| Muscovy duck reovirus (MDRV) is a highly pathogenic virus causing acute infectious disease in ducklings that are from 2 to 4 weeks old. However the molecular pathogenic mechanism of MDRV infection is still not clear. In the study, the transcriptome of 10 day old Muscovy duck was sequencing by new generation sequencing method from liver and spleen, and it was successful assembly and annotation. Then the differentiation expression genes (DEGs) in liver and spleen were screened and analyzed between MDRV infection duck and healthy duck. The biology function and pathway also were identified. At the last, the innate immune function, apoptosis procession and hepatic steatosis were confirmed by Q-PCR or Western blot. The main results are as follows:1. In this study, the total length of 17G reads were obtained, and 65,535 Unigenes were assembled, the average length of each read was up to 987bp.41,651 valid Unigenes were received by COG annotation, which include 25 biological functions.2. Results of expression profile showed that one thousand three hundred and fifty two (1,352) DEGs were up-regulated and four thousand nine hundred and six (4,906) DEGs were down-regulated in infected duck spleens. These DEGs included forty eight biological functions by GO enrichment analysis. Significant enrichment analysis with KEGG, two hundred and twenty-nine pathways were obtained.3. Four thousand one hundred and ninety (4,190) DEGs were up-regulated and one thousand one hundred and thirteen (1,113) DEGs were down-regulated in infected duck livers. These DEGs were classified into forty eight biology function subjected to GO functional classification annotation. Significant enrichment analysis with KEGG, two hundred and thirty seven pathways were obtained.4 Compared with DEGs in liver and spleen of MDRV infection duck, the results showed that the DEGs in the spleen were up regulation, but that in the liver were down regulation. There were 1,350 common DEGs. These data will help to provide the molecular basis on elucidating the pathogenesis of MDRV infection.5. The results of KEGG analysis showed that the gene expression of RIG-I, MDA5, TLR-1, TLR-2 and TLR-4 were up-regulated to indentify the MDRV intection. Then they activated IRF7 protein to enhance the IFN secretion and regulate NF-?B signal pathway to enhance the IL6 secretion. So the innate immune function was enhanced by JAK-STAT signal pathway. Results were accurate and reliable by S YRB-PCR.6. Apoptosis induced by MDRV in the liver was studied by TUNEL method and flow cytometry. The results showed that the spleen infected with MDRV enhanced the apoptosis lever. At the same time, we analyzed the pathway of apoptosis induced by MDRV through the transcriptome technology (KEGG). Results showed that the apoptosis induced by MDRV was relevant with Fas signaling pathway, IL-1R signaling pathway and PI3K signaling pathway, which were verified by Western blot and Q-PCR.7. In this experiment we found that there were remarkable fatty degeneration in Muscovy duck liver infection of MDRV by the pathological tissue section technique and the content of free fatty acids was significantly improved (P<0.01) compared with control duck. We considered that the fatty degeneration of liver induced by MDRV was related with the increase of free fatty acids. The molecular mechanism of MDRV infection induced hepatic steatosis was related with the fat digestion and absorption, biosynthesis of unsaturated fatty acids, fatty acid degradation three biochemical metabolic pathway of fatty acid by RNA-SEQ and fluorescence quantitative PCR.In conclusion, the 10 day old Muscovy duck transcriptome feature was obtained in this study, and the molecular mechanism of the innate immune function, apoptosis and fatty degeneration were illuminated, which indicated that the host mobilized multi genes and multi pathway to take part in regulation on the inhibition of MDRV invasion. |
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