鸭坦布苏病毒鸡胚弱化毒株的选育

doi:10.3864/j.issn.0578-1752.2016.14.016

Abstract

Abstract: 【Objective】 Tembusu virus BZ-2010 strain was continuously passaged in specific-pathogen-free embryonic (SPF) eggs in order to select a live attenuated vaccine candidate of good safety and immunogenicity properties. 【Method】 Tembusu virus BZ-2010 strain was cultured for 120 passages in SPF eggs. The safety of the 120th passage viral strain was evaluated with 1-day-old SPF ducklings and 30-week-old egg-laying ducks. The property of virulent return of VC2 viral strain was evaluated with 1-day-old SPF ducklings. The neutralizing antibodies were detected after the 18-week-old breeding ducks were immunized with VC2 strain. The protective effects were evaluated after the 25-week-old breeding ducks were immunized with VC2 strain. E gene and NS4A gene of BZ_2010 and VC2 strains were amplified by RT-PCR and sequenced. 【Result】 The average death time of SPF eggs was shortened by passage virus and viral titer was increased with the escalation of passage times in SPF chicken embryonic eggs. ELD₅₀ of the 20th virus was 10-5.3/0.1mL and ELD₅₀ of the 120th virus was 10-5.8/0.1mL.The viral titer reached the plateau at passage 80 and remained unchanged further passages. The experimental ducks showed no clinical symptoms after 1-day-old ducklings and 30-week-old breeding ducks were immunized with VC2 strain by subcutaneous injection and by intramuscular injection, respectively. The results showed that VC2 strain had a good safety. No symptoms appeared in 1-day-old ducklings in which VC2 strain were cultured for 5 passages. 1-day-old ducklings were infected with the 5th tissue suspension and no symptoms were observed in liver pathological section by microscope. The results showed that VC2 strain had a good stability. Sequence analysis revealed that the E protein of Tembusu VC2 evolved amino acid changes in positions 86, 157, 189, 301, and 302, respectively. The NS4A protein of Tembusu VC2 only had one amino acid change in position 54 in that phenylalanine was replaced by Leucine. The level of antibodies rose very quickly, reached the plateau at the 4th week and remained a long time. Ducks were challenged by TMUV virulent strain at 2 and 50 weeks after immunization with VC2 strain in the experimental group. There was no symptom, normal stool, and regular egg production in the vaccinated group after challenge of virulent strain. The results showed that VC2 strain could provide complete protection for the challenge of TMUV virulent strain.【Conclusions】 An attenuated strain of TMUV with good immunogenicity and high safety was acquired through serial passages of SPF chicken embryos. The level of antibodies rose very quickly and remained a long time after immunization of the VC2 attenuated strain. The toxicity attack experiments showed that VC2 could provide complete protection for the challenge of TMUV virulent strain.

Key words: duck Tembusu virus, chicken embryo, attenuation, live vaccine

YU Ke-xiang, MA Xiu-li, YUAN Xiao-yuan, LIU Cun-xia, HU Feng, LING Hong-li, LI Yu-feng, HUANG Bing. Selection of a Live Chicken Embryo Attenuated Duck Tembusu Virus Vaccine[J].Scientia Agricultura Sinica, 2016, 49(14): 2822-2829.

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References

[1] 李玉峰，马秀丽，于可响，王友令，高巍，黄兵，徐怀英，吴静，王生雨，王莉莉，秦卓明. 一种从鸭新分离的黄病毒研究初报. 畜牧兽医学报，2011, 42(6): 885-891.

LI Y F, MA X L, YU K X, WANG Y L, GAO W, HUANG B, XU H Y, WU J, WANG S Y, WANG L L, QIN Z M. A brief report of flaviviruses newly isolated from duck. Acta Veterianaria et Zootechnica Sinaca, 2011, 42(6): 885-891. (in Chinese)

[2] 滕巧泱，颜丕熙，张旭，闫丽萍，李泽君. 一种新的黄病毒导致蛋鸭产蛋下降及死亡. 中国动物传染病学报，2010, 18(6): 1-4.

TENG Q Y, YAN P X, ZHANG X, YAN L P, LI Z J. A novel flavivirus causing duck egg drops and death. Chinese Journal of Animal Infectious Desease, 2010, 18(6): 1-4. (in Chinese)

[3] 万春和，施少华，程龙飞，陈红梅，傅光华，张大丙，林芳，林建生，黄瑜. 一种引起种(蛋)鸭产蛋骤降新病毒的分离与初步鉴定. 福建农业学报，2010, 25(6): 663-666.

Wan C H, Shi S H, Chen L F, Chen H M, Fu G H, Zhang D B, Lin F, Lin J S, Huang Y. A newly identified flavivirus virus causing abrupt egg-laying reduction in ducks. Fujian Journal of Agricultural Sciences, 2010, 25(6): 663-666. (in Chinese)

[4] 曹贞贞，张存，黄瑜，刁有祥，叶伟成，刘月焕，韩婧文，马国明，张冬冬，许丰，王丹，姜甜甜，袁媛，谢小雨，高绪慧，唐熠，施少华，万春和，张晨，何玢，杨梦婕，陆新浩，张冰，张国中，马学军，张大丙. 鸭出血性卵巢炎的初步研究. 中国兽医杂志，2010, 46(12): 3-6.

CAO Z Z, ZHANG CUN, HUANG Y, DIAO Y X, YE W C, LIU Y H, HAN J W, MA G M, ZHANG D D, XU F, WANG D, JIANG T T, YUAN Y, XIE X Y, GAO X H, TANG Y, SHI S H, WANG C H, ZHANG C, HE F, YANG M J, LU X H, ZHANG B, ZHANG G Z, MA X J, ZHANG D B. Preliminary studies on duck hemorrhagic ovaritis. Chinese Journal of Veterinary Medicine, 2010, 46(12): 3-6. (in Chinese)

[5] 刘志刚，孙青松，姚蓉，刘冰心，晁行周，邹忠，刘立峰，吴彦，郑爱芳，赵苏红，金梅林. 鸭坦布苏病毒研究进展. 中国动物传染病学报，2013, 21(1): 81-86.

LIU Z G, SUN Q S, YAO R, LIU B X, CHAO X Z, ZOU Z, LIU L F, WU Y, ZHONG A F, ZHAO S H, JIN M L. Research progress on duck tembusu virus. Chinese Journal of Animal Infectious Diseases, 2013, 21(1): 81-86. (in Chinese)

[6] 朱丽萍，颜世敢. 鸭坦布苏病毒研究进展. 中国预防兽医学报，2012, 34(1): 79-82.

ZHU L P, YAN S G. Research progress on duck tembusu virus. Chinese Journal of Preventive Veterinary Medicine, 2012, 34(1): 79-82. (in Chinese)

[7] JIANG T, LIU J, DENG Y Q, SU J L, XU L J, LIU Z H, LI X F, YU X D, ZHU S Y, GAO G F, QIN E D, QIN C F. Development of RT-LAMP and real-time RT-PCR assays for the rapid detection of the new duck Tembusu-like BYD virus. Archives of Virology, 2012, 157(12): 2273-2280.

[8] SU J, LI S, HU X, YU X, WANG Y, LIU P, LU X, ZHANG G, HU X, LIU D, LI X, SU W, LU H, MOK N S, WANG P, WANG M, TIAN K, GAO G F. Duck egg-drop syndrome caused by BYD virus, a new Tembusu-related flavivirus. PLoS ONE, 2011, 6(3): e18106.

[9] LI G, GAO X, XIAO Y, LIU S, PENG S, LI X, SHI Y, ZHANG Y, YU L, WU X, YAN P, YAN L, TENG Q, TONG G, LI Z. Development of a live attenuated vaccine candidate against duck Tembusu viral disease. Virology, 2014, 450: 233-242.

[10] LIU M, CHEN S, CHEN Y, LIU C, CHEN S, YIN X, LI G, ZHANG Y. Adapted Tembusu-like virus in chickens and geese in China. Journal of clinical microbiology, 2012, 50(8): 2807-2809.

[11] YU K, SHENG Z Z, HUANG B, MA X, LI Y, YUAN X, QIN Z, WANG D, CHAKRAVARTY S, LI F, SONG M, SUN H. Structural, antigenic, and evolutionary characterizations of the envelope protein of newly emerging duck Tembusu virus. PLoS ONE, 2013, 8(8): e71319.

[12] 张琳，逯茂洋，胡北侠，蒋一男，许传田，杨少华，张贝，张秀美. 4株鸭坦布苏病毒包膜蛋白基因的分子进化分析及表达. 中国兽医学报，2013, 33(2): 175-180.

ZHANG L, LU M Y, HU B X, JIANG Y N, XU C T, YANG S H, ZHANG B, ZHANG X M. Molecular evolution and expression of envelop protein of duck Tembusu virus isolated from China, Chinese Journal of Veterinary Science, 2013, 33(2): 175-180. (in Chinese)

[13] LI S, ZHANG L, WANG Y, WANG S, SUN H, SU W, HE W, HAN B, SU J. An infection full-length cDNA clone of duck Tembusu virus, a newly emerging flavivirus causing duck egg drop syndrome in China. Virus research, 2013, 171(1): 238-241.

[14] LI L, AN H, SUN M, DONG J, YUAN J, HU Q. Identification and genomic analysis of two duck-origin Tembusu virus strains in southern China. Virus Genes, 2012, 45(1): 105-112.

[15] LI X, LI G, TENG Q, YU L, WU X, LI Z. Development of a blocking ELISA for detection of serum neutralizing antibodies against newly emerged duck Tembusu virus. PLoS ONE, 2012, 7(12): e53026.

[16] MULLER D A, YOUNG P R. The flavivirus NS1 protein: molecular and structural biology, immunology, role in pathogenesis and application as a diagnostic biomarker. Antiviral Rescearch, 2013, 98(2): 192-208.

[17] HUANG Q, LI Q, JOY J, CHEN A S, RUIZ-CARRILLO D, HILL J, LESCAR J, KANG C. Lyso-myristoyl phosphatidylcholine micelles sustain the activity of Dengue non-structural (NS) protein 3 protease domain fused with the full-length NS2B. Protein Expression and Purification, 2013, 92(2): 156-162.

[18] MODIS Y, OGATA S, CLEMENTS D, HARRISON S C. A ligand-binding pocket in the dengue virus envelop eglycoprotein. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100 (12): 6986-6991.

[19] YAMAGUCHI Y, NUKUI Y, TAJIMA S, NEROME R, KATO F, WATANABE H, TAKASAKI T, KURANE I. An amino acid substitution (V3I) in the Japanese encephalitis virus NS4A protein increases its virulence in mice, but not its growth rate in vitro. Journal of General Virology, 2011, 92(7): 1601-1606.

[20] REY F A, HEINZ F X, MANDL C, KUNZ C, HARRISON S C. The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature, 1995, 375: 291-298.

[21] Murray C L, Jones C T, Rice C M. Architects of assembly: roles of Flaviviridae non-structural proteins in virion morphogenesis. Nature reviews microbiology, 2008, 6(9): 699-708.

[22] MARKOFF L, PANG X, HOUNG H S H S, FALGOUT B, OLSEN R, JONES E, POLO S. Derivation and characterization of a dengue type 1 host range-restricted mutant virus that is attenuated and highly immunogenic in monkeys. Journal of Virology, 2002, 76: 3318-3328.

[23] Potapova U V, Feranchuk S I, Potapov V V, Kulakova N V, Kondratov I G, Leonova G N, Belikov S I. NS2B/NS3 protease: allosteric effect of mutations associated with the pathogenicity of tick-borne encephalitis virus. Journal of Biomolecular Structure and Dynamics, 2012, 30(6): 638-651.

[24] LINDENBACH B D, RICE C M. Genetic interaction of flavivirus nonstructural proteins NS1 and NS4A as a determinant of replicase flmction. Journal of Virology, 1999, 73(6): 4611-4621.

[25] ARROYO J, GUIRAKHOO F, FENNER S, ZHANG Z X，MONATH T P，CHAMBERS T J. Molecular basis for attenuation of neurovirulence of a yellow fever Virus/Japanese encephalitis virus chimera vaccine (ChimeriVax-JE). Journal of virology, 2001, 75: 934-942.

[26] CHEN W J, WU H R, CHIOU S S. E/NS1 modifications of dengue 2 virus after serial passages inmammalian and/or mosquito cells. Intervirology, 2003, 46(5): 289-295.

[27] ZHANG S, LI L, WOODSON S E, HUANG C Y, KINNEY R M, BARRETT A D, BEASLEY D W. A mutation in the envelope protein fusion loop attenuates mouse neuroinvasiveness of the NY99 strain of West Nile virus. Virology, 2006, 353: 35-40.

[28] NI H, BURNS N J, CHANG G J, ZHANG M J, WILLS M R, TRENT D W, SANDERS P G, BARRETT A D. Comparison of nucleotide and deduced amino acid sequence of the 5’ non-coding region and structural protein genes of the wild-type Japanese encephalitis virus strain SA14 and its attenuated vaccine derivatives. Journal of General Virology, 1994, 75(6): 1505-1510.

[29] NI H, CHANG G J, XIE H, TRENT D W, BARRETT A D. Molecular basis of attenuation of neurovirulence of wild-type Japanese encephalitis virus strain SA14. Journal of General Virology, 1995, 76(2): 409-413.

[30] AIHARA S, RAO C M, YU Y X, LEE T, WATANABE K, KOMIYA T, SUMIYOSHI H, HASHIMOTO H, NOMOTO A. Identification of mutations that occurred on the genome of Japanese encephalitis virus during the attenuation process. Virus Genes, 1991, 5(2): 95-109.

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