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

Abstract

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

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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References 40

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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

Virulence, E Gene Sequence and Antigenic Difference of 4 Duck Tembusu Virus Isolations

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