CAJ | 학술논문

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.
용계배성섬유세포대래자야외적5개전염성법씨낭병독주(IBDV-JD1、JD2、NB、HZ1、HZ2)진행분리,측정이화특성、치병성,동시진행혈청아형측정급A편단기인조적극륭분석.시험소용5개법씨낭조직현액재계배성섬유세포맹전2～14대후괄응세포병산생세포병변.세포괄응적IBDV독주적이화화형태특정여경전전염성법씨낭병독주일치.제IBDV-HZ1、HZ2속경전IBDV혈청형외,IBDV-JD1、JD2화NB독주분속불동적혈청아형.인공감염실험결과현시,분리적IBDV독주산생여야외병례상사적림상증상화병변,출현법씨낭려포수질적림파세포변성、배사화소실.기인조서렬분석현시,IBDV-NB독주A편단유3 264개핵감산조성,편마유145개안기산잔기조성적VP5화유1 012개안기산잔기조성적다취단백.여래자GenBank적IBDV Ⅰ형독주비교,NB독주A편단편마적다취단백여JD1독주적동원성최고,체99.5%,VP2여JD1、CEF94、D78적동원성위99.8%,VP3여JD1적동원성위99.2%,VP4여JD1적동원성위100%,VP5여JD1,HZ2,P2,CEF94,CT,Cu-1화D78독주적동원성위99.3%.NB독주VP2단백적제253、280、284위안기산잔기여IBDV변이독주화경전독주일치,단불동우IBDV초강독주.저사결과암시IBDV적항원표위시구상의뢰성표위,IBDV혈청아형적형성여IBDV약독역묘병독주밀절상관.
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.