Autophagy Induced By Infectious Pancreatic Necrosis Virus Promotes Its Multiplication In The Chinook Salmon Embryo Cell Line

Infectious pancreatic necrosis（IPN）is a highly pathogenic,highly fatal viral disease caused by Infectious pancreatic necrosis virus（IPNV）that mainly infects the fry and fingerlings of salmonids.Widely popular in Europe and many countries in Asia,it is an important quarantine object for the import and export of fish eggs in countries.Cell autophagy has a dual effect in the process of pathogenic microorganism infection,and some pathogenic microorganisms can induce autophagy to promote the proliferation of themselves,and the multiplication of certain pathogenic microorganisms is suppressed by cell autophagy.In order to investigate the interaction between IPNV and autophagy in Chinook salmon embryo cells（CHSE-214）,in this study,CHSE-214 cells were first treated with rapamycin（100 n M,500 n M,1000 n M）,3-MA（1 m M,5 m M,10 m M）or PBS.The cells treated with PBS were used as negative control.Samples were collected after 72hours of incubation,and the appropriate concentrations of rapamycin and 3-MA were screened by MTT to exclude the effects of chemical drug toxicity on the accuracy of the experiment.Subsequently,this study investigated whether IPNV infected chse-214 cells induced autophagy by transmission electron microscopy,indirect immunofluorescence and Western blotting.Autophagosomes were observed by transmission electron microscopy after CHSE-214 cells infected with IPNV or treated with PBS,rapamycin.Green fluorescent protein and LC3 fusion expression vector（p EGFP-LC3）were transferred into CHSE-214 cells for 24 hours,and the point-like fluorescence aggregation of LC3 molecules was observed by laser confocal detection after PBS,rapamycin treatment or IPNV（MOI=0.1）infection of cells for 24 hours.The PBS treatment group was the negative control,while the rapamycin treatment group was the positive control.CHSE-214 cells were pretreated with PBS or rapamycin 4 h and infected with IPNV（MOI=0.1）,and collected cell samples from different treatment groups at 24 h and 48 h after infection to detect the LC3 protein phenotype changes and the degradation of p62 by Western blotting.Then the effect of autophagy on IPNV was detected by Reverse Transcription Quantitative real-time PCR（RT-q PCR）,Western blotting,TCID₅₀and other methods.Cells were pretreated with PBS,rapamycin,or3-MA 4 h in advance and then IPNV with MOI=0.1 was inoculated.Cell samples from different treatment groups were collected 12 h,24 h,36 h,48 h,and 72 h after infection.RT-q PCR,western blotting,TCID₅₀and other methods were used to detect changes in IPNV at nucleic acid level,protein level and virus titer and the cytopathic condition was observed under a microscope.MTT results showed that the cell relative cell viability of the rapamycin treated group and the 1 m M,5 m M 3-MA treated group was not significantly different from that of the PBS treated group,and the 10 m M 3-MA treated group was significantly lower than that of the PBS treated group（P<0.05）.Therefore,500 n M rapamycin and 5 m M 3-MA were used in this study for subsequent experiments.Transmission electron microscopy results showed that the typical bilayer membrane autophagosome structure could be seen in the cells treated with rapamycin and infected with IPNV,but not found in the negative control,indicating that IPNV infection induced autophagy in CHSE-214 cells.Laser confocal microscopy results showed that green fluorescent spot-like aggregation of LC3 molecules could be observed in the rapamycin-treated group and IPNV-infected cells,and analysis of the immunofluorescence microscopy field（n=10）using Image J software showed that there was no significant difference in the level of EGFP-LC3 spotted aggregation between rapamycin-treated and IPNV-infected cells,and it was significantly higher than that of PBS-treated cells（P<0.05）.The results further verified the generation of autophagy induced by IPNV-infected host cells.Western blotting results showed that cells in the rapamycin-treated group and the IPNV-infected group could observe a clear conversion of LC3-I to LC3-II,and the ratios of LC3-II/LC3-I,LC3-II/β-tubulin were significantly higher than PBS-treated group（P<0.05）.The ratios of LC3-II/LC3-I and LC3-II/β-tubulin in the IPNV-infected group at 48 h were significantly higher than those at 24 h,which were significantly lower than those in the rapamycin-treated group（P<0.05）.The level of p62 protein in rapamycin-treated and IPNV-infected cells was significantly lower than that in the PBS-treated group（P<0.05）.In addition,the level of p62 protein in the IPNV-infected cells at 48 h was significantly lower than 24 h,which were significantly higher than those in the rapamycin-treated group（P<0.05）.Western blotting results further confirmed the autophagy induced by IPNV infection in CHSE-214 cells.The results of viral load analysis showed that VP1 and VP2 m RNA expression levels in rapamycin-treated cell samples collected at different time were significantly higher than those in the PBS-treated group（P<0.05）.The m RNA replication level of VP1 and VP2 genes in 3-MA-treated group were significantly lower than that in the PBS-treated group P<0.05).In addition,Western blotting results showed a similar trend in VP2 protein expression levels.This indicates that autophagy promotes the replication of IPNV m RNA and the protein expression of virus.The virus titer results showed that the virus titers of the rapamycin-treated group at 48 h and 72 h were significantly higher than those of the PBS-treated group（P<0.05）,and the virus titers of the 3-MA-treated group were significantly lower than those of the PBS-treated group（P<0.05）,indicating that autophagy could improve the yield of IPNV.The microscopic results showed that after 60 hours of cell treatment,compared with cells without any treatment,the virus infected with IPNV appeared cytopathic phenomenon;and the degree of cytopathy in the IPNV+Rapamycin treatment group was higher than that in the IPNV+PBS treatment group The degree of cell lesions in the IPNV+3-MA treatment group was lower than that in the IPNV+PBS treatment group.Increasing cell autophagy activity increased the number of diseased cells and increased virus yield.This result is consistent with the titer measurement results.In summary,this study proved that IPNV infection induced autophagy in CHSE-214 cells,and that autophagy promoted the multiplication of IPNV at the nucleic acid and protein levels,thereby increasing the yield of IPNV.This study provides a new perspective for exploring the pathogenesis of IPNV and a new direction for the research of anti-IPNV drugs.