Abstract：Bovine viral diarrhea virus (BVDV) infection causes Bovine viral diarrhea/mucosal disease (BVD/MD), which can cause persistent infection and diarrheal mucosal disease in calves. The fatality rate of mucosal disease is as high as 100%, which is a serious threat to large-scale breeding and safety of biological products. In the previous study, the Turbo ID system was performed to capture the differentially expressed genes post-infection with BVDV, and the results showed differentially expressed genes such as UDP-glucose-glycoprotein-glucosyltransferase 1 (UGGT1). To explore the effect of UGGT1 gene on the replication of Bovine viral diarrhea virus, CRISPR/Cas9 was used to knock down UGGT1 gene in Madin-Darby bovine kidney (MDBK) cells in this study. Western blot was used to identify the knockdown of UGGT1 gene. Cell morphology changes were observed under a microscope and cell proliferation was counted. The effects of UGGT1 gene on BVDV replication were detected by immunofluorescence staining, qPCR, cytopathic effects (CPE) and virus titer in UGGT1 knockdown cells and Scramble cells infected with BVDV TC strain. The results showed that the UGGT1 gene of MDBK cells was successfully knocked down. There was no difference in the morphology and growth rate of UGGT1 knockdown cells, Scramble cells and wild-type MDBK cells. Compared with the negative control Scramble, BVDV infected UGGT1 knockdown cells 36 h after labeling double, the green fluorescence of double strand RNA (dsRNA) was significantly reduced; the level of 5'UTR RNA was significantly reduced after 24 h of infection (P<0.05), and the difference was extremely significant at 48 h (P<0.01); BVDV titer decreased significantly after 36 h of BVDV infection (P<0.05); After 36 h of infection, a large number of cells in Scramble fell off, and UGGT1 knockdown cells were significantly lower than those in Scramble control. This study showed that UGGT1 gene knockdown could inhibit the replication of BVDV, which provides an important basis for the establishment of new methods to prevent and control BVDV.
史慧君, 陈俊贞, 葛丽娟, 权冉, 塞力克·杰恩斯, 李丹, 杨莉, 付强. UGGT1基因敲低对牛病毒性腹泻病毒复制的影响[J]. 农业生物技术学报, 2021, 29(10): 1968-1977.
SHI Hui-Jun, CHEN Jun-Zhen, GE Li-Juan, QUAN Ran, SAILIKE·Jie-En-Si, LI Dan, YANG Li, FU Qiang. Effect of UGGT1 Gene Knockdown on the Replication of Bovine viral diarrhea virus. 农业生物技术学报, 2021, 29(10): 1968-1977.
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