传染性法氏囊病毒的抗原及分子特征

用鸡胚成纤维细胞对来自野外的 5 个传染性法氏囊病毒株 (IBDV-JD1 、 JD2 、 NB 、 HZ1 、 HZ2) 进行分离，测定理化特性、致病性，同时进行血清亚型测定及 A 片段基因组的克隆分析 . 试验所用 5 个法氏囊组织悬液在鸡胚成纤维细胞盲传 2~14 代后适应细胞并产生细胞病变 . 细胞适应的 IBDV 毒株的理化和形态特征与经典传染性法氏囊病毒株一致 . 除 IBDV-HZ1 、 HZ2 属经典 IBDV 血清型外， IBDV-JD1 、 JD2 和 NB 毒株分属不同的血清亚型 . 人工感染实验结果显示，分离的 IBDV 毒株产生与野外病例相似的临床症状和病变，出现法氏囊滤泡髓质的淋巴细胞变性、坏死和消失 . 基因组序列分析显示， IBDV-NB 毒株 A 片段由 3 264 个核苷酸组成，编码由 145 个氨基酸残基组成的 VP5 和由 1 012 个氨基酸残基组成的多聚蛋白 . 与来自 GenBank 的 IBDV Ⅰ型毒株比较， NB 毒株 A 片段编码的多聚蛋白与 JD1 毒株的同源性最高，达 99.5% ， VP2 与 JD1 、 CEF94 、 D78 的同源性为 99.8% ， VP3 与 JD1 的同源性为 99.2% ， VP4 与 JD1 的同源性为 100% ， VP5 与 JD1 ， HZ2 ， P2 ， CEF94 ， CT ， Cu-1 和 D78 毒株的同源性为 99.3%. NB 毒株 VP2 蛋白的第 253 、 280 、 284 位氨基酸残基与 IBDV 变异毒株和经典毒株一致，但不同于 IBDV 超强毒株 . 这些结果暗示 IBDV 的抗原表位是构象依赖性表位， IBDV 血清亚型的形成与 IBDV 弱毒疫苗病毒株密切相关 .

Antigenic and Molecular Characterization of Infectious Bursal Disease Virus in China From Layer Chicken Flocks

Five field isolates of infectious bursal disease virus (IBDV) were isolated from bursae of sick young chickens by inoculating chicken embryo fibroblasts (CEF) while the morphological and physiochemical properities of these isolates were detected. Subtype of the virus isolates were analysed by cross virus neutralization. Virulence of IBDV isolates was determined with specific pathogen-free (SPF) chicken inoculation. Nucleotide sequence of genomic segnent A of the isolate NB was further sequenced and analysed. All bursal suspensions produced cytopathic effects (CPE) in CEF after2～14 passages. The physiochemical and morphological properties of IBDV isolates were in accordance with that of typical IBDV. The three CEF-adapted virus isolates JD1, JD2 and NB were divided into 3 subtypes of serotype Ⅰ IBDV except IBDV isolates HZ1 and HZ2 belonged to classical IBDV. In a pathogenicity experiment, the clinical signs and bursal atrophy resembling those of field outbreaks were produced and microscopic bursa lesions revealed that there was degeneration, necrosis and disappearance of lymphocytes in the medullary area of bursal follicles. Sequence data demonstrated that genomic A-segment of the isolate NB was composed of 3 259 nucleotides, encoding VP5 of 145 amino acid residues and the polyprotein (VP243) of 1 012 amino acid residues. Compared with serotype Ⅰ IBDV strains from GenBank, within serotype Ⅰ IBDV strains, the isolate NB has the highest identity to the polyprotein of the isolate JD1(99.5%), VP2 of the isolates JD1, CEF94 and D78 (99.8%), VP3 of the isolate JD1 (99.2%), VP4 of the isolate JD1 (100%)and VP5 of the isolates JD1, HZ2, P2, CEF94, CT, Cu-1 and D78 (99.3%). In the VP2-predicted amino acid sequence of the isolate NB, amino acid residues at positions 253, 280 and 284 were consistent with and other published variant and classical IBDV strains, and different from very virulent IBDV. These results indicated that antigenic epitopes of IBDV are conformational and subtype forming of IBDV isolates originated from classical IBDV attenuated vaccine strains.