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| Effect of TRAF2 and SNED1 Gene Knockdown on the Replication of Bovine viral diarrhea virus |
| LIU Xin-Yi1,2,*, QUAN Ran1,2,*, LIU Yu-Cheng3, CHEN Jun-Zhen1,2, NI Hui-Ying1,2, FU Qiang1,2,**, SHI Hui-Jun1,2,** |
1 College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China;
2 Xinjiang Key Laboratory of Herbivore Drug Research and Creation, Urumqi 830052, China;
3 State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding/Institute of Animal Husbandry and Veterinary Medicine, Xinjiang Academy of Agricultural Sciences, Shihezi 832000, China |
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Abstract Bovine viral diarrhea/mucosal disease (BVD/MD) is a contact infectious disease caused by Bovine viral diarrhea virus (BVDV), which will lead to persistent infection, and infected calves (Bos taurus) will continuously expel the virus to the outside. Our research group used RNA- protein interaction detection (RaPID) technology to screen tumor necrosis factor receptor associated factor 2, (TRAF2) and Sushi, Nidogen and EGF like domains 1 (SNED1) genes. In order to determine the effects of TRAF2 and SNED1 genes on BVDV replication, in this study, the small interfering RNA (siRNA) technique was used to knock down the TRAF2 and SNED1 genes in madin-darby bovine kidney cells (MDBK), and the knock-down efficiency of TRAF2 and SNED1 genes and the changes of BVDV 5'UTR RNA level of BVDV infected cells were detected by qPCR. The accumulation of double stranded RNA (dsRNA), an intermediate product of BVDV replication, was detected by immunofluorescence staining. The cytopathic effect (CPE) was observed by fluorescence inverted microscope, and the virus titer of offspring was calculated by Karber method. The results showed that the mRNA levels of TRAF2 and SNED1 were significantly reduced (P<0.05), which indicated that TRAF2 KD and SNED1 KD cells were successfully constructed. In BVDV infected control group (NC), CPE phenomena such as plaque, shedding and death began to appear at 36 h, and CPE phenomena of TRAF2 KD cells and SNED1 KD cells weakened. Compared with NC, after BVDV infected TRAF2 KD and SNED1 KD cells, the level of BVDV 5'UTR RNA decreased significantly at 24, 36 and 48 h (P<0.05). The formation and accumulation of dsRNA, an intermediate product of BVDV replication, were detected by immunofluorescence staining. The green fluorescence intensity in TRAF2 KD and SNED1 KD cells decreased significantly at 36 and 48 h. 48 h after BVDV infected TRAF2 KD and SNED1 KD cells, the virus titer of the offspring decreased extremely significantly (P<0.01). The above results showed that knocking down TRAF2 and SNED1 would inhibit BVDV replication, and this study provides an important basis for revealing the pathogenesis of BVDV.
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Received: 12 September 2023
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Corresponding Authors:
** 466183013@qq.com; shihuijunmm@163.com
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| About author:: * These authors contributed equally to this work |
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[1] 陈楠楠. 2023. 槲皮素通过调控HSP70表达和氧化应激抑制BVDV复制的分子机制研究[D]. 博士学位论文, 黑龙江八一农垦大学, 导师: 朱战波, pp. 1-92.
(Chen N N.2023. Studies of the molecular mechanism by which quercetin suppresses BVDV replication through the regulation of HSP70 expression and oxidative stress[D]. Thesis for Ph. D., Heilongjiang Bayi Agricultural University, Supervisor: Zhu Z B, pp. 1-92)
[2] 梁霄勇, 季新成, 冉多良. 2016. 牛病毒性腹泻病毒TC株的分离鉴定及E0基因的序列分析[J]. 畜牧与兽医, 48(7): 36-40.
(Liang X Y, Ji X C, Ran D L.2016. Isolation and identification of Bovine viral diarrhea virus TC strain and sequence analysis of E0 gene[J]. Animal Husbandry ＆ Veterinary Medicine, 48(7): 36-40.)
[3] 田瑞鑫, 李胜男, 胡新艳, 等. 2020. 牛miR-29b影响牛病毒性腹泻病毒感染BALB/c小鼠的作用[J]. 中国兽医学报, 40(02): 264-271.
(Tian R X, Li S N, Hu X Y, et al.2020. Effect of bovine miRNA-29b on Bovine viral diarrhea virus infection in BALB/c mice[J]. Chinese Journal of Veterinary Science, 40(02): 264-271.)
[4] 张俊杰, 凌宗帅, 黄凯, 等. 2010. 北京地区规模化奶牛场牛病毒性腹泻病血清学调查[J]. 中国奶牛, 10: 41-42.
(Zhang J J, Ling Z S, Huang K, et al.2010. Serological survey of bovine viral diarrhea in Beijing dairy herds[J]. China Dairy Cattle, 10: 41-42.)
[5] 赵雅楠, 何伟, 邵泉林, 等. 2023. 可电离脂质纳米粒用于siRNA递送的研究进展[J]. 药学学报, 58(08): 2292-2299.
(Zhao Y N, He W, Shao Q L, et al.2023. Research progress of ionizable lipid nanoparticles for siRNA delivery[J]. Acta Pharmaceutica Sinica, 58(08): 2292-2299.)
[6] Bradley J R, Pober J S.2001. Tumor necrosis factor receptor-associated factors (TRAFs)[J]. Oncogene, 20(44): 6482-6491.
[7] Charras G, Sahai E.2014. Physical influences of the extracellular environment on cell migration[J]. Nature Reviews Molecular Cell Biology, 15(12): 813-824.
[8] Chi S, Chen S, Jia W, et al.2022. Non-structural proteins of Bovine viral diarrhea virus[J]. Virus Genes, 58(6): 491-500.
[9] Fan W, Wang Y, Jiang S, et al.2022. Identification of key proteins of cytopathic biotype Bovine viral diarrhoea virus involved in activating NF-κB pathway in BVDV-induced inflammatory response[J]. Virulence, 13(1): 1884-1899.
[10] Grech A P, Amesbury M, Chan T, et al.2004. TRAF2 differentially regulates the canonical and noncanonical pathways of NF-kappaB activation in mature B cells[J]. Immunity, 21(5): 629-642.
[11] Habelhah H, Takahashi S, Cho S G, et al.2004. Ubiquitination and translocation of TRAF2 is required for activation of JNK but not of p38 or NF-kappaB[J]. Embo Journal, 23(2): 322-332.
[12] Haga I R, Pechenick Jowers T, Griffiths S J, et al.2014. TRAF2 facilitates Vaccinia virus replication by promoting rapid virus entry[J]. Journal Of Virology, 88(7): 3664-3677.
[13] Khodakaram T A, Farjanikish G H.2017. Persistent Bovine viral diarrhea virus (BVDV) infection in cattle herds[J]. Iranian Journal Of Veterinary Research, 18(3): 154-163.
[14] Lambeth L S, Moore R J, Muralitharan M S, et al.2007. Suppression of Bovine viral diarrhea virus replication by small interfering RNA and short hairpin RNA-mediated RNA interference[J]. Veterinary Microbiology, 119(2-4): 132-143.
[15] Lee T H, Huang Q, Oikemus S, et al.2003. The death domain kinase RIP1 is essential for tumor necrosis factor alpha signaling to p38 mitogen-activated protein kinase[J]. Molecular And Cellular Biology, 23(22): 8377-8385.
[16] Ma X, Rawnsley D R, Kovacs A, et al.2021. TRAF2, an innate immune sensor, reciprocally regulates mitophagy and inflammation to maintain cardiac myocyte homeostasis[J]. JACC Basic Translational Science, 7(3): 223-243.
[17] Mathapati B S, Mishra N, Rajukumar K, et al.2010. Entry of Bovine viral diarrhea virus into ovine cells occurs through clathrin-dependent endocytosis and low pH-dependent fusion[J]. In Vitro Cellular & Developmental Biology-Animal, 46(5): 403-407.
[18] Mishra N, Rajukumar K, Kalaiyarasu S, et al.2011. Small interfering RNAs targeting viral structural envelope protein genes and the 5'UTR inhibit replication of Bovine viral diarrhea virus in MDBK cells[J]. Acta Virologica, 55(3): 279-282.
[19] Naba A, Clauser K R, Lamar J M, et al.2014. Extracellular matrix signatures of human mammary carcinoma identify novel metastasis promoters[J]. Elife, 11(3): e01308.
[20] Pomerantz J L, Baltimore D.1999. NF-kappaB activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase[J]. Embo Journal, 18(23): 6694-6704.
[21] Rothe M, Wong S C, Henzel W J, et al.1994. A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor[J]. Cell, 78(4): 681-692.
[22] Song Q J, Weng X G, Cai D J, et al.2016. Forsythoside a inhibits BVDV replication via TRAF2-dependent CD28-4-1BB signaling in bovine PBMCs[J]. PLOS ONE, 11(9): e0162791.
[23] Weissmueller S, Manchado E, Saborowski M, et al.2014. Mutant p53 drives pancreatic cancer metastasis through cell-autonomous PDGF receptor β signaling[J]. Cell, 157(2): 382-394. |
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