一株新型鸭呼肠孤病毒的分离及抗体检测的间接ELISA建立

doi:10.11843/j.issn.0366-6964.2024.12.028

摘要/Abstract

摘要：

新型鸭呼肠孤病毒(Novel duck reovirus, NDRV)能造成多品种雏鸭肝、脾的出血坏死，并造成免疫抑制，给我国的水禽养殖业造成了严重经济损失，但市场上仍无商品化检测试剂盒。基于此，本研究通过蚀斑纯化从临床病料中成功分离到一株纯净的新型鸭呼肠孤病毒(命名为E232株)，并以原核表达的σC蛋白为包被原建立了能够检测NDRV血清抗体的间接ELISA。遗传进化分析结果显示，E232株属于NDRV，且与其他NDRV毒株的核苷酸同源性为96.4%~98.9%。动物回归试验结果表明，E232株发病率达到100%，发病雏鸭的脾出现严重肿胀、出血、坏死，泄殖腔排毒量和咽部排毒量均在感染后第5天达到高峰。建立的间接ELISA具有良好的特异性，与其他鸭病病原阳性血清均无交叉反应；检测NDRV阳性血清最低检出效价为1∶6 400，批内重复变异系数为1.53%~7.24%，批间重复变异系数为1.84%~8.30%，检测236份临床血清样品的阳性率为80.93%。综上，本研究E232株的成功分离，丰富了我国NDRV流行病学信息库；间接ELISA的成功建立，为临床NDRV防控提供了良好的技术手段。

关键词: 新型鸭呼肠孤病毒, 分离鉴定, σC蛋白, 间接ELISA

Abstract:

Novel duck reovirus (NDRV), which can infect ducklings of many breeds, leads to hemorrhagic necrosis of liver and spleen and cause immunosuppression, and has caused serious economic losses to Chinese waterfowl farming industry. However, there are still no commercial test kits on the market. In this study, a pure new duck reovirus was successfully isolated from clinical materials by plaque purification, named as E232 strain. And by using σC protein expressed in prokaryotes as the coating antigen, an indirect ELISA was established to detect NDRV serum antibodies. The genetic evolution results show that E232 belongs to NDRV and has 96.4%-98.9% nucleotide homology with other NDRV strains. Animal regression test showed that the incidence of E232 is 100%. The spleens of the affected ducklings showed severe swelling, bleeding and necrosis, and the cloacal detoxification and pharynx detoxification reached the peak on the 5th day after infection. This assay detected NDRV antibody titer in positive sera at 1∶6 400 dilution, the coefficients of variation of intra batch and inter batch repeatability tests were 1.53%-7.24% and 1.84%-8.30%. It showed 80.93% positive rate for 236 clinical serum samples but did not react with any other disease positive sera. In conclusion, the successful isolation of the E232 strain in this study has enriched the epidemiological information database of NDRV in China. The successful establishment of the indirect ELISA provides a good technical means for the prevention and control of NDRV.

Key words: novel duck reovirus, isolation and identification, σC protein, indirect ELISA

中图分类号:

S852.659.4

徐晨晨, 马旭杰, 宋素泉, 闫丽萍. 一株新型鸭呼肠孤病毒的分离及抗体检测的间接ELISA建立[J]. 畜牧兽医学报, 2024, 55(12): 5651-5662.

XU Chenchen, MA Xujie, SONG Suquan, YAN Liping. Isolation of a Novel Duck Reovirus Strain and Establishment of Indirect ELISA for Detecting Antibodies[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(12): 5651-5662.

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毒株 Strains	毒株类型 Type of strains	GenBank登录号 GenBank accession number	毒株 Strains	毒株类型 Type of strains	GenBank登录号 GenBank accession number
YL	NDRV	MZ733722.1	YB	MDRV	KX831117.1
SY	NDRV	MK955827.1	C4	MDRV	DQ066924.1
LRS-GD20	NDRV	OM930751.1	ZJ99	MDRV	AY619690.1
XT18	NDRV	MK749407.1	815-12	MDRV	KC508656.1
GL18	NDRV	MW924628.1	HN21	MDRV	OK626889.1
LY20	NDRV	ON009436.1	S1133	ARV	AF330703.1
QR	NDRV	MZ736874.1	P100	ARV	DQ868791.1
SD-12	NDRV	KJ879930.1	GX	ARV	KJ476705.1
TH11	NDRV	KC493571.1	C-98	ARV	EF057397.1
091	NDRV	JX478256.1	Fahey-Crawley	ARV	DQ868789.1
ZJ2000M	MDRV	KF306091.1	GuangxiR1	ARV	KC183744.1
S12	MDRV	DQ643970.1	SD10-1	ARV	KP288863.1
MW9710	MDRV	AY580159.1	B-98	ARV	DQ643974.1
S14	MDRV	DQ066925.1	1733	ARV	AF004857.1

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