Abstract:

【Objective】 The aim of this study was to determine the signal pathways (PERK, IRE1 and ATF6) of unfolded protein response induced by tembusu virus in ducklings, so as to provide a theoretical basis for elucidating the pathogenesis of TMUV. [Method] 1-day-old SPF ducklings were infected intraperitoneally with TMUV (JS804). Ducklings inoculated in the same manner with equal volume of RMPI-1640 were used as negative control. Five ducklings from each group were euthanized at 12, 24, 36 h and 48 h post infection, and their brains, livers and hearts were collected. The total RNAs were extracted from collected tissues by using total RNA extraction kit. Then the total RNAs were reverse transcribed into cDNA. Specific target genes representing the three known pathways of unfolded protein response were selected, and the primers were designed based on the published GenBank sequence. The relative expression of target genes was quantified by real time PCR. Real time PCR data were analyzed using the comparative Ct method (2^-ΔΔCt). GAPDH was chosen as a reference gene for internal control. 【Result】 In three organs from infected ducklings, it was observed that the viral titers were the highest in the liver, lower in the heart, and the lowest in the brain. The unfolded protein response was characterized by upregulated expression of GRP78. The relative expression of GRP78 in the brain and liver were persistently upregulated and reached a peak at 36 h post infection (4.21 fold and 10.14 fold, respectively). GRP78 expression in the heart was upregulated transiently at 36 h post infection (1.32 fold). ATF4 expression represented the activation of PERK pathway. The ATF4 expression in the liver and brain were persistently upregulated from 24 h and 36 h post infection to 48 h post infection respectively, and peaked at 36 h post infection (2.71 fold and 6.02 fold, respectively). However, the upregulation of ATF4 expression in the heart was observed at 36 h post infection (1.57 fold). The activation of IRE pathway was characterized by XBP1s. In the liver, the expression level of XBP1s increased most significantly (9 fold). In the brain, the expression level of EDEM enhanced most significantly (3.87 fold) and persistently upregulated from 12 to 48 h post infection. Comparing to negative control, the expression of ATF6 pathway marker GRP94 and XBP1u were upregulated in three tissues, which reached a peaked at 36 h post infection, although expression profiles of GRP94 and XBP1u were different at indicated time points. 【Conclusion】 It was the first report that TMUV infection induced three branches of unfolded protein response in ducklings, and these results might be helpful for understanding the interaction between tembusu virus infection and host response.

Key words: Tembusu virus, SPF ducklings, endoplasmic reticulum, unfolded protein response, signaling pathway