鸡传染性法氏囊病病毒研究进展

传染性法氏囊病(Infection bursal disease, IBD)是由鸡传染性法氏囊病毒(Infectious bursal disease virus, IBDV)引起的鸡和火鸡的一种高度接触性传染病,给世界各国的禽养殖业带来了巨大损失。自IBDV发现至今新的变异株不断出现,分子结构的改变导致病毒致病力的改变及宿主对疫苗应答的改变,使得传统的疫苗已不能控制其流行,因此各国学者对其基因组结构和功能进行了广泛深入的研究,并积极研制新型有效的疫苗以达到防治的目的。 1 基因组结构和功能 IBDV基因组由A(3.2kb)、B(2.8kb)两个双链RNA节段构成。A编码形成VP2蛋白(37-40k…     

小牛血清对鸡传染性法氏囊病病毒增殖的抑制机制

本试验研究了小牛血清(CS)对法氏囊炎病毒(IBDV)蚀斑形成的抑制机制。CS与鸡胚细胞(CEF)作用后,CS中的抑制因子能被细胞吸收。这说明血清中的抑制因子可附着在CEF上。细胞预先用CS处理,则吸附病毒的能力明显降低。还发现,若把CS加入琼脂培养液中,则能抑制IBDV的蚀斑形成。这说明CS能抑制病毒对其周围细胞的感染。CS对IBDV蚀斑形成的抑制机制,不是由于抑制因子直接中和了病毒,而是因为抑制因子附着在细胞表面,占据了细胞的病毒受体,从而阻止了病毒附着于细咆,以致抑制了病毒蚀斑的形成。     

传染性法氏囊病病毒在次代鸡胚成纤维细胞上的增殖

研究了用次代鸡胚成纤维细胞（SCEF）增殖传染性法氏囊病病毒（IBDV）的可能性。在研究了原代鸡胚成纤维细胞（PCEF）与SCEF的生长特性的基础上,就各种培养方式采用PCEF与SCEF增殖IBDV进行了比较。结果表明,可用SCEF代替PCEF进行IBDV的增殖培养。     

分析细胞代谢确定传染性法氏囊病病毒在鸡胚细胞上的增殖

研究了接种病毒后的鸡胚成纤维细胞(CEF)代谢与传染性法氏囊病病毒(IBDV)增殖的关系,结果表明,感染细胞对未感染细胞葡萄糖消耗的相对增加量α可作为IBDV增殖的参数。Α随感染过程而增加,当α达到最大值不再改变时,IBDV增殖也达最大。此点可作为培养IBDV时的收毒点     

传染性法氏囊病病毒的自然重组

传染性法氏囊病病毒(Infectious Bursal Disease Virus,IBDV)是双RNA病毒科(Birnaviridae)的典型代表,其引起的传染性法氏囊病(Infectious Bursal Disease,IBD)是危害养禽业的一种重要免疫抑制病和致死性传染病。IBDV的自然重组给疫病防控带来了新风险。本文综述了IBDV基因组节段重组和基因内重组的主要类型,分析了其形成机制及生物学意义,提出了该类病毒遗传演化研究以及疫病综合防控的新思路。     

鸡传染性法氏囊病病毒超强毒株GX8/99株的致病性

鸡传染性法氏囊病病毒(IBDV)超强毒株GX8/99,系1999年从广西一自然发病鸡群采集到.用原始病鸡法氏囊悬液连续3次人工感染SPF鸡后,再取其法氏囊制备悬液,分装,在-70℃保存.以此悬液经卵黄囊接种10日龄SPF鸡胚,测定鸡胚的半数致死量(ELD50).随着鸡的日龄和接种剂量的不同,其致死率有很大差异.对28～30日龄SPF鸡,病毒接种量为200个ELD50时,感染后7日内死亡率最高可达73%～90.5%(11/15和19/21);感染量为20个ELD50时,死亡率亦可达53%～92%(8/15和23/25).以500个ELD50感染28～104日龄的SPF鸡,死亡率均在55.1%～67.2%;甚至113～120日龄的SPF鸡,感染后仍有10%～15%致死率.但128日龄的SPF鸡感染后既不引起死亡也不表现任何症状,但抗体全部转阳.人工接种发病死亡的鸡,其法氏囊的出血程度也随感染量和年龄而异.50日龄鸡接种2000个ELD50后,死亡的鸡100%(12/12)法氏囊严重出血;而40日龄鸡感染200个ELD50后,死亡鸡中仅17%(3/18)发生出血.在2、3、4周龄带有母源抗体的商品代蛋鸡,以2000个ELD50病毒接种后,只引起10%(2/20)、35%(7/20)和35%(7/20)的死亡率.但在5周龄商品代蛋鸡,仅接触感染的致死率可达61.3%(98/160).另一批商品代蛋鸡,在4周龄和5周龄人工接种200个ELD50病毒后,死亡率分别是81.6%～94.3%(62/76～33/35)和93.9%～94%(31/33～47/50).通过总共1200多羽鸡的试验表明,GX-8/99株是一个超强毒IBDV毒株,表现为高死亡率(最高可达94%),易感年龄延长至4月龄,中枢性免疫器官法氏囊出血严重和胸腺明显萎缩.     

鸡传染性法氏囊病病毒研究进展

传染性法氏囊病(Infection bursal disease, IBD)是由鸡传染性法氏囊病毒(Infectious bursal disease virus, IBDV)引起的鸡和火鸡的一种高度接触性传染病,给世界各国的禽养殖业带来了巨大损失.自IBDV发现至今新的变异株不断出现,分子结构的改变导致病毒致病力的改变及宿主对疫苗应答的改变,使得传统的疫苗已不能控制其流行,因此各国学者对其基因组结构和功能进行了广泛深入的研究,并积极研制新型有效的疫苗以达到防治的目的.     

表达绿色荧光蛋白报告基因重组禽腺联病毒载体系统的构建与鉴定

为了开展重组禽腺联病毒(Recombinant avian adeno-associated virus,rAAAV)介导的基因转移研究,用限制性内切酶消化含AAAV全基因组的重组质粒pCR-AAAV,去除AAAV Rep和Cap蛋白编码序列,将PCR扩增的绿色荧光蛋白(Green fluorescent protein,GFP)报告基因插入AAAV末端反向重复序列(Inverted terminal repeats,ITR)之间,获得表达GFP基因的AAAV转移载体pAITR-GFP;以含AAAV全基因组的质粒为模板,用PCR分别扩增AAAV Rep、Cap和Rep-Cap蛋白基因,将Rep和Cap基因分别插入真核细胞双表达载体pVITRO2-mcs的两个多克隆位点,将Rep-Cap蛋白基因插入真核细胞表达载体pcDNA3,获得AAAV辅助质粒pVITRO2-ARC和pcDNA-ARC;将pAITR-GFP、pVITRO2-ARC或pcDNA-ARC与腺病毒辅助质粒pHelper组成三质粒转染系统,用磷酸钙沉淀法共转染AAV-293细胞,获得了表达GFP的rAAAV.经SDS-聚丙烯酰胺凝胶电泳分离后,纯化病毒出现分子量正确的VP1、VP2、VP3结构蛋白;经PCR检测证明,重组病毒中含有GFP报告基因;用重组病毒分别感染鸡胚成纤维细胞(CEF)和鸡胚肝CEL细胞,可以观察到GFP报告基因的表达,表达时间持续两周以上.这些试验结果表明,成功建立了辅助病毒非依赖性rAAAV体外包装体系,为禽源细胞的基因转移研究和禽重组活载体疫苗的研制打下了基础.     

传染性法氏囊病病毒分子生物学研究进展

传染性法氏囊病病毒(Infectious bursal disense virus,IBDV)是引起鸡的最严重的传染病之一,给养鸡业造成了巨大的经济损失,其危害除了直接引起临床症状外,更为重要的是破坏机体的免疫器官,主要是破坏B淋巴细胞前体,引起严重的免疫抑制,使鸡对其他病原的免疫力丧失,结果导致死亡。 IBDV最初被认为是呼肠孤病毒科的成员,主要是因为其在鸡胚肾细胞上培养出现     

重组禽腺联病毒介导的输卵管暂态生物反应器的初步研究

将输卵管特异表达启动子调控的人组织激肽释放酶（human tissue kallikrein,hKLK1）表达盒插入至AAAV转移载体pAITR中,与AAAV包装载体pcDNA-ARC及腺病毒辅助质粒pHelper三质粒利用磷酸钙沉淀法共转染AAV-293细胞,制备输卵管特异表达hKLK1的rAAAV。将获得的重组病毒以每只鸡2×1010病毒颗粒数翅静脉注射正常产蛋母鸡,RT-PCR结果显示hKLK1只在输卵管部位表达;酶活性检测结果表明：注射后第2天就可以检测到rhKLK1的活性,第3周表达量最高,达107.3U/ml,表达时间持续6周之久;用含rhKLK1的蛋清灌喂自发性高血压大鼠（SHR）,可使其血压下降70mmHg,5天后回升到饲喂前水平。以上结果表明重组禽腺联病毒介导的鸡输卵管暂态生物反应器不仅具有很好的组织特异性,而且可指导外源基因长期稳定的表达。     

含禽流感病毒M2e 氨基端抗原表位的重组传染性法氏囊病毒VP2 蛋白的免疫原性鉴定

【目的】构建传染性法氏囊病毒VP2蛋白展示禽流感M2e抗原表位的重组蛋白,研发预防H5或H9亚型禽流感和传染性法氏囊的基因工程疫苗。【方法】根据现有禽流感疫苗株M2e的氨基端12个氨基酸多肽序列(nM2e)序列,结合GenBank中H5和H9亚型禽流感病毒nM2e的比对结果,确定nM2e序列。用融合PCR分别将1拷贝H5或H9的nM2e序列插入IBD B87株VP2基因的PBC区,获得VP2BCnM2e重组基因。将重组基因克隆至杆状病毒表达系统,转染Sf9细胞进行表达。经间接免疫荧光和Western blotting检测Sf9细胞表达重组基因后,扩繁重组病毒,制备疫苗,间隔4周对非免鸡作2次重复免疫,用间接ELISA和鸡胚成纤维细胞中的病毒血清中和试验检测血清中VP2和nM2e的抗体效价。【结果】成功构建含H5或H9 nM2e的VP2BCnM2e重组基因,该重组基因在Sf9细胞中得到表达。经免疫鸡,两重组蛋白均能激发针对VP2和nM2e的抗体,VP2BCnM2eH5组抗体效价高于VP2BCnM2eH9组。【结论】两重组蛋白均具有免疫原性,VP2BCnM2eH5免疫原性更佳。     

Exchange of the VP5 of infectious bursal disease virus in a serotype I strain with that of a serotype II strain reduced the viral replication and cytotoxicity

Infectious bursal disease virus (IBDV), belonging to Avibirnavirus genus in the Birnaviridae family, consists of two segments of double-strand RNA. There are two distinct serotypes of IBDV, the pathogenic serotype I and the non-pathogenic serotype II. Comparison of the deduced amino acid sequences of a panel of VP5 genes retrieved from GenBank revealed a high identity among strains within the serotype I or serotype II group but a low identity between strains across two serotypes. In this study, we rescued two mosaic viruses, rGtGxVP5 and rGt2382VP5 by exchanging the VP5 gene of a cell culture-adapted serotype I Gt strain with its counterpart of the very virulent IBDV Gx strain, or a non-pathogenic 23/82 strain of the serotype II. In comparison to the parental strain rGt virus, the rGtGxVP5 showed the similar viral replication, cytotoxicity and the ability of inducing apoptosis; however, the other mosaic virus rGt2382VP5 had a lower titer and a reduced cytotoxicity. Although exchange of VP5 within serotype I group did not alter the viral replication and cytotoxicity of Gt strain, exchange of VP5 in the serotype I with that of a serotype II reduced the viral replication and cytotoxicity on chicken embryo fibroblast (CEF) cells. Therefore, the VP5 of serotype II may be one of the factors responsible for the distinct pathogenic features of two serotypes.     

Synthesis of reassortant infectious bursal disease virus in chickens injected directly with infectious clones from different virus strains

The infectious bursal disease virus (IBDV), a member of the Birnaviridae family, containing a bisegmented double-stranded RNA genome, encodes four structural viral proteins, VP1, VP2, VP3, and VP4, as well as a non-structural protein, VP5. In the present paper, the segment A from two IBDV strains,field isolate ZJ2000 and attenuated strain HZ2, were inserted into one NaeⅠ site by site-directed silent mutagenesis and subcloned into the eukaryotic expression plasmid pCI under the control of the human cytomegalovirus (hCMV) immediate early enhancer and promoter to construct the recombinant plasmids pCI-AKZJ2000 and pCI-AKHZ2, respectively. Each of the two recombinants was combined with another recombinant pCI plasmid containing the marked segment B of strain HZ2 (pCI-mB), and injected intramuscularly into nonimmunized chickens. Two chimeric IBDV strains were recovered from the chickens. Two out of eight chickens in each of two groups showed the bursal histopathological change. The reassortant virus derived from pCI-AKZJ2000/pCI-mB can infect chicken embryos and shows relatively low virulence. We have developed a novel virus reverse genetic approach for the study of IBDV. The results also form the basis for investigating the role of VP1 in viral replication and pathogenecity.     

Vaccination against very virulent infectious bursal disease virus using recombinant T4 bacteriophage displaying viral protein VP2

In order to develop a desirable inexpensive, effective and safe vaccine against the very virulent infectious bursal disease virus （vvIBDV）, we tried to take advantage of the emerging T4 bacteriophage surface protein display system. The major immunogen protein VP2 from the vvIBDV strain HK46 was fused to the nonessential T4 phage surface capsid protein, a small outer capsid （SOC） protein, resulting in the 49 kDa SOC-VP2 fusion protein, which was verified by sodium dodecylsulfate polyacrylamide gel electrophoresis and Western blot. Immunoelectromicroscopy showed that the recombinant VP2 protein was successfully displayed on the surface of the T4 phage. The recombinant VP2 protein is antigenic and showed reactivities to various monoclonal antibodies （mAbs） against IBDV, whereas the wild-type phage T4 could not react to any mAb. In addition, the recombinant VP2 protein is immunogenic and elicited specific antibodies in immunized specific pathogen free （SPF） chickens. More significantly, immunization of SPF chickens with the recombinant T4-VP2 phage protected them from infection by the vvIBDV strain HK46. When challenged with the vvIBDV strain HK46 at a dose of 100 of 50% lethaldose （LD50） per chicken 4 weeks after the booster was given, the group vaccinated with the T4-VP2 recombinant phage showed no clinical signs of disease or death, whereas the unvaccinated group and the group vaccinated with the wild-type T4 phage exhibited 100% clinical signs of disease and bursal damages, and 30%-40% mortality. Collectively, the data herein showed that the T4-displayed VP2 protein might be an inexpensive, effective and safe vaccine candidate against vvIBDV.     

表达鸡传染性法氏囊病毒VP2蛋白的干酪乳杆菌免疫保护效力

利用干酪乳杆菌作为传染性法氏囊病毒(IBDV)VP2抗原传递系统,探讨口服雏鸡的免疫次数、免疫剂量、免疫途径和攻毒保护效果。用pLA-VP2重组干酪乳杆菌对5日龄雏鸡进行二次和三次免疫,并设108、109、1010 CFU/mL的重组干酪乳杆菌组,间接ELISA检测血清IgG和小肠洗液sIgA,末免后7 d攻毒,计算保护效果。根据确定的2次免疫和109 CFU/mL免疫剂量免疫5日龄雏鸡,分别口服、滴鼻/点眼pLA-VP2/L.casei,口服、肌注商品活苗及口服pLA/L.casei和PBS为对照,监测IgG和sIgA抗体水平;末免后7 d检测脾淋巴细胞增殖情况并攻毒,7 d后剖检,观察法氏囊损伤程度并记录病变得分和保护率。结果表明各组的特异性sIgA、IgG抗体水平显著高于对照组(P0.01);口服pLA-VP2/L.casei组的淋巴细胞刺激指数显著高于其他组(P0.01),保护率高达83.3%,免疫保护效果优于滴鼻/点眼组。因此,构建的重组干酪乳杆菌的安全性优于商品活苗,可以作为IBDV候选疫苗。     

Synonymous codon usage of the VP2 gene of a very virulent infectious bursal disease virus isolate serial passaged in chicken embryos

Three very virulent infectious bursal disease virus (vvIBDV) strains were isolated from a single farm and shown to be phylogenetically related to the vvIBDV isolate UK661. In this study, a comparative analysis of the synonymous codon usage in the hypervariable region of theVP2 (vVP2) gene of the vvIBDV strains was done on viruses serially passaged in chicken embryos. Sequencing demonstrated that codons change during the serial passage in the vVP2 gene of the viruses. Nine codon mutations resulted in amino acids changes. The amino acid changes were I256V, I296L 6in isolate XA1989, A222P, I242V, Q253H, I256V in isolate XA1998, and Q253H, I256V, I296L in isolate XA2004. Three of the nine amino acid changes occurred at residue 256. The codons of the amino acids A232, N233, I234, T269, T283 and H338 changed to the synonymous codons in XA1989 after the 16th passage, in XA1998 after the 24th passage and in XA2004 22nd passage viruses. These mutations change the key amino acid residues Q253H and I256V in the domains which are essential for its virulence, and the synonymous codons were observed compared to classical virulent IBDV. The results indicated that the codon changes during the serial passage comprised of synonymous codon usage in the vVP2 gene of IBDV, and this synonymous codon bias was correlated with pathotypes. The extent of synonymous codon usage bias in the IBDV-vVP2 gene maybe influence the gene expression level and secondary structure of protein as well as hydrophobicity, therefore the results provide useful perspectives for evolution and understanding of the pathogenesis of IBDV.     

Expression of immunogenic VP2 protein of infectious bursal disease virus in Arabidopsis thaliana

VP2 protein is the major host-protective immunogen of infectious bursal disease virus (IBDV) of chickens. Transgenic lines of Arabidopsis thaliana expressing recombinant VP2 were developed. The VP2 gene of an IBDV antigenic variant E strain was isolated, amplified by RT-PCR and introduced into a plant expression vector, pE1857, having a strong promoter for plant expression. A resulting construct with a Bar gene cassette for bialaphos selection in plant (rpE-VP2) was introduced into Agrobacterium tumefaciens by electroporation. Agrobacterium containing the rpE-VP2 construct was used to transform Ar. thaliana and transgenic plants were selected using bialaphos. The presence of VP2 transgene in plants was confirmed by PCR and Southern blot analysis and its expression was confirmed by RT-PCR. Western blot analysis and antigen-capture ELISA assay using monoclonal anti-VP2 were used to determine the expression of VP2 protein in transgenic plants. The level of VP2 protein in the leaf extracts of selected transgenic plants varied from 0.5% to 4.8% of the total soluble protein. Recombinant VP2 protein produced in plants induced antibody response against IBDV in orally-fed chickens.     

鸡传染性法氏囊病病毒Gt株VP5基因缺失株感染性克隆的构建

传染性法氏囊病病毒 (IBDV)是双链,双节段RNA病毒,其基因组由A、B两个节段组成,编码结构蛋白VP1-VP4和非结构蛋白VP5。【目的】利用反向遗传操作构建拯救VP5基因缺失重组IBDV。【方法】利用体外定点突变技术,缺失IBDV Gt株VP5基因,通过多重PCR在基因组两端分别引入锤头状核酶序列(HamRz)和丁肝病毒核酶序列(HdvRz)。将带有核酶序列的IBDV基因组插入载体pCAGG的b肌动蛋白启动子下游,构建了IBDV感染性克隆pCAGGmGtA △VP5HRT,将该感染性克隆与pCAGGmGtBHRT共转染DFⅠ细胞。【结果】RT-PCR和间接免疫荧光均显示获得重组病毒,将其命名为rmGtA △VP5。IBDV VP5基因缺失感染性克隆的成功构建为从分子水平上深入研究vp5基因功能奠定了基础。     

Proteome dynamics in primary target organ of infectious bursal disease virus

Viruses induce dramatic changes in target tissue during pathogenesis, including host cellular responses that either limit or support the pathogen. The infectious bursal disease virus (IBDV) targets primarily the bursa of Fabricius (BF) of chickens, causing severe immunodeficiency. Here, we characterized the cellular proteome changes of the BF caused by IBDV replication in vivo using 2DE followed MALDI-TOF MS identification. Comparative analysis of multiple 2DE gels revealed that the majority of protein expression changes appeared between 24 and 96 h after IBDV infection. MS identified 54 altered cell proteins, 12 of which were notably upregulated by IBDV infection. Meanwhile, the other 42 cellular proteins were considerably suppressed by IBDV infection and are involved in protein degradation, energy metabolism, stress response, host macromolecular biosynthesis, and transport process. The upregulation of β-actin and downregulation of dynamin during IBDV infection were also confirmed by Western blot and immunofluorescence analysis. These altered protein expressions provide a response profile of chicken BF to virulent IBDV infection. Further functional study on these altered proteins may lead to better understanding of pathogenic mechanisms of virulent IBDV infection and to new potential therapeutic targets.