Structure of Infectious Bursal Disease Virus

Infectious bursal disease virus of chickens was purified, and its structure was examined by the negative-staining technique in the electron microscope. The buoyant density of infectious bursal disease virus in CsCl was found to be 1.34 g/cm(3). The morphological details suggest that the capsid of the virion consists of a single layer of 32 capsomeres arranged in 5:3:2 symmetry. The virion measured about 55 nm in diameter and had no envelope.     

Biophysical and biochemical characterization of five animal viruses with bisegmented double-stranded RNA genomes.

Infectious pancreatic necrosis virus of fish, infectious bursal disease virus of chickens, Tellina virus and oyster virus of bivalve molluscs, and drosophila X virus of Drosophila melanogaster are naked icosahedral viruses with an electron microscopic diameter of 58 to 60 nm. The genome of each of these viruses consists of two segments of double-stranded RNA (molecular weight range between 2.6 x 10(6) and 2.2 x 10(6), and the virion, capsid proteins fall into three size class categories (large, medium, and small; ranging from 100,000 to 27,000) as determined by polyacrylamide slab gel electrophoresis. The hydrodynamic properties of the five viruses are similar as determined by analytical ultracentrifugation and laser quasi-elastic, light-scattering spectroscopy. The calculated particle weights range between 55 x 10(6) and 81 x 10(6). Tryptic peptide comparisons of 125I-labeled virion proteins showed that five viruses are different from each other, although there was considerable overlap in the peptide maps of the three aquatic viruses, indicting a degree of relatedness. Cross-neutralization tests indicated that drosophila X, infectious pancreatic necrosis, and infectious bursal disease viruses were different from each other and from oyster and Tellina viruses. The same test showed oyster and Tellina viruses to be related. The biochemical and biophysical properties of the five viruses cannt be included in the family Reoviridae or in any of the present virus genera.     

Structural and growth characteristics of infectious bursal disease virus.

The infectious bursal disease virus is not enveloped and has a diameter of 60 nm and a density of about 1.32 g/ml. It contains two pieces of single-stranded RNA with molecular weights close to 2 X 10(6). The capsid is made up of four major polypeptides with molecular weights of 110,000, 50,000, 35,000, and 25,000. The virus replicates in chicken embryo fibroblasts rather than in epitheloid cells. After an eclipse period of 4 h, virus production reaches a maximum about 12 h later. The virus has no structural or biological similarities with defined avian reoviruses, and it cannot be classified in one of the established taxonomic groups.     

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.     

Three-dimensional structure of infectious bursal disease virus determined by electron cryomicroscopy.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae group, is a commercially important pathogen of chickens. From electron micrographs of frozen, hydrated, unstained specimens, we have computed a three-dimensional map of IBDV at about 2 nm resolution. The map shows that the structure of the virus is based on a T=13 lattice and that the subunits are predominantly trimer clustered. The subunits close to the fivefold symmetry axes are at a larger radius than those close to the two- or threefold axes, giving the capsid a markedly nonspherical shape. The trimer units on the outer surface protrude from a continuous shell of density. On the inner surface, the trimers appear as Y-shaped units, but the set of units surrounding the fivefold axes appears to be missing. It is likely that the outer trimers correspond to the protein VP2, carrying the dominant neutralizing epitope, and the inner trimers correspond to protein VP3, which has a basic carboxy-terminal tail expected to interact with the packaged RNA.     

Monoclonal antibodies against different epitopes of a 40 Kd capsid protein of infectious bursal disease viruses.

Nine monoclonal antibodies (Mab) against a 40 Kd capsid protein of infectious bursal disease virus (IBDV) strain P3009 were isolated. They were characterized by enzyme-linked immunosorbent assay, indirect fluorescent antibody staining and virus neutralization. They were divided into two groups concerning virus neutralization. Group I Mabs were able to neutralize virus infectivity; however, group II Mabs were not. Competitive binding assays using these Mabs demonstrated the existence of two distinct antigenic regions (A and B) on the 40 Kd protein. Region A was recognized by group I Mabs and region B was by group II Mabs. The binding reaction with group I Mabs was affected by denaturing of the viral proteins, indicating that the antigenic region involving neutralization was conformation-dependent. The results of enzyme-linked immunosorbent assays and virus neutralization tests suggested that group I Mabs might react with one epitope within region A and group II Mabs with 2 or 3 epitopes within region B.     

The genome of infectious bursal disease virus consists of two segments of double-stranded RNA.

The RNA of infectious bursal disease virus was reexamined in a detailed analysis. It could be established that its genome consists of two segments of double-stranded RNA. The RNA is RNase resistant and has a sedimentation coefficient of 14S and a buoyant density of 1.62 g/ml. The purine/pyrimidine ratio is nearly 1; the guanine plus cytosine content is 55.3%; the Tm is 95.5 degrees C. The molecular weights of the two double-stranded segments were determined to be 2.2 x 10(6) and 2.5 x 10(6).     

鸡传染性法氏囊病上海超强毒株NH99的分离与鉴定

通过对上海近郊某鸡场数群 10日龄左右的病鸡的临床症状、病理变化、病毒分离、纯化、动物回归、血清学检测及病毒核酸纯化、VP2基因序列的测定与分析 ,确认上海地区以发病日龄早 ,发病率、死亡率高为特征的鸡传染病为超强毒型鸡传染性法氏囊病 ,病原具有鸡传染性法氏囊病病毒超强毒 (vvIBDV)的分子特征 ,为vvIBDV。     

Processing of infectious bursal disease virus (IBDV) polyprotein and self-assembly of IBDV-like particles in Hi-5 cells

The capsid of infectious bursal disease virus (IBDV), with a size of 60-65 nm, is formed by an initial processing of polyprotein (pVP2-VP4-VP3) by VP4, subsequent assemblage of pVP2 and VP3, and the maturation of VP2. In Sf9 cells, the processing of polyprotein expressed was restrained in the stage of VP2 maturation, leading to a limited production of capsid, i.e., IBDV-like particles (VLPs). In the present study, another insect cell line, High-Five (Hi-5) cells, was demonstrated to efficiently produce VLPs. Meanwhile, in this system, polyprotein was processed to pVP2 and VP3 protein and pVP2 was further processed to the matured form of VP2. Consequently, Hi-5 cells are better in terms of polyprotein processing and formation of VLPs than Sf9. In addition to the processing of pVP2, VP3 was also degraded. With insufficient intact VP3 protein present for the formation of VLPs, the excessive VP2 form subviral particles (SVPs) with a size of about 25 nm. The ratio of VLPs to SVPs is dependent on the multiplicity of infections (MOIs) used, and an optimal MOI is found for the production of both particles. VLPs were separated from SVPs with a combination of ultracentrifugation and gel-filtration chromatography, and a large number of purified particles of both were obtained. In conclusion, the insect cell lines and MOIs were optimized for the production of VLPs, and pure VLPs with morphology similar to that of the wild-type viruses can be effectively prepared. The efficient production and purification of VLPs benefits not only the development of an antiviral vaccine against IBDV but also the understanding of the structure of this avian virus that is economically important.     

Biophysical studies of infectious pancreatic necrosis virus.

The molecular weight of infectious pancreatic necrosis virus (IPNV) has been determined by analytical ultracentrifugation and dynamic light scattering. The sedimentation coefficient of the virus was found to be 435S. The average value for molecular weight is (55 +/- 7) x 106. The virus genome consists of two segments of double-stranded RNA (molecular weights, 2.5 x 106 and 2.3 x 106), which represents 8.7% of the virion mass. The capsid protein moiety of IPNV consists of four species of polypeptides, as determined by polyacrylamide gel electrophoresis. The number of molecules of each polypeptide in the virion has been determined. There are 22 molecules of the internal polypeptide alpha (molecular weight, 90,000), 544 molecules of the outer capsid polypeptide beta (molecular weight, 57,000), and 550 and 122 molecules, respectively, of the internal polypeptides gamma1 (molecular weight, 29,000) and gamma2 (molecular weight, 27,000). IPNV top component contains only the beta polypeptide species, and its molecular weight is estimated to be 31 x 106. The hydrodynamic diameter and electron microscopic diameter (calculated by catalase crystal-calibrated electron microscopy) of IPNV was compared with those of reovirus and encephalomyocarditis virus. Due to the swelling of the outer capsid, reovirus particles were found to be much larger when hydrated (96-nm diameter) than when dehydrated (76-nm diameter), having a large water content content and low average density. In contrast, IPNV particles are more rigid, having nearly the same average diameter under hydrous (64 nm) as under anhydrous conditions (59.3 nm). Encephalomyocarditis virus has a very low water content and does not shrink at all when prepared for electron microscopy.     

传染性法氏囊病毒的抗原及分子特征

用鸡胚成纤维细胞对来自野外的 5 个传染性法氏囊病毒株 (IBDV-JD1 、 JD2 、 NB 、 HZ1 、 HZ2) 进行分离,测定理化特性、致病性,同时进行血清亚型测定及 A 片段基因组的克隆分析 . 试验所用 5 个法氏囊组织悬液在鸡胚成纤维细胞盲传 2~14 代后适应细胞并产生细胞病变 . 细胞适应的 IBDV 毒株的理化和形态特征与经典传染性法氏囊病毒株一致 . 除 IBDV-HZ1 、 HZ2 属经典 IBDV 血清型外, IBDV-JD1 、 JD2 和 NB 毒株分属不同的血清亚型 . 人工感染实验结果显示,分离的 IBDV 毒株产生与野外病例相似的临床症状和病变,出现法氏囊滤泡髓质的淋巴细胞变性、坏死和消失 . 基因组序列分析显示, IBDV-NB 毒株 A 片段由 3 264 个核苷酸组成,编码由 145 个氨基酸残基组成的 VP5 和由 1 012 个氨基酸残基组成的多聚蛋白 . 与来自 GenBank 的 IBDV Ⅰ型毒株比较, NB 毒株 A 片段编码的多聚蛋白与 JD1 毒株的同源性最高,达 99.5% , VP2 与 JD1 、 CEF94 、 D78 的同源性为 99.8% , VP3 与 JD1 的同源性为 99.2% , VP4 与 JD1 的同源性为 100% , VP5 与 JD1 , HZ2 , P2 , CEF94 , CT , Cu-1 和 D78 毒株的同源性为 99.3%. NB 毒株 VP2 蛋白的第 253 、 280 、 284 位氨基酸残基与 IBDV 变异毒株和经典毒株一致,但不同于 IBDV 超强毒株 . 这些结果暗示 IBDV 的抗原表位是构象依赖性表位, IBDV 血清亚型的形成与 IBDV 弱毒疫苗病毒株密切相关 .     

传染性法氏囊病病毒VP3抗原表位的鉴定

利用肽扫描技术对4株IBDV VP3的单克隆抗体(HRB-3F、HRB-7B、HRB-7C和HRB-10E)的抗原表位进行了研究.通过Western blot和ELISA鉴定,将HRB-3F和HRB-7B的抗原表位定位于VP3 109～119 aa(位于IB-DV聚合蛋白的864～874 aa),HRB-7C和HRB-10E的抗原表位定位于VP3 177～190 aa(位于IBDV聚合蛋白的932～945 aa).进一步检测其反应原性及免疫原性,结果表明,这两个表位均能与抗IBDV阳性血清反应.将这两个表位短肽免疫BALB/c小鼠,其血清可以和IBDV反应,具有较好的免疫原性.与D6948、HK46和UK661等多株IBDV相应区域的同源性进行了比较,结果显示,这两个表位在多种毒株中同源性为100%.通过IBDV VP3抗原表位的研究,筛出两个新的保守线性表位并进行精确定位,对进一步分析IBDV结构与功能以及建立以表位为基础的抗原抗体诊断方法具有重要的意义.     

A non-canonical lon proteinase lacking the ATPase domain employs the ser-Lys catalytic dyad to exercise broad control over the life cycle of a double-stranded RNA virus

We have identified a region related to the protease domain of bacterial and organelle ATP-dependent Lon proteases in virus protein 4 (VP4) of infectious bursal disease virus strain P2 (IBDVP2), a two-segmented double-stranded RNA virus. Unlike canonical Lons, IBDVP2 VP4 possesses a proteinase activity though it lacks an ATPase domain. Ser652 and Lys692 of IBDVP2 VP4 are conserved across the Lon/VP4 family and are essential for catalysis. Lys692 has the properties of a general base, increasing the nucleophilicity of Ser652; a similar catalytic dyad may function in the other Lons. VP4 can cleave in trans and is responsible for the interdomain proteolytic autoprocessing of the pVP2- VP4-VP3 polyprotein encoded by RNA segment A. VP2, which is later derived from pVP2, and VP3 are major capsid proteins of birnaviruses. Results of the characterization of a range of the IBDVP2 VP4 mutants in cell cultures implicate VP4 in trans-activation of the synthesis of VP1, putative RNA-dependent RNA polymerase encoded by RNA segment B, and in cleavage rate-dependent control of process(es) crucial for the generation of the infectious virus progeny.     

Peptide map comparison of the proteins of infectious bursal disease virus.

The genome of infectious bursal disease virus consists of two segments of double-stranded RNA of 2.5 X 10(6) and 2.2 X 10(6) molecular weight. Polyacrylamide gel electrophoresis of purified virus resolved four structural polypeptides: VP-1 (90,000), VP-2 (41,000), VP-3 (35,000), and VP-4 (28,000). Peptide map comparisons of radioiodinated virion proteins indicated no precursor-product relationship between them. The possible relationship between the size of the virus genome and the number and sizes of different viral proteins is discussed.     

Structural polymorphism of the major capsid protein of a double-stranded RNA virus: an amphipathic alpha helix as a molecular switch

The infectious bursal disease virus T=13 viral particle is composed of two major proteins, VP2 and VP3. Here, we show that the molecular basis of the conformational flexibility of the major capsid protein precursor, pVP2, is an amphipatic alpha helix formed by the sequence GFKDIIRAIR. VP2 containing this alpha helix is able to assemble into the T=13 capsid only when expressed as a chimeric protein with an N-terminal His tag. An amphiphilic alpha helix, which acts as a conformational switch, is thus responsible for the inherent structural polymorphism of VP2. The His tag mimics the VP3 C-terminal region closely and acts as a molecular triggering factor. Using cryo-electron microscopy difference imaging, both polypeptide elements were detected on the capsid inner surface. We propose that electrostatic interactions between these two morphogenic elements are transmitted to VP2 to acquire the competent conformations for capsid assembly.     

Biosynthesis of virus-specific proteins in cells infected with infectious bursal disease virus and their significance as structural elements for infectious virus and incomplete particles.

It has previously been shown that infectious bursal disease virus is a naked icosahedral particle with a diameter of about 60 nm and a genome consisting of two segments of double-stranded RNA (Müller et al., J. Virol. 31:584-589, 1979). One of the two major structural polypeptides (molecular weight, 40,000) of this virus could not be found in lysates of infected cells; it is derived from a precursor polypeptide demonstrable inside the cells in relatively large quantities and seems to be processed during virus assembly or later. The precursor molecule is regularly present in the infectious virus particle (buoyant density, 1.33 g/ml) in minor proportions, but it represents an outstanding structural element of incomplete noninfectious particles ("top components"; buoyant density, 1.29 g/ml) which contain viral RNA. This type of incomplete particles is mainly produced by chicken embryo fibroblasts in contrast to lymphoid cells from the bursa of Fabricius. Precursor-product relationships also seem to exist in the biosynthesis of the other viral polypeptides. In contrast to some other viruses with a segmented double-stranded RNA genome, none of the structural proteins of infectious bursal disease virus is appreciably glycosylated.     

Folding and processing of the capsid protein precursor P1 is kinetically retarded in neutralization site 3B mutants of poliovirus.

Poliovirus mutants in neutralizing antigenic site 3B were constructed by replacing the glutamic acid residue at amino acid 74 of capsid protein VP2 (VP2074E), using site-specific mutagenesis methods. All viable mutants display small-plaque phenotypes. Characterization of these mutants indicates that capsid assembly is perturbed. Although the defect in capsid assembly reduces the yield of mutant virus particles per cell, the resultant assembled particle is wild-type-like in structure and infectivity. Analyses of capsid assembly intermediates show a transient accumulation of the unprocessed capsid protein precursor, P1, indicating that cleavage of the mutant P1 by the 3CD protease is retarded. The mutant VP0-VP3-VP1 complex generated upon P1 cleavage appears assembly competent, forming pentamer and empty capsid assembly intermediates and infectious virion particles. Although the structure of the infectious mutant virus is virtually identical with that of the wild-type virus, the thermal stability of the mutant virus is dramatically increased over that of the wild-type virus. Thus, mutations at this residue are pleiotropic, altering the kinetics of capsid assembly and generating a virus that is more thermostable and more resistant to neutralization by the site 3B monoclonal antibodies.     

Host proteolytic activity is necessary for infectious bursal disease virus capsid protein assembly

In many viruses, a precursor particle, or procapsid, is assembled and undergoes massive chemical and physical modification to produce the infectious capsid. Capsid assembly and maturation are finely tuned processes in which viral and host factors participate. We show that the precursor of the VP2 capsid protein (pVP2) of the infectious bursal disease virus (IBDV), a double-stranded RNA virus, is processed at the C-terminal domain (CTD) by a host protease, the puromycin-sensitive aminopeptidase (PurSA). The pVP2 CTD (71 residues) has an important role in determining the various conformations of VP2 (441 residues) that build the T = 13 complex capsid. pVP2 CTD activity is controlled by co- and posttranslational proteolytic modifications of different targets by the VP4 viral protease and by VP2 itself to yield the mature VP2-441 species. Puromycin-sensitive aminopeptidase is responsible for the peptidase activity that cleaves the Arg-452-Arg-453 bond to generate the intermediate pVP2-452 polypeptide. A pVP2 R453A substitution abrogates PurSA activity. We used a baculovirus-based system to express the IBDV polyprotein in insect cells and found inefficient formation of virus-like particles similar to IBDV virions, which correlates with the absence of puromycin-sensitive aminopeptidase in these cells. Virus-like particle assembly was nonetheless rescued efficiently by coexpression of chicken PurSA or pVP2-452 protein. Silencing or pharmacological inhibition of puromycin-sensitive aminopeptidase activity in cell lines permissive for IBDV replication caused a major blockade in assembly and/or maturation of infectious IBDV particles, as virus yields were reduced markedly. PurSA activity is thus essential for IBDV replication.     

Protective Vaccination against Infectious Bursal Disease Virus with Whole Recombinant Kluyveromyces lactis Yeast Expressing the Viral VP2 Subunit

Here we report on vaccination approaches against infectious bursal disease (IBD) of poultry that were performed with complete yeast of the species Kluyveromyces lactis (K. lactis). Employing a genetic system that enables the rapid production of stably transfected recombinant K. lactis, we generated yeast strains that expressed defined quantities of the virus capsid forming protein VP2 of infectious bursal disease virus (IBDV). Both, subcutaneous as well as oral vaccination regiments with the heat-inactivated but otherwise untreated yeast induced IBDV-neutralizing antibodies in mice and chickens. A full protection against a subsequent IBDV infection was achieved by subcutaneous inoculation of only milligram amounts of yeast per chicken. Oral vaccination also generated protection: while mortality was observed in control animals after virus challenge, none of the vaccinees died and ca. one-tenth were protected as indicated by the absence of lesions in the bursa of Fabricius. Recombinant K. lactis was thus indicated as a potent tool for the induction of a protective immune response by different applications. Subcutaneously applied K. lactis that expresses the IBDV VP2 was shown to function as an efficacious anti-IBD subunit vaccine.