畜牧兽医学报 ›› 2021, Vol. 52 ›› Issue (5): 1208-1217.doi: 10.11843/j.issn.0366-6964.2021.05.007
杨文兵1, 邹亚文1, 蒋一凡2, 余婉婷3, 杨毅1, 邬静1*, 王乃东1*
收稿日期:
2020-10-12
出版日期:
2021-05-23
发布日期:
2021-05-22
通讯作者:
王乃东,主要从事兽医临床微生物组学与生物技术研究,E-mail:naidongwang@huanu.edu.cn;邬静,主要从事动物非传染性群发病与动物保健研究,E-mail:12145090@qq.com
作者简介:
杨文兵(1996-),男,山西太原人,硕士生,主要从事兽医临床微生物组学与生物技术研究,E-mail:wenbingyang@stu.hunau.edu.cn;邹亚文(1991-),男,湖南岳阳人,博士生,主要从事动物亚单位疫苗研究,E-mail:1558636333@qq.com。
基金资助:
YANG Wenbing1, ZOU Yawen1, JIANG Yifan2, YU Wanting3, YANG Yi1, WU Jing1*, WANG Naidong1*
Received:
2020-10-12
Online:
2021-05-23
Published:
2021-05-22
摘要: 非洲猪瘟(African swine fever,ASF)是一种由非洲猪瘟病毒(African swine fever virus,ASFV)引起的猪高致死性传染病。ASFV编码蛋白p30、p54和p72等具有较高的免疫原性,且部分氨基酸序列较为保守,常被作为血清学诊断靶点,用于评价ASF不同阶段或发病程度的抗体水平变化,ASF血清学诊断靶点相关深入研究有助于其感染检测、致病机制和机体免疫系统反应等探究。本文系统阐述用于ASF血清学诊断的主要候选蛋白及其靶点的检测应用进展,并分析候选靶点的抗原表位(区域)及其抗体结合的特点,探讨其作为ASF血清学诊断新靶点的应用潜力。
中图分类号:
杨文兵, 邹亚文, 蒋一凡, 余婉婷, 杨毅, 邬静, 王乃东. 非洲猪瘟血清学诊断靶点的研究进展[J]. 畜牧兽医学报, 2021, 52(5): 1208-1217.
YANG Wenbing, ZOU Yawen, JIANG Yifan, YU Wanting, YANG Yi, WU Jing, WANG Naidong. Advances Research on African Swine Fever Serological Diagnostic Targets[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1208-1217.
[1] | GAUDREAULT N N, MADDEN D W, WILSON W C, et al. African swine fever virus: An emerging DNA arbovirus[J]. Front Vet Sci, 2020, 7:215. |
[2] | REVILLA Y, PÉREZ-NÚÑEZ D, RICHT J A. African swine fever virus biology and vaccine approaches[J]. Adv Virus Res, 2018, 100:41-74. |
[3] | DIXON L K, ISLAM M, NASH R, et al. African swine fever virus evasion of host defences[J]. Virus Res, 2019, 266:25-33. |
[4] | PENRITH M L, VOSLOO W. Review of African swine fever: transmission, spread and control[J]. J S Afr Vet Assoc, 2009, 80(2):58-62. |
[5] | MONTGOMERY R E. On a form of swine fever occur-ring in British East Africa (Kenya Colony)[J]. J Comp Pathol Therapeut, 1921, 34:159-191. |
[6] | 张睿, 黄旖童, 鲍晨沂, 等. 非洲猪瘟流行病学及其在中国扩散的因素分析[J]. 病毒学报, 2019, 35(3):512-522.ZHANG R, HUANG Y T, BAO C Y, et al. Epidemiology of African swine fever and analysis of risk factors of its spread in China: An overview[J]. Chinese Journal of Virology, 2019, 35(3):512-522. (in Chinese) |
[7] | LADDOMADA A, ROLESU S, LOI F, et al. Surveillance and control of African swine fever in free-ranging pigs in Sardinia[J]. Transbound Emerg Dis, 2019, 66(3):1114-1119. |
[8] | PETRINI S, FELIZIANI F, CASCIARI C, et al. Survival of African swine fever virus (ASFV) in various traditional Italian dry-cured meat products[J]. Prev Vet Med, 2019, 162:126-130. |
[9] | GE S Q, LI J M, FAN X X, et al. Molecular characterization of African swine fever virus, China, 2018[J]. Emerg Infect Dis, 2018, 24(11):2131-2133. |
[10] | ZHOU X T, LI N, LUO Y Z, et al. Emergence of African swine fever in China, 2018[J]. Transbound Emerg Dis, 2018, 65(6):1482-1484. |
[11] | 姜睿娇, 张鹏飞, 朱光恒, 等. 非洲猪瘟检测技术进展[J]. 病毒学报, 2019, 35(3):523-532.JIANG R J, ZHANG P F, ZHU G H, et al. Diagnostic assays for African swine fever[J]. Chinese Journal of Virology, 2019, 35(3):523-532. (in Chinese) |
[12] | LI L, REN Z W, WANG Q H, et al. Infection of African swine fever in wild boar, China, 2018[J]. Transbound Emerg Dis, 2019, 66(3):1395-1398. |
[13] | GOATLEY L C, REIS A L, PORTUGAL R, et al. A pool of eight virally vectored African swine fever antigens protect pigs against fatal disease[J]. Vaccines, 2020, 8(2):234. |
[14] | WU K K, LIU J M, WANG L X, et al. Current state of global African swine fever vaccine development under the prevalence and transmission of ASF in China[J]. Vaccines, 2020, 8(3):531. |
[15] | BELLINI S, RUTILI D, GUBERTI V. Preventive measures aimed at minimizing the risk of African swine fever virus spread in pig farming systems[J]. Acta Vet Scand, 2016, 58(1):82. |
[16] | GALLARDO C, FERNÁNDEZ-PINERO J, ARIAS M. African swine fever (ASF) diagnosis, an essential tool in the epidemiological investigation[J]. Virus Res, 2019, 271:197676. |
[17] | 王西西. 非洲猪瘟病毒蛋白对cGAS-STING信号通路抑制作用研究[D]. 北京:中国农业科学院, 2019.WANG X X. Inhibition mechanisms of African swine fever virus protein on cGAS-STING-mediated signaling pathway[D]. Beijing:Chinese Academy of Agricultural Sciences, 2019. (in Chinese) |
[18] | JIA N, OU Y W, PEJSAK Z, et al. Roles of African swine fever virus structural proteins in viral infection[J]. J Vet Res, 2017, 61(2):135-143. |
[19] | LITHGOW P, TAKAMATSU H, WERLING D, et al. Correlation of cell surface marker expression with African swine fever virus infection[J]. Vet Microbiol, 2014, 168(2-4):413-419. |
[20] | GÓMEZ-PUERTAS P, RODRÍGUEZ F, OVIEDO J M, et al. The African swine fever virus proteins p54 and p30 are involved in two distinct steps of virus attachment and both contribute to the antibody-mediated protective immune response[J]. Virology, 1998, 243(2):461-471. |
[21] | BARDERAS M G, RODRÍGUEZ F, GÓMEZ-PUERTAS P, et al. Antigenic and immunogenic properties of a chimera of two immunodominant African swine fever virus proteins[J]. Arch Virol, 2001, 146(9):1681-1691. |
[22] | RODRÍGUEZ J M, GARCÍA-ESCUDERO R, SALAS M L, et al. African swine fever virus structural protein p54 is essential for the recruitment of envelope precursors to assembly sites[J]. J Virol, 2004, 78(8):4299-4313. |
[23] | HERNÁEZ B, DÍAZ-GIL G, GARCÍA-GALLO M, et al. The African swine fever virus dynein-binding protein p54 induces infected cell apoptosis[J]. FEBS Lett, 2004, 569(1-3):224-228. |
[24] | ALCARAZ C, RODRIGUEZ F, OVIEDO J M, et al. Highly specific confirmatory western blot test for African swine fever virus antibody detection using the recombinant virus protein p54[J]. J Virol Methods, 1995, 52(1-2):111-119. |
[25] | LIU Q, MA B T, QIAN N C, et al. Structure of the African swine fever virus major capsid protein p72[J]. Cell Res, 2019, 29(11):953-955. |
[26] | NEILAN J G, ZSAK L, LU Z, et al. Neutralizing antibodies to African swine fever virus proteins p30, p54, and p72 are not sufficient for antibody-mediated protection[J]. Virology, 2004, 319(2):337-342. |
[27] | RODRÍGUEZ J M, YÁÑEZ R J, ALMAZÁN F, et al. African swine fever virus encodes a CD2 homolog responsible for the adhesion of erythrocytes to infected cells[J]. J Virol, 1993, 67(9):5312-5320. |
[28] | SANNA G, DEI GIUDICI S, BACCIU D, et al. Improved strategy for molecular characterization of African swine fever viruses from Sardinia, based on analysis of p30, CD2v and I73R/I329L variable regions[J]. Transbound Emerg Dis, 2017, 64(4):1280-1286. |
[29] | RUIZ-GONZALVO F, RODRÍGUEZ F, ESCRIBANO J M. Functional and immunological properties of the baculovirus-expressed hemagglutinin of African swine fever virus[J]. Virology, 1996, 218(1):285-289. |
[30] | 周晓慧, 肖景景, 张鑫宇, 等. 非洲猪瘟病毒强免疫原性重组CD2v抗原的制备与初步应用[J]. 畜牧兽医学报, 2020, 51(10):2472-2480.ZHOU X H, XIAO J J, ZHANG X Y, et al. Preparation and preliminary application of highly immunogenic recombinant CD2v antigen of African swine fever virus[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(10):2472-2480. (in Chinese) |
[31] | 钟秋萍, 于婉琪, 薄宗义, 等. 非洲猪瘟病毒外膜蛋白CD2v的原核表达及其免疫特性的研究[J]. 中国兽医科学, 2020, 50(6):689-695.ZHONG Q P, YU W Q, BO Z Y, et al. Prokaryotic expression and immunological characterization of African swine fever virus out membrane CD2v protein[J]. Chinese Veterinary Science, 2020, 50(6):689-695. (in Chinese) |
[32] | 于浩洋, 吴绍强, 王彩霞, 等. 非洲猪瘟CD2v胞外区基因片段的真核表达及其多克隆抗体的制备[J]. 中国兽医科学, 2021, 51(1):45-52.YU H Y, WU S Q, WANG C X, et al. Eukaryotic expression the CD2v extracellular gene fragment of African swine fever virus and characterization of its polyclonal antibodies[J]. Chinese Veterinary Science, 2021, 51(1):45-52. (in Chinese) |
[33] | MONTEAGUDO P L, LACASTA A, LÓPEZ E, et al. BA71ΔCD2:a new recombinant live attenuated African swine fever virus with cross-protective capabilities[J]. J Virol, 2017, 91(21):e01058-17. |
[34] | BORCA M V, CARRILLO C, ZSAK L, et al. Deletion of a CD2-like gene, 8-DR, from African swine fever virus affects viral infection in domestic swine[J]. J Virol, 1998, 72(4):2881-2889. |
[35] | BERGERON H C, GLAS P S, SCHUMANN K R. Diagnostic specificity of the African swine fever virus antibody detection enzyme-linked immunosorbent assay in feral and domestic pigs in the United States[J]. Transbound Emerg Dis, 2017, 64(6):1665-1668. |
[36] | STRIZHAKOVA O M, LYSKA V M, MALOGOLOVKIN A S, et al. Validation of an ELISA kit for detection of antibodies against:ASF virus in blood or spleen of domestic pigs and wild boars[J]. Agric Biol, 2016, 51(6):845-852. |
[37] | ASAMBE A, SACKEY A K B, TEKDEK L B. Prevalence of African swine fever virus and classical swine fever virus antibodies in pigs in Benue State, Nigeria[J]. Trop Anim Health Prod, 2018, 50(3):689-692. |
[38] | GALLARDO C, FERNÁNDEZ-PINERO J, PELAYO V, et al. Genetic variation among African swine fever genotype Ⅱ viruses, eastern and central Europe[J]. Emerg Infect Dis, 2014, 20(9):1544-1547. |
[39] | NIETO-PELEGRÍN E, RIVERA-ARROYO B, SÁNCHEZ-VIZCAÍNO J M. First detection of antibodies against African swine fever virus in Faeces samples[J]. Transbound Emerg Dis, 2015, 62(6):594-602. |
[40] | GIMÉNEZ-LIROLA L G, MUR L, RIVERA B, et al. Detection of African swine fever virus antibodies in serum and oral fluid specimens using a recombinant protein 30(p30) dual matrix indirect ELISA[J]. PLoS One, 2016, 11(9):e0161230. |
[41] | 张蕾, 董春娜, 李静, 等. 非洲猪瘟病毒间接ELISA抗体检测方法的建立[J]. 病毒学报, 2020, 36(4):670-674.ZHANG L, DONG C N, LI J, et al. Development of an ELISA based on synthetic peptides for detection of antibodies against the African swine fever virus[J]. Chinese Journal of Virology, 2020, 36(4):670-674. (in Chinese) |
[42] | CARLSON J, ZANI L, SCHWAIGER T, et al. Simplifying sampling for African swine fever surveillance: Assessment of antibody and pathogen detection from blood swabs[J]. Transbound Emerg Dis, 2018, 65(1):e165-e172. |
[43] | 林彦星, 曹琛福, 张彩虹, 等. 非洲猪瘟病毒抗体量子点检测试纸条的研制[J]. 中国兽医科学, 2017, 47(10):1214-1220.LIN Y X, CAO C F, ZHANG C H, et al. Establishment of a quantum dots-based immunochromatographic strip for detection of the antibodies against African swine fever virus[J]. Chinese Veterinary Science, 2017, 47(10):1214-1220. (in Chinese) |
[44] | MUR L, IGOLKIN A, VARENTSOVA A, et al. Detection of African swine fever antibodies in experimental and field samples from the Russian federation:Implications for control[J]. Transbound Emerg Dis, 2016, 63(5):e436-e440. |
[45] | AIRA C, RUIZ T, DIXON L, et al. Bead-based multiplex assay for the simultaneous detection of antibodies to African swine fever virus and classical swine fever virus[J]. Front Vet Sci, 2019, 6:306. |
[46] | MURGIA M V, MOGLER M, CERTOMA A, et al. Evaluation of an African swine fever (ASF) vaccine strategy incorporating priming with an alphavirus-expressed antigen followed by boosting with attenuated ASF virus[J]. Arch Virol, 2019, 164(2):359-370. |
[47] | GALLARDO C, BLANCO E, RODRÍGUEZ J M, et al. Antigenic properties and diagnostic potential of African swine fever virus protein pp62 expressed in insect cells[J]. J Clin Microbiol, 2006, 44(3):950-956. |
[48] | AFAYOA M, OLAHO-MUKANI W, OKUNI J B, et al. Development and evaluation of an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) for the diagnosis of African swine fever[J]. Virol Mycol, 2015, 4(2):145. |
[49] | SASTRE P, GALLARDO C, MONEDERO A, et al. Development of a novel lateral flow assay for detection of African swine fever in blood[J]. BMC Vet Res, 2016, 12:206. |
[50] | 吴海涛, 成大荣, 吴萌, 等. 非洲猪瘟病毒胶体金免疫层析试纸条的研制[J]. 黑龙江畜牧兽医, 2018(17):126-128, 238.WU H T, CHENG D R, WU M, et al. Preparation of colloidal gold immunochromatographic test strip of African swine fever virus[J]. Heilongjiang Animal Science and Veterinary Medicine, 2018(17):126-128, 238. (in Chinese) |
[51] | SZEREDI L, BAKCSA E, ZÁDORI Z, et al. Detection of African swine fever virus in cell culture and wild boar tissues using a commercially available monoclonal antibody[J]. J Virol Methods, 2020, 282:113886. |
[52] | PETROVAN V, YUAN F F, LI Y H, et al. Development and characterization of monoclonal antibodies against p30 protein of African swine fever virus[J]. Virus Res, 2019, 269:197632. |
[53] | WU P, LOWE A D, RODRÍGUEZ Y Y, et al. Antigenic regions of African swine fever virus phosphoprotein P30[J/OL]. Transboundary and Emerging Diseases, 2020, doi:10. 1111/tbed. 13533. |
[54] | VLAD P. Antigenic characterization of African swine fever virus (ASFV) p30 and p54 proteins[D]. Manhattan: Kansas State University, 2019. |
[55] | PETROVAN V, MURGIA M V, WU P, et al. Epitope mapping of African swine fever virus (ASFV) structural protein, p54[J]. Virus Res, 2020, 279:197871. |
[56] | HEIMERMAN M E, MURGIA M V, WU P, et al. Linear epitopes in African swine fever virus p72 recognized by monoclonal antibodies prepared against baculovirus-expressed antigen[J]. J Vet Diagn Invest, 2018, 30(3):406-412. |
[57] | QUEMBO C J, JORI F, VOSLOO W, et al. Genetic characterization of African swine fever virus isolates from soft ticks at the wildlife/domestic interface in Mozambique and identification of a novel genotype[J]. Transbound Emerg Dis, 2018, 65(2):420-431. |
[58] | BLOME S, FRANZKE K, BEER M. African swine fever-A review of current knowledge[J]. Virus Res, 2020, 287:198099. |
[59] | 张洪亮, 金铭, 赵越, 等. 非洲猪瘟病毒免疫学及疫苗研究进展[J]. 病毒学报, 2019, 35(3):533-541.ZHANG H L, JIN M, ZHAO Y, et al. Research progress in the immunology and vaccine of the African swine fever virus[J]. Chinese Journal of Virology, 2019, 35(3):533-541. (in Chinese) |
[60] | 白晨雨, 王同燕, 赵少若, 等. 非洲猪瘟病毒p62蛋白单克隆抗体的制备及初步应用[J]. 畜牧兽医学报, 2020, 51(5): 1074-1082.BAI C Y, WANG T Y, ZHAO S R, et al. Preparation of monoclonal antibodies against recombinant p62 protein of African swine fever virus and its preliminary application[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(5): 1074-1082. (in Chinese) |
[61] | ADENAIKE E A, TEKDEK L B, KAZEEM H M, et al. Antibody detection in suspected carriers of African swine fever virus in tarka local government area of Benue State, Nigeria[J]. Int J Prod Res, 2017, 29(2):289-293. |
[62] | ANANDARAO R, SWAMINATHAN S, FERNANDO S, et al. Recombinant multiepitope protein for early detection of dengue infections[J]. Clin Vaccine Immunol, 2006, 13(1):59-67. |
[63] | HE J, XIU B S, WANG G H, et al. Double-antigen sandwich ELISA for the detection of anti-hepatitis C virus antibodies[J]. J Virol Methods, 2011, 171(1):163-168. |
[64] | SU Q D, GUO M Z, JIA Z Y, et al. Epitope-based recombinant diagnostic antigen to distinguish natural infection from vaccination with hepatitis A virus vaccines[J]. J Virol Methods, 2016, 233:41-45. |
[65] | LAMPE K, GOTTSTEIN B, BECKER T, et al. Immunization of rhesus macaques with Echinococcus multilocularis recombinant 14-3-3 antigen leads to specific antibody response[J]. Parasitol Res, 2017, 116(1):435-439. |
[66] | MIRZAPOUR A, SEYYED TABAEI S J, BANDEHPOUR M, et al. Designing a recombinant multi-epitope antigen of Echinococcus granulosus to diagnose human cystic echinococcosis[J]. Iran J Parasitol, 2020, 15(1):1-10. |
[67] | MOHAN C M, DEY S, RAI A, et al. Recombinant haemagglutinin neuraminidase antigen-based single serum dilution ELISA for rapid serological profiling of Newcastle disease virus[J]. J Virol Methods, 2006, 138(1-2):117-122. |
[68] | ZHANG W B, WEN H, LI J, et al. Immunology and Immunodiagnosis of cystic echinococcosis: an update[J]. Clin Dev Immunol, 2012, 2012:101895. |
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