Ginkgo Biloba Exocarp Polysaccharide Inhibits The Action Of Newcastle Disease Virus

In order to study the effect of GBEP on NDV,Ginkgo biloba enocarp polysaccharides(GBEP)was extracted by water extraction and alcohol precipitation.The content of GBEP and its characterization was determined by phenol-sulfuric acid method,iodine-potassium iodide experiment and Fourier transform infrared spectroscopy.On the basis of this,different concentrations of GBEP were inoculated with DF-1 cells and 9-11 day-old chicken embryos to determine the safe concentration of GBEP on cells and chicken embryos.And then compare the role of different forms of administration on NDV(administer and then add the virus,add the virus and then administer and mix the virus and drug).In addition,NDV dilutions were added to DF-1 cells.The total RNA of cells and viruses was extracted at different time intervals.RT-PCR was used to quantitatively analyze the expression levels of NDV in different time periods.Analyze the effects of NDV on DF-1 cells on the adsorption,proliferation and release.The changes of cell surface were observed by scanning electron microscopy(SEM),and the proliferation of NDV cells was observed by indirect immunofluorescence assay.Finally,GBEP was added to different stages of virus replication at different concentrations(20,10,5?g/mL).The expression of NDV was quantitatively analyzed by RT-PCR.and the immunofluorescence method was used to investigate the effect of NDV on DF-1 cells in the presence of replication.Results:GBEP has a sugar content of 51.9%and does not contain starch,The infrared spectrum showed a typical polysaccharide map,which was strongly absorbed at 1250.6 cm-1.GBEP had no significant effect on DF-1 cell growth at no more than 50 ?g/mL,no more than 4 mg per chicken embryo does not affect the normal development of chicken embryos.In the terms of anti-NDV,the OD value of the 1.25 ?g/mL GBEP group was significantly higher than that of the group which administer and then add the virus(P<0.01)on the DF-1 cells,there was no significant difference compared with the same dose of ribavirin group(P>0.05),the group that add the virus and then administer,the OD value of GBEP group was significantly higher than that of the control group(P<0.05),but less than the same dose of ribavirin,and the effect was increased with the increase of dose.The concentration of GBEP in the mixed group(P<0.01),and there was no significant difference(P<0.05)in the same dose of ribavirin group(P<0.01).In the chicken embryo test,the embryonic development and HA blood coagulation of the 0.8 mg GBEP/pc group were similar to that of the ribavirin group,and the chicken embryo died at 48 h in the control group.Five of the chicken embryos were killed in 48h and all of them were died in 60h in the group which add the virus and then administer.One of the chicken embryos was died at the same time in the group which administer and then add the virus,while no death in the mixed group and the length of embryos was significantly longer than that of the control group(P<0.05).The results of NDV detection on the replication cycle of DF-1 cells showed that NDV dilution was added to DF-1 cells,the adsorption capacity of cells was about 110min,and the presence of virus particles was found on the cell surface.The multiplication of virus reached its maximum in the cells and showed a significant time effect when its dilution was added to the cells for about 13 hours.When the virus was added to the cells for 6 h,NDV was detected in the cell culture supernatant,and the NDV content reached the maximum at 42 h,indicating that the virus was released after 6 h.The adsorption capacity of NDV to DF-1 cells was significantly lower than that of the control group(P<0.01)when different concentrations of GBEP and NDV were simultaneously added to the cells.It was also possible to significantly reduce the proliferation of NDV cells(P<0.01)by adding different concentrations of GBEP after virus entry into cells.In addition,the cells and the virus were incubated and then added to the cells.The adsorption capacity of NDV cells was also significantly lower than that of the control group(P<0.01).The results of RT-PCR showed that the expression of NDV-F gene in each drug group was lower than that in the control group.Immunofluorescence assay showed that the fluorescence intensity of each group was lower than that of the control group.Conclusion:The above results indicate that GBEP is an acid polysaccharide containing sulfuric acid groups,which has the effect of inhibiting and inactivating NDV on DF-1 cells and chicken embryos,and the effect on DF-1 cells is better than that of chicken embryos.In addition,GBEP can be significantly Reduce the adsorption and intracellular proliferation of DF-1 cells by NDV.