4株鸭坦布苏病毒的毒力、E基因序列和抗原差异性

doi:10.3864/j.issn.0578-1752.2019.23.021

摘要/Abstract

摘要：

【目的】比较鸭坦布苏病毒（duck tembusu virus, DTMUV）分离株的毒力、分析E蛋白基因序列、研究抗原差异性,为鸭坦布苏病防控提供依据。【方法】将DTMUV-HB(2011)、DTMUV-AH(2014)、DTMUV-GX1(2012)、DTMUV-GX2(2015) 4株病毒接种10日龄易感鸭胚进行增殖,并利用6日龄SPF鸡胚测定ELD₅₀。根据测定的病毒含量,将分离毒株稀释为100ELD₅₀/0.5mL,人工感染40只180日龄健康北京鸭,进行临床症状观察、病毒分离、大体剖检,分析分离毒株的毒力有无差异。提取8株DTMUV分离病毒株的RNA,通过RT-PCR扩增E基因,分析比较不同地区的DTMUV的E基因核苷酸和氨基酸序列相似性,构建进化树。利用实验室建立的鸭坦布苏病毒血凝抑制试验分别测定4株病毒阳性血清对4株病毒的HI效价。同时采用固定病毒稀释血清法,利用C6/36细胞测定4株病毒阳性血清对4株DTMUV的血清中和效价。通过交叉血凝抑制试验和血清交叉中和试验比较4株病毒的抗原差异性（R值）。【结果】 (1)各毒株的病毒含量范围在10 ^4.7—10 ^5.3 ELD₅₀/0.1mL。人工感染鸭试验,攻毒后3 d鸭采食量和产蛋量均显著下降,各组病毒分离阳性率均在85%以上,剖检病变主要表现为卵泡变形、出血。(2)分离株序列分析结果表明,核苷酸序列相似性为95.7%—100%,推导氨基酸序列相似性在98.2%以上。遗传进化分析表明共同构成了一个进化分支。(3)4株病毒及其阳性血清进行血凝抑制交叉试验,计算的抗原性差异R值在0.79—1.12之间;进行细胞中和交叉试验,计算的R值在0.79—1.20之间。【结论】分离的4株DTMUV在毒力、E蛋白基因和抗原性上未见显著差异。

关键词: 鸭坦布苏病毒, 毒力, E基因, 抗原性

Abstract:

【Objective】The research aimed to compare the virulence of Duck Tembusu Virus (DTMUV), and to analysis its envelope protein gene sequence analysis and antigenic difference. 【Method】Susceptible 10-day-old duck embryos were inoculated with four different DTMUV strains, including DTMUV-HB isolated in 2011, DTMUV-AH isolated in 2014, DTMUV-GX1 isolated in 2012 and DTMUV-GX2 isolated in 2015. The median embryo lethal dose (ELD₅₀) of the four strains was measured with 6-day-old SPF chicken embryos. According to that, forty 180-day-old healthy Peking duck were challenged respectively with four strains which were diluted into 100 ELD₅₀/0.5 mL. The clinical, virological, pathological features of different DTMUV strains infection in ducks were characterized. The viral RNA of eight DTMUV strains were extracted from the allantoic fluid, and then the E gene were amplified by RT-PCR and sequenced. Then the similarity analysis of nucleotide and amino acid sequence and phylogenetic analysis were carried out. The HI titers of 4 antisera against 4 DTMUV strains were determined with duck Tembusu virus hemagglutination inhibition test. The neutralization titers of 4 antisera against 4 DTMUV strains were determined by neutralization assay using C6/36 cell lines. We analyzed the antigenic difference of 4 DTMUV strains by R value, which contained cross hemagglutination inhibition test and cell cross neutralization test. 【Result】(1) The median embryo lethal dose (ELD₅₀) were 10 ^-4.7-10 ^-5.3/0.1ml. The artificial infection test suggested that, feed intake and egg production of the challenged group decreased significantly on 3 days post inoculation (dpi), and the virus positive isolation rate were more than 85% on 2 dpi. The gross lesions of the reproductive system were mainly deformed and hemorrhaged follicular by necropsying on 8 dpi. (2) The results of sequence analysis showed that nucleotide sequence similarity was 95.7% - 100%, and the similarity of amino acid sequence was 98.2%. Genetic evolutionary analysis illustrated that all the DTMUV isolates in this study gathered into the same clade. (3) The R value showed antigenic difference of cross hemagglutination inhibition test were 0.79-1.12, and that of cell cross neutralization test were 0.79-1.20. 【Conclusion】There were no significant difference in virulence, E gene sequence and antigenicity of four DTMUV strains isolated in this study.

Key words: Duck Tembusu virus, virulence, envelope protein gene, antigenicity

杨志远,段会娟,王小蕾,刘立新,赵际成,潘洁,刘月焕,林健. 4株鸭坦布苏病毒的毒力、E基因序列和抗原差异性[J]. 中国农业科学, 2019, 52(23): 4406-4414.

YANG ZhiYuan,DUAN HuiJuan,WANG XiaoLei,LIU LiXin,ZHAO JiCheng,PAN Jie,LIU YueHuan,LIN Jian. Virulence, E Gene Sequence and Antigenic Difference of 4 Duck Tembusu Virus Isolations[J]. Scientia Agricultura Sinica, 2019, 52(23): 4406-4414.

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动物数量（只） Animal	攻毒用毒株 Virus	攻毒途径及剂量 Routes and dose	病毒分离阳性率 Positive rate of virus isolation
8	DTMUV-HB	肌肉注射 Intramuscular injection 0.5mL (100ELD₅₀)	100%(8^A/8^B)
8	DTMUV-AH		87.5%(7/8)
8	DTMUV-GX1		87.5%(7/8)
8	DTMUV-GX2		100%(8/8)
8	/	/	0%(0/8)

毒株 Virus	4株DTMUV阳性血清HI效价 Positive sera HI titer of 4 DTMUV isolates
毒株 Virus	DTMUV-HB	DTMUV-AH	DTMUV-GX1	DTMUV-GX2
DTMUV-HB	1:806.3	1:806.3	1:806.3	1:640.0
DTMUV-AH	1:160.0	1:201.6	1:201.6	1:100.8
DTMUV-GX1	1:127.0	1:127.0	1:100.8	1:100.8
DTMUV-GX2	1:201.6	1:254.0	1:254.0	1:201.6

R值 R value	DTMUV-HB	DTMUV-AH	DTMUV-GX-1	DTMUV-GX-2
DTMUV-HB	**	0.79	0.91	1.20
DTMUV-AH	0.89	**	0.82	0.91
DTMUV-GX-1	1.12	1.12	**	0.93
DTMUV-GX-2	0.89	0.79	1.12	**

毒株 Virus	4株DTMUV阳性血清中和效价 Positive sera neutralization titer of 4 DTMUV isolates
毒株 Virus	DTMUV-HB	DTMUV-AH	DTMUV-GX1	DTMUV-GX2
DTMUV-HB	1:54.6	1:29.3	1:34.9	1:55.4
DTMUV-AH	1:44.3	1:38.3	1:34.9	1:48.9
DTMUV-GX1	1:59.3	1:33.4	1:45.3	1:45.4
DTMUV-GX2	1:62.0	1:28.4	1:37.7	1:44.0