表达禽腺病毒血清4型Fiber2蛋白的重组耐热新城疫病毒的构建及免疫效果评估

doi:10.16431/j.cnki.1671-7236.2022.02.027

摘要/Abstract

摘要： 【目的】研究针对新城疫病毒(Newcastle disease virus, NDV)和禽腺病毒(Fowl adenovirus, FAdV)的耐热基因工程疫苗。【方法】利用反向遗传学操作技术将NDV耐热株的HN基因替换到LaSota疫苗株上, 再将禽腺病毒血清4型(Fowl adenovirus serotype 4, FAdV-4)的Fiber2基因插入到其基因组上, 构建表达Fiber2蛋白的重组耐热NDV质粒pTS-HN-Fiber2。通过病毒拯救技术拯救重组NDV rTS-HN-Fiber2, 并测定其生物学特性和作为疫苗候选株的免疫原性和攻毒保护性。【结果】 rTS-HN-Fiber2的鸡胚平均致死时间>168 h, 且脑内接种致病指数为0, 属于弱毒的范畴; 在细胞上的生长曲线结果表明, rTS-HN-Fiber2与亲本LaSota株有相似的生长曲线, 但最终的生长滴度略低于LaSota株; rTS-HN-Fiber2在56 ℃处理15 min后, 病毒滴度下降约10³ TCID₅₀/mL, 而LaSota株56 ℃处理5 min几乎无感染性; 间接免疫荧光试验结果表明, rTS-HN-Fiber2能表达Fiber2蛋白。免疫和攻毒试验结果显示, rTS-HN-Fiber2能产生NDV抗体, 且能显著提高雏鸡在FAdV-4强毒下的存活率, 减轻FAdV-4强毒引起的组织病变, 降低组织中的病毒载量。【结论】本研究成功构建了表达FAdV-4 Fiber2蛋白的重组耐热NDV, 该病毒保持了亲本LaSota株的弱毒生物学特性, 但热稳定性有显著提升; 重组NDV免疫雏鸡可产生针对NDV和FAdV-4强毒的保护, 该重组NDV可作为开发针对FAdV-4和NDV二联基因工程疫苗的候选病毒株。

关键词: 禽腺病毒血清4型(FAdV-4); 新城疫病毒(NDV); Fiber2蛋白; 重组病毒; 耐热

Abstract: 【Objective】 This study was aimed to develop a heat-resistant genetically engineered vaccine against Newcastle disease virus (NDV) and Fowl adenovirus (FAdV).【Method】 Using reverse genetic manipulation techniques, the HN gene of NDV heat-resistant strain was substituted into the LaSota vaccine strain and then the Fiber2 gene of FAdV serotype 4 (FAdV-4) was inserted into its genome to generate a recombinant heat-resistant NDV plasmid pTS-HN-Fiber2, expressing Fiber2 protein.Recombinant NDV rTS-HN-Fiber2 was rescued by virus rescue technology, and its biological characteristics, immunogenicity and challenge protection as a vaccine candidate was determined.【Result】 The mean death time of rTS-HN-Fiber2 was > 168 h and the intracerebral pathogenicity index was 0, which belonged to the category of lentogenic strain.The results of growth curve on BHK-21 cells showed that rTS-HN-Fiber2 had similar growth curve with LaSota strain, but the final growth titer was slightly lower than LaSota strain.The titer of rTS-HN-Fiber2 decreased by about 10³ TCID₅₀/mL after treatment at 56 ℃ for 15 min, while NDV LaSota strain showed almost no infection after treatment at 56 ℃ for 5 min.Indirect immunofluorescence assay result showed that rTS-HN-Fiber2 could express Fiber2 protein efficiently.The results of immunization and challenge test showed that rTS-HN-Fiber2 could produce NDV antibody, significantly improve the survival rate of chicks exposed to FAdV-4, reduce the tissue lesions caused by FAdV-4 virulence and reduce the virus load in the tissues.【Conclusion】 The recombinant heat-resistant NDV expressing FAdV-4 Fiber2 protein was successfully constructed.The recombinant virus maintained the lentogenic strain characteristics of the LaSota strain, but the thermal stability was significantly improved.Chickens immunized with recombinant NDV could produce protection against NDV and FAdV-4.The recombinant NDV could be used as a candidate virus strain for the development of dual genetic engineering vaccine against FAdV-4 and NDV.

Key words: Fowl adenovirus serotype 4 (FAdV-4); Newcastle disease virus (NDV); Fiber2 protein; recombinant virus; heat-resisting

中图分类号:

S852.62

商雨, 李林涛, 李丽, 徐英英, 冯贺龙, 汪宏才, 张蓉蓉, 曾哲, 王红琳, 罗青平, 邵华斌, 温国元. 表达禽腺病毒血清4型Fiber2蛋白的重组耐热新城疫病毒的构建及免疫效果评估[J]. 中国畜牧兽医, 2022, 49(2): 650-659.

SHANG Yu, LI Lintao, LI Li, XU Yingying, FENG Helong, WANG Hongcai, ZHANG Rongrong, ZENG Zhe, WANG Honglin, LUO Qingping, SHAO Huabin, WEN Guoyuan. Construction of Recombinant Heat-resistant Newcastle Disease Virus Expressing Fiber2 Protein of Fowl Adenovirus Serotype 4 and Evaluation of Its Immune Effect[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 650-659.

参考文献

[1] HESS M. Detection and differentiation of Avian adenoviruses: A review[J]. Avian Pathology, 2000, 29(3): 195-206.
[2] LI P H, ZHENG P P, ZHANG T F, et al. Fowl adenovirus serotype 4: Epidemiology, pathogenesis, diagnostic detection, and vaccine strategies[J]. Poultry Science, 2017, 96(8): 2630-2640.
[3] MAZAHERI A, PRUSAS C, VOSS M, et al. Some strains of serotype 4 Fowl adenoviruses cause inclusion body hepatitis and hydropericardium syndrome in chickens[J]. Avian Pathology, 1998, 27(3): 269-276.
[4] MANSOOR M K, HUSSAIN I, ARSHAD M, et al. Preparation and evaluation of chicken embryo-adapted Fowl adenovirus serotype 4 vaccine in broiler chickens[J]. Tropical Animal Health and Production, 2011, 43(2): 331-338.
[5] SHAH M S, ASHRAF A, KHAN M I, et al. Molecular cloning, expression and characterization of 100K gene of Fowl adenovirus-4 for prevention and control of hydropericardium syndrome[J]. Biologicals, 2016, 44(1): 19-23.
[6] KIM J N, BYUN S H, KIM M J, et al. Outbreaks of hydropericardium syndrome and molecular characterization of Korean fowl adenoviral isolates[J]. Avian Diseases, 2008, 52(3): 526-530.
[7] CHEEMA A E, AHMAD J, AFZAL M. An Adenovirus infection of poultry in Pakistan[J]. Revue Scientifique et Technique, 1989, 8(3): 789-795.
[8] ZHAO J, ZHONG Q, ZHAO Y, et al. Pathogenicity and complete genome characterization of Fowl adenoviruses isolated from chickens associated with inclusion body hepatitis and hydropericardium syndrome in China[J]. PLoS One, 2015, 10(7): e0133073.
[9] LI L, LUO L, LUO Q, et al. Genome sequence of a Fowl adenovirus serotype 4 strain lethal to chickens, isolated from China[J]. Genome Announcements, 2016, 4(2): e00140-16.
[10] 宋一诺, 邱阳, 刘奎昊, 等. 2019—2020年山东省中西部地区鸭源禽腺病毒病原学检测及遗传进化分析[J]. 中国动物检疫, 2021, 38(6): 6-12.
SONG Y N, QIU Y, LIU K H, et al. Pathogenic detection of duck-derived FAdV in central and Western Shandong from 2019 to 2020 and analysis on its genetic evolution[J]. China Animal Health Inspection, 2021, 38(6): 6-12. (in Chinese)
[11] 尹德晶, 张云丹, 岳筠, 等. 禽腺病毒血清4型贵州株全基因序列分析[J]. 中国畜牧兽医, 2021, 48(1): 44-54.
YIN D J, ZHANG Y D, YUE J, et al. Whole genomic sequencing analysis of Fowl adenovirus serotype 4 from Guizhou[J]. China Animal Husbandry & Veterinary Medicine, 2021, 48(1): 44-54. (in Chinese)
[12] MAREK A, NOLTE V, SCHACHNER A, et al. Two fiber genes of nearly equal lengths are a common and distinctive feature of Fowl adenovirus C members[J]. Veterinary Microbiology, 2012, 156(3-4): 411-417.
[13] WANG W, LIU Q, LI T, et al. Fiber-1, not fiber-2, directly mediates the infection of the pathogenic serotype 4 Fowl adenovirus via its shaft and knob domains[J]. Journal of Virology, 2020, 94(17): e00954-20.
[14] ZHANG Y, LIU R, TIAN K, et al. Fiber2 and hexon genes are closely associated with the virulence of the emerging and highly pathogenic fowl adenovirus 4[J]. Emerging Microbes & Infections, 2018, 7(1): 199.
[15] SOHAIMI N M, BEJO M H, OMAR A R, et al. Hexon and fiber gene changes in an attenuated Fowl adenovirus isolate from Malaysia in embryonated chicken eggs and its infectivity in chickens[J]. Journal of Veterinary Science, 2018, 19(6): 759-770.
[16] LIU R, ZHANG Y, GUO H, et al. The increased virulence of hypervirulent Fowl adenovirus 4 is independent of fiber-1 and penton[J]. Research in Veterinary Science, 2020, 131: 31-37.
[17] WANG X, TANG Q, CHU Z, et al. Immune protection efficacy of FAdV-4 surface proteins fiber-1, fiber-2, hexon and penton base[J]. Virus Research, 2018, 245: 1-6.
[18] TIAN K Y, GUO H F, LI N, et al. Protection of chickens against hepatitis-hydropericardium syndrome and Newcastle disease with a recombinant Newcastle disease virus vaccine expressing the Fowl adenovirus serotype 4 fiber-2 protein[J]. Vaccine, 2020, 38(8): 1989-1997.
[19] SCHACHNER A, MAREK A, JASKULSKA B, et al. Recombinant FAdV-4 fiber-2 protein protects chickens against hepatitis-hydropericardium syndrome (HHS)[J]. Vaccine, 2014, 32(9): 1086-1092.
[20] WANG P, ZHANG J, WANG W, et al. A novel monoclonal antibody efficiently blocks the infection of serotype 4 Fowl adenovirus by targeting fiber-2[J]. Veterinary Research, 2018, 49(1): 29.
[21] CHEN L, YIN L, ZHOU Q, et al. Immunogenicity and protective efficacy of recombinant fiber-2 protein in protecting SPF chickens against Fowl adenovirus 4[J]. Vaccine, 2018, 36(9): 1203-1208.
[22] 匡露寒, 季国荣, 杨灿灿, 等. 基于抗原蛋白Fiber-2及细胞因子佐剂的Ⅰ群4型禽腺病毒亚单位疫苗的开发[J]. 中国兽医科学, 2020, 50(4): 452-458.
KUANG L H, JI G R, YANG C C, et al. Development of group Ⅰ serotype 4 Fowl adenovirus subunit vaccine based on antigen protein Fiber-2 and cytokine adjuvants[J]. Chinese Veterinary Science, 2020, 50(4): 452-458. (in Chinese)
[23] BELLO M B, YUSOFF K, IDERIS A, et al. Diagnostic and vaccination approaches for Newcastle disease virus in poultry: The current and emerging perspectives[J]. Biomed Research International, 2018, 2018: 7278459.
[24] KIM S H, SAMAL S K. Newcastle disease virus as a vaccine vector for development of human and veterinary vaccines[J]. Viruses, 2016, 8(7): 183.
[25] WEN G, HU X, ZHAO K, et al. Molecular basis for the thermostability of Newcastle disease virus[J]. Scientific Reports, 2016, 6: 22492.
[26] 郭浩然. 表达禽腺病毒Fiber2基因重组新城疫病毒的拯救及免疫效果评价[D]. 合肥: 安徽农业大学, 2019.
GUO H R. Recovery and evaluation of immune responses induced by a recombinant Newcastle disease virus expressing Fowl adenoviruses antigen Fiber2[D]. Hefei: Anhui Agricultural University, 2019. (in Chinese)