1群4型禽腺病毒的分离鉴定及Fiber-2蛋白的免疫效果分析

doi:10.11843/j.issn.0366-6964.2020.10.014

Abstract

Abstract: The aim of this study was to identify pathogens, which caused pericardial effusion, hepatomegaly and bleeding at a chicken farm in Henan province, and furthermore to analyze effectiveness of immunogenic proteins of the pathogen. This study employed virus isolation, serological assays, PCR and sequencing analysis, animal experimentation, E. coli expression and protein purification, immunogenicity and challenge test and other methods. The results showed that virus was isolated in 7-day-old SPF chicken embryonated eggs, inoculated via the yolk sac route by two blind passages. Viral confirmation was carried out using PCR techniques, and showed a 900-bp-long fragment which shared a 100% homology with the Hexon gene of the serotype C4 strain. Serum neutralization results indicated that this isolate avian adenovirus was the group I type 4 avian adenovirus, named HN-ZK strain. The virus could induce CPE on primary hepatocytes of chicken embryo, and its titer was 10^7.5 TCID₅₀·0.1 mL^-1. Animal experimentation illustrated that the isolated virus caused 100% (10/10) typical symptoms and pathogenicity in 35-day-old SPF chickens. Furthermore, the DNA of the isolate virus was used as a template to express Fiber-2 fragment, with a molecular weight of 33 kDa by using the E. coli expression system, and the protein was concentrated and purified to 300 mg·mL^-1 after centrifugation and purification. SPF chickens were immunized with different doses of the purified Fiber-2 protein, and showed that a dose of 20 μg per chick was completely resistant to challenge of the virulent virus, suggesting the purified Fiber-2 protein has better immunogenicity. This study can provide data for diagnosing the hydropericardium hepatitis syndrome, and developing a genetic engineering subunit vaccine.

Key words: group I avian adenovirus, identification, fiber-2 protein, subunit vaccine

CLC Number:

S852.659.1

CHENG Zengqing, FAN Gencheng, DOU Xiaolong, LIU Hongxiang, SHEN Maoxin, XU Quangang, YANG Hanchun. Isolation and Identification of Group I Type 4 Avian Adenovirus and Analysis in Immune Effect of Fiber-2 Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(10): 2463-2471.

References 28

[1]	王婉,王彦红,刘伊,等. 鸡包涵体肝炎病原检测及病理学观察[J]. 畜牧兽医学报, 2015, 46(2):273-278.WANG W,WANG Y H,LIU Y,et al. Pathogenic detection and pathological observation of inclusion body hepatitis in chicken[J]. Acta Veterinaria et Zootechnica Sinica, 2015, 46(2):273-278. (in Chinese)
[2]	JADHAO S J, DEEPAK J N, KATARIA J M, et al. Characterisation of fowl adenoviruses from chickens affected with infectious hydropericardium during 1994-1998 in India.[J]. Comparative Study Indian J Exp Biol, 2003, 41(4):321-327.
[3]	HESS M, RAUE R, PRUSAS C. Epidemiological studies on fowl adenoviruses isolated from cases of infectious hydropericardium[J]. Avian Pathol, 1999, 28(5):433-439.
[4]	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 Pathol, 1998, 27(3):269-276.
[5]	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, 11(8):e0161744.
[6]	LOUIS N, FENDER P, BARGE A, et al. Cell-binding domain of adenovirus serotype 2 fiber[J]. J Virol, 1994, 68(6):4104-4106.
[7]	HENRY L J, XIA D, WILKE M E, et al. Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli[J]. J Virol, 1994, 68(8):5239-5246.
[8]	GELDERBLOM H, MAICHLE-LAUPPE I. The fibers of fowl adenoviruses[J]. Arch Virol, 1982, 72(4):289-298.
[9]	AFZAL M, MUNEER R, STEIN G. Studies on the aetiology of hydropericardium syndrome (Angara disease) in broilers[J]. Vet Rec, 1991, 128(25):591-593.
[10]	HESS M, CUZANGE A, RUIGROK R W, et al. The avian adenovirus penton:two fibres and one base[J]. J Mol Biol, 1995, 252(4):379-385.
[11]	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]. Vet Microbiol, 2012, 156(3-4):411-417.
[12]	唐雪,马海田. 一种改良的鸡胚原代肝细胞的分离培养方法[J].南京农业大学学报, 2008, 31(4):154-156.TANG X, MA H T. Cultivation and identification of chicken embryo hepatocytes in primary culture[J].Journal of Nanjing Agricultural University, 2008, 31(4):154-156.(in Chinese)
[13]	REED L J, MUENCH H. A simple method of estimating fifty percent endo-points[J]. Am J Hyg, 1938, 27:493-797.
[14]	ANJUM A D, SABIR M A, IQBAL Z. Hydropericarditis syndrome in broiler chickens in Pakistan[J]. Vet Rec, 1989,124(10):247-248.
[15]	ABDUL-AZIZ T A, AL-ATTAR M A. New syndrome in Iraqi chickes[J]. Vet Rec, 1991, 129(12):272.
[16]	ABDUL-AZIZ T A, HASAN S Y. Hydropericar-dium syndrome in broiler chickens:its contagious nature and pathology[J]. Res Vet Sci,1995,59(3):219-221.
[17]	ABE T, NAKAMURA K, TOJO H, et al. Histology, immunohistochemistry, and ultrastructure of hydropericardium syndrome in adult broiler breeders and broiler chicks[J]. Avian Dis, 1998, 42(3):606-612.
[18]	DAHIYA S, SRIVASTAVA R N, HESS M, et al. Fowl adenovirus serotype 4 associated with outbreaks of infectious hydropericardium in Haryana, India[J]. Avian Dis, 2002, 46(1):230-233.
[19]	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 Dis, 2008, 52(3):526-530.
[20]	GAHÉRY-SÉGARD H, FARACE F, GODFRIN D, et al. Immune response to recombinant capsid proteins of adenovirus in humans:antifiber and anti-penton base antibodies have a synergistic effect on neutralizing activity[J]. J Virol, 1998, 72(3):2388-2397.
[21]	BRADLEY R R, LYNCH D M, IAMPIETRO M J, et al. Adenovirus serotype 5 neutralizing antibodies target both hexon and fiber following vaccination and natural infection[J]. J Virol, 2012, 86(1):625-629.
[22]	YU B, DONG J, WANG C, et al. Characteristics of neutralizing antibodies to adenovirus capsid proteins in human and animal sera[J]. Virology, 2013, 437(2):118-123.
[23]	FINGERUT E, GUTTER B, GALLILI G, et al. A subunit vaccine against the adenovirus egg-drop syndrome using part of its fiber protein[J]. Vaccine, 2003, 21(21-22):2761-2766.
[24]	MAUTNER V, WILLCOX H N. Adenovirus antigens:a model system in mice for subunit vaccination[J]. J Gen Virol, 1974, 25(3):325-336.
[25]	SHAH M S, ASHRAF A, RAHMAN M, et al. A subunit vaccine against hydropericardium syndrome using adenovirus penton capsid protein[J]. Vaccine, 2012, 30(50):7153-7156
[26]	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.
[27]	PITCOVSKI J, FINGERUT E, GALLILI G, et al. A subunit vaccine against hemorrhagic enteritis adenovirus[J]. Vaccine, 2005, 23(38):4697-4702.
[28]	SCHONEWILLE E, JASPERS R, PAUL G, et al. Specific-pathogen-free chickens vaccinated with a live FAdV-4 vaccine are fully protected against a severe challenge even in the absence of neutralizing antibodies[J]. Avian Dis, 2010, 54(2):905-910.

Isolation and Identification of Group I Type 4 Avian Adenovirus and Analysis in Immune Effect of Fiber-2 Protein

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 28

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	CHEN Yuetong, LIU Xiaohan, WANG Zhiyang, ZHAO Yuxin, ZHOU Tiezhong, HU Zengjin, ZHU Yue, WANG Shaohui, TIAN Mingxing, DING Siyu, QI Jingjing, YU Shengqing. Isolation, Identification, Pathogenicity and Drug Susceptibility of Mycoplasma gallisepticum from Dead Chicken Embryos in Large-scale Chicken Farms in Guangdong Province [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 290-299.
[2]	YANG Heng, LI Zhanhong, SONG Zi'ang, GAO Lin, LI Zhuoran, LIAO Defang, XIAO Lei, LI Huachun. Development and Application of Serotyping Real-time Quantitative RT-PCR Methods for Palyam Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 395-400.
[3]	YU Zhou, YANG Baigao, ZHANG Hang, XU Xi, ZHANG Peipei, FENG Xiaoyi, CAO Jianhua, NIU Yifan, DU Weihua, HAO Haisheng, ZHU Huabin, ABULIZI·Wusiman, ZHAO Xueming. Research Progress of Estrus Markers in Dairy Buffalo [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3623-3630.
[4]	YANG Yue, WANG Rui, GAN Yuan, HAO Fei, XIE Xing, ZHANG Lei, SHAO Guoqing, MENG Qingguo, CHEN Rong, FENG Zhixin. Purification and Identification of SIgA in Porcine Colostrum based on Tandem Affinity Chromatography [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3884-3894.
[5]	LIU Xinhuan, YUN Jialei, MAO Li, LI Jizong, HAO Fei, HE Miaofeng, YANG Leilei, ZHANG Wenwen, CHENG Zilong, SUN Min, LIU Maojun, WANG Shaohui, BAI Juan, LI Wenliang. Isolation, Identification, Virulence Genes and Drug Resistance Analysis of Escherichia coli Isolated from Diarrheal Goat and Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3445-3454.
[6]	WANG Jiali, ZHOU Ning, CHEN Xi, YUE Hua, TANG Cheng. Isolation, Identification and Pathogenicity of Canine Adenovirus Type 2 Strain with Natural Deletion in E3 Gene [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2982-2990.
[7]	CHENG Xiangning, ZHANG Yun, HE Jiawei, CHEN Zhengkun, JIN Meilan, DING Honglei, WANG Qinghua. Isolation, Identification and Antifungal Susceptibility Analysis of Candida albicans Isolated from Pigeons [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 3002-3011.
[8]	ZHAO Feifei, LI Jie, HAN Ning, XIE Shiting, ZENG Zhenling. Antibacterial Drug Resistance Analysis of Klebsiella pneumoniae Isolated from Slaughterhouse [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 3044-3053.
[9]	LIU Hongyi, LUO Tingyu, LI Changwen, YU Haibo, LU Xiaoye, CHEN Hongyan, XIA Changyou, GAO Caixia. Identification of SLA-1 Alleles and Analysis of Molecular Genetic Characteristics in Rongchang Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 3064-3077.
[10]	XUAN Ting, YANG Kaiyi, CAI Jinshuang, GENG Yan, LI Yufeng. Analysis of the Immune Characteristics of the Six Recombinant Proteins of Glaesserella parasuis in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2073-2082.
[11]	YUAN Sheng, LI Anqi, Lü Wenke, YANG Lulu, ZHOU Feng, HUANG Liangzong, BAI Aiquan, WEN Feng, HUANG Shujian, GUO Jinyue. Analysis of Variation in Major Virulence-related Genes of a Strain of Pseudorabies Virus and Its Pathogenicity to Rabbits [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2195-2199.
[12]	LIU Jiankui, XU Ye, LIU Chen, YU Hui, YANG Yuan, HE Le, LI Jiarui, DAN Huijuan, DAI Ailing, YANG Xiaoyan, WEI Chunhua. Genomic Characteristics of Porcine Reproductive and Respiratory Syndrome Virus in Fujian Province from 2017 to 2021 based on Whole Genome [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1579-1589.
[13]	XU Xinyan, ZHENG Yating, LIU Di, MA Bo, LIU Jiasen, QU Liandong. Isolation and Identification of a Feline Herpesvirus-1 Strain [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1713-1720.
[14]	CHI Lili, WANG Junna, ZHANG Yuming, LIU Jian, CHEN Zhiyuan, LI Shufan, YIN Yanbo, XU Shouzhen. Prokaryotic Expression and Immunogenicity Analysis of Truncated Fusion Protein of FAdV-4 Fiber2 and FAdV-8b Fiber [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5162-5170.
[15]	DONG Zhihao, SHI Yuxin, GUO Guanhua, YUAN Kaimin, XIU Haoyu, WANG Chao, BAI Junyan, WANG Dong. Comparative Analysis of Salivary Compounds in Different Estrous Stages of Cows [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4636-4652.