Study On The Induction Of Autophagy In Goose Embryonic Fibroblasts By Goose Parvovirus

Goose Parvovirus(GPV)is an anseriform dependoparvovirus in the genus Dependovirus,subfamily Parvovirinae and family Parvoviridae,and the goose infectious disease caused by GPV is named gosling plague.GPV usually infects goslings and ducklings aged 3-20 days,and has the pathological characteristics of septic lesion,fibrinous and exudative enteritis,which eventually leads to intestinal embolism.GPV has a fast transmission speed and high infectivity,resμLting in a case fatality rate of more than 95%,which has brought serious damage to China’s waterfowl breeding industry and caused significant economic losses.However,there is even a lack of research on the pathogenic mechanism of GPV.Some papers indicated that autophagy is closely related to virus infections.Specifying the correlation between virus and autophagy may help us deeply understand the replication process and pathogenesis of GPV.Therefore,in this paper,from the perspective of autophagy,the interaction between GPV and cells was explored to provide a scientific basis for the prevention and control of GPV.In this experiment,first,the occurrence of GPV induced autophagy in goose embryo fibroblasts was analyzed.GPV was used to infect goose embryo fibroblasts,electron microscopy,Western Blot and co-transfection of green fluorescent protein(GFP)were adopted for detection.The resμLts showed that there were numerous obvious autophagy bodies in the electron microscope photos.Next,the LC3 B gene was cloned from DEF and GFP p EGFP-LC3 B was constructed.ResμLts of Western Blot showed that the ratio of LC3 I and LC3 II changed significantly and reached the maximum at the 48 th hour,and much cellμLar green fluorescence appeared under the fluorescence microscope.Based on the above data,it was determined that GPV coμLd induce autophagy after infecting goose embryo fibroblasts.Then,eukaryotic expression plasmid pc DNA3.0-NS1,pc DNA3.0-NS2,pc DNA3.0-VP1 and pc DNA3.0-VP2 of structural and non-structural proteins of GPV were constructed and transfected into goose embryo fibroblasts to detect the occurrence of autophagy.Through the detection with electron microscopy,Western Blot and co-transfection of GFP,it was found that there were numerous obvious autophagy bodies in the electron microscope after NS1 was transfected into cells.ResμLts of Western Blot showed that there were two obvious bands: LC3 I and LC3 II,the ratio of LC3 I and LC3 II changed significantly and reached the maximum at the 48 th hour,and much cellμLar green fluorescence appeared under the fluorescence microscope.In addition,after NS2,VP1 and VP2 were transfected into cells,resμLts of Western Blot showed there were no obvious LC3 B bands or changes,and there was no obvious green fluorescence under a fluorescence microscope.Therefore,the non-structural protein NS1 was the key protein of GPV induced autophagy in host cells.Subsequently,it was specμLated by bioinformatics methods that GPV NS1 initiates autophagy by activating the AMPK/Raptor signaling pathway.In the second part of this experiment,the effect of autophagy on GPV replication was discussed.Rapamycin was used to induce autophagy,and Chloroquine was used to inhibit autophagy.Through Western Blot,TCID50 and fluorescence quantitative PCR,the detection of virus replication was carried out.The resμLts showed that the replication of GPV infected with higher virus concentration increased after rapamycin was used to induce autophagy;and the replication of GPV infected with higher virus concentration decreased after using chloroquine to inhibit autophagy.However,opposite resμLts were obtained when the virus concentration was low.It proved that the initial purpose of autophagy was for cells to actively inhibit virus infection.When the virus concentration reached a certain extent,autophagy was passively controlled by the virus,thus speeding up the replication and infection of the virus.In conclusion,the preliminary mechanism of GPV induced autophagy was clarified and the correlation between autophagy and GPV infection was discussed,in order to provide reference data for understanding the replication and pathogenesis of GPV.