Viral genotyping of infectious bursal disease viruses isolated from the 2002 acute outbreak in Spain and comparison with previous isolates

An infectious bursal disease (IBD) outbreak occurred in the east region of Spain in the spring of 2002 and rapidly spread thorough the whole country, although proper vaccination programs were applied. In this report, 33 infectious bursal disease viruses (IBDVs) isolated from this outbreak were characterized by nucleotide sequencing of the VP2 gene hypervariable region and were compared with reference IBD strains and the 1990s Spanish IBDVs in order to determine possible emergence of IBDV isolates with modified antigenic or virulent properties. Moreover, histopathologic and immunohistochemical studies of those cases where bursal tissues were available were carried out. Of the 33 isolates, 23 were identified as very virulent IBDVs (vvIBDVs), whereas the other 10 isolates were classified as attenuated or intermediate virulence classical strains and could possibly be IBDV live vaccine strains used in the immunization of these chickens. Results of this study indicate that wIBDV isolates from the 2002 Spanish outbreak are closely related with those from the 1990s outbreak. However, acute IBD cases have not been reported in Spain during these 10 yr. Genetic, management, and environmental factors likely related with IBD reemergence in Spain are discussed. Moreover, our results indicate that good correlation exists between the IBDV subtype present in the field and the degree of lesions in bursa tissue, as well as the immunohistochemistry staining.     

Prokaryotic Expression and Bioinformatics Analysis of VP2 Gene of Infectious Bursal Disease Virus C4 Strain

This study was aimed to investigate the relationship between the virulence characteristics of infectious bursal disease virus(IBDV) C4 strain and its VP2 amino acid sequence. The RNA of IBDV C4 strain was extracted,and its VP2 gene was amplified by RT-PCR.VP2 nucleotide sequences and deduced amino acids of different virulent IBDV strains were compared. At the same time, prokaryotic expression vector pET-32a(+) was used to express the VP2 gene. The expression of recombinant VP2 protein was detected by SDS-PAGE and Western blotting. The results showed that the VP2 gene of IBDV C4 strain belonged to the very virulent infectious bursal disease virus (vvIBDV) in evolutionary relationship, the VP2 nucleotides homology between IBDV C4 strain and other vvIBDV strains were 98.1% to 98.7%, and there were no mutations in S-W-S-A-S-G-S (326-332 amino acids) and 222(A), 256(I), 294(I) and 299(S). The VP2 amino acid sequence of IBDV C4 strain was consistent with the characteristics of other vvIBDV strains. However, there were three differences amino acids sites at 201(D/G), 281(G/R) and 313(V/A) between the amino acids of the C4 strain and the very virulent strain UK661. And the change of 281(R) was in the small hydrophilic region of 279 to 290, which was related to the antigenicity of the virus; The recombinant VP2 protein molecular weight expressed in Escherichia coli BL21 was about 67 ku. This study provided a basis for further research on antigenic changes resulting from amino acid variation of 201(G), 281 (R) and 313(A). These results indicated that the VP2 gene of the IBDV C4 strain was consistent with the major characteristics of the vvIBDV strain VP2 gene. The difference of three amino acid sites in the vvIBDV strain C4 might be related to the evolution of virulence of IBDV strain in China.     

传染性法氏囊病病毒C4毒株VP2基因的原核表达及生物信息学分析

试验旨在研究一株传染性法氏囊病病毒（IBDV）河南分离株的毒力特征及其与VP2氨基酸序列特征的关系。通过提取IBDV C4株RNA,利用RT-PCR扩增其VP2基因,与其他不同毒力IBDV毒株进行核苷酸及推导的氨基酸序列比对分析,同时使用pET-32a（+）原核表达载体表达VP2基因,用SDS-PAGE和Western blotting检测重组VP2蛋白的表达。结果显示,扩增的IBDV C4株的VP2基因序列在进化关系上属于超强毒力IBDV（vvIBDV）分类,与选取的vvIBDV毒株代表毒株核苷酸序列同源性在98.1%~98.7%之间,其七肽区为S-W-S-A-S-G-S（第326-332位氨基酸）符合超强毒株特征,且222（A）、256（I）、294（I）和299（S）位氨基酸与超强毒力毒株的4个特征性氨基酸一致;但IBDV C4毒株的VP2蛋白氨基酸序列与超强毒力毒株代表毒株UK661相比,201（D/G）、281（G/R）、313（V/A）位氨基酸不同,其中281位氨基酸的改变处于279-290的小亲水区内,与病毒抗原性有关;构建的pET-32a（+）-VP2原核表达载体在大肠杆菌BL21感受态细胞上表达出分子质量约67 ku的重组VP2蛋白,为进一步比较201（G）、281（R）、313（A）位氨基酸差异导致的抗原特性改变提供了研究基础。本试验结果表明,IBDV C4株VP2基因与vvIBDV毒株VP2基因的主要特性一致,但也有3处氨基酸与代表毒株UK661存在差异,这些改变可能与中国IBDV毒株毒力的进化有关。     

IBDV地方变异株VP2基因的克隆与特性鉴定

应用 RT- PCR技术对 1株分离于地方免疫鸡群中暴发的传染性法氏囊病病例的传染性法氏囊病病毒 ( infec-tious bursal disease virus,IBDV) HN0 2 6株的 VP2基因进行了克隆与序列分析 ,并与相应毒株的 VP2高变区核苷酸及氨基酸序列进行了比较研究。结果表明 ,HN0 2 6株的核苷酸和氨基酸序列与变异株 Var- A的同源性最高 ,分别达97.3%和 97.6 % ;其次为超强毒株 UK6 6 1 ,分别为 95 .7%和 95 .2 % ;而和弱毒株 PBG98的同源率仅有 92 .8%和90 .3%。其中 ,在第一、二亲水区 ,HN0 2 6株均有 1个氨基酸发生了变化 ,即第 2 2 2位转变为 E、第 31 8位转变为 D。更重要的是 ,其 2 4 9位和 2 5 4位上的氨基酸分别为 K和 S,这些均为变异株的特性。另外 ,HN0 2 6株的七肽区保持SWSASGS不变 ,且第 2 79位和 2 84位氨基酸分别为 D和 A,又完全具备强毒株的特性。因此 ,初步确定所分离的HN0 2 6为有较强致病力的变异株 IBDV。     

传染性法氏囊病病毒强毒株HB／11的分离与鉴定

2011年8月,从河南省疑似传染性法氏囊病鸡群采集病料,通过鸡胚接种、琼脂扩散试验和RT-PCR等方法,证实分离的病毒为传染性法氏囊病病毒（Infectiousbursaldiseasevirus,IBDV）,命名为HB／11株。序列分析表明,HB／11株VP2基因的核苷酸序列与GenBank中发表的部分国内外IBDV超强毒株VP2基因序列相似性在99％左右;HB／11株VP2高变区基因含有七肽基序为SWSASGS,在222、253、256、279、284、294和299位上的氨基酸残基分别是A、Q、I、D、A、I和S,具有IBDV强毒的分子特征。动物回归试验表明,30日龄鸡群接种该毒株后引起鸡群发病率为100％,死亡率为70％,剖检可见鸡传染性法氏囊病典型病变。因此该病毒为IBDV强毒株。     

Molecular characterisation of infectious bursal disease viruses isolated in recent acute outbreaks in Slovenia

In 2004 and then in 2006 several outbreaks of infectious bursal disease (IBD) were reported in broiler and broiler breeder flocks in Slovenia. In this report ten recently emerged IBD viruses (IBDV) were characterised by sequence analysis of the VP2 hypervariable region and compared to previous Slovene IBDV strains from 1995/1996 and to some representative serotype 1 IBDV strains of different pathotypes. On the basis of nucleotide and amino acid identities, phylogenetic analyses and the presence of very virulent IBDV (vvIBDV) conserved amino acid substitutions, all Slovene isolates from recent outbreaks were identified as vvIBDV. Although some unique nucleotide exchanges and amino acid substitutions have been observed, the results of this study indicated that recent vvIBDV isolates are closely related with those from outbreaks in the 1990s. However, acute IBD has not been reported in commercial flocks in Slovenia for some years. This could lead to the conclusion that poor biosecurity and relaxed vaccination could be responsible for the re-emergence of vvIBDV.     

不同时期8株IBDV地方株VP2基因变异分析

对分离于洛阳地区1991和2001年前后间隔达10年之久的两个时期的8株IBDV进行了VP2基因的克隆与序列分析。结果发现，8个地方株虽均属于IBDV超强毒株，但各个毒株间也有一定的差异。根据它们的核苷酸和氨基酸同源性高低可明显分为3群，分别位于系统进化树上超强毒区的3个小分支上。第1群包括1991年分离的L912、L014和L916三株；第2群包括L913、L017和L018三株；第3群包括2001年分离的L015和L016二株。群内各毒株间同源性较高，在98．3％～100％之间；群间各毒株的同源性则相对较低，在95％～97．9％之间，其中最低的为L015和L017、L016和L018二对，同源性均只有95％。进一步的序列分析表明，8个地方分离株均具有vvIBDV所具有的特征。其中，1991年的4株在各亲水区和七肽区的氨基酸和经典株及传统的超强毒株相比均无明显的变化；而2001年的4个分离株虽仍符合超强毒株的特点，但在相应亲水区均有1～2个氨基酸发生替换，特别是L015和L016株还出现了4个其它各毒株均没有的氨基酸位点变化。