一株牛肠病毒F型的全基因组分析及抗体检测间接ELISA方法的建立

doi:10.11843/j.issn.0366-6964.2025.02.030

摘要/Abstract

摘要：

本研究从牛肠病毒(BEV)阳性粪便中通过接种BHK细胞进行蚀斑纯化分离得到一株F型肠道病毒，经过RT-PCR鉴定正确后送生物公司进行全基因测序以及进行TCID₅₀的测定，以MEGA11.0等生物信息学软件对BEV全基因进行分析并构建遗传进化树。以原核表达方式得到含BEV分离株VP3基因的重组蛋白，作为包被抗原，建立检测抗体的间接ELISA方法，并对建立的ELISA方法的特异性、灵敏性、重复性进行测定与用于临床样品的检测。结果显示，BEV分离株的TCID₅₀为10^-6.26·mL^-1，病毒分离株全长为7 439 nt，ORF大小为6 504 bp，编码2 168个氨基酸，与F型肠道病毒BEV4遗传关系最近且核苷酸相似性最高，故分离到的病毒为F型肠道病毒，暂命名为BEV-QD，与其他F型毒株相比，BEV-QD株存在氨基酸的突变以及缺失并且大多集中在P2、P3区。本次建立的抗体检测间接ELISA方法重复性良好；阳性血清稀释1 600倍后结果仍呈现阳性，灵敏性良好；与BVDV等阳性血清不发生特异性结合，特异性良好；检测40份临床样品中，13份为阳性，阳性率为32.5%。本研究分离到一株F型牛肠病毒突变株，建立了一种抗体间接ELISA方法。为牛肠病毒的检测和防控提供了基础手段。

关键词: 牛肠道病毒, 分离鉴定, TCID₅₀, 全基因组分析, ELISA

Abstract:

In this study, a strain of F-type enterovirus was isolated from bovine enterovirus(BEV) positive feces through plaque purification by inoculating BHK cells. After being correctly identified by RT-PCR, it was sent to a biological company for whole-genome sequencing and TCID₅₀ determination. The whole genome of BEV was analyzed and a genetic evolution tree was constructed using bioinformatics software such as MEGA11.0. The recombinant protein containing the VP3 gene of the BEV isolate was obtained through prokaryotic expression and used as a coating antigen to establish an indirect ELISA method for detecting antibodies. The specificity, sensitivity, and repeatability of the established ELISA method were measured and used for the detection of clinical samples. The results showed that the TCID₅₀ of the BEV isolate was 10^-6.26·mL^-1, and the full length of the virus isolate was 7 439 nt, with an ORF size of 6 504 bp, encoding 2 168 amino acids. It had the closest genetic relationship and the highest nucleotide homology with F-type enterovirus BEV4. Therefore, the isolated virus was an F-type enterovirus, temporarily named BEV-QD. Compared with other F-type strains, the BEV-QD strain had amino acid mutations and deletions, mostly concentrated in the P2 and P3 regions. The indirect ELISA method for antibody detection established in this study had good repeatability; the results remained positive after the positive serum was diluted 1 600 times, indicating good sensitivity; it did not specifically bind to positive sera such as BVDV, indicating good specificity; among the 40 clinical samples tested, 13 were positive, with a positive rate of 32.5%. This study isolated an F-type bovine enterovirus mutant strain and established an indirect ELISA method for antibody detection, providing a basic means for the detection and prevention of bovine enterovirus.

Key words: bovine enterovirus, isolation and identification, TCID₅₀, whole-genome analysis, ELISA

中图分类号:

S855.3

刘健, 于泽海, 张玫瑜, 李丹, 王君, 刘芳琴, 张群, 徐守振. 一株牛肠病毒F型的全基因组分析及抗体检测间接ELISA方法的建立[J]. 畜牧兽医学报, 2025, 56(2): 814-825.

LIU Jian, YU Zehai, ZHANG Meiyu, LI Dan, WANG Jun, LIU Fangqin, ZHANG Qun, XU Shouzhen. Full-genome Analysis of a Bovine Enterovirus Type F and the Establishment of an Indirect ELISA Method for Antibody Detection[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 814-825.

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毒株 Strains	登录号 Accession numbers	国家 Country	分型 Species	宿主 Host
HeN-YR91	MN598018. 1	China	F	牛
HeN-B62	MN598011.1	China	F	牛
BEV-261	DQ092770.1	USA	F	牛
BEVVG527	D00214.1	USA	E	牛
NX-FY40	MN598039.1	China	E	牛
JL-JY42	MN598032.1	China	E	牛
NX-b-DR47	MN598037.1	China	E	牛
SL305	AF123433.1	Australia	E	牛
E1-NSWL7	OP020147.1	Australia	E	牛
BEV-7414	NC001859	USA	E	牛
PS87F3	DQ092794.1	USA	F	牛
PS89F2	DQ092795.1	USA	F	牛
Porcine enterovirus type 9	Y14459	United Kingdom	G	猪
Ovine enterovirus TB4-OEV	JQ277724	Hungary	G	羊
Human enterovirus 88 strain BAN01-10398	AY843306	USA	B	人
F4	AY462106.1	USA	F	牛
A71F5	MG674288.1	China	EV-A	人
BEV4	ON986117.1	China	F	牛
128135	HQ424437.2	Greece	EV-A	人

功能区 Function region	位置 Position	核酸数目/ bp Nucleotide number
5’UTR	1-822 bp	822
VP4	823-1 029 bp	207
VP2	1 030-1 767 bp	738
VP3	1 768-2 496 bp	729
VP1	2 497-3 321 bp	825
2A	3 322-3 822 bp	501
2B	3 723-4 119 bp	297
2C	4 120-5 106 bp	987
3A	5 107-5 373 bp	267
3B	5 374-5 442 bp	69
3C	5 443-5 991 bp	549
3D	5 992-7 326 bp	1 335
3’UTR	7 327-7 439bp	113

毒株Strain	全基因组 Whole genome	ORF	5’UTR	VP1	VP2	VP3
HeN-YR91	81.0	78.9	95.6	72.5	73.3	72.7
HeN-B62	79.0	77.8	85.9	66.8	71.5	70.6
BEV-261	79.4	78.0	89.2	70.3	72.9	74.5
BEVVG527	68.5	67.2	79.0	54.9	67.3	60.7
NX-FY40	69.3	67.9	77.8	55.6	66.3	64.0
JL-JY42	70.2	68.6	80.6	53.0	65.4	64.3
NX-b-DR47	68.8	67.3	75.4	55.2	68.4	62.9
SL305	68.7	67.6	76.6	60.1	66.0	63.5
E1-NSWL7	68.8	67.5	78.7	54.7	64.9	62.1
BEV-7414	68.5	67.2	79.0	54.9	67.3	60.7
PS87	78.3	76.6	89.8	65.8	69.5	69.7
PS89	80.0	78.8	88.0	74.5	74.1	73.3
Porcine enterovirus type 9	62.8	63.4	45.2	54.4	62.6	60.2
Ovine enterovirus TB4-OEV	65.3	63.5	78.0	51.8	63.1	63.2
Human enterovirus 88 strain BAN01-10398	59.0	60.8	47.8	20.1	60.0	54.8
F4	75.1	74.3	76.7	60.2	70.2	68.2
A71F5	58.8	59.5	44.0	36.7	62.3	51.2
BEV4	88.4	87.6	94.7	87.5	86.9	89.0
128135	59.1	60.7	48.8	44.7	62.5	59.2

区域 Area	功能区 Function region	氨基酸突变位置 Amino acid mutation location	氨基酸Amino acid
区域 Area	功能区 Function region	氨基酸突变位置 Amino acid mutation location	突变前Before mutation	突变后After mutation
P1区	VP2	167	F	Y
	VP2	224	P	N
	VP3	375	N/S	A
	VP1	616	T	L
	VP1	640	D/N	T
P2区	2A	902	M/N	S
		922	L	I
		964	P/F	L
	2B	1 054	L	V
	2C	1 308	P	S
	2C	1 320	P/T	I
P3区	3C	1 541	C	S
	3C	1 626	H/D	Y
	3D	1 844	C	R
		1 911	P/S	L
		1 935	A	V
		1 955	P	L
		1 957	P	L

血清 Serum	OD_{450 nm}(${\bar x}$ ± s)	变异系数/% CV	结果判定 Result determination
BEV抗体阳性血清	0.996±0.032	3.213	+
Map阳性血清	0.340±0.038	11.173	-
BVDV阳性血清	0.353±0.047	4.798	-
IBRV阳性血清	0.324±0.047	14.370	-
MB阳性血清	0.393±0.034	8.781	-