鸭坦布苏病毒的血凝性

doi:10.3864/j.issn.0578-1752.2019.23.022

摘要/Abstract

摘要：

【目的】研究鸭坦布苏病毒是否具有血凝性,确定其凝集的红细胞种类和血凝反应条件。【方法】鸭坦布苏病毒以不同稀释度经脑内途径接种1—3日龄的BALB/c乳鼠,观察乳鼠发病情况;收集发病乳鼠鼠脑,参照文献记载的方法用蔗糖-丙酮提纯病毒液;以适当灭活剂灭活病毒,观察灭活后的病毒液性状,并以6日龄SPF鸡胚进行灭活检验;参照文献记载的方法初步测定病毒液与鸡、鸭、鹅、鸽、猪红细胞悬液的反应性;比较并确定鸭坦布苏病毒进行血凝反应的适宜红细胞悬液浓度;比较pH6.0—7.0的反应体系对病毒血凝效价的影响;比较4℃、室温和37℃对病毒血凝反应结果的影响。【结果】乳鼠接种鸭坦布苏病毒后6日,1﹕10稀释接种组的乳鼠出现严重的临床症状,表现为四肢抽搐,瘫痪等。大部分乳鼠处于濒死状态,1﹕50和1﹕100稀释接种组的乳鼠症状稍轻于1﹕10稀释接种组乳鼠的症状;提纯后鸭坦布苏病毒液呈淡红色澄明液体,用甲醛灭活后病毒液性状无明显改变,但用β-丙内酯后产生大量絮状沉淀,性状发生明显改变;病毒液可以凝集鸡、鸭、鹅、鸽、猪红细胞悬液;不同浓度红细胞悬液的比较结果显示,病毒液与0.33%的红细胞悬液作用时凝集图形清晰稳定;通过不同pH反应体系的比较,发现病毒液在pH 6.0—6.8时均可发生血凝反应,当pH为6.0—6.2时凝集效价最高;通过不同反应温度的比较,证实病毒液在4℃、室温和37℃时均具有血凝性,但4℃时的血凝反应时间明显延长。【结论】乳鼠增殖的鸭坦布苏病毒提纯后具有广谱的血凝性,可以凝集鸡、鸭、鹅、鸽和猪的红细胞,血凝性稳定,在4℃、室温和37℃下均可以与鹅红细胞凝集,以0.33%浓度的红细胞悬液血凝为宜,反应最适pH为6.0—6.2。

关键词: 鸭坦布苏病毒, 血凝, 反应条件

Abstract:

【Objective】 The objective of this paper was to study the hemagglutinating activity of duck Tembusu virus (DTMUV). 【Method】The DTMUV was inoculated intracerebrally in the 1-3-day-old sucking mice of BALB/c in different dilution to observe the morbidity. The brains of sucking mice were collected and purified according to the referenced methods. The purified virus fluid was inactivated by suitable inactivator, followed by the inactivated inspection in 6-day-old SPF chicken embryo. The virus fluid was evaluated the hemagglutinating activity with the erythrocyte suspension of chicken, duck, goose, pigeon and swine. The concentration of the erythrocyte suspension used in the hemagglutination test was compared to select the proper concentration. The pH value of the reaction system between 6.0-7.0 and the reaction temperature including 4℃, room temperature and 37℃ were compared. 【Result】 In the 6 day after inoculation , the sucking mice inoculated with 1:10 dilution appeared severe clinical symptoms including twitch, paralysis, and most of them were in agonal stage, while the symptoms of the sucking mice inoculated with 1:50 and 1:100 dilution were slighter. The purified DTMUV fluid was pinkey and clarified. It could be inactivated by formaldehyde but not β-propiolactone that would produce lots of flocks and change the property of the fluid. The DTMUV could hemagglutinate with the erythrocyte suspension of chicken, duck, goose, pigeon and swine, and the agglutination graphics was clear and stable when the concentration of the erythrocyte suspension was 0.33%. The hemagglutinating activity of DTMUV could be showed when the reaction system pH value was 6.0-6.8, while the highest hemagglutination titer appeared during the pH 6.0-6.2. Furthermore, the hemagglutinating activity could be appeared at 4℃, room temperature and 37℃, respectively, although the time required for hemagglutination at 4℃ was much longer. 【Conclusion】The DTMUV proliferated in the sucking mice of BALB/c had a broad spectrum of hemagglutinating activity that could hemagglutinate with the erythrocyte suspension of chicken, duck, goose, pigeon and swine. The hemagglutinating activity was stable and could hemagglutinate with the goose erythrocyte at 4℃, room temperature and 37℃. The suitable reaction conditions were 0.33% concentration of the erythrocyte suspension and pH 6.0-6.2 of the reaction system.

Key words: duck Tembusu virus, hemagglutinating activity, reaction conditions

王小蕾, 刘月焕, 段会娟, 刘立新, 杨志远, 赵际成, 潘洁, 刘瑞华, 赵文奇, 田方杰, 吕金宝, 林健. 鸭坦布苏病毒的血凝性[J]. 中国农业科学, 2019, 52(23): 4415-4422.

WANG XiaoLei, LIU YueHuan, DUAN HuiJuan, LIU LiXin, YANG ZhiYuan, ZHAO JiCheng, PAN Jie, LIU RuiHua, ZHAO WenQi, TIAN FangJie, Lü JinBao, LIN Jian. Hemagglutinating Activity of Duck Tembusu Virus[J]. Scientia Agricultura Sinica, 2019, 52(23): 4415-4422.

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稀释度 Dilution	接种数（只） No. of sucking mice	接种后不同时间（日）出现临床症状的鼠数 No. of mice appearing clinical symptoms at each day post inoculation
稀释度 Dilution	接种数（只） No. of sucking mice	1	2	3	4	5	6
1:10	20	N	N	N	N	A	C
1:50	193	N	N	N	N	B	C
1:100	120	N	N	N	N	B	C

灭活剂 Inactivator	终浓度 Final concentration (%)	灭活时间Inactivation hours (h)
灭活剂 Inactivator	终浓度 Final concentration (%)	24	48	72	96	120
甲醛 Formaldehyde	0.05	0^A/5^B	0/5	0/5	0/5	0/5
	0.1	0/5	0/5	0/5	0/5	0/5
	0.15	0/5	0/5	0/5	0/5	0/5
β-丙内酯 β-Propiolactone	0.0125	ND	ND	ND	ND	ND
	0.025	ND	ND	ND	ND	ND
	0.05	ND	ND	ND	ND	ND

动物 Animal	日龄 Age (d)	性别 Gender	HA效价 HA titer	凝集图形 Agglutination graphics
猪（长白猪） Swine (Landrace)	60	公母混合（3头） Mixed of male and female	1:256	红细胞对照不完全下沉,形成环状 The erythrocyte control sank not completely and formed a ring
鸡（SPF） Chicken (SPF chicken)	5	公（3只）Male	1:256	均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination
	360	公（3只）Male	1:256		均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination
	鸭（北京鸭） Duck (Peking duck)	60	母（3只）Female	1:512		均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination
	鸽（信鸽） Pigeon (Carrier pigeon)	360	公母混合（3只） Mixed of male and female	1:512	均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination
鹅（白鹅） Goose (White goose)	180	公母混合（3只） Mixed of male and female	1:512	均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination

鹅红细胞浓度 Concentration of goose erythrocyte	HA效价 HA titer	凝集图形 Agglutination graphics
0.2%	1:1024	红细胞对照孔下沉点小,结果不易判定 The erythrocyte control sank as a tiny point so that the results were difficult to interpretation
0.33%	1:512	均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination
0.5%	1:256	均一薄膜状凝集,清晰,有解凝迹象 Clear homogeneous thin-film agglutination
1%	1:128	均一薄膜状凝集,解凝快,不稳定 Clear homogeneous thin-film agglutination with fast dissolving

pH值 pH value	HA效价 HA titer	凝集图形 Agglutination graphics
6.0	1:512	均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination
6.2	1:512	均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination
6.4	1:256	均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination
6.6	1:256	均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination
6.8	1:128	均一薄膜状凝集,清晰稳定 Clear and stable homogeneous thin-film agglutination
7.0	1:64	易解凝,结果不易判定 The thin-film agglutination was easy to dissolve so that the result was difficult to interpretation