| Hand, foot, and mouth disease (HFMD) is a serious global epidemic disease. Recurring outbreaks of EV71 have been reported in several countries, particularly in mainland of China, Taiwan, Singapore and other regions of Aisa since 1990s. HFMD mostly affects infants and children younger than 5 years of age and it is capable of developing into dangrous neurological complications, such as encephalitis, meningitis and pulmonary edema, which could lead to serious injury or even fatal. Therefore, it is still a major public health issue and a threat to children health. Enterovirus 71 (EV71) and Coxsackie virus A 16 (CA16), which belong to a pathogenic serotype of the Picornaviridae family, are the principal etiological pathogens of HFMD. In recent years, accumulating studies on HFMD are mainly focused on EV71 infection and there is lack of enough attention to CA16 infection. Although both EV71 and CA16 are major etiological pathogens leading to HFMD, they differ in clinical manifestations. EV71 infection is thought to easily progress to serious complications, while CA16 infection generally results in mild clinical symptoms. Nevertheless, patients with CA16 infection who developed into severe stage also presented the basic characteristics and clinical symptoms similar to that of EV71 infection. Additionally, increasing attentions have been paid to repeated infection phenomenon elicited by CA16. Moreover, it had been reported that several HFMD outbreaks with a high lethality evoked by EV71 and CA16 circulating alternatively or together. Although EV71 inactived vaccine has come into the market, there is still a need to further investigate on the underlying mechanisms of the difference clinical symptoms between EV71 and CA16 infection and the immunology of EV71 and CA16 infeciton.Based on the successful establishent of CA16 infection model in rehesus monkey infant by our previous study, global gene expression analysis of peripheral blood mononuclear cells in rhesus monkey infants with CA16 infection-induced HFMD has been made in the first part of this thesis. The results demonstrate there are 948 differentially expressed genes at all 4 time points tested during CA16 infection and these differentially expressed genes primarily divided into 5 GO groups, including cell communication, cell cycle, immune system process, regulation of transcription and metabolic process. These results suggested that all of the five functional categories were closely associated with the progressive pathogenesis of CA16 infection. In addition, co-expression network analysis of the top 10 differentially expressed genes in 7-1st dpi rhesus monkey infants has been made and found that IL-6, IL-8, IL-18, which involved in inflammatory response and immune response, were key regulatory genes. To further explore the underlying regulatory mechanisms associated with the immune response after CA16 infection, a PANTHER pathway analysis pie chart was created for the 115 differentially expressed immune system process genes that were identified in ontology analysis. Of these pathways, inflammation mediated by chemokine and cytokine signaling pathways and the interleukin signaling pathway comprise a relatively large proportion. Finally, arounding the anti-viral innate immune response releated to the mobilization of type I interferon (I-IFN), genes referred to IFN-I production and autophagy were analysed and these genes were down-regulated after CA16 infection.In the second part of this thesis, we further analyzed the changes of IFN-I production related genes expressed in normal human airway epithelial cell (16HBE) after EV71 and CA16 infection. The results show that the gene expression level of TLR3, MAVS, MDA5, MyD88, IRF7, IFNa and IFNβ were significantly increased after EV71 infection. However, TLR3 and IRF3 were significantly decreased after CA16 infection, while TLR7, MAVS, RIG-I, MyD88, IRF7, IFNa and IFNβ are no significant differences. Additionally, viral copy number and viral titer suggest that CA16 replicates more efficiently than EV71 infection in 16HBE. These results indicate that EV71 could activate these signalling pathways of IFN-I production, but IFN-I producion could not resist EV71 infection, and EV71 still could replicate in 16HBE. However, CA16 infection does not trigger the production of IFN-I. Hence, we inferred that CA16 infection could escape from the innate immune response of 16HBE by an unknown mechnism and then rapidly enhanced viral proliferate in 16HBE. These results may reveal that EV71 and CA16 could strengthen their infection by different means.The third part of this thesis, we explored the reason about the difference of IFN-I production induced by EV71 and CA16 infection. We fouced on whether autophagy caused by EV71 and CA16 infection have effects on IFN-I production and the expression of viral receptors. Endogenous LC3 and exogenous LC3 both demonstrated that EV71 could induce a complete autophagy while CA16 induced incomplete autophagy. After administrating autophagy inhibitor 3-MA, both EV71 and CA16 infection could markedly surpress cell viability and the viral PFUs also decrease. In addition, after inhibiting autophagy phenomenon, the gene and protein expression of TLR7, MyD88, IRF7 were significantly up-regulated. Therefore, it was suggested that EV71 and CA16 infection could restrain the production of IFN-I mediated by TLR7 signalling pathway via inducing autophagy in 16HBE, thereafter promoting vial replicaiton and virues packaging and maturation. Moreover, it was found that TLR7 and endosome marker, M6PR, could co-loated in 16HBE, and after blocking autophagy, the expression of TLR7 was remarkably heightened. These results further comfirm that EV71 and CA16 infection could inhibit TLR7 signaling pathway by inducing the occurrence of autophagy. Meanwhile, the expression of SCABR2 and CD162 were notably down-regulated once autophagy induced by EV71 and CA16 were hindered, these suggesting that EV71 and CA16 infection could reinforce the expression of viral receptors through triggering autophagy, which may cooperate EV71 and CA16 to escape from the antiviral immune response via suppressing IFN-I production and promoting viral receptors expression.In summary, this study firstly reveals the differentially expressed genes profile of CA16 infection by global gene expression analysis of peripheral blood mononuclear cells in rhesus monkey infants. We found that EV71 and CA16 induce differential levels of IFN-I expression by differential pattern-recognition receptors, and that both of EV71 and CA16 could surpress the production of IFN-I and promote the expression of viral receptors via inducing autophagy in 16HBE. Thus this study provides clues to investigate the mechanisms of infection and pathogensis by CA16 and partly explains the mechanism of CA16 repeated infection. Importanting, this study provides a new target to treat HFMD caused by EV71 and CA16. |
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