The Effect Of Coxsackievirus A10-Induced Autophagy On Virus Replication In Vitro And In Vivo

Objective:In this thesis,Coxsackie Virus group A type 10(CV-A10)was used as the research object to explore whether it could induce autophagy on 16HBE and RD cells.Based on this speculation,we aim to explore the relationship between CV-A10-induced autophagy and virus replication in vitro and in vivo by using cell and the established CV-A10-infected ICR mouse model.Methods:In cell model,to identify whether CV-A10-infected 16HBE and RD cells could induce autophagy,western blot was used to detect the transformation of autophagy maker LC3-II protein,as well as the expression of autophagy protein P62 and Beclinl in CV-A10 infected 16HBE and RD cell.Immunofluorescence was used to detect the localization of GFP-RFP-LC3 in cells and transmission electron microscopy was used to detect the formation of autophagosome.Western blot was used to detect the expression of autophagy maker LC3-II protein after using the autophagy inhibitor 3-methyladenine(3-MA).In addition,to explore the effect of autophagy on viral replication,microplate CPE method,Real-time PCR and Western blot were used to detect the changes of viral titer,viral load and the expression of the CV-A10 VP1 between the 3-MA treated group and the untreated group.In animal model,based on the established CV-A10-infected ICR suckling mice model,hematoxylin-eosin staining was used to observe the pathological injury of each tissue.Pathological sections of each tissues of ICR suckling mice were prepared to detect the distribution of virus in each tissue using immunofluorescence method.The CV-A10 VP1 expression in brain tissue of neonatal ICR suckling mice was detected by Western blot.Results:Western blot analysis showed that the expression of LC3-? in CV-A10 infected 16HBE and RD cells were higher than that in the control group.GFP-RFP-LC3 plasmid was transfected into 16HBE and RD cell and the aggregation of red and green autophagic positive fluorescent particles were observed after CV-A10 infection.The autophagosomes were observed by transmission electron microscope.The expression of autophagy-related protein Beclinl increased with the increase of infection time,while the expression of P62 protein decreased with the increase of infection time.After treatment with autophagy inhibitor 3-MA,the expression of LC3-? protein decreased and the CV-A10-induced autophagy was inhibited.And the viral titer,viral RNA load and the expression of CV-A10 VP1 protein were all lower than those in the untreated group.The survival rate and the body weight of suckling mice treated with 3-MA were significantly higher than those of the untreated group.The CV-A10 infection model of 1-day-old ICR suckling mice was established.Hematoxylin-eosin staining showed that the pathological damage of brain,heart,lung,intestine and muscle tissue in the 3-MA treatment group was less severe than those in the untreated group.Immunofluorescence results showed that the virus particles in the brain,heart,lung,intestine and muscle tissue of mice were lower than those in the untreated group,and the pathological changes in the tissue of mice were reduced.The result of Real-time PCR showed that the viral load of brain tissue of suckling mice in the 3-MA treated group was lower than that in the untreated group at 3,5 and 8 days after virus infection.Conclusion:CV-A10 can induce autophagy in 16HBE and RD cells.P62 and Beclinl are involved in the autophagy process.The autophagy inhibitor 3-MA could inhibit CV-A10-induced autophagy,and it can aslo inhibit virus replication.3-MA can also inhibit the replication of CV-A10 in suckling mice,mitigate pathological lesion and improved the survival rate of them basing on the the established CV-A10-infected ICR mouse model.