Inhibition of TiLV Replication by AT2-R in Nile Tilapia Oreochromis niloticus
WEN Jing1,2, ZHENG Shucheng2, KE Zishan2, WANG Yingying2, LI Yingying2, MO Xubing2, ZHANG Defeng2, YIN Jiyuan2, ZHOU Wenli1, WANG Qing2
1. College of Fisheries, Tianjin Agricultural University, Tianjin 300384, China; 2. Guangdong Provincial Key Laboratory of Aquatic Animal Immunology and Sustainable Aquaculture, Key Laboratory of Fishery Drug Development, Ministry of Agriculture and Rural Affairs, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, China
Abstract：In order to explore the function of type-2 angiotensin Ⅱ receptor (AT2-R) of Nile tilapia Oreochromis niloticus in the process of tilapia lake virus (TiLV) infection, the complete open reading frame (ORF) sequence of AT2-R gene of Nile tilapia AT2-R (OnAT2-R) was cloned by reverse transcription PCR (RT-PCR) and the tissue distribution and phase expression were analyzed in OnAT2-R by real-time fluorescence quantitative PCR (qRT-PCR). Meanwhile, the recombinant eukaryotic expression vector pEYFP-OnAT2-R was constructed for subcellular localization analysis in tilapia brain (TiB) cells which were infected with TiLV after overexpression of OnAT2-R gene, and the relative expression of TiLV-S8 segment was detected by qRT-PCR. The results showed that the OnAT2-R gene ORF sequence of the Nile tilapia was found to be 1338 bp in length, encoding 445 amino acids with a 7-transmembrane region. The OnAT2-R gene was found in all tissues examined in the Nile tilapia, with the maximal expression level in liver, followed by muscle and skin, and the minimal expression level in spleen and stomach. The relative expression level of OnAT2-R gene in the liver was shown to be increased first and then decreased in the Nile tilapia with TiLV infection, while the relative expression of OnAT2-R gene in the muscle was increased and then decreased, followed by increased again. Furthermore, the pEYFP-OnAT2-R was successfully constructed and the subcellular localization analysis showed that OnAT2-R protein was mainly localized on the cell membrane. The mRNA level of TiLV-S8 segment was significantly reduced in TiB cells overexpressed with OnAT2-R gene followed by TiLV infection. The finding indicates that OnAT2-R is a potential cell surface molecular receptor located on the cell membrane and has the effect on inhibition of TiLV replication, which will provide theoretical references with further in-depth research on pathogenesis of TiLV infection and antiviral mechanism of tilapia.
[1] DE GASPARO M, CATT K J, INAGAMI T, et al. International union of pharmacology. ⅩⅩⅢ. The angiotensin Ⅱ receptors[J]. Pharmacological Reviews,2000,52(3):415-472. [2] ZHANG H T, HAN G W, BATYUK A, et al. Structural basis for selectivity and diversity in angiotensin Ⅱ receptors[J]. Nature,2017,544(7650):327-332. [3] FENG Y H, SAAD Y, KARNIK S S. Reversible inactivation of AT(2) angiotensin Ⅱ receptor from cysteine-disulfide bond exchange[J]. FEBS Letters,2000,484(2):133-138. [4] KARNIK S S, UNAL H, KEMP J R, et al. International union of basic and clinical pharmacology. XCIX. Angiotensin receptors:interpreters of pathophysiological angiotensinergic Stimuli[corrected[J]. Pharmacological Reviews,2015,67(4):754-819. [5] QIN Q, WANG J H, YAN Y L, et al. Angiotensin Ⅱ induces the differentiation of mouse epicardial progenitor cells into vascular smooth muscle-like cells[J]. Biochemical and Biophysical Research Communications,2016,480(4):696-701. [6] ANAND U, FACER P, YIANGOU Y, et al. Angiotensin Ⅱ type 2 receptor (AT2 R) localization and antagonist-mediated inhibition of capsaicin responses and neurite outgrowth in human and rat sensory neurons[J]. European Journal of Pain,2013,17(7):1012-1026. [7] SZCZEPANSKA-SADOWSKA E, CZARZASTA K, CUDNOCH-JEDRZEJEWSKA A. Dysregulation of the renin-angiotensin system and the vasopressinergic system interactions in cardiovascular disorders[J]. Current Hypertension Reports,2018,20(3):19. [8] KEMP B A, HOWELL N L, GILDEA J J, et al. AT2 receptor activation induces natriuresis and lowers blood pressure[J]. Circulation Research,2014,115(3):388-399. [9] 段世超,费扬,黄植,等.血管紧张素Ⅱ2型受体——肿瘤细胞凋亡诱导因子[J].分子诊断与治疗杂志,2012(4):279-283. [10] WONG M K S, TAKEI Y. Angiotensin AT2 receptor activates the cyclic-AMP signaling pathway in eel[J]. Molecular and Cellular Endocrinology, 2013,365(2):292-302. [11] IMBROGNO S, GAROFALO F, AMELIO D, et al. Humoral control of cardiac remodeling in fish:role of Angiotensin II[J]. General and Comparative Endocrinology,2013,194:189-197. [12] FILICE M, BARCA A, AMELIO D, et al. Morpho-functional remodelling of the adult zebrafish (Danio rerio) heart in response to waterborne angiotensin Ⅱ exposure[J]. General and Comparative Endocrinology,2021,301:113663. [13] NICHOLSON P, FATHI M A, FISCHER A, et al. Detection of tilapia Lake Virus in Egyptian fish farms experiencing high mortalities in 2015[J]. Journal of Fish Diseases,2017,40(12):1925-1928. [14] SURACHETPONG W, JANETANAKIT T, NONTHABENJAWAN N, et al. Outbreaks of tilapia lake virus infection, Thailand, 2015—2016[J]. Emerging Infectious Diseases,2017,23(6):1031-1033. [15] 雷燕,赵振峰,唐绍林,等.国内养殖罗非鱼首次检出罗湖病毒[J].海洋与渔业·水产前沿,2017(7):72. [16] DEL-POZO J, MISHRA N, KABUUSU R, et al. Syncytial hepatitis of tilapia (Oreochromis niloticus L.) is associated with orthomyxovirus-like virions in hepatocytes[J]. Veterinary Pathology,2017,54(1):164-170. [17] BACHARACH E, MISHRA N, BRIESE T, et al. Characterization of a novel orthomyxo-like virus causing mass die-offs of tilapia[J]. mBio,2016,7(2):e00431-16. [18] AL-HUSSINEE L, SUBRAMANIAM K, AHASAN M S, et al. Complete genome sequence of a tilapia lake virus isolate obtained from Nile tilapia (Oreochromis niloticus)[J]. Genome Announcements,2018,6(26):e00580-18. [19] SUBRAMANIAM K, FERGUSON H W, KABUUSU R, et al. Genome sequence of tilapia lake virus associated with syncytial hepatitis of tilapia in an Ecuadorian aquaculture facility[J]. Microbiology Resource Announcements,2019,8(18):e00084-19. [20] PULIDO L L H, MORA C M, HUNG A L, et al. Tilapia lake virus (TiLV) from Peru is genetically close to the Israeli isolates[J]. Aquaculture,2019,510:61-65. [21] AHASAN M S, KELEHER W, GIRAY C, et al. Genomic characterization of tilapia lake virus isolates recovered from moribund Nile tilapia (Oreochromis niloticus) on a farm in the United States[J]. Microbiology Resource Announcements,2020,9(4):e01368-19. [22] CHAPUT D L, BASS D, ALAM M M, et al. The segment matters:probable reassortment of tilapia lake virus (TiLV) complicates phylogenetic analysis and inference of geographical origin of new isolate from Bangladesh[J]. Viruses,2020,12(3):258. [23] ACHARYA V, CHAKRABORTY H J, ROUT A K, et al. Structural characterization of open reading frame-encoded functional genes from tilapia lake virus (TiLV)[J]. Molecular Biotechnology,2019,61(12):945-957. [24] AICH N, PAUL A, CHOUDHURY T G, et al. tilapia Lake Virus (TiLV) disease:current status of understanding[J]. Aquaculture and Fisheries,2022,7(1):7-17. [25] KING A M Q, LEFKOWITZ E J, MUSHEGIAN A R, et al. Changes to taxonomy and the International code of virus classification and nomenclature ratified by the International Committee on Taxonomy of Viruses (2018)[J]. Archives of Virology,2018,163(9):2601-2631. [26] CHENGULA A A, MUTOLOKI S, EVENSEN Ø, et al. tilapia lake virus does not hemagglutinate avian and piscine erythrocytes and NH4Cl does not inhibit viral replication in vitro[J]. Viruses,2019,11(12):1152. [27] ABU RASS R, KUSTIN T, ZAMOSTIANO R, et al. Inferring protein function in an emerging virus:detection of the nucleoprotein in tilapia lake virus[J]. Journal of Virology,2022,96(6):e0175721. [28] LUEANGYANGYUEN A, SENAPIN S, DONG H T, et al. Expression and purification of S5196-272 and S6200-317 proteins from tilapia Lake Virus (TiLV) and their potential use as vaccines[J]. Protein Expression and Purification,2022,190:106013. [29] HU H Z, ZENG W W, WANG Y Y, et al. Development and application of a recombinant protein-based indirect ELISA for detection of anti-tilapia lake virus IgM in sera from tilapia[J]. Aquaculture,2020,520:734756. [30] 王雅慧,王英英,王庆,等.罗非鱼湖病毒S10基因编码蛋白的表达及单克隆抗体的制备[J].广东农业科学,2021,48(11):112-118. [31] WANG Y Y, WANG Q, LI Y Y, et al. Integrated analysis of mRNA-miRNA expression in tilapia infected with tilapia lake virus (TiLV) and identifies primarily immuneresponse genes[J]. Fish & Shellfish Immunology,2020,99:208-226. [32] PORRELLO E R, DELBRIDGE L M D, THOMAS W G. The angiotensin Ⅱ type 2 (AT2) receptor:an enigmatic seven transmembrane receptor[J]. Frontiers in Bioscience (Landmark Edition),2009,14(3):958-972. [33] YU L, ZHENG M Q, WANG W, et al. Developmental changes in AT1 and AT2 receptor-protein expression in rats[J]. Journal of the Renin-Angiotensin-Aldosterone System:JRAAS,2010,11(4):214-221. [34] XIONG Q Q, HUANG H J, WANG N, et al. Metabolite-sensing G protein coupled receptor TGR5 protects host from viral infection through amplifying type I interferon responses[J]. Frontiers in Immunology,2018,9:2289. [35] MAINOU B A, ASHBROOK A W, SMITH E C, et al. Serotonin receptor agonist 5-nonyloxytryptamine alters the kinetics of reovirus cell entry[J]. Journal of Virology,2015,89(17):8701-8712. [36] LI R M, TAN B H, YAN Y, et al. Extracellular UDP and P2Y6 function as a danger signal to protect mice from vesicular stomatitis virus infection through an increase in IFN-β production[J]. Journal of Immunology,2014,193(9):4515-4526. [37] ZHANG C F, YAN Y, HE H W, et al. IFN-stimulated P2Y13 protects mice from viral infection by suppressing the cAMP/EPAC1 signaling pathway[J]. Journal of Molecular Cell Biology,2019,11(5):395-407. [38] HUANG H J, ZHANG N, XIONG Q Q, et al. Elimination of GPR146-mediated antiviral function through IRF3/HES1-signalling pathway[J]. Immunology,2017,152(1):102-114. [39] LEONHARDT J, VILLELA D C, TEICHMANN A, et al. Evidence for heterodimerization and functional interaction of the angiotensin type 2 receptor and the receptor MAS[J]. Hypertension,2017,69(6):1128-1135. [40] NAMSOLLECK P, MOLL G N. Does activation of the protective Renin-Angiotensin System have therapeutic potential in COVID-19?[J]. Molecular Medicine,2020,26(1):80.
2024, Vol. 43 
