1株传染性法氏囊病病毒的分离鉴定及VP2基因序列分析

doi:10.16431/j.cnki.1671-7236.2022.03.022

摘要/Abstract

摘要： 【目的】研究1株传染性法氏囊病病毒（Infectious bursal disease virus，IBDV）广西分离株的毒力特征及其与VP2基因序列特征的关系，为IBDV的流行病学研究和疫病防控提供参考。【方法】通过PCR、鸡胚传代培养、DF-1细胞的适应培养及琼脂扩散试验等方法分离鉴定病毒，并扩增其VP2基因，利用Mega 7.0、MegAlign软件将其与其他不同毒力IBDV毒株进行核苷酸及氨基酸序列比对分析，并进行SPF鸡致病性试验，用实时荧光定量PCR方法测定IBDV拷贝数变化情况。【结果】成功分离到1株IBDV毒株，命名为GX20210126株。该毒株接种SPF鸡胚可导致鸡胚出现出血、矮化和肝脏发黄、出血及针尖状坏死；且GX20210126对DF-1具有良好的细胞适应性，可引起显著细胞病变。琼脂扩散试验显示，该毒株具有良好的抗原性，病毒液能与IBDV阳性血清之间形成白色沉淀线。VP2基因进化分析显示GX20210126株与国际标准超强毒株在同一个大的分支上，氨基酸序列相似性在86.8%~99.6%之间，其中与UK661株相似性最高，为99.6%，仅有2处氨基酸位点发生改变，即D279N和I272T。以EID₅₀为10^-3.8/0.1 mL，0.5 mL/只的剂量点眼、滴鼻接种8日龄SPF鸡，攻毒后鸡出现羽毛蓬松、精神萎靡、拉白绿色水样粪便等临床症状，剖检可见肌肉、肝脏出血，肾脏尿酸盐沉积，法氏囊萎缩，IBDV拷贝数在感染后第5天达到峰值。【结论】 IBDV广西流行株GX20210126具有超强毒株基因序列特征，人工感染鸡群可导致IBDV典型临床症状和病理变化。

关键词: 传染性法氏囊病病毒(IBDV); VP2蛋白; 分离鉴定; 序列分析

Abstract: 【Objective】 The purpose of this study was to study the virulence characteristics of a strain of Infectious bursal disease virus (IBDV) isolated from Guangxi and its relationship with the sequence characteristics of VP2 gene, so as to provide reference for the epidemiological research and epidemic prevention and control of IBDV.【Method】 The virus was isolated and identified by PCR, chicken embryo subculture, adaptive culture of DF-1 cells and agar diffusion test, and its VP2 gene was amplified.The nucleotide and amino acid sequences were compared with other IBDV strains with different virulence by Mega 7.0 and MegAlign softwares, and the pathogenicity to SPF chicken was tested.The changes of IBDV copy number were determined by Real-time PCR.【Result】 One strain of IBDV was successfully isolated, and named as GX20210126 strain.SPF chicken embryos inoculated with this strain could cause bleeding, dwarfing, liver yellowing, bleeding and acitoid necrosis.Moreover, GX20210126 had good cell adaptability to DF-1 and could cause significant cytopathic changes.The agar diffusion test showed that the strain had good antigenicity, and a white precipitation line could be formed between the virus liquid and the IBDV-positive serum.VP2 gene evolution analysis showed that the GX20210126 strain was on the same branch with the international standard super virulent strain, and the amino acid similarity was between 86.8% and 99.6%.It had the highest similarity with the UK661 strain, and the amino acid similarity was 99.6%.Only two amino acid positions changed, which were D279N and I272T.The 8-day-old SPF chickens were inoculated with EID₅₀ 10^-3.8/0.1 mL, 0.5 mL per chicken.After inoculation, the chickens showed fluffiness, droopiness, white and green water feces, and other clinical symptoms.Necropsy showed muscle and liver hemorrhage, kidney urate deposition, and bursa atrophy.What was more, IBDV copy number peaked on the 5th day.【Conclusion】 The IBDV epidemic strain GX20210126 in Guangxi had the genetic sequence characteristics of a super virulent strain.Artificial infection of chickens could lead to typical clinical symptoms and pathological changes of IBDV.

Key words: Infectious bursal disease virus (IBDV); VP2 protein; isolation and identification; sequence analysis

中图分类号:

S852.65

何献铭, 任广彩, 叶俊贤, 兰虹, 刘郁夫, 熊挺, 杨泽坤, 徐婷, 陈瑞爱. 1株传染性法氏囊病病毒的分离鉴定及VP2基因序列分析[J]. 中国畜牧兽医, 2022, 49(3): 1005-1014.

HE Xianming, REN Guangcai, YE Junxian, LAN Hong, LIU Yufu, XIONG Ting, YANG Zekun, XU Ting, CHEN Ruiai. Isolation and Identification of a Strain of Infectious Bursal Disease Virus and Sequence Analysis of VP2 Gene[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 1005-1014.

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