Differentiation of infectious bursal disease viruses isolated in Croatia

Infectious bursal disease viruses (IBDVs) in 26 IBDV-positive bursa samples collected in Croatia during the period 1996-2000 and in two commercially available vaccines were differentiated by the presence or absence of the CfoI, SacI, SspI, StuI, and TaqI restriction sites in the 422-bp fragment of segment A of the VP2 gene (nt 732-1153). The fragments from 14 (54%) field isolates were TaqI+ StuI+ SspI+ and SacI- CfoI-, indicating their very virulent (vv) character. The presence of CfoI restriction site in 10 (38%) field isolates is uncommon for vvIBDV strains. It was detected in only the 88180 vvIBDV strain. Nevertheless, these isolates can be classified as vv strains according to TaqI+ StuI+ SspI+ SacI- restrictions. Two SacI+ StuI+ CfoI+ TaqI- SspI- field isolates (8%) could be classified as non-vvIBDVs. The StuI+ restriction is common to vvIBDV strains. However, the StuI recognition sequence is present in the F52/70 classic European and 002-73 attenuated strains as well. The SacI+ CfoI+ StuI- SspI- restrictions and the lack of the TaqI restriction at nt position 832 show that the IBDV in GUMBOKAL IM-SPF vaccine corresponds to the attenuated and/or vaccine strains. The TaqI restriction at nt position 875 suggests that the IBDV in GUMBOKAL SPF vaccine could belong to the mild strains.     

靶向传染性法氏囊病病毒VP1基因siRNA对病毒复制的抑制

根据传染性法氏囊病病毒（IBDV）VP1基因保守区序列,选择设计了3个靶向VP1的小干扰RNA序列（siR-NA）：VP1-siRNA668、VP1-siRNA1034和VP1-siRNA2250,化学合成DNA序列,体外转录合成siRNA,转染鸡胚成纤维细胞（CEF）后接种IBDV,分析siRNA对病毒复制的影响。结果显示,病毒接种后72h,3个VP1-siRNA转染组未出现明显的细胞病变（CPE）,病毒滴度分别为102.75,102.00,101.75 TCID50/0.1mL,而siRNACON转染和单纯IBDV接种对照组均出现明显CPE,病毒滴度均为106 TCID50/0.1mL;用荧光定量RT-PCR分析VP1基因水平,3个VP1-siRNA转染组比对照组分别降低了73.10%,81.79%,90.28%。结果表明,本试验选择设计的3个siRNA具有明显抑制IBDV复制功能,其中2 250位点的siRNA抑制效果最明显,推测该区域是VP1基因的重要功能区,是新型抗IBDV药物与疫苗设计的重要靶标。     

Differentiation between antibodies to serotypes 1 and 2 infectious bursal disease viruses in chicken sera

The usefulness of the virus neutralization (VN) test, the enzyme-linked immunosorbent assay (ELISA), and the agar gel precipitin (AGP) test in differentiating antibodies to infectious bursal disease virus serotypes 1 and 2 was investigated. Sera examined were from chickens that were challenged with live virus or inoculated with inactivated oil-emulsion IBDV vaccines or were both challenged and inoculated. Antibodies to serotypes 1 and 2 were differentiated by the VN test but not by the ELISA, and the AGP test was less than satisfactory.     

Lysis of chicken lymphocytes by infectious bursal disease viruses

Infectious bursal disease virus types 1 and 2 were able to induce direct lysis of chicken bursal cells, thymus cells, and peripheral blood lymphocytes in chromium-release assays. These two viruses were unable to lyse two established lymphoblastoid cell lines, although IBDV-1 was capable of multiplying in MSB-1 cells.     

传染性法氏囊病安徽近期毒株VP2基因在昆虫细胞中的表达

为真核表达传染性法氏囊病病毒(IBDV)VP2基因,将2007年12月从安徽境内发生的传染性法氏囊病(IBD)免疫预防失败的病鸡法氏囊组织中克隆的IBDV VP2基因插入到pFastBac1供体质粒中,转化E.coilDH10Bac感受态细胞,经抗性筛选,获得重组杆状病毒表达质粒rBac-VP2,用脂质体法转染SD细胞.对rBac-VP2感染的Sf9细胞,用间接免疫荧光试验(IFA)检测,具有特异性荧光;用western blot分析,在50 ku处出现1条特异蛋白条带;电镜观察重组VP2蛋白能够自组装成病毒样颗粒.本实验为研制针对近期流行IBDV的新型病毒样颗粒疫苗和新型检测试剂奠定了基础.     

Antigenic diversity of infectious bursal disease viruses

Statistically significant antigenic differences were detected among serotype I infectious bursal disease viruses (IBDV) using the virus-neutralization test. Eight serotype I commercial vaccine strains, five serotype I field strains, and two serotype II field strains were tested. Hyperimmune guinea pig antisera against heterologous and homologous IBDV strains were used in cross-neutralization tests. Relatedness values were calculated from geometric mean antibody titers based on a minimum of three tests. Six subtypes were distinguished among the 13 serotype I strains tested.     

鸡传染性腔上囊病病毒VP2基因在昆虫细胞中的表达

为了深入研究鸡传染性腔上囊病病毒（IBDV）VP2基因的结构和功能,利用Bac-to-Bac系统研制出含有VP2基因的重组杆状病毒rBac-VP2,将rBac-VP2感染Sf9细胞,并用抗IBDV VP2特异性单克隆抗体经间接免疫荧光试验检测,证实感染重组病毒Sf9细胞能高效表达IBDV VP2基因产物;Western-blotting分析结果表明,VP2基因表达产物的分子质量约为40 ku.     

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.     

Immunogenicity of infectious bursal disease viruses in chickens.

Cross-protective properties of infectious bursal disease viruses (IBDVs) were studied. Viruses represented different subtypes of serotype 1, including recently isolated viruses (variants), and a serotype 2 virus. Chickens were vaccinated at 3 weeks of age with inactivated vaccines containing 10(5), 10(6), 10(7), or 10(8) mean tissue-culture infectious dose of a given virus and challenged 2 weeks later using either 10(2) or 10(3.5) mean embryo infectious dose (EID50) of either a standard virus or a variant serotype 1 virus. Protection was evaluated at 5 and 10 days post-challenge, based on gross and microscopic lesions, body weight, and bursa/body-weight ratios. The serotype 2 virus did not confer protection on birds challenged with the serotype 1 viruses. Vaccines made of variant viruses at the low doses protected chickens challenged with the high or low doses of either the standard or the variant viruses. Vaccines made of the standard or variant strains at low doses protected against high or low challenge doses of the standard strain. Vaccines made of the high dose of any of the standard strains protected chickens against the variant virus when the low challenge dose (10(2) EID50) was used, but not when the high challenge dose (10(3.5) EID50) was used. The lowest dose of the standard viruses vaccines required to confer protection against the variant virus varied depending on the strain. Results indicated that protection depended on the strain and dose of both the vaccine and challenge viruses and that the variant strains and standard strains share a common protective antigen(s).     

传染性腔上囊病病毒VP3蛋白模拟表位的筛选

为了研究传染性腔上囊病病毒（IBDV）的致病机理及研制有效的IBDV疫苗，利用噬菌体展示技术对IBDV VP3抗原表位进行了筛选。以4株IBDV VP3单克隆抗体HRB-3F、HRB-7B、HRB-7C和HRB-10E作为筛选分子，对噬菌体随机15肽库进行3轮吸附-洗脱-扩增淘洗，从每株单克隆抗体筛选到的噬菌斑中随机挑取20个单克隆噬菌斑，通过间接ELISA和竞争抑制ELISA检测，共选出13个单克隆噬菌斑，经噬菌体gⅢ部分基因的核苷酸序列测定，确定了8个15肽为IBDV抗原表位。这8个15肽在一级结构上没有3个以上连续氨基酸与IBDV GX（Gen—Bank登录号：AY444873）VP3的氨基酸序列相同，但二级结构上均以β折叠为主，并且与单抗的结合可被VP3蛋白有效地抑制。证实，筛选的是IBDV VP3的模拟表位。     

Prevention of avian lymphoid leukosis by induction of bursal atrophy with infectious bursal disease viruses.

Five groups of genetically susceptible chickens were inoculated at hatching with lymphoid leukosis virus; four of these were given infectious bursal viruses of varying virulence at 14 days of age and one group was not inoculated (control). All chickens in the control group developed evidence of lymphoid leukosis by 180 days. Two groups given relatively virulent bursal disease viruses, which destroyed bursal lymphoid cells, did not develop lymphoid leukosis. Treatment with avirulent vaccines had no visible effect on bursal morphology and did not significantly alter the incidence of lymphoid leukosis in two other groups, although the time of development was delayed. Results of our study show that viral-induced destruction of the bursa of Fabricius eliminates the development of lymphoid leukosis but that infection without bursal destruction has little effect on lymphoid leukosis.     

鸡传染性法氏囊病病毒VP2蛋白可溶性原核表达及免疫原性分析

为在大肠杆菌中表达出高可溶性的VP2-LS3蛋白笼纳米颗粒并检测其免疫原性,将扩增的7种融合标签(GST、NusA、MBP、PpiB、γ-crystallin、ArsC和Grifin)片段连在VP2-LS3基因的N端,构建7个带有不同标签的重组原核表达载体。再将其分别转化至大肠杆菌BL21(DE3),用IPTG诱导表达,表达产物经SDS-PAGE电泳分析,筛选出显著促进VP2-LS3蛋白可溶性表达的融合标签,并进行大量诱导表达、纯化。纯化得到的VP2-LS3重组蛋白分别进行TEV酶酶切、电镜观察分析及免疫家兔,并对获得的兔抗VP2血清进行Western blot和间接ELISA分析。结果显示,与其他6个标签相比,MBP标签促进VP2-LS3蛋白的可溶性表达具有显著性,可溶性表达水平达到69.5%;TEV酶可以把His6-MBP标签蛋白与VP2-LS3蛋白分开,获得高纯度的VP2-LS3蛋白;镜检显示形成了LS3蛋白笼纳米颗粒;Western blot结果表明,表达的VP2-LS3重组蛋白可与兔抗血清发生特异性反应,而不与阴性对照血清发生反应;间接ELISA检测制备的兔抗VP2血清效价达到1∶12 800。本研究获得了高可溶性的VP2-LS3蛋白笼纳米颗粒,为传染性法氏囊病病毒(IBDV)基因工程疫苗的研发奠定基础。