山西省鸡传染性法氏囊病病毒的分离鉴定

为了解山西省鸡传染性法氏囊病病毒(IBDV)流行毒株的遗传变异规律,将近5年来采集自山西不同地区发病鸡群的法氏囊组织病料,通过接种易感雏鸡分离出15株IBDV。用绒毛尿囊膜接种法测定鸡胚半数致死量,其ELD50在10~(-2.18)~10~(-2.91);测定所有分离株对非免疫鸡的致死率,结果为63.3%~86.7%,属于超强毒株;利用RT-PCR扩增病毒VP2基因并进行序列分析。结果表明,所分离的15株病毒均具有传染性法氏囊病病毒超强毒株的主要分子特征,即222A(WS2为S)、249Q、254G、256I、279D、284A、294I、299S及七肽区SWSASGS,综合其对非免疫鸡的致死率,确认所有分离毒株属于超强毒病毒株。说明在山西省境内存在传染性法氏囊病病毒超强毒株。     

中国部分地区传染性法氏囊病病毒分子流病学调查

为调查我国鸡传染性法氏囊病病毒(IBDV)的流行情况,从我国11个省(市、自治区)采集法氏囊病料,分离鉴定鸡传染性法氏囊病病毒20株,运用RT-PCR方法对分离株的VP2基因进行扩增、测序和序列分析.研究表明,20株分离毒株中有19株具有IBDV超强毒株的分子特征,即222A、256I、294I和299S,与我国IBDV超强毒Gx株的核苷酸同源率在96.7%～99.4%,推导氨基酸同源率在98.2%～100% 遗传进化分析表明,所有分离毒株具有共同的祖先.     

传染性法氏囊病病毒自然重配株全基因组序列分析

从具有新的流行病学特征的传染性法氏囊病（IBD）发病鸡群中分离到2株传染性法氏囊病病毒（IBDV）,分别命名为QL和ZZ—ll,对4周龄SPF鸡的致死率分别为94％和86％。为分析该毒株的分子生物学特征,对其全基因组序列进行了测定,2个病毒株基因组A节段长度为3260bp、B节段长度2827bp。病毒演化分析结果显示2个病毒基因组A节段的核苷酸序列与已发表的强毒株序列的同源性分别为96．8％～98．1％和96．9％～98．4％,处在IB—DV超强毒株分支上;而B节段与已发表的弱毒株序列的同源性分别为89．7％～90．4％和90．0％～90．7％,位于弱毒株分支上。IBDV超强毒株和弱毒株序列特征氨基酸残基与基序分析表明,QL和ZZ—11两个病毒株的A节段VP2基因的氨基酸残基为222A、249Q、253Q、254G、256I、294I和299S,七肽基序为SWSASGS,均符合超强毒株的分子特征;而B节段777～782位核苷酸序列为GGTGCC,没有KpnI酶切位点,具有弱毒株的序列特点。以上分析结果表明,QL和ZZ—11为IBDV自然重配株,A节段源于超强毒株,而B节段源于弱毒株。     

传染性法氏囊病病毒自然重配株全基因组序列分析

从具有新的流行病学特征的传染性法氏囊病(IBD)发病鸡群中分离到2株传染性法氏囊病病毒(IBDV),分别命名为QL和ZZ-11,对4周龄SPF鸡的致死率分别为94%和86%。为分析该毒株的分子生物学特征,对其全基因组序列进行了测定,2个病毒株基因组A节段长度为3 260bp、B节段长度2 827bp。病毒演化分析结果显示2个病毒基因组A节段的核苷酸序列与已发表的强毒株序列的同源性分别为96.8%⁹8.1%和96.9%98.1%和96.9%⁹8.4%,处在IBDV超强毒株分支上;而B节段与已发表的弱毒株序列的同源性分别为89.7%98.4%,处在IBDV超强毒株分支上;而B节段与已发表的弱毒株序列的同源性分别为89.7%⁹0.4%和90.0%90.4%和90.0%⁹0.7%,位于弱毒株分支上。IBDV超强毒株和弱毒株序列特征氨基酸残基与基序分析表明,QL和ZZ-11两个病毒株的A节段VP2基因的氨基酸残基为222A、249Q、253Q、254G、256I、294I和299S,七肽基序为SWSASGS,均符合超强毒株的分子特征;而B节段77790.7%,位于弱毒株分支上。IBDV超强毒株和弱毒株序列特征氨基酸残基与基序分析表明,QL和ZZ-11两个病毒株的A节段VP2基因的氨基酸残基为222A、249Q、253Q、254G、256I、294I和299S,七肽基序为SWSASGS,均符合超强毒株的分子特征;而B节段777⁷82位核苷酸序列为GGTGCC,没有KpnⅠ酶切位点,具有弱毒株的序列特点。以上分析结果表明,QL和ZZ-11为IBDV自然重配株,A节段源于超强毒株,而B节段源于弱毒株。     

鸡传染性法氏囊病病毒弱毒株在鸡体连续传代后毒力增强的分子机理研究

将两株鸡传染性法氏囊病病毒（IBDV）弱毒株在SPF鸡体内连续传代至第5、第6代时,出现明显的法氏囊萎缩和B：B指数下降,表明IBDV弱毒株在鸡体内连续传代后毒力增强。为进一步阐释哪些基因位点导致了上述毒力的变化,本试验测定了基础弱毒株及其在鸡体内传代后各个代次毒的基因组序列,比对分析后发现VP2蛋白253位氨基酸发生了由H到Q或N的变异,表明VP2蛋白253位氨基酸的替换可能会增强传染性法氏囊病病毒在鸡体内的致病性。     

4株鸡传染性法氏囊病病毒VP2基因序列分析

试验从发病鸡场分离获得4株鸡传染性法氏囊病病毒（infectious bursal disease virus,IBDV）,分别为河北株（HB）、湖北株（HUB）、山东株（SD）、山西株（SX）。通过特异性引物扩增VP2基因并进行测序,序列分析结果显示,4株病毒中,HUB、SD和SX的七肽区为S-W-S-A-S-G-S（aa 326S-332S）,其222（A）、256（I）、294（I）和299（S）位是传染性法氏囊病病毒超强毒株（vvIBDV）的4个特征性氨基酸,确定这3株病毒均为超强毒株;而HB株七肽区为S-W-S-A-R-G-S（aa 326S-332S）,其222（P）、256（V）、294（L）和299（N）位氨基酸显示该毒株属于弱毒株。进化树分析结果显示,HUB、SD和SX 3株病毒与2011年中国分离的大部分IBDV毒株亲缘关系较近,同源性在96%以上,同时与经典毒株Cro-Po/00、超强毒株GX和UK661亲缘关系很近;而HB株与减毒疫苗株D78株和B87株的亲缘关系近。本试验结果表明,IBDV目前在中国流行呈混发型,但以超强毒株为主。     

广西某鸡群呼吸道综合征的病原学分析

本研究对临床上患呼吸道感染的病鸡进行病原检测,分离到1株传染性法氏囊病病毒(infectious bursal disease virus,IBDV)和1株传染性支气管炎病毒(infectious bronchitis virus,IBV),此外还从病鸡肝脏等组织中分离到金黄色葡萄球菌和大肠杆菌。对IBDV分离株NN160401的VP1-b、v VP2基因以及IBV分离株GX160421的S1基因分别进行扩增、测序以及序列的相似性比较和遗传进化树分析。结果发现,分离株NN160401为基因重排毒株,其VP1-b与HLJ-0504株的序列相似,v VP2则具有IBDV超强毒株特征;分离株GX160421的S1基因序列与广西分离株亲缘关系较近,属于近年报道的New-typeⅡ,但与其他参考株及疫苗株H120的核苷酸相似性仅为58.8%~67.2%。综合分析表明,该病鸡群为早期感染IBDV造成了机体的免疫抑制,后来继发IBV的感染并伴发了细菌感染。     

基因自然重排鸡传染性法氏囊病病毒的分子特征

为了对某鸡群疑似传染性法氏囊病(IBD)的病例进行分子诊断、病毒分离和分子特征分析，本试验通过核酸检测调查该鸡群法氏囊组织和病毒分离物中传染性法氏囊病病毒(IBDV)的感染情况，将分离得到的毒株命名为GL1906,继而对该病毒基因组双节段的VP2高变区(vVP2)序列和VP1-b序列进行分析。结果显示，GL1906 vVP2基因的特征性氨基酸位点在222A、256I、284A、294I和299S上均符合超强毒株(vvIBDV)的特征，但279N符合致弱毒株的特征；VP1-b基因在242D、390L、393E与弱毒株一致，287A与vvIBDV一致，此外第777～782位核苷酸序列为GGTGCC,与弱毒株一致；GL1906 vVP2的核苷酸、氨基酸序列与vvIBDV的同源性最高，在系统进化树中与vvIBDV同为A3分支；GL1906 VP1-b的核苷酸、氨基酸序列与B节段属于独特来源的NN1172同源性最高，在系统进化树中同属于B3独特分支。结果表明，本试验分离株GL1906的基因组双节段vVP2和VP1-b具有不同的来源，是基因型为A3B3的基因自然重排毒株。     

鸡传染性法氏囊病病毒SD株毒力测定

从一个免疫失败鸡场中分离到一株鸡传染性法氏囊病病毒野毒株,命名为SD株.经血清学试验、鸡胚接种、病毒形态观察等证实了分离物为鸡传染性法氏囊病病毒(IBDV).测定病毒效价ELD50达到10-6.5/0.2 mL;动物回归试验表明,该SD株接种4周龄鸡后引起鸡群发病率为100%,病死率达85%,剖检可见法氏囊呈"紫葡萄样...     

传染性法氏囊病病毒的分离与鉴定

从中国广东省发生传染性法氏囊病的鸡场分离了8株传染性囊病病毒(Infectious bursal disease virus,IBDV)毒株,运用RT-PCR对分离毒株进行了鉴定,测定了8个分离株的ELD50以及其对SPF鸡的致死率。结果表明:这8株分离毒对SPF鸡的致死率均等于或超过60%,属于超强毒。运用RT-PCR方法对分离毒株进行了鉴定并对各毒株VP2基因高变区进行扩增测序,分析结果表明:这8株分离株均为IBDV超强毒株,同时也说明当前在广东省引起鸡传染性法氏囊病的主要毒株类型多为超强毒IBDV。     

表达传染性法氏囊病病毒VP2基因的重组鸡痘病毒的构建及其免疫保护性研究

以脂质体转染技术构建了表达鸡传染性法氏囊病病毒（ＩＢＤＶ）ＶＰ２基因的重组鸡痘病毒ＦＰＶ－ＶＰ２，该病毒在鸡胚成纤维细胞及鸡体内均能稳定产生子代病毒，经翅皮下５×１０５ＰＦＵ／羽免疫１日龄ＳＰＦ鸡，免疫后４周以１００ＬＤ５０／羽ＩＢＤＶ超强毒株Ｇ株攻毒，获得了５／６的保护，但不能有效预防临床发病及法氏囊受损萎缩。实验结果证明了ＶＰ２是ＩＢＤＶ的宿主保护性抗原，提示Ｔ细胞介导的免疫可能在ＩＢＤＶ的免疫中起着较为重要的作用。本研究为ＩＢＤＶ重组病毒疫苗研制进行了有益探索。     

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。     

传染性法氏囊病病毒JS株VP2基因真核表达载体的构建及其应用

应用RT-PCR方法从鸡传染性法氏囊病病毒(IBDV)JS株中扩增出VP2基因,并克隆入T-easy载体。序列测定分析结果表明IBDVJS株的VP2基因与国际标准强毒株的核苷酸序列同源性达98%,氨基酸序列同源性达99%以上。随后将VP2基因克隆入真核表达载体pcDNA3.1/zeo( ),构建成功真核表达质粒pcD-VP2,pcD-VP2体外转染COS-1细胞,能在COS-1细胞中表达。利用pcD-VP2质粒进行动物实验,结果表明雏鸡免疫14d后在体内可检测到特异性抗体,pcD-VP2的真核表达质粒二次免疫诱导鸡产生对IBDV强毒攻击的保护率为67%,这一结果提示VP2基因具有重要开发应用价值。     

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。     

Induction of protective immunity in chickens immunised with plasmid DNA encoding infectious bursal disease virus antigens

Direct DNA inoculations were used to determine the efficacy of gene immunisation of chickens to elicit protective immune responses against infectious bursal disease virus (IBDV). The vp2 gene of IBDV strains GP40 and D78, and the vp2-vp4-vp3 encoding segment of strain D78 were cloned in an expression vector which consisted of human cytomegalovirus (HCMV) immediate early enhancer and promoter, adenovirus tripartite leader sequences and SV40 polyadenylation signal. For purification of vaccine-quality plasmid DNA from E. coli, an effective method was developed. Chickens were vaccinated by inoculation of DNA by two routes (intramuscular and intraperitoneal). Two weeks later, chickens were boosted with DNA, and at 2 weeks post-boost, they were challenged with virulent IBDV strain. Low to undetectable levels of IBDV-specific antibodies and no protection were observed with DNA encoding VP2. However, plasmids encoding VP2-VP4-VP3 induced IBDV-specific antibodies and protection in the chickens. DNA immunisation opens a new approach to the development of gene vaccines for chickens against infectious diseases.     

Molecular characterization and phylogenetic analysis of infectious bursal disease viruses isolated from chicken in South China in 2011

Infectious bursal disease virus (IBDV) is a double-stranded RNA virus that causes immunosuppressive disease in young chickens. Thousands of cases of IBDV infection are reported each year in South China, and these infections can result in considerable economic losses to the poultry industry. To monitor variations of the virus during the outbreaks, 30 IBDVs were identified from vaccinated chicken flocks from nine provinces in South China in 2011. VP2 fragments from different virus strains were sequenced and analyzed by comparison with the published sequences of IBDV strains from China and around the world. Phylogenetic analysis of hypervariable regions of the VP2 (vVP2) gene showed that 29 of the isolates were very virulent (vv) IBDVs, and were closely related to vvIBDV strains from Europe and Asia. Alignment analysis of the deduced amino acid (aa) sequences of vVP2 showed the 29 vv isolates had high uniformity, indicated low variability and slow evolution of the virus. The non-vvIBDV isolate JX2-11 was associated with higher than expected mortality, and had high deduced aa sequence similarity (99.2 %) with the attenuated vaccine strain B87 (BJ). The present study has demonstrated the continued circulation of IBDV strains in South China, and emphasizes the importance of reinforcing IBDV surveillance.     

Pathogenicity and molecular analysis of an infectious bursal disease virus isolated from Malaysian village chickens

The characteristics of the pathogenic infectious bursal disease virus (IBDV) that infected avian species other than commercial chickens were largely unknown. In this study, by using in vivo and molecular methods, we had characterized an IBDV isolate (named 94268) isolated from an infectious bursal disease (IBD) outbreak in Malaysian village chickens--the adulterated descendant of the Southeast Asian jungle fowl (Gallus bankiva) that were commonly reared in the backyard. The 94268 isolate was grouped as the very virulent IBDV (vvIBDV) strain because it caused severe lesions and a high mortality rate in village chickens (>88%) and experimentally infected specific-pathogen-free chickens (>66%). In addition, it possessed all of the vvIBDV molecular markers in its VP2 gene. Phylogenetic analysis using distance, maximum parsimony, and maximum likelihood methods revealed that 94268 was monophyletic with other vvIBDV isolates and closely related to the Malaysian vvIBDV isolates. Given that the VP2 gene of 94268 isolate was almost identical and evolutionarily closely related to other field IBDV isolates that affected the commercial chickens, we therefore concluded that IBD infections had spread across the farm boundary. IBD infection in the village chicken may represent an important part of the IBD epidemiology because these birds could harbor the vvIBDV strain and should not be overlooked in the control and prevention of the disease.     

传染性法氏囊病超强毒Gx株的致弱研究

本研究成功将鸡传染性法氏囊病超强毒vvIBDVGx株通过SPF鸡胚的快速培育及鸡胚成纤维细胞传代致弱，揭示了vvIBDV从超强毒力向弱毒力转化过程中，其主要结构蛋白VP2基因核苷酸及推导的氨基酸序列的变化规律。对不同代次细胞毒进行了序列分析。发现细胞毒在第7代以前，VP2基因序列没有改变。与欧洲标准超强毒氨基酸同源性达100%；细胞毒第8代，有个别核苷酸发生了改变。但没有影响氨基酸序列；细胞毒第9代是变化复杂的过渡代；10代毒VP2基因与欧洲标准弱毒Cu-1氨基酸序列同源性达97%；以后的细胞适应毒至20代。其VP2基因序列不再改变。致病性实验表明原代毒对4周龄SPF鸡致死率为64%。细胞毒第5代的致死率为60%，而20代毒对鸡无致病性。在鸡体内连续传代6代不返强。