Effect of Knocking Out the Mda5 Gene by CRISPR/Cas9 Technology on the Replication of Newcastle Disease and Infectious Bursal Virus

doi:10.13560/j.cnki.biotech.bull.1985.2022-0087

Abstract

Abstract:

The melanoma-differentiation-associated gene 5（Mda5）of DF-1 was knocked down using CRISPR/Cas9 technology to explore the effect of the Mda5 gene on the replication of Newcastle disease virus（NDV）and infectious bursal disease virus（IBDV）replication. CRISPR/Cas9 gene editing technology was applied to construct Mda5 knockout DF-1 cell lines，and real-time fluorescence quantitative PCR and immunofluorescence were used to detect the RNA levels of viruses，cytopathic conditions and mRNA levels of antiviral-related genes after IBDV and NDV infection of Mda5 knockout cells. Mda5 knockout DF-1 cells were obtained；there were no significant differences in cell morphology，apposition ability and proliferation ability compared to control cells. Compared to control cells，Mda5 knockout DF-1 cells infected with IBDV showed significant down-regulation of IFN-β and PKR expression，no significant differences in CH25H expression and viral replication levels. After infection with NDV，IFN-β expression and viral replication levels were significantly down-regulated，CH25H expression was significantly up-regulated，and PKR expression was not significantly different after infection with NDV. In DF-1 while applying CRISPR/Cas9 technology to knockout Mda5，though IFN-β significantly responded to IBDV and NDV infection，failed to significantly inhibit viral replication，suggesting that Mda5 is not a key pattern recognition receptor for antiviral replication.

Key words: Mda5, Newcastle disease virus, infectious bursa disease virus, CRISPR/Cas9, knockout

ZHONG Jing, SUN Ling-ling, ZHANG Shu, MENG Yuan, ZHI Yi-fei, TU Li-qing, XU Tian-peng, PU Li-ping, LU Yang-qing. Effect of Knocking Out the Mda5 Gene by CRISPR/Cas9 Technology on the Replication of Newcastle Disease and Infectious Bursal Virus[J]. Biotechnology Bulletin, 2022, 38(11): 90-96.

Figures/Tables 7

Table 1 sgRNA1-6 primer sequences

引物名称Primer name	序列Sequence（5'-3'）
sg RNA1	F：CACCGTGTAGAGGAAGCGCTCGTCT R：AAACAGACGAGCGCTTCCTCTACAC
sg RNA2	F：CACCGCTGCTATGCGCCGTGGAACG R：AAACCGTTCCACGGCGCATAGCAGC
sg RNA3	F：CACCGTCGCGGCGGCCACGTTCCA
	R：AAACTGGAACGTGGCCGCCGCGAC
sg RNA4	F：CACCGTGAACCATCCGGGGTCGCGG R：AAACCCGCGACCCCGGATGGTTCAC
sg RNA5	F：CACCgCAGTGAACCATCCGGGGTCG R：AAACCGACCCCGGATGGTTCACTGC
sg RNA6	F：CACCGCTGGGGTTCACGTAGCAAG R：AAACCTTGCTACGTGAACCCCAGC

Table 2 qPCR primer sequences

引物名称Primer name	序列Sequence（5'-3'）
IFN-β qPCR F	TGCAACCATCTTCGTCACCA
IFN-β qPCR R	GGAGGTGGAGCCGTATTCTG
PKR qPCR F	CCTATGCAATCAAACGAGTTGAG
PKR qPCR R	GTCCCTTCCCAGCTGCAATA
qPCR（CH25H）F qPCR（CH25H）R	ATCCATTCCTCCTCGGATGC AAAGGCACAAGTCGGTGAGT

Fig. 1 Results of recombinant plasmid construction A：sgRNA skeleton；B：sgRNA expression vector sequencing framework

Fig. 2 Establishment of MDA5 KO cell line A：24 h after MDA5 KO transfection；B：PCR identification results graph of MDA5 KO cells；C：sequencing results of PCR products from MDA5 KO cells

Fig. 3 Comparison of morphology and growth rate between MDA5 KO and ordinary DF-1 A：24 h after conventional DF-1 and MDA5 KO were thawed；B：72 h after conventional DF-1 and MDA5 KO were thawed；C：count results of conventional DF-1 and MDA5 KO at 24 h after thawing；D：growth curves of ordinary DF-1 and MDA5 KO 1-3 d after thawing

Fig. 4 Results of IBDV infection with MDA5 KO A：Comparison of normal DF-1 before and after infection with IBDV.B：Comparison of MDA5 KO before and after infection with IBDV. C：Results of IFN-β gene detection after IBDV infection. D：PKR gene test results after IBDV infection. E：Results of CH25H gene detection after IBDV infection. F：Results of IBDV gene detection after IBDV infection

Fig. 5 Results of NDV infection with MDA5 KO A：Comparison of normal DF-1 before and after infection with NDV. B：Comparison of MDA5 KO before and after infection with NDV. C：Results of IFN-β gene detection after NDV infection. D：PKR gene test results after NDV infection. E：Results of CH25H gene detection after NDV infection. F：Results of NDV gene detection after NDV infection

References 23

[1]	文开. 鸡新城疫的病原学、流行病学及综合防控措施[J]. 中国畜禽种业, 2021, 17(9):180-181.
	Wen K. Etiology, epidemiology and comprehensive prevention and control measures of Newcastle disease in chicken[J]. Chin Livest Poult Breed, 2021, 17(9):180-181.
[2]	覃周岚. 鸡传染性法氏囊炎病因及防控[J]. 中国畜禽种业, 2017, 13(3):159.
	Qin ZL. Etiology and prevention and control of infectious bursal disease in chickens[J]. Chin Livest Poult Breed, 2017, 13(3):159.
[3]	Zhang WX, Zuo EW, He Y, et al. Promoter structures and differential responses to viral and non-viral inducers of chicken melanoma differentiation-associated gene 5[J]. Mol Immunol, 2016, 76:1-6. doi: 10.1016/j.molimm.2016.06.006 URL
[4]	de Oliveira Mann CC, Hornung V. Molecular mechanisms of nonself nucleic acid recognition by the innate immune system[J]. Eur J Immunol, 2021, 51(8):1897-1910. doi: 10.1002/eji.202049116 URL
[5]	Yin X, Riva L, Pu Y, et al. MDA5 governs the innate immune response to SARS-CoV-2 in lung epithelial cells[J]. Cell Rep, 2021, 34(2):108628.
[6]	Lee CC, Wu CC, Lin TL. Characterization of chicken melanoma differentiation-associated gene 5(MDA5)from alternative translation initiation[J]. Comp Immunol Microbiol Infect Dis, 2012, 35(4):335-343. doi: 10.1016/j.cimid.2012.02.004 URL
[7]	Zhang WX, Zuo EW, He Y, et al. Promoter structures and differential responses to viral and non-viral inducers of chicken melanoma differentiation-associated gene 5[J]. Mol Immunol, 2016, 76:1-6. doi: 10.1016/j.molimm.2016.06.006 URL
[8]	Karpala AJ, Stewart C, McKay J, et al. Characterization of chicken Mda5 activity:regulation of IFN-β in the absence of RIG-I functionality[J]. J Immunol, 2011, 186(9):5397-5405. doi: 10.4049/jimmunol.1003712 pmid: 21444763
[9]	Kint J, Fernandez-Gutierrez M, Maier HJ, et al. Activation of the chicken type I interferon response by infectious bronchitis coronavirus[J]. J Virol, 2015, 89(2):1156-1167. doi: 10.1128/JVI.02671-14 pmid: 25378498
[10]	Xiang B, Zhu WX, Li YL, et al. Immune responses of mature chicken bone-marrow-derived dendritic cells infected with Newcastle disease virus strains with differing pathogenicity[J]. Arch Virol, 2018, 163(6):1407-1417. doi: 10.1007/s00705-018-3745-6 pmid: 29397456
[11]	韩青松. 鸡MDA5调控新城疫病毒免疫反应机制研究[D]. 杨凌: 西北农林科技大学, 2019.
	Han QS. Study on the mechanism of chicken MDA5 regulating chicken immune response to Newcastle disease virus[D]. Yangling: Northwest A & F University, 2019.
[12]	Lee CC, Wu CC, Lin TL. Chicken melanoma differentiation-associated gene 5(MDA5)recognizes infectious bursal disease virus infection and triggers MDA5-related innate immunity[J]. Arch Virol, 2014, 159(7):1671-1686. doi: 10.1007/s00705-014-1983-9 URL
[13]	张蕾, 张海波, 章敬旗, 等. CRISPR/Cas9系统在家禽中应用研究进展[J]. 中国畜牧兽医, 2020, 47(1):140-147.
	Zhang L, Zhang HB, Zhang JQ, et al. Research progress on application of CRISPR/Cas9 system in poultry[J]. China Animal Husb Vet Med, 2020, 47(1):140-147.
[14]	Cheng YQ, Lun MX, Liu YX, et al. CRISPR/Cas9-mediated chicken TBK1 gene knockout and its essential role in STING-mediated IFN-β induction in chicken cells[J]. Front Immunol, 2019, 9:3010. doi: 10.3389/fimmu.2018.03010 URL
[15]	刘欢欢, 谢丽君, 邵志勇, 等. MDA5在先天性免疫抗病毒作用中的研究进展[J]. 中国畜牧兽医, 2015, 42(1):230-233.
	Liu HH, Xie LJ, Shao ZY, et al. Research progress on antiviral effect of MDA5 in innate immunity[J]. China Animal Husb Vet Med, 2015, 42(1):230-233.
[16]	Liao ZH, Dai ZK, Cai CY, et al. Knockout of Atg5 inhibits proliferation and promotes apoptosis of DF-1 cells[J]. In Vitro Cell Dev Biol Anim, 2019, 55(5):341-348. doi: 10.1007/s11626-019-00342-7 URL
[17]	Kasumba DM, Grandvaux N. Therapeutic targeting of RIG-I and MDA5 might not lead to the same Rome[J]. Trends Pharmacol Sci, 2019, 40(2):116-127. doi: S0165-6147(18)30228-1 pmid: 30606502
[18]	Barber Megan R W, Aldridge Jerry R, Webster Robert G, Magor Katharine E. Association of RIG-I with innate immunity of ducks to influenza.[J]. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(13).
[19]	Thompson MR, Sharma S, Atianand M, et al. Interferon γ-inducible protein(IFI)16 transcriptionally regulates type i interferons and other interferon-stimulated genes and controls the interferon response to both DNA and RNA viruses[J]. J Biol Chem, 2014, 289(34):23568-23581. doi: 10.1074/jbc.M114.554147 pmid: 25002588
[20]	翟景波, 吕昌龙. RLRs家族中RIG-I和MDA-5的研究进展[J]. 微生物学免疫学进展, 2017, 45(1):54-59.
	Zhai J. B.; Lv, C. L. Progress on study of RIG-I and MDA5 in RLRs family[J]Proceedings in Microbiology Immunology, 2017, 45(1):54-59.
[21]	Carty M, Guy C, Bowie AG. Detection of viral infections by innate immunity[J]. Biochem Pharmacol, 2021, 183:114316.
[22]	王振兴. 黄芪甲苷经AMPK/mTOR信号通路调控细胞自噬与凋亡保护PM2. 5诱导急性肺损伤的机制研究[D]. 成都: 成都中医药大学, 2019.
	Wang ZX. Astragaloside Ⅳ regulates autophagy and apoptosis in PM2. 5-induced acute lung injury via AMPK/mTOR pathway[D]. Chengdu: Chengdu University of TCM, 2019.
[23]	孔正茹. 禽网状内皮组织增生病病毒诱导宿主天然免疫反应及感染法氏囊转录组学的研究[D]. 扬州: 扬州大学, 2019.
	Kong ZR. Host innate immune response induced by avian reticuloendotheliosis virus and transcriptomics study of virus infected Bursa[D]. Yangzhou: Yangzhou University, 2019.