Synthesis of bluetongue virus (BTV) corelike particles by a recombinant baculovirus expressing the two major structural core proteins of BTV.

The L3 and M7 genes of bluetongue virus (BTV), which encode the two major core proteins of the virus (VP3 and VP7, respectively), were inserted into a baculovirus dual-expression transfer vector and a recombinant baculovirus expressing both foreign genes isolated following in vivo recombination with wild-type Autographa californica nuclear polyhedrosis virus DNA. Spodoptera frugiperda insect cells infected with the recombinant synthesized large amounts of BTV corelike particles. These particles have been shown to be similar to authentic BTV cores in terms of size, appearance, stoichiometric arrangement of VP3 to VP7 (ratio, 2:15), and the predominance of VP7 on the surface of the particles. In infected insect cells, the corelike particles were observed in paracrystalline arrays. The formation of these structures indicates that neither the BTV double-stranded viral RNA species nor the associated minor core proteins are necessary for assembly of cores in insect cells. Furthermore, the three BTV nonstructural proteins NS1, NS2, and NS3, are not required to assist or direct the formation of empty corelike particles from VP3 and VP7.     

Baculovirus multigene expression vectors and their use for understanding the assembly process of architecturally complex virus particles

The baculovirus expression vector is a eukaryotic DNA viral vector for the cloning and expression of foreign genes in cultured lepidopteran insect cells and insects. It has become an important tool for the large-scale production of recombinant proteins for a variety of applications including the structure-function analysis of genes and their gene products. We have developed a number of baculovirus multigene expression vectors and utilized these to understand the assembly process of multicomponent capsid structures of large viruses such as bluetongue virus (BTV), a member of the Orbivirus genus within the family Reoviridae. BTV is some 810 Å in diameter and comprised of two protein shells containing four major proteins, VP2, VP5, VP7 and VP3, surrounding a genome of ten double-stranded RNA segments and three minor proteins (VP2, VP4 and VP6). BTV is the etiological agent of a sheep disease that is sometimes fatal in certain parts of the world (e.g., Africa, Asia, and the Americas). Using baculovirus multigene vectors, we have co-expressed various combinations of BTV genes in insect cells and produced structures that mimic the various stages of BTV assembly. For example, co-expressed VP3 and VP7 form BTV core-like particles, while co-expressed VP2, VP5, VP7 and VP3 form BTV virus-like particles. Using deletion, point and domain switching analyses of each protein, we have been able to identify certain sequences in the VP7 and VP3 proteins that are essential for the assembly of core-like particles. These expression and biochemical studies have been complemented by collaboration studies using cryoelectron microscopy and image processing analyses to provide the three-dimensional structure of the expressed particles. In addition and with other associates, we have used X-ray crystallography of VP7 to deduce its atomic structure. Extensive studies on the immune responses elicited by these self-assembled particles, and chimeric derivatives involving various foreign antigens, have been carried out. Finally, using as little as 10 μg of the self-assembled virus-like particles, we have shown that they can confer long-lasting protection in sheep against BTV.     

在昆虫细胞中同时表达蓝舌病毒VP3与VP7蛋白可装配成核心样颗粒

将蓝舌病毒（ＢＴＶ）１３型Ｓ７与Ｌ３基因同时插入杆状病毒双表达载体ｐＥａｓｔＢａｃＤｕａｌ,获得重组杆状病毒ｒｖＢａｃＢＴＶＰ３７。该病毒在昆虫细胞中同时高水平表达ＢＴＶ１３ ＶＰ３与ＶＰ７蛋白,可以高效自动装配出２０面体的６０￣７０ｎｍ空心颗粒。分析表明,所获颗粒为空心的ＢＴＶ核心样颗粒（ＣＬＰ）,其成分为ＶＰ３与ＶＰ７,不含ＢＴＶ其它任何蛋白与核酸。这种装配需要ＶＰ３与ＶＰ７的共同参与,二者缺     

Development of recombinant vaccines against bluetongue

Bluetongue virus is the aetiological agent of bluetongue, a disease of domestic and wild ruminants. Twenty-four serotypes are recognized. Novel subunit vaccines, that complement existing modified live polyvalent vaccines, are being developed. Serotype-specific viral neutralizing antibodies that are able to protect sheep against virulent homologous virus challenge can be induced by immunizing with the BTV outer capsid protein VP2 purified from virions or with VP2 expressed by baculovirus recombinants. Presentation of VP2 on virus-like particles, which assemble upon co-expression of the four major structural viral proteins (VP2, VP5, VP3 and VP7), improves the protective effect of VP2. Sheep immunized with core-like particles, comprised of VP3 and VP7, developed only limited clinical signs after virulent virus challenge, demonstrating that not only the outer capsid proteins, but also the core proteins are involved in protection against bluetongue.     

Synthesis and characterization of chimeric particles between epizootic hemorrhagic disease virus and bluetongue virus: functional domains are conserved on the VP3 protein.

A functional assay has been developed to determine the conservative nature of the interacting sites of various structural proteins of orbiviruses by using baculovirus expression vectors. For this investigation, proteins of two serologically related orbiviruses, bluetongue virus (BTV) and the less studied epizootic hemorrhagic disease virus (EHDV), were used to synthesize chimeric particles. The results demonstrate that the inner capsid protein VP3 of EHDV-1 can replace VP3 protein of BTV in formation of the single-shelled corelike particles and the double-shelled viruslike particles. Moreover, we have demonstrated that all three minor core proteins (VP1, VP4, and VP6) can be incorporated into the homologous and chimeric corelike and viruslike particles, indicating that the functional epitopes of the VP3 protein are conserved for the morphological events of the virus. This is the first evidence of assembly of seven structural proteins of the virus by a baculovirus expression system. Confirmation at the molecular level was obtained by determining the EHDV-1 L3 gene nucleic sequence and by comparing it with sequences available for BTV. The analysis revealed a high degree homology between the two proteins: 20% difference, 50% of which is conservative. The consequences for Orbivirus phylogeny and the possibility of gene reassortments are discussed.     

Expression and functional characterization of bluetongue virus VP2 protein: role in cell entry

Segment 2 of bluetongue virus (BTV) serotype 10, which encodes the outer capsid protein VP2, was tagged with the S-peptide fragment of RNase A and expressed by a recombinant baculovirus. The recombinant protein was subsequently purified to homogeneity by virtue of the S tag, and the oligomeric nature of the purified protein was determined. The data obtained indicated that the majority of the protein forms a dimer and, to a lesser extent, some trimer. The recombinant protein was used to determine various biological functions of VP2. The purified VP2 was shown to have virus hemagglutinin activity and was antigenically indistinguishable from the VP2 of the virion. Whether VP2 is responsible for BTV entry into permissive cells was subsequently assessed by cell surface attachment and internalization studies with an immunofluorescence assay system. The results demonstrated that VP2 alone is responsible for virus entry into mammalian cells. By competition assay, it appeared that both VP2 and the BTV virion attached to the same cell surface molecule(s). The purified VP2 also had a strong affinity for binding to glycophorin A, a sialoglycoprotein component of erythrocytes, indicating that VP2 may be responsible for BTV transmission by the Culicoides vector to vertebrate hosts during blood feeding. Further, by various enzymatic treatments of BTV-permissive L929 cells, preliminary data have been obtained which indicated that the BTV receptor molecule(s) is likely to be a glycoprotein and that either the protein moiety of the glycoprotein or a second protein molecule could also serve as a coreceptor for BTV infection.     

同时表达蓝舌病毒四个主要结构蛋白可装配成病毒样颗粒

为研制蓝舌病毒（ｂｌｕｅｔｏｎｇｕｅ ｖｉｒｕｓ,ＢＴＶ）基因工程疫苗和进一步研究ＢＴＶ结构与功能的关系,对ＢＴＶ病毒样颗粒（ＶＬＰ）的装配进行了研究。同时在昆虫细胞中表达ＢＴＶ主要结构蛋白ＶＰ７、ＶＰ３、ＶＰ２与ＶＰ５,将细胞裂解液超速离心纯化后,发现主要存在两 形态的颗粒：一种与前文报道的病毒核心颗粒（ＣＬＰ）相同,直径约为６０ｎｍ￣７０ｎｍ,蛋白壳厚１０ｎｍ￣１５ｎｍ;另一种大小为７０ｎｍ￣     

Expression of two related nonstructural proteins of bluetongue virus (BTV) type 10 in insect cells by a recombinant baculovirus: production of polyclonal ascitic fluid and characterization of the gene product in BTV-infected BHK cells.

In vitro translation of bluetongue virus (BTV) double-stranded RNA in the rabbit reticulocyte lysate system has shown segment 10 (S10) to code for two related proteins, NS3 and NS3A. The presence of both products in vivo, however, has remained unconfirmed owing to the very low level of synthesis of the S10 gene product(s) in BTV-infected BHK cells. In the present work, a cDNA copy of BTV type 10 (BTV-10) S10 RNA was inserted into Autographa californica nuclear polyhedrosis baculovirus (AcNPV) in lieu of the 5' coding region of the AcNPV polyhedrin gene. Spodoptera frugiperda cells infected with the recombinant baculovirus synthesized two polypeptides, which were shown to represent NS3 and NS3A by Western blot (immunoblot) and peptide map analysis. Antibodies raised to the expressed NS3 by immunization of mice detected both NS3 and NS3A in BTV-10-infected BHK cells but not in purified BTV-10 virus particles. In contrast to in vitro translation of BTV S10 RNA in which NS3 and NS3A are synthesized in equimolar amounts, NS3 was the principle product both in the baculovirus expression system and in vivo in BTV-infected cells. The results indicate the caution which should be exercised when using the rabbit reticulocyte lysate system to predict the pattern of protein synthesis from a gene with alternative start codons. The expressed NS3 and NS3A proteins reacted strongly with sera from sheep infected with homologous and heterologous BTV serotypes, suggesting that the S10 gene products are highly conserved group-specific antigens.     

Development of baculovirus triple and quadruple expression vectors: co-expression of three or four bluetongue virus proteins and the synthesis of bluetongue virus-like particles in insect cells.

Baculovirus multiple gene transfer vectors pAcAB3 and pAcAB4 have been developed to facilitate the insertion of three or four foreign genes respectively into the Autographa californica nuclear polyhedrosis virus (AcNPV) genome by a single co-transfection experiment. The pAcAB3 vector contains a polyhedrin promoter and two p10 promoters on either side of the polyhedrin promoter but in opposite orientations. The pAcAB4 vector has an additional polyhedrin promoter in opposite orientation to the first copy that is in juxtaposition to the first p10 promoter. Each of these derived vectors (pAcAB3, pAcAB4) have been used for the simultaneous expression of three or four bluetongue virus (BTV) genes respectively. When Spodoptera frugiperda cells were infected with the recombinant virus (AcBT-3/2/7/5) expressing the four major structural genes of BTV, double-capsid, virus-like particles consisting of VP2, VP3, VP5 and VP7 of BTV were assembled.     

蓝舌病病毒vp7基因的表达及其产物在c—ELISA中的应用

获得稳定、高效的具有良好抗原性的蓝舌病毒(Bluetongue virus,BTV)vp7基因重组抗原。将BTV编码群特异性抗原VP7的S7基因片段克隆至pMD18-T质粒载体中,构建S7克隆重组质粒,进行核苷酸序列分析。与已报道的多株BTV编码VP7的基因比较后发现,所测定毒株的核苷酸序列与BTV10型的S7基因同源性高达98．7％,推测的氨基酸同源性为99．3％,证实为BTV的S7基因。然后亚克隆插入pBAD/Thio TOPO表达载体,转化LGM194细胞,经抗性培养、PCR、限制性内切酶分析、测序鉴定,筛选获得BTV S7基因片段正向插入、有正确读码框的阳性克隆,成功构建了BTV群特异性抗原VP7的重组表达载体。经L-araboinose诱导表达,可稳定、高效地表达VP7蛋白抗原。SDS-PAGE、ELISA试验表明,表达蛋白为融合蛋白,具有反应原性,分子量约54．5kD,重组蛋白的获得率为1．52mg／g湿菌,其表达产量约占菌体总蛋白的12％左右,相当于93．5mg／L菌液。融合蛋白中含有BTV VP7特异性蛋白抗原,可作为c-ELISA包被抗原,为蓝舌病的免疫血清学诊断试剂的制备和分子生物学研究打下了坚实基础。     

Detection of Bluetongue Virus Group-specific Antigen Using Monoclonal Antibody Based Sandwich ELISA

A monoclonal antibody (MAb) specific for the bluetongue virus (BTV) group specific antigen (VP7) was characterized for its reactivity with purified virus and recombinant BTV VP7 (rVP7) protein and its suitability for use in the sandwich ELISA.The MAb,designated as 5B5 was specific to VP7 and belongs to IgG2a subclass and was selected for the development of the sELISA in this study.The MAb had a titer of 1:25 with BTV and 1:2 with the rVP7 protein.The sELISA is based on capturing of BTV antigen with VP7 spec...     

Identification of bluetongue virus VP6 protein as a nucleic acid-binding protein and the localization of VP6 in virus-infected vertebrate cells.

Recently the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has been effectively adapted as a highly efficient vector in insect cells for the expression of various genes. A cDNA sequence of RNA segment 9 of bluetongue virus serotype 10 (BTV-10, an orbivirus member of the Reoviridae family) encoding a minor core protein (VP6) has been inserted into the BamHI site of the pAcYM1 transfer vector derived from AcNPV. Spodoptera frugiperda cells were cotransfected with the derived vector in the presence of authentic AcNPV DNA to produce recombinant viruses. These synthesized significant amounts of a protein (representing ca. 50% of the stained cellular protein) similar in size and antigenicity to the authentic BTV VP6. The expressed protein was identified as a nucleic acid-binding protein by using an RNA overlay-protein blot assay. A polyclonal anti-VP6 serum prepared by using the expressed VP6 protein has been used in an immunogold procedure to locate VP6 in BTV-infected mammalian cells. Gold was found to be associated with the matrix of virus inclusion bodies (VIB), with viruslike particles in the VIB, as well as with mature virion particles that were in close proximity to the VIB or were released from cells and adsorbed to cell surfaces. The recombinant virus antigen has also been used to identify antibodies to different BTV serotypes in infected sheep sera, indicating the potential of the expressed protein as a group-reactive antigen for the diagnosis of BTV infections.     

Expression and functional characterization of bluetongue virus VP5 protein: role in cellular permeabilization

Segment 5 of bluetongue virus (BTV) serotype 10, which encodes the outer capsid protein VP5, was tagged with glutathione S-transferase and expressed by a recombinant baculovirus. The recombinant protein was subsequently purified to homogeneity, and its possible biological role in virus infection was investigated. Purified VP5 was able to bind mammalian cells but was not internalized, which indicates it is not involved in receptor-mediated endocytosis. The purified VP5 protein was shown to be able to permeabilize mammalian and Culicoides insect cells, inducing cytotoxicity. Sequence analysis revealed that VP5 possesses characteristic structural features (including two amino-terminal amphipathic helices) compatible with virus penetration activity. To assess the role of each feature in the observed cytotoxicity, a series of deleted VP5 molecules were generated, and their expression and biological activity was compared with the parental molecule. VP5 derivatives that included the two amphipathic helices exhibited cytotoxicity, while those that omitted these sequences did not. To confirm their role in membrane destabilization two synthetic peptides (amino acids [aa] 1 to 20 and aa 22 to 41) encompassing the two helices and an additional peptide representing the adjacent downstream sequences were also assessed for their effect on the cell membrane. Both helices, but not the downstream VP5 sequence, exhibited cytotoxicity with the most-amino-terminal helix (aa 1 to 20) showing a higher activity than the adjacent peptide (aa 22 to 41). Purified VP5 was shown to readily form trimers in solution, a feature of many proteins involved in membrane penetration. Taken together, these data support a role for VP5 in virus-cell penetration consistent with its revelation in the entry vesicle subsequent to cell binding and endocytosis.     

Expression of largest RNA segment and synthesis of VP1 protein of bluetongue virus in insect cells by recombinant baculovirus: association of VP1 protein with RNA polymerase activity.

The bluetongue virus core particles have been shown to contain an RNA-directed RNA polymerase (1). To identify the protein responsible for the virion RNA polymerase activity, the complete 3.9 Kb DNA clone representing the largest RNA segment 1 (L1) of bluetongue virus (BTV-10) was placed under control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). The derived recombinant virus was used to infect Spodoptera frugiperda cells. As demonstrated by stained polyacrylamide gel electrophoresis and by the use of bluetongue virus antibody, infected insect cells synthesized the largest protein of BTV-10 (VP1, 150 k Da). Antibody raised in rabbit to recombinant VP1 protein recognized bluetongue virus VP1 protein. The recombinant virus infected cell lysate had significantly inducible levels of RNA polymerase enzymatic activity as determined by a poly (U)-oligo (A) polymerase assay. The availability of enzymatically active bluetongue virus RNA polymerase provides a system in which we can precisely delineate the role this protein plays in the regulation of bluetongue replication.     

A single point mutation in the VP7 major core protein of bluetongue virus prevents the formation of core-like particles.

To understand the assembly process of bluetongue virus (BTV), we have established a functional assay which allows us to produce and manipulate BTV core-like particles (CLPs) composed of the viral VP7 and VP3 proteins. A cDNA clone encoding the 349-amino-acid VP7 protein has been manipulated to generate deletion, extension, and site-specific mutants. Each mutant was coexpressed with the BTV VP3 protein to generate CLPs. Deletion and extension mutants involving the VP7 carboxy terminus prevented CLP formation, while an extension mutant involving an 11-amino-acid rabies virus sequence added to the amino terminus of VP7 allowed CLP formation. Substitution of either of two cysteine residues of VP7 (Cys-15 or Cys-65) by serine also did not prevent CLP formation; however, substitution of the single lysine residue of VP7 (Lys-255) by leucine abrogated CLP formation, indicating a critical role for this lysine.     

Protection of IFNAR (?/?) Mice against Bluetongue Virus Serotype 8, by Heterologous (DNA/rMVA) and Homologous (rMVA/rMVA) Vaccination,Expressing Outer-Capsid Protein VP2

The protective efficacy of recombinant vaccines expressing serotype 8 bluetongue virus (BTV-8) capsid proteins was tested in a mouse model. The recombinant vaccines comprised plasmid DNA or Modified Vaccinia Ankara viruses encoding BTV VP2, VP5 or VP7 proteins. These constructs were administered alone or in combination using either a homologous prime boost vaccination regime (rMVA/rMVA) or a heterologous vaccination regime (DNA/rMVA). The DNA/rMVA or rMVA/rMVA prime-boost were administered at a three week interval and all of the animals that received VP2 generated neutralising antibodies. The vaccinated and non-vaccinated-control mice were subsequently challenged with a lethal dose of BTV-8. Mice vaccinated with VP7 alone were not protected. However, mice vaccinated with DNA/rMVA or rMVA/rMVA expressing VP2, VP5 and VP7 or VP2 alone were all protected.     

Use of baculovirus expression vectors: development of diagnostic reagents, vaccines and morphological counterparts of bluetongue virus

The productivity and flexibility of insect baculovirus expression vectors and the ability of the baculovirus genome to incorporate (and express) large amounts of foreign DNA allows this system to be used for both single and multiple gene expression. Using the system, bluetongue virus (BTV) genes have been expressed to develop diagnostic reagents and vaccines as well as to understand the basic structures of the virions. BTV which causes disease in ruminants in many parts of the world, consists of 10 double-stranded RNA segments enclosed by double capsids that are composed by 7 structural proteins. Since each protein is encoded by a single RNA species, DNA clones of all 10 RNA species were synthesized and individually expressed in baculovirus vectors at high levels. This has yielded proteins that have been shown to be excellent diagnostic and vaccine reagents. In addition, multiple expression vectors have been used to synthesize morphological structures (viral and subviral) representing BTV.     

Use of baculovirus expression vectors: development of diagnostic reagents, vaccines and morphological counterparts of bluetongue virus

Abstract The productivity and flexibility of insect baculovirus expression vectors and the ability of the baculovirus genome to incorporate (and express) large amounts of foreign DNA allows this system to be used for both single and multiple gene expression. Using the system, bluetongue virus (BTV) genes have been expressed to develop diagnostic reagents and vaccines as well as to understand the basic structures of virions. BTV which causes disease in ruminants in many parts of the world, consists of 10 double-stranded RNA segments enclosed by double capsids that are composed by 7 structural proteins. Since each protein is encoded by a single RNA species, DNA clones of all 10 RNA species were synthesized and individually expressed in baculovirus vectors at high levels. This has yielded proteins that have been shown to be excellent diagnostic and vaccine reagents. In addition, multiple expression vectors have been used to synthesize morphological structures (viral and subviral) representing BTV.     

Optimized production of recombinant bluetongue core-like particles produced by the baculovirus expression system.

The baculovirus-expression vector system (BEVS) has been widely used for the experimental production of many human and animal single- and multi-unit vaccines, heterologous proteins, and viral insecticides. In this work, the production of recombinant bluetongue virus core-like particles (CLPs), using Sf9 cells in shaker-suspension culture with the SF900 II medium (GIBCO, NY), has been studied. This system involved the simultaneous production of two proteins, VP7 and VP3, and was shown to achieve high volumetric productivities. The key parameters of the time of infection (TOI), and the multiplicity of infection (MOI) were studied. The results show that the peak-volumetric yields and cell-specific yields achieved using low MOIs at low-cell densities were the same as those obtained following infections with a high MOI at high-cell densities. This work establishes the feasibility of using low MOIs in the baculovirus system to produce complex multiprotein particles.     

Detection of bluetongue virus group-specific antigen using monoclonal antibody based sandwich ELISA

A monoclonal antibody (MAb) specific for the bluetongue virus (BTV) group specific antigen (VP7) was characterized for its reactivity with purified virus and recombinant BTV VP7 (rVP7) protein and its suitability for use in the sandwich ELISA. The MAb, designated as 5B5 was specific to VP7 and belongs to IgG2a subclass and was selected for the development of the sELISA in this study. The MAb had a titer of 1:25 with BTV and 1:2 with the rVP7 protein. The sELISA is based on capturing of BTV antigen with VP7 specific MAb followed by detection using BTV polyclonal antiserum raised in rabbits. The assay was evaluated with six cell culture adapted serotypes of BTV that have been isolated from India, 1, 2, 15, 17, 18 and 23. The assay could detect BTV antigen as early as day 8 in blood. It was also successfully applied for the detection of BTV group specific antigen in clinical samples of blood, washed RBCs, buffy coat and plasma. A total of 102 field samples from animals, suspected of being infected with BTV, were tested and 29.42% were positive. The blood samples were also amplified in cell culture which improved the sensitivity of the assay. Results confirmed that the sELISA is rapid and specific.