Autophagy Response Of Spodoptera Frugiperda To AcMNPV Infection

Autophagy is considered to be an important part of innate immunity by capturing and degrading various pathogens such as bacteria and viruses.However,in the long-term evolution,virues have evolved to own the ability to evade the host immunity or use cell autophagy to benefit self-replication.Our preliminarily research showed that Autographa californic multiple nucleopolyhedrovirus（AcMNPV）infection can cause autophagic response in Sf9 cells,a cell line of Lepidoptera insect Spodoptera frugiperda,but the effect of autophagy on virus proliferation and the molecular mechanism of autophagy has not been elucidated.The present study is aimed to obtain evidence that AcMNPV infection triggers autophagy in Sf9 cells by various experimental methods,to explore the effect of autophagy level in host cells on the AcMNPV proliferation,and to further analyze of the gene expression changes in the fat body of Spodoptera frugiperda caused by viruses infection based on the transcriptome data.All the above research could lay a foundation for exploring the molecular mechanism of virus-induced autophagy.The main results are as follows:1.The recombinant virus EGFP-AcMNPV was constructed by the Bac-to-Bac system,infected Sf9 cells（MOI:8）,green fluorescence was observed at 24h post infection.And the fluorescence was significantly enhanced at 24h to 48h,indicating that the virus had replicated and reproduced extensively.Real-time quantitative PCR technology was used to detect the expression changes of Atgs in Sf9 cells after EGFP-AcMNPV infection.The results implied that AcMNPV infection could elicit an autophagy response in Sf9 cells.2.By Lyso Tracker Red staining,the confocal laser microscopy was used to perform microscopic examination and statistical analysis of autophagolysosomes occurring in the AcMNPV infects Sf9 cells,showing that the number of autophagolysosomes increased in infected Sf9 cells.ATG8 protein level was monitored by Western blot.Show that the conversion of ATG8 to ATG8-PE obviously increased,indicating that AcMNPV infection could induce autophagic responses in Sf9 cells.3.The autophagy inducer Rapamycin the autophagy inhibitor Chloroquine（CQ）were used to treat Sf9 cells with AcMNPV infection,to detect the effect of changes in autophagy level on virus proliferation.When Sf9 cells were treated with an autophagy inducer,the treated cells showed a significant increase in green fluorescence compared with the control cells,This indicates viral replication and proliferation was increased through fluorescence microscopy observation;TCID₅₀ detected virus titer and Western blot analysis the EGFP protein expression,proved that it’s good for virus proliferation with the drug treatment,indicated that enhanced autophagy in Sf9 cells facilitated virus replication.In the contrast,the results of autophagy inhibitor CQ treatment were opposite,the virus replication and proliferation were reduced.The above results indicated that the virus uses the host cell autophagy to promote its own proliferation in the process of infecting Sf9 cells.4.At 30min to 6h in the early stage of virus infection,there was no significant change in total AKT expression.And the level of p-AKT was increased rapidly.LY294002,a specific inhibitor of PI3K/AKT pathway could increase the conversion of ATG8-PE form,and increased the replication of the virus accordingly.The results indicated that in the early stage of viral infection,PI3K/AKT signaling pathway was involved in the process of viral infection triggering autophagy.5.The nucleus of Sf cells became larger and the cell morphology was abnormal at 48hours after AcMNPV infection.Cell flow cytometry results showed that the cells began to apoptosis 24h after infection and obvious apoptosis occurred at 36h to 48h infection,This indicated that autophagy and apoptosis were two types of programmed death with AcMNPV infection Sf9 cells.6.Transcriptome sequencing was performed by the fat body of Spodoptera frugiperda larvae which had been injected with AcMNPV.Numberous genes such as AMP,Inhibitor of Apoptosis domain,PI3/4K,and transmembrane receptor were significantly up-regulated at24h after virus infection.KEGG enrichment analysis showed that lysosome,autophagy,apoptosis,Toll and Imd,PI3K/AKT,m TOR and other signaling pathways had been enriched.The above research helps us to further understand the relationship between AcMNPV and Spodoptera frugiperda,and provides a theoretical basis for the use of viral insecticides in the control of Lepidoptera pests.