependent endocytosis pathways,and the endocytic pathway for MDRV invading cells has not yet been studied.Hence Fluorescent labeling techniques and chemical inhibitors were used to explore the endocytic pathway of Muscovy duck reovirus invading cells.The test results are as follows:1.Inhibitors on cell proliferation-toxicity detectionTo ensure that the growth status of cells will not change significantly after drug treatment,CCK-8 kit was used to detect the appropriate safe concentrations of CPZ,M-β-CD and Dynasore.In a 96-well plate grow with enough DF-1/Vero cells,different concentrations of M-β-CD,CPZ,and Dynasore were added,cell activity was measured using a Cell Counting Kit-8 kit after drug treatment,and the DF-1/Vero cells was observed under a microscope.Combined with Morphological changes of DF-1/Vero cells,to determine M-β-CD 0.8 mM,1.6 mM,3.2 mM;CPZ 4μM,8 μM,16 μM;Dynasore 15.5 nM,31 nM,62 nM,etc.As the safe drug concentration of further experiment to DF-1/Nero cells.2.Preliminary investigation on the endocytic pathway of Muscovy duck reovirus invading DF-1/Vero cellsCPZ and M-β-CD can inhibit clathrin and caveolin mediated endocytosis,and Dynasore can inhibit dynamin-2 protein.In this experiment,DF-1/Vero cells were divide into different group with different drugs CPZ,M-(3-CD,Dynasore,CPZ + M-β-CD,CPZ +Dynasore,M-β-CD + Dynasore,and then infected with MDRV.At the same time,set the corresponding negative control group and positive control group and collected samples after treated for 24 hours.Real-time RT-PCR and Westren-Blot methods were used to determine the copy number of intracellular p10.8 gene and(A protein expression amount of Muscoy duck reovirus.The results show:(1)After pretreatment of DF-1/Nero cells with different drug concentrations as M-β-CD 0.8 mM,1.6 mM,3.2 mM for 30 min,infected with MDRV,and the corresponding negative control group and positive control group were set.The results showed that in DF-1/Vero cells,compared with the positive control group,as the M-β-CD drug concentration increasing,the intracellular MDRV p10.8 gene copy number and the σA protein expression amount had significantly downward trend.It indicated that the replication of the virus in the cell is negatively correlated with the concentration of M-P-CD.(2)After pretreated DF-1/Vero cells with different CPZ concentration about 4 pM,8 μM,16 μM,infected wuth MDRV,and the corresponding negative control group and positive control group were set.The results showed that in DF-1/Vero cells,compared with the positive control group,although different concentrations of drug decreased the intracellular virus copy number of p10.8 gene and the σA protein expression,the amount of intracellular virus was reduced.And do not vary with the concentration of the drug,indicating that there is no significant correlation between the amount of virus in cells and the drug concentration.(3)DF-1/Vero cells were pretreated with Dynasore 15.5 nM,31 nM,and 62 nM for 30 min,then infected with MDRV,the corresponding negative and positive control groups were set.The results showed that:In DF-1/ero cells,after the pretreatment of Dynasore,the drug group intracellular virus p10.8 gene copy number and σA protein expression was significantly lower than the positive control group,and in different concentration of drugs,the intracellular viral copies of the p10.8 gene and the σA protein were also significantly different.It indicated that the replication of the virus in cells is affected by Dynasore.(4)In DF-1 cells,the p10.8 gene copy number and σA expression of the intracellular virus,were compared between the drug pretreated group and the individual virus positive control group after pretreated with both M-β-CD and Dynasore drugs.The protein expression level decreased significantly with the increase of drug concentration.However,CPZ pretreatment had no significant effect on the invasion of MDRV cells.In CPZ + M-β-CD and CPZ + Dynasore group added with M-β-CD and Dynasore,the amount of intracellular virus also decreased significantly.In the M-β-CD + Dynasore group,the amount of intracellular virus decreased significantly.The same results were also obtained in Vero cells,indicating that MDRV invading DF-1/Vero cells requires the participation of caveolin and activator proteins.(5)The MDRV σA protein and caveolin-1 were marked by different type of immunofluorescent.The results showed that,as time went by,the virus gradually migrated from the outside of the membrane to the nucleus,and a small part of the virus entered the nucleus.Taken together,MDRV invading DF-1/Vero cells through endocytic pathways mediated by caveolin.3.Preliminary investigation on the effect of cytoskeleton on Muscovy duck reovirus invading cellsPretreated cells with different concentrations of microtubule inhibitor Nocodazole and microfilament inhibitor Cytochalasin D,then infected with MDRV,set negative control group and positive control group.Collected cells 24 hours later,detected ntracellular virus p10.8 gene copy number and σA protein expression by Real-time RT-PCR and Westren-Blot Methods.The amount of virus showed a decreasing trend on the concentration of Nocodazole,That microtubules are also involved in the virus into the cell pathway.4.Preliminary exploration of the release environment of Muscovy duck reovirus in cells.Viruses in endosomes must regulate the pH of endosomes to activate conformational changes in the virus,otherwise they will be presented to lysosomes for degradation.It is known that CRVs require a low pH environment in the endosomes,but if the same species MDRV also require a low pH environment is not also clear.It is known that NH4C1 can regulate the pH of intracellular lysosomes.This experiment used different concentrations of NH4C1 to pretreat cells to change the acidic environment of the endosomes in varying degrees,followed by infected with MDRV,and set negative control group and positive control group,received samples 24 hours later.The changes in the amount of intracellular virus were detected to confirm the effect of pH changes in the endosomes on the release of the virus.The results showed that with the increase of drug concentration,the intracellular virus p10.8 gene copy number and the expression of σA protein were significantly inhibited.It can be speculated that MDRV needs a low pH environment to facilitate the virus to penetrate the cytoplasmic membrane and complete the shelling.Conclusion:Muscovy duck reovirus invading DF-1/Vero cells is mediated by caveolin and requires an endocytic pathway involving dynamin proteins,through regulates a low pH environment to release virus into cytoplasm and replicate. |
PDF Full Text Request