禽呼肠孤病毒感染对鸡法氏囊及免疫应答的影响

研究LY株禽呼肠孤病毒(ARV)感染1日龄SPF鸡后对法氏囊发育影响,对传染性法氏囊病毒(IBDV)、禽流感病毒(AIV)、新城疫病毒(NDV)疫苗免疫诱发的抗体的影响,及对强毒株IBDV致病作用的影响。结果表明,LY株ARV感染1日龄SPF鸡可引起法氏囊萎缩和部分淋巴细胞减少,但对增重及AIV和NDV疫苗免疫后抗体滴度却没有显著影响。ARV感染可降低弱毒IBDV疫苗免疫后的抗体反应,但对随后IBDV强毒株攻毒的抵抗力却与对照鸡无显著差异。经IBDV弱毒疫苗免疫后,再接种强毒株IBDV,不会引起死亡,但却仍能显著抑制对AIV、NDV疫苗免疫后的抗体滴度。然而,对于1~7日龄经ARV感染的鸡,IBDV强毒的这种免疫抑制作用又显著低于未经ARV感染的对照鸡。     

传染性法氏囊病不同代次细胞毒免疫效果的研究

将从山东省东营分离到的1株传染性法氏囊病病毒(IBDV)野毒(暂命名IBDV SDDY株,经鉴定该株与IBDV STC株具有较高的同源性)经SPF鸡胚传代,然后转为细胞培养,取第20代、21代、25代毒,以1倍剂量(3000TCID50/0.2mL)和5倍剂量不同的免疫剂量,分别在7日龄、14日龄对商品代海蓝褐蛋鸡进行免疫,并于免疫前用IBD-ELISA试剂盒检测IBDV母源抗体水平,于35日龄用传染性法氏囊病病毒超强毒(very virulent Infectious Bursal Disease Virus,vvIBDV)GX 8/99攻毒,在攻毒前再次检测IBDV的抗体水平,攻毒后观察记录各分组鸡的致病率和死亡率,并计算免疫器官体重指数,观察免疫器官的组织损伤情况.试验结果表明3代毒都具有较高免疫原性,但是20代毒仍具有较大的毒性,7日龄接种会引起法氏囊的萎缩,造成持续的组织损伤;21代毒、25代毒保护率高,无免疫抑制,是比较理想的疫苗来源;7日龄免疫较14日龄免疫更易造成组织损伤和免疫抑制,14日龄免疫较为合适.     

传染性法氏囊病不同代次细胞毒免疫效果的研究

将从山东省东营分离到的1株传染性法氏囊病病毒(IBDV)野毒(暂命名 IBDV SDDY株,经鉴定该株与 IB DV STC株具有较高的同源性)经 SPF 鸡胚传代,然后转为细胞培养,取第 20 代、21 代、25 代毒,以 1 倍剂量(3000TCID50/0.2mL )和5倍剂量不同的免疫剂量,分别在7日龄、14日龄对商品代海蓝褐蛋鸡进行免疫,并于免疫前用 IBD ELISA试剂盒检测 IBDV母源抗体水平,于 35 日龄用传染性法氏囊病病毒超强毒 (very virulent In fectious Bursal Disease Virus, vvIBDV )GX 8/99攻毒,在攻毒前再次检测IBDV的抗体水平,攻毒后观察记录各分组鸡的致病率和死亡率,并计算免疫器官体重指数,观察免疫器官的组织损伤情况。试验结果表明:3 代毒都具有较高免疫原性,但是20代毒仍具有较大的毒性,7 日龄接种会引起法氏囊的萎缩,造成持续的组织损伤;21 代毒、25代毒保护率高,无免疫抑制,是比较理想的疫苗来源;7日龄免疫较14日龄免疫更易造成组织损伤和免疫抑制,14日龄免疫较为合适。     

雏鸡感染CAV后对IBD发病特征的影响

鸡传染性法氏江病（IBD）是引起鸡的一种急性、高度传染性病毒病,以损伤体液免疫机能为主要特征;鸡传染性贫血病（CM）特征是幼龄鸡的淋巴组织萎缩和再生障碍性贫血,以侵害细胞免疫为主。现地IBDV与CAV混合感染的现象常有发生,但由于CIA症状不典型,不易察觉。本试验拟在人工感染条件F观察CAV对IBD的影响。实验分为3组：空白对照轨IBDV感染组、IBDV～CAV混合感染组,每组设10只SPF雏鸡。1日龄接种CAV,3日龄接种IBDV。分别于6‘13日龄测定并计算各级鸡的法氏囊、胸腺和体重的比值,分离外周血、法氏囊和胸腺中的淋巴…     

重组禽腺联病毒介导的miRNA抑制传染性法氏囊病病毒在鸡胚内的复制

【目的】在鸡胚水平上探索VP1和VP2基因特异miRNA抑制传染性法氏囊病病毒(infectious bursaldisease virus,IBDV)复制的可行性。【方法与结果】将表达VP1基因特异miRNA重组载体pAITR-RFPmiVP1或VP2基因特异miRNA重组载体pAITR-RFPmiVP2E与禽腺联病毒(avian adeno-associated virus,AAAV)包装载体pcDNA-ARC和腺病毒辅助载体pHelper共转染AAV-293细胞,获得重组病毒rAAAV-RFPmiVP1和rAAAV-RFPmiVP2E,用同样方法获得不表达miRNA的rAAAV-RFP和表达对照miRNA的rAAAV-RFPmiVP2con。电镜观察显示重组病毒具有典型的AAAV颗粒形态;PCR检测结果表明其基因组中含miRNA表达盒;经poly(A)加尾RT-PCR检测证明重组病毒感染细胞能表达基因特异的miRNA。分别将重组病毒经卵黄囊途径接种8日龄SPF鸡胚,然后经绒毛尿囊膜途径用Lukert株IBDV攻毒,收获鸡胚进行IBDV组织细胞半数感染剂量(TCID50)测定。结果在攻毒后第3天,rAAAV-RFP和rAAAV-RFPmiVP2con接种组的IBDV TCID50为8.0 log10,rAAAV-RFPmiVP1和rAAAV-RFPmiVP2E接种组的IBDV TCID50分别下降到1.0和1.5 log10;在攻毒后第6天,rAAAV-RFP和rAAAV-RFPmiVP2con接种组的IBDV TCID50仍为8.0 log10,rAAAV-RFPmiVP1和rAAAV-RFPmiVP2E接种组的TCID50分别下降到0.8和2.0 log10。【结论】rAAAV是有效的miRNA鸡胚导入载体,表达的VP1和VP2基因特异miRNA能有效阻断IBDV复制。     

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     

表达IBDV VP2融合蛋白的重组MDV的构建及其免疫特性

将增强型绿色荧光蛋白基因(eGFP)与鸡传染性法氏囊病病毒(IBDV)的VP2基因融合,插入马立克氏病毒(MDV)CVI988/Rispens的非必需区US10片段中,成功构建表达VP2融合蛋白的MDVCVI988转移载体pUC18-US10-VP2。将转移载体质粒与CVI988/Rispens疫苗毒共转染鸡胚成纤维细胞(CEF),筛选获得表达VP2融合蛋白的重组MDV(rMDV)。聚合酶链式反应(PCR)和间接免疫荧光实验(IFA)证明,rMDV传至第31代仍能稳定表达VP2融合蛋白。用rMDV免疫SPF鸡,进行IBDV攻毒保护试验,1日龄SPF鸡分别用1000PFU、2000PFU、5000PFU的rMDV进行免疫,33日龄用100LD50的IBDVJS超强毒进行攻毒,鸡的免疫保护率分别为50%、60%、80%。值得注意的是,5000PFU的rMDV一次免疫1日龄SPF鸡,其法氏囊组织病理损伤等级与IBD中等毒力活疫苗常规二次免疫相当(2·0/1·5),其保护效果无显著差异(p>0·05),而与非重组病毒免疫组相比较,保护效果差异显著(P<0·01),这表明构建的表达IBDVVP2融合蛋白的rMDV可以有效地为SPF鸡提供免疫保护作用。     

NASBA快速检测禽流感H5亚型病毒

采用建立的依赖核酸序列的扩增(Nucleic acid sequence-based amplification,NASBA)对禽流感病毒3株H5亚型、1株H1、H3、H6亚型、3株禽流感H9亚型、5株不同宿主来源的新城疫病毒、鸭肝炎病毒、鸭瘟病毒、SPF鸡胚尿囊液及禽流感(H9)疫苗、新城疫疫苗、传染性法氏囊病疫苗、传染性支气管炎疫苗进行检测,结果NASBA(H5试剂)仅检测到禽流感病毒H5亚型,表明方法的特异性强.采用已知禽流感病毒A/Chicken/HK/1000/97(H5N1)的鸡胚尿囊液(ELD5010-7.5/mL),经10倍连续稀释,将经典的鸡胚病原分离法和NASBA进行比较,二种方法的灵敏度相当.用A/Chicken/HK/1000/97(H5N1)病毒人工感染SPF鸡、商品鸡,采用NASBA和病原分离法同时对人工感染鸡的粪拭子、血液进行了动态检测;采集感染死亡鸡的组织脏器,共检测了101个组织脏器,两种方法的符合率为90%(87/97).     

超强毒IBDV细胞克隆化毒回归鸡后的致病性与VP2基因高变区的分子变化

为了研究超强毒鸡传染性法氏囊病病毒(vvIBDV)的致病性及其VP2基因高变区的分子变化,以vvIBDV GX8/99株囊毒为研究对象,将该毒在SPF鸡胚连传10代后,再在鸡胚成纤维细胞(CEF)上连续盲传.该病毒在CEF上传至22代时开始引发CEF细胞病变,随之在96孔细胞培养板上用无限稀释法连续克隆2次后获得了4个病毒克隆,再将该4个病毒克隆分别连续回传3～5周龄SPF鸡10代.分别比较4个克隆毒及其回传SPF鸡后不同传代毒,对4～6周龄SPF鸡的致病性及VP2高变区氨基酸分子的变化,结果表明,4个克隆化毒的细胞培养毒对SPF鸡只有0～6.7%的致死率,不同克隆毒的SPF鸡传代毒对SPF鸡的致病性却都逐渐增强,但程度差异很大,其中克隆#5在回鸡1、5和10代后的致死率从0分别增加到10%、20%和27%;克隆#4从6.7%增加至13%、17%和23%;但克隆#1和#3的致病性变化相对较小.相对于原始囊毒及鸡胚毒,4个克隆化毒在测序的VP2高变区的约145个氨基酸中,有10个位点发生了相同的变异,变得与适应细胞的疫苗毒D78株基本一致,在回鸡传代导致对鸡毒力增强的过程中,这10个位点中大多数氨基酸不再变化,只有第253位和256位氨基酸从囊毒的Q和I变为细胞适应毒的H和V后,有些病毒克隆回鸡至第10代时又变为原囊毒的Q和I,这表明VP2高变区大多数氨基酸的变异可能与病毒的致病性关系不密切,而与对细胞培养或组织的亲和性的关系更为密切.本研究最重要的意义在于建立的超强毒GX8/99株细胞克隆化毒及其相应的回鸡传代毒系列,为研究vvIBDV其它基因变异与致病性及其它生物学特性的关系,提供了一个新的思路和必要的研究材料.     

NASBA快速检测禽流感H5亚型病毒

采用建立的依赖核酸序列的扩增(Nucleicacidsequencebasedamplification,NASBA)对禽流感病毒3株H5亚型、1株H1、H3、H6亚型、3株禽流感H9亚型、5株不同宿主来源的新城疫病毒、鸭肝炎病毒、鸭瘟病毒、SPF鸡胚尿囊液及禽流感(H9)疫苗、新城疫疫苗、传染性法氏囊病疫苗、传染性支气管炎疫苗进行检测,结果NASBA(H5试剂)仅检测到禽流感病毒H5亚型,表明方法的特异性强。采用已知禽流感病毒A/Chicken/HK/1000/97(H5N1)的鸡胚尿囊液(ELD5010-7.5/mL),经10倍连续稀释,将经典的鸡胚病原分离法和NASBA进行比较,二种方法的灵敏度相当。用A/Chicken/HK/1000/97(H5N1)病毒人工感染SPF鸡、商品鸡,采用NASBA和病原分离法同时对人工感染鸡的粪拭子、血液进行了动态检测;采集感染死亡鸡的组织脏器,共检测了101个组织脏器,两种方法的符合率为90%(87/97)。     

传染性法氏囊病病毒多表位模拟肽串联基因的表达与免疫原性分析

用5株传染性法氏囊病病毒(IBDV)单克隆抗体(mAb)从噬菌体随机肽库中得到了5个含有不同IBDV抗原表位的模拟肽序列。在此基础上,将5个抗原表位用GGGS四肽连接构建多表位基因5epis。将该基因合成、克隆后,构建原核表达质粒pET-5epis,在大肠杆菌中表达重组多表位蛋白r5EPIS,经SDS-PAGE分析,r5EPIS占菌体总蛋白的15%、分子量10kDa。用IBDV单克隆抗体和多克隆抗体对r5EPIS进行免疫印迹对比分析,结果表明,r5EPIS具有IBDV特异性和免疫反应性。将r5EPIS经皮下注射免疫兔,400μg/只/次,免疫2次,间隔7d,用IBDV间接ELISA检测血清抗体,第一次免疫7d后抗体效价为1∶4000,第二次免疫14d后抗体效价升高到1∶256000,说明r5EPIS可诱导机体产生IBDV特异性抗体。用r5EPIS加免疫佐剂经肌肉注射免疫鸡,50μg/只/次,免疫2次后,血清抗体效价可达到1∶12800,用200个ELD50IBDV超强毒株GX8/99攻击实验鸡,r5EPIS免疫组全部存活,而单用佐剂对照组的死亡率为86.7%(13/15),证明r5EPIS可诱导机体产生抗IBDV感染的保护性免疫应答,预示构建的5epis可作为IBD多表位疫苗研究的候选基因。     

抗IBDV独特型抗体杂交瘤细胞株的建立及其产物生物学特性测定

用纯化的抗IBDV IgG免疫Balb/c小鼠,取其脾细胞与SP2/0细胞在聚乙二醇（PEG）作用下融合,ELISA法检测筛选,经有限稀释法克隆3次,获得2株（5F₄株,2B₆株）分泌抗IBDV独特型抗体的杂交瘤细胞株,其能诱生Balb/c小鼠产生ELISA抗体效价分别为1∶12 800和1∶25 600的含抗IBDV独特型抗体的腹水。用此独特型抗体与福氏完全佐剂和福氏不完全佐剂乳化制备成抗IBDV独特型抗体疫苗,免疫接种SPF鸡和普通京白公鸡,然后用IBDV强毒株(SD株)2000 ELD50攻毒,SPF鸡免疫组50只,有5只发病、2只死亡;对照组10只全部发病,8只死亡。普通京白鸡免疫组30只,有7只发病, 1只死亡;对照组10只全部发病,6只死亡。经X²检验,SPF鸡X²＝34.15,普通鸡X²＝16.68,查X²值表得X²_(1)0.01＝6.63, SPF鸡X²和普通鸡X2均大于X²_{(1) 0.01}（P＜0.01）,由此表明抗IBDV独特型抗体疫苗具有很好的免疫原性,对易感日龄的SPF鸡和普通鸡均具有极其明显的保护作用。从而证实了抗IBDV独特型抗体疫苗有潜在的研究和应用价值。     

Genetic reassortment of infectious bursal disease virus in nature

Infectious bursal disease virus (IBDV), a double-stranded RNA virus, is a member of the Birnaviridae family. Four pathotypes of IBDV, attenuated, virulent, antigenic variant, and very virulent (vvIBDV), have been identified. We isolated and characterized the genomic reassortant IBDV strain ZJ2000 from severe field outbreaks in commercial flocks. Full-length genomic sequence analysis showed that ZJ2000 is a natural genetic reassortant virus with segments A and B derived from attenuated and very virulent strains of IBDV, respectively. ZJ2000 exhibited delayed replication kinetics as compared to attenuated strains. However, ZJ2000 was pathogenic to specific pathogen free (SPF) chickens and chicken embryos. Similar to a standard virulent IBDV strain, ZJ2000 caused 26.7% mortality, 100% morbidity, and severe bursal lesions at both gross and histopathological levels. Taken together, our data provide direct evidence for genetic reassortment of IBDV in nature, which may play an important role in the evolution, virulence, and host range of IBDV. Our data also suggest that VP2 is not the sole determinant of IBDV virulence, and that the RNA-dependent RNA polymerase protein, VP1, may play an important role in IBDV virulence. The discovery of reassortant viruses in nature suggests an additional risk of using live IBDV vaccines, which could act as genetic donors for genome reassortment.     

敲除meq的鸡马立克氏病毒强毒株对超强毒的免疫保护作用

【目的】比较和评价一株敲除了meq基因的马立克氏病毒(MDV)的致病性及其诱发的保护性免疫作用。【方法】将1日龄SPF鸡150只随机分为5组,每组30只,分别饲养于正压过滤空气的SPF动物饲养隔离罩内。1日龄时,第1和第5组鸡以2000PFU/只的剂量腹腔接种GX0101Δmeq,第2组鸡以2000PFU/只的剂量腹腔接种CVI988/Rispens疫苗株,第3和第4组鸡不接种任何病毒作为对照组。免疫接种5d后,第1、2、3组分别以500PFU/只的剂量攻击MDV超强毒株vvrMd5。饲养90d,观察死亡情况,对各组死亡鸡只剖检,并取疑似马立克特有病变脏器做石蜡切片,于攻毒后90d处死全部存活鸡并随机取心脏、肝脏、脾脏做病理切片。【结果】单独接种GX0101Δmeq的第5组没有任何马立克氏病临床症状和特有的组织学病变,接种GX0101Δmeq再感染超强毒株vvrMd5的第1组也没有马立克病特有的组织学病变,但CVI988/Rispens免疫后感染超强毒株vvrMd5的第2组显示马立克病特有病变的病理切片比例为9/42,单独接种超强毒株vvrMd5的第3组死亡率为87%,死亡鸡出现可眼观典型肿瘤率为25%,免疫接种GX0101Δmeq和CVI988/Rispens的第1组和第2组对超强毒株vvrMd5攻击的保护指数分别为100%和89%。【结论】本实验构建的MDVmeq基因缺失株-GX0101Δmeq可在体外稳定复制,不仅对SPF鸡没有致病性和致瘤性,而且能诱导比CVI988/Rispens疫苗株更好的对超强毒MDV的免疫保护效果。     

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.     

Protective Oral Vaccination against Infectious bursal disease virus Using the Major Viral Antigenic Protein VP2 Produced in Pichia pastoris

Infectious bursal disease virus (IBDV) causes economically important immunosuppressive disease in young chickens. The self-assembling capsid protein (VP2) from IBDV strain IR01 was expressed in Pichia pastoris resulting in the formation of homomeric, 23-nm infectious bursal disease subviral particles (IBD-SVPs) with a yield of 76 mg/l before and 38 mg/l after purification. Anti-IBDV antibodies were detected in chickens injected with purified IBD-SVPs or fed with either purified IBD-SVPs or inactivated P. pastoris cells containing IBD-VP2 (cell-encapsulated). Challenge studies using the heterologous classical IBDV strain (MB3) showed that intramuscular vaccination with 20 µg purified IBD-SVPs conferred full protection, achieved complete virus clearance and prevented bursal damage and atrophy, compared with only 40% protection, 0–10% virus clearance accompanied by severe atrophy and substantial bursal damage in mock-vaccinated and challenge controls. The commercial IBDV vaccine also conferred full protection and achieved complete virus clearance, albeit with partial bursal atrophy. Oral administration of 500 µg purified IBD-SVPs with and without adjuvant conferred 100% protection but achieved only 60% virus clearance with adjuvant and none without it. Moderate bursal damage was observed in both cases but the inclusion of adjuvant resulted in bursal atrophy similar to that observed with live-attenuated vaccine and parenteral administration of 20 µg purified IBD-SVPs. The oral administration of 250 mg P. pastoris cells containing IBD-VP2 resulted in 100% protection with adjuvant and 60% without, accompanied by moderate bursal damage and atrophy in both groups, whereas 25 mg P. pastoris cells containing IBD-VP2 resulted in 90–100% protection with moderate bursal lesions and severe atrophy. Finally, the oral delivery of 50 µg purified IBD-SVPs achieved 40–60% protection with severe bursal lesions and atrophy. Both oral and parenteral administration of yeast-derived IBD-VP2 can therefore induce a specific and protective immune response against IBDV without affecting the growth rate of chickens.     

Characteristics of bursal T lymphocytes induced by infectious bursal disease virus

Infectious bursal disease virus (IBDV) is an avian lymphotropic virus that causes immunosuppression. When specific-pathogen-free chickens were exposed to a pathogenic strain of IBDV (IM), the virus rapidly destroyed B cells in the bursa of Fabricius. Extensive viral replication was accompanied by an infiltration of T cells in the bursa. We studied the characteristics of intrabursal T lymphocytes in IBDV-infected chickens and examined whether T cells were involved in virus clearance. Flow cytometric analysis of single-cell suspensions of the bursal tissue revealed that T cells were first detectable at 4 days postinoculation (p.i.). At 7 days p.i., 65% of bursal cells were T cells and 7% were B cells. After virus infection, the numbers of bursal T cells expressing activation markers Ia and CD25 were significantly increased (P<0.03). In addition, IBDV-induced bursal T cells produced elevated levels of interleukin-6-like factor and nitric oxide-inducing factor in vitro. Spleen and bursal cells of IBDV-infected chickens had upregulated gamma interferon gene expression in comparison with virus-free chickens. In IBDV-infected chickens, bursal T cells proliferated in vitro upon stimulation with purified IBDV in a dose-dependent manner (P<0.02), whereas virus-specific T-cell expansion was not detected in the spleen. Cyclosporin A treatment, which reduced the number of circulating T cells and compromised T-cell mitogenesis, increased viral burden in the bursae of IBDV-infected chickens. The results suggest that intrabursal T cells and T-cell-mediated responses may be important in viral clearance and promoting recovery from infection.