Changes in VP2 gene during the attenuation of very virulent infectious bursal disease virus strain Gx isolated in China

Very virulent (vv) infectious bursal disease virus (IBDV) Gx strain with high pathogenicity was attenuated through replication in specific-pathogen-free (SPF) chicken embryos and in chicken embryo fibroblast (CEF) cell cultures. The changes in VP2 nucleotide and the deduced amino acid sequences were obtained during attenuation of vvIBDV in CEF culture. Sequence analysis of selected passages from numbers 0 to 20 in CEFs (designated here Gx to CEF-20) showed that no changes were detectable in the VP2 gene before CEF-7. There were a few changes in the nucleotide sequence of the VP2 gene but no amino acid substitutions at CEF-8. The virus of CEF-9 was an intermediate with some amino acid changes that possibly were related to virulence. CEF-10 virus had become similar to CU-1 strain. The VP2 gene sequence remained the same from CEF-10 to CEF-20. The results of pathogenicity tests showed that the mortalities of Gx, CEF-5, CEF-8, and CEF-9 in 4-wk-old SPF chickens were 64%, 60%, 60%, and 32%, respectively; whereas CEF-10, CEF-15, and CEF-20 were nonpathogenic. Virus neutralization tests with Gx strain showed that the antigenicities are similar from Gx to CEF-20.     

鸡传染性法氏囊病病毒超强株VP2基因重组新城疫病毒胚胎免疫安全性及效力试验

为研制能够同时预防传染性法氏囊病(IBD)和新城疫(ND)的疫苗,本研究选用表达IBD病毒(IBDV)超强毒株(vvIBDV)VP2基因的重组ND病毒(NDV)LaSota疫苗株(rL-VP2),测定其半数鸡胚感染量、平均鸡胚致死时间、脑内接种指数和静脉内致病指数等指标,按不同剂量接种18胚龄SPF鸡胚,分别于出雏后第9d、第14d和第21d采血,用微量凝集法和ELISA方法测定血清中抗NDV抗体和抗IBDV抗体水平,并于出雏后28d用NDV强毒(F48E9株)和vvIBDVGx株攻毒,评估重组疫苗的胚胎免疫效果。结果显示:按104EID50/枚剂量进行免疫不会影响SPF鸡胚出雏率和出雏后21d存活率,并且该免疫组雏鸡能够对NDV强毒和vvIBDV攻毒提供安全的免疫保护。     

传染性法氏囊病超强病毒株VP2基因原核表达及其免疫原性检测

为检测传染性法氏囊病超强毒株(vvIBDV)重组VP2蛋白的免疫原性,本研究利用RT-PCR方法扩增vvIBDV的结构蛋白VP2基因,并将其克隆到表达载体pGEX-4T-3中,构建重组表达质粒pGEX-VP2。将其转化受体菌E.coli BL21(DE3)plysS,经IPTG诱导后,SDS-PAGE电泳和western blot分析表明,表达的重组蛋白约69 ku,并以包涵体形式存在。表达的重组蛋白经纯化后,免疫6周龄BALB/c小鼠制备免疫血清,ELISA分析表明制备的血清效价在1∶5 120以上,表明vvIBDV VP2具有良好的免疫原性,为建立vvIBDV的ELISA检测方法提供了试验依据。     

鸡传染性法氏囊病毒江苏地方株的分离及其VP2基因分析

用SPF鸡胚从江苏溧水某鸡场病死鸡的法氏囊组织中分离到1株法氏囊病毒(IBDV-LS株),该病毒不能凝集鸡的红细胞,琼扩试验证明与IBDV标准阳性血清发生反应,应用RT-PCR扩增IBDV VP2基因,测序后并与GenBank中的IBDV已知序列进行比较。结果表明,分离的IB-DV-LS与国内外超强毒的核苷酸同源性在94.6%以上,推导氨基酸同源性96.6%,并与超强毒株处于进化树同一分支;说明IBDV-LS属IBDV超强毒株,而亲水区内个别氨基酸的替换,提示该毒株已发生一定的变异。     

广西地区传染性囊病病毒超强毒株的分离鉴定及其VP2基因高变区的序列分析

研究通过鸡胚绒毛尿囊膜(CAM)接种、琼脂扩散试验、致细胞病变和RT-PCR等方法,成功从广西疑似病鸡的腔上囊组织中获得了8个传染性囊病病毒(IBDV)毒株,分离株可致攻毒鸡发病率100%,死亡率60%～90%;分子特征上,8个分离株VP2高变区在酶切位点和特征性氨基酸上均属于vvIBDV的特征.同源性分析表明,8个分离株之间在核苷酸和氨基酸上的同源性分别为96.8%～99.1%和95.9%～99.3%,与其他参考vvIBDV毒株的同源性分别为94%～97.7%和92.5%～97.3%,而与常用疫苗株Bursine-2、B87(in),变异株GLS,经典强毒株的同源性仅有90%左右.遗传进化树上,8个分离株与国内外参考vvIBDV同处于一个大分支,与疫苗株和经典强毒株的距离较远,8个分离株与中国内地毒株SD1/97的亲缘关系最近.研究表明,广西仍然存在vvIBDV的流行.     

Molecular characterization of two Bangladeshi infectious bursal disease virus isolates using the hypervariable sequence of VP2 as a genetic marker

Two Bangladeshi infectious bursal disease virus (IBDV) isolates collected in 2007, termed GB1 and GB3, were subjected to comparative sequencing and phylogenetic analyses. Sequence analysis of a 474-bp hypervariable region in the VP2 gene revealed that among four major amino acid substitutions observed in the strains, two were unique to GB1 and GB3 (Ser217Leu and Ala270Thr) while one substitution was only found in GB1 (Asn299Ser). Among IBDVs from Bangladesh including GB1 and GB3, the rate of identity and homology was around 97~99%. The amino acid sequences of GB1 and GB3 differ from those of previous Bangladeshi IBDV isolates and contain amino acid substitutions Pro222Ala and Asn299Ser (in GB3 only). Phylogenetic analysis revealed that GB1 and GB3 are grouped with other very virulent IBDVs of European and American origin in contrast to two previously isolated Bangladeshi IBDV strains (GenBank accession Nos. {"type":"entrez-nucleotide","attrs":{"text":"AF362776","term_id":"14582985","term_text":"AF362776"}}AF362776 and {"type":"entrez-nucleotide","attrs":{"text":"AF260317","term_id":"13957666","term_text":"AF260317"}}AF260317), which belong to the Asian group. It was concluded that GB1 and GB3 belong to a very virulent group of IBDVs. However, amino acid sequences of GB1 and GB3 differ from those of the other Bangladeshi IBDVs by one or two amino acids encoded in the hypervariable region of the VP2 gene.     

鸡传染性法氏囊病病毒超强毒株VP2基因突变对细胞嗜性改变的研究

为研究鸡传染性法氏囊病病毒超强毒株(vvIBDV)细胞嗜性改变的分子基础,本研究通过定点突变、重叠延伸PCR(SOE-PCR)等技术,以vvIBDV HLJ-0504株为骨架构建了两组感染性克隆,并借助已建立的反向遗传操作技术进行病毒拯救。IFA检测、电镜观察及RT-PCR鉴定均显示:Q253H/A284T双点突变的pCAGGHLJ0504A889/980HRT和pCAGGHLJ0504BHRT共转染DF1细胞成功拯救出重组病毒(rHLJ0504HT),而未进行双点突变的pCAGGHLJ0504AHRT和pCAGGHLJ0504BHRT共转染组未获得重组病毒。上述结果表明,双点突变Q253H/A284T能使vvIBDV HLJ-0504适应非允许细胞CEF,但未进行Q253H/A284T双点突变的vvIBDV HLJ-0504不能感染非允许细胞CEF,因此,该研究表明VP2的Q253H/A284T两个氨基酸突变是vvIBDV细胞嗜性改变的分子基础。     

鸡传染性法氏囊病超强毒Gx株感染性分子克隆的构建

本研究以鸡传染性法氏囊病超强毒Gx株(野毒株)基因组为模板,用蛋白酶K法提取病毒基因组核酸dsRNA,在cDNA克隆的5'端上游引入了T7启动子序列,采用Long-accurateRT-PCR(LA-PCR)一步法扩增并克隆了病毒基因组A节段与B节段全长cDNA。序列测定结果表明,基因组A节段全长共3267个核苷酸,包括5'及3'端的非编码区和两个部分重叠的开放阅读框,基因组B节段全长共2843个核苷酸,包括5'及3'端的非编码区和一个开放阅读框。将IBDV-Gx株的A节段全长基因组及B节段全长基因组分别克隆入pMD18-T载体,构建成pMD-A、pMD-B两个带有T7启动子的重组质粒。两个重组质粒线性化后,进行体外转录,然后共电转染于鸡胚成纤维细胞37℃培养72h并传代。收获的细胞传代培养物分别用RT-PCR、间接免疫荧光、蚀斑试验等方法进行鉴定,结果用RT-PCR扩增出了VP3及VP5基因片段,间接免疫荧光检测到了特异性的荧光抗体,蚀斑试验结果表明蚀斑形成单位为3×103PFU/mL。     

Direct evidence of reassortment and mutant spectrum analysis of a very virulent infectious bursal disease virus

The complete genomic sequence of very virulent infectious bursal disease virus (vvIBDV) Gx strain was determined, including the sequences of segment A, encoding the precursor polyprotein, and segment B, encoding the viral RNA polymerase (VP1) and 5'- and 3'-untranslating regions. Alignment of segment A of Gx with the sequences of 12 other vvIBDV strains showed 97.5% to 99.0% amino acid identity, whereas alignment of segment B of Gx with nine other vvIBDV strains revealed high sequence divergence, ranging from 10.3% to 11%. Phylogenetic analysis of segments A and B showed that they were in different branches, indicating that the reassortment occurred in this strain and that segment A and segment B derived from different pathotype strains. The mutant spectrum analysis of quasispecies virus demonstrated that the mean minimum mutation frequency in VP1 was 8.78-fold higher than in the polyprotein. The most frequent mutations were in the first 1986 nucleotides (nonsynonymous mutations) and the last 660 nucleotides (synonymous mutations), indicating that the 219 amino acid residues in the C-terminal of the VP1 form a functional region.     

传染性法氏囊病病毒超强毒株HLJ-0504全基因组克隆及其新特征分析

为了解传染性法氏囊病病毒(IBDV)HLJ-0504株的分子生物学特征,本研究利用融合PCR技术获得HLJ-0504株全基因组序列。核苷酸和推导氨基酸序列遗传演化分析发现,HLJ-0504A节段位于IBDV超强毒的分枝上,而B节段则介于超强毒株和减毒株之间,形成一个独立分枝,属于一株新的自然重组病毒。VP2抗原性预测分析表明,HLJ-0504株VP2第Ⅰ亲水区中的氨基酸发生突变(D212N),该突变可能导致了HLJ-0504株抗原性发生漂变。本实验结果为深入研究IBDV的分子特征奠定了基础。     

Baculovirus virions displaying infectious bursal disease virus VP2 protein protect chickens against infectious bursal disease virus infection

Infectious bursal disease (IBD) is an acute and contagious viral infection of young chickens caused by IBD virus (IBDV). The VP2 protein of IBDV is the only antigen for inducing neutralizing antibodies and protective immunity in the natural host. In the current study, we have succeeded in construction of one recombinant baculovirus BacSC-VP2 expressing His6-tagged VP2 with the baculovirus envelope protein gp64 transmembrane domain (TM) and cytoplasmic domain (CTD). The His6-tagged recombinant VP2 was expressed and anchored on the plasma membrane of Sf-9 cells, as examined by western blot and confocal microscopy. Immunogold electron microscopy demonstrated that the VP2 protein of IBDV was successfully displayed on the viral surface. Vaccination of chickens with the VP2-pseudotyped baculovirus vaccine (BacSC-VP2) elicited significantly higher levels of VP2-specific enzyme-linked immunosorbent assay antibodies and neutralizing antibodies than the control groups. IBDV-specific proliferation of lymphocytes was observed in chickens immunized with the recombinant BacSC-VP2. An in vivo challenge study of the recombinant baculovirus BacSC-VP2 showed effective protection against a very virulent (vv) IBDV infection in chickens. In addition, mortality and gross and histopathological findings in the bursa demonstrated the efficacy of the vaccine in reducing virulence of the disease. These results indicate that the recombinant baculovirus BacSC-VP2 can be a potential vaccine against IBDV infections.