1株牛结节性皮肤病病毒的分离鉴定与生物学特性研究

doi:10.16431/j.cnki.1671-7236.2025.06.031

摘要/Abstract

摘要： 【目的】了解山东地区牛结节性皮肤病病毒(Lumpy skin disease virus，LSDV)流行株的病原学特性。【方法】采集山东某牛场疑似结节性皮肤病(lumpy skin disease，LSD)牛的皮肤结节组织，用原代羔羊睾丸细胞(primary lamb testicular cells，PLT)进行病毒分离；通过PCR扩增LSDV G蛋白偶联受体(G-protein-coupled chemokine receptor,GPCR)基因并测序，利用抗LSDV特异性血清进行间接免疫荧光试验(IFA)鉴定病毒特异性；对分离毒株进行基因组测序并分析其遗传变异情况；在不同细胞中对分离毒株连续传代并测定病毒滴度，绘制生长曲线，进一步测定其在不同细胞中的增殖能力；通过抗体中和试验测定分离毒株对山羊痘疫苗抗体的中和指数。【结果】病牛皮肤结节组织样品经PLT细胞培养，盲传3代后出现细胞变圆、聚集、脱落为特征的细胞病变效应(cytopathic effect，CPE)。PCR扩增获得大小约1 010 bp的分离毒株GPCR基因特异性条带，且该基因序列与国内外LSDV毒株相似性为99.3%～100%。分离毒株可与抗LSDV特异性血清结合，免疫荧光染色后出现特异性绿色荧光斑。基因组测序比对结果显示，分离毒株与2019—2022年流行的7株国内以及2022年泰国毒株遗传距离较近。分离毒株传代20～30代时，病毒滴度可稳定在10^-6.08至10^-6.17 TCID₅₀/mL。生长曲线显示，该分离毒株滴度在96 h达到峰值(10^-6.16 TCID₅₀/mL)，144 h下降至10^-5.60 TCID₅₀/mL。分离毒株在MBDK和BHK-21细胞系上均能连续增殖3代，其病毒滴度可分别达10^-4.85和10^-4.63 TCID₅₀/mL。山羊痘疫苗抗体对该毒株的中和指数(276)显著低于山羊痘疫苗毒株(1 833)(P＜0.05)。【结论】从病牛皮肤结节组织中成功分离到1株LSDV，该毒株与国内除西藏地区外的其他地区的毒株遗传距离较近，在MBDK和BHK-21细胞中可稳定传代培养，且山羊痘疫苗抗体对该毒株的中和能力较差。

关键词: 结节性皮肤病病毒(LSDV); 分离鉴定; 生物学特性; 疫苗

Abstract: 【Objective】 The objective of this study was to investigate the etiological characteristics of bovine Lumpy skin disease virus (LSDV) in Shandong.【Method】 Skin nodules of suspected lumpy skin disease (LSD) cattle were collected from a cattle farm in Shandong province,and primary lamb testicular cells (PLT) were used for virus isolation.The LSDV G-protein-coupled chemokine receptor (GPCR) gene was amplified by PCR and sequenced,and the virus specificity was identified by indirect immunofluorescence assay (IFA) using anti-LSDV specific serum.The genome of the isolated strain was sequenced and its genetic variation was analyzed.The isolated strain was passed continuously in different cells,the virus titers were measured,and the growth curve was drawn.The proliferation ability in different cells was further determined.The neutralization index of the isolated strain against the antibodies of goat pox vaccine was determined by antibody neutralization test.【Result】 The nodular tissue samples of diseased cattle were cultured by PLT cells,and after blind transmission for 3 generations,the cytopathic effect (CPE) characterized by cell roundness,aggregation and shedding occurred.PCR amplification obtained 1 010 bp specific band of GPCR gene of the isolated strain，and the similarity of this gene sequence with LSDV strains at home and abroad was 99.3%-100%.The isolated strain could bind to the specific serum against LSDV,and the specific green fluorescent spot appears after immunofluorescence staining.The results of genome sequencing showed that the isolated strain was genetically close to the 7 domestic strains in 2019-2022 and Thailand strain in 2022.The virus titer was stable at 10^-6.08 to 10^-6.17 TCID₅₀/mL when the isolates were passed through 20-30 generations.The growth curve showed that the titer of the isolated strain peaked at 96 h (10^-6.16 TCID₅₀/mL) and decreased to 10^-5.60 TCID₅₀/mL at 144 h.The isolated strain could prolifate continuously on MBDK and BHK-21 cell lines for 3 generations,and the virus titers could reach 10^-4.85 and 10^-4.63 TCID₅₀/mL,respectively.The neutralization index of goat pox vaccine antibody against this strain (276) was significantly lower than that of goat pox vaccine strain (1 833) (P＜ 0.05).【Conclusion】 A strain of LSDV was successfully isolated from the skin nodular tissue of infected cattle.This strain was genetically close to other strains in China except Tibet,and could be stably cultured in MBDK and BHK-21 cells.Moreover,the neutralization ability of goat pox vaccine antibody against this strain was poor.

Key words: Lumpy skin disease virus (LSDV); isolation and identification; biological characteristics; vaccine

中图分类号:

S858.23

赵一龙, 唐娜, 井长华, 徐晴晴, 尹秀升, 王海明, 孙婧, 林盼盼, 董林, 刘吉山, 曹荣峰. 1株牛结节性皮肤病病毒的分离鉴定与生物学特性研究[J]. 中国畜牧兽医, 2025, 52(6): 2790-2799.

ZHAO Yilong, TANG Na, JING Changhua, XU Qingqing, YIN Xiusheng, WANG Haiming, SUN Jing, LIN Panpan, DONG Lin, LIU Jishan, CAO Rongfeng. Isolation,Identification and Biological Characteristics of a Strain of Lumpy Skin Disease Virus[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2790-2799.

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