The Experts below are selected from a list of 285 Experts worldwide ranked by ideXlab platform
Jiyong Zhou - One of the best experts on this subject based on the ideXlab platform.
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itraq based quantitative subcellular proteomic analysis of avibirnavirus infected cells
Electrophoresis, 2015Co-Authors: Boli Hu, Jiyong Zhou, Aifang Du, Xiaojuan Zheng, Yina ZhangAbstract:: Infectious bursal disease virus (IBDV) enters the host cells via endocytic pathway to achieve viral replication in the cytoplasm. Here, we performed LC-MS/MS coupled with isobaric tags for relative and absolute quantification labeling of differentially abundant Proteins of IBDV-infected cells using a subcellular fractionation strategy. We show that the viral infection regulates the abundance and/or subcellular localization of 3211 Proteins during early infection. In total, 23 cellular Proteins in the cytoplasmic proteome and 34 in the nuclear proteome were significantly altered after virus infection. These differentially abundant Proteins are involved in such biological processes as immune response, signal transduction, RNA processing, macromolecular biosynthesis, energy metabolism, virus binding, and cellular apoptosis. Moreover, transcriptional profiles of the 25 genes corresponding to the identified Proteins were analyzed by quantitative real-time RT-PCR. Ingenuity Pathway Analysis clustered the differentially abundant Proteins primarily into the mTOR pathway, PI3K/Akt pathway, and interferon-β signaling cascades. Confocal microscopy showed colocalization of the viral Protein VP3 with host Proteins heterogeneous nuclear ribonucleoProtein H1, nuclear factor 45, apoptosis inhibitor 5, nuclear Protein localization Protein 4 and DEAD-box RNA helicase 42 during the virus infection. Together, these identified subcellular constituents provide important information for understanding host-IBDV interactions and underlying mechanisms of IBDV infection and pathogenesis.
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inhibition of antiviral innate immunity by birnavirus VP3 Protein via blockage of viral double stranded rna binding to the host cytoplasmic rna detector mda5
Journal of Virology, 2014Co-Authors: Chengjin Ye, Boli Hu, Jiyong Zhou, Lun Wang, Xingmeng LuAbstract:Chicken MDA5 (chMDA5), the sole known pattern recognition receptor for cytoplasmic viral RNA in chickens, initiates type I interferon (IFN) production. Infectious bursal disease virus (IBDV) evades host innate immunity, but the mechanism is unclear. We report here that IBDV inhibited antiviral innate immunity via the chMDA5-dependent signaling pathway. IBDV infection did not induce efficient type I interferon (IFN) production but antagonized the antiviral activity of beta interferon (IFN-β) in DF-1 cells pretreated with IFN-α/β. Dual-luciferase assays and inducible expression systems demonstrated that IBDV Protein VP3 significantly inhibited IFN-β expression stimulated by naked IBDV genomic double-stranded RNA (dsRNA). The VP3 Protein competed strongly with chMDA5 to bind IBDV genomic dsRNA in vitro and in vivo, and VP3 from other birnaviruses also bound dsRNA. Site-directed mutagenesis confirmed that deletion of the VP3 dsRNA binding domain restored IFN-β expression. Our data demonstrate that VP3 inhibits antiviral innate immunity by blocking binding of viral genomic dsRNA to MDA5. IMPORTANCE MDA5, a known pattern recognition receptor and cytoplasmic viral RNA sensor, plays a critical role in host antiviral innate immunity. Many pathogens escape or inhibit the host antiviral immune response, but the mechanisms involved are unclear for most pathogens. We report here that birnaviruses inhibit host antiviral innate immunity via the MDA5-dependent signaling pathway. The antiviral innate immune system involving IFN-β did not function effectively during birnavirus infection, and the viral Protein VP3 significantly inhibited IFN-β expression stimulated by naked viral genomic dsRNA. We also show that VP3 blocks MDA5 binding to viral genomic dsRNA in vitro and in vivo. Our data reveal that birnavirus-encoded viral Protein VP3 is an inhibitor of the antiviral innate immune response and inhibits the antiviral innate immune response via the MDA5-dependent signaling pathway.
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Inhibition of Antiviral Innate Immunity by Birnavirus VP3 Protein via Blockage of Viral Double-Stranded RNA Binding to the Host Cytoplasmic RNA Detector MDA5
Journal of virology, 2014Co-Authors: Lu Jia, Lun Wang, Yanting Sun, Jiyong ZhouAbstract:Chicken MDA5 (chMDA5), the sole known pattern recognition receptor for cytoplasmic viral RNA in chickens, initiates type I interferon (IFN) production. Infectious bursal disease virus (IBDV) evades host innate immunity, but the mechanism is unclear. We report here that IBDV inhibited antiviral innate immunity via the chMDA5-dependent signaling pathway. IBDV infection did not induce efficient type I interferon (IFN) production but antagonized the antiviral activity of beta interferon (IFN-β) in DF-1 cells pretreated with IFN-α/β. Dual-luciferase assays and inducible expression systems demonstrated that IBDV Protein VP3 significantly inhibited IFN-β expression stimulated by naked IBDV genomic double-stranded RNA (dsRNA). The VP3 Protein competed strongly with chMDA5 to bind IBDV genomic dsRNA in vitro and in vivo, and VP3 from other birnaviruses also bound dsRNA. Site-directed mutagenesis confirmed that deletion of the VP3 dsRNA binding domain restored IFN-β expression. Our data demonstrate that VP3 inhibits antiviral innate immunity by blocking binding of viral genomic dsRNA to MDA5. IMPORTANCE MDA5, a known pattern recognition receptor and cytoplasmic viral RNA sensor, plays a critical role in host antiviral innate immunity. Many pathogens escape or inhibit the host antiviral immune response, but the mechanisms involved are unclear for most pathogens. We report here that birnaviruses inhibit host antiviral innate immunity via the MDA5-dependent signaling pathway. The antiviral innate immune system involving IFN-β did not function effectively during birnavirus infection, and the viral Protein VP3 significantly inhibited IFN-β expression stimulated by naked viral genomic dsRNA. We also show that VP3 blocks MDA5 binding to viral genomic dsRNA in vitro and in vivo. Our data reveal that birnavirus-encoded viral Protein VP3 is an inhibitor of the antiviral innate immune response and inhibits the antiviral innate immune response via the MDA5-dependent signaling pathway.
Luz Mayorca - One of the best experts on this subject based on the ideXlab platform.
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The simian virus 40 minor structural Protein VP3, but not Vp2, is essential for infectious virion formation.
Journal of General Virology, 2003Co-Authors: Editte Gharakhanian, Luz Muñoz, Luz MayorcaAbstract:The SV40 capsid is composed of pentameric capsomeres of the major structural Protein Vp1. The two minor structural Proteins, Vp2 and VP3, interact with the capsid. Here, the roles of Vp2 and VP3 were explored during the course of SV40 infection. Start codons of Vp2, VP3, or both Vp2 and VP3, were destroyed by site-directed mutagenesis, and mutant genomes were transfected into CV-1 cells. SV40ΔVp2 produced plaques and infectious virion particles with titres indistinguishable from wild-type. SV40ΔVP3 and SV40 ΔVp2/VP3 were defective in plaque formation and rendered no infectious particles. All three mutants showed normal nuclear localization of T-Ag and Vp1; they also showed packaging of SV40 DNA by nuclease digestion assays. Thus, VP3 is essential for formation of infectious SV40 particles, whereas Vp2 is not. One critical role of full-length VP3 appears to be in virus–cell interactions at post-packaging steps of a permissive infection.
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The simian virus 40 minor structural Protein VP3, but not Vp2, is essential for infectious virion formation.
The Journal of general virology, 2003Co-Authors: Editte Gharakhanian, Luz Muñoz, Luz MayorcaAbstract:The SV40 capsid is composed of pentameric capsomeres of the major structural Protein Vp1. The two minor structural Proteins, Vp2 and VP3, interact with the capsid. Here, the roles of Vp2 and VP3 were explored during the course of SV40 infection. Start codons of Vp2, VP3, or both Vp2 and VP3, were destroyed by site-directed mutagenesis, and mutant genomes were transfected into CV-1 cells. SV40DeltaVp2 produced plaques and infectious virion particles with titres indistinguishable from wild-type. SV40DeltaVP3 and SV40 DeltaVp2/VP3 were defective in plaque formation and rendered no infectious particles. All three mutants showed normal nuclear localization of T-Ag and Vp1; they also showed packaging of SV40 DNA by nuclease digestion assays. Thus, VP3 is essential for formation of infectious SV40 particles, whereas Vp2 is not. One critical role of full-length VP3 appears to be in virus-cell interactions at post-packaging steps of a permissive infection.
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Short Communication The simian virus 40 minor structural Protein VP3, but not Vp2, is essential for infectious virion formation
2003Co-Authors: Editte Gharakhanian, Luz MayorcaAbstract:The SV40 capsid is composed of pentameric capsomeres of the major structural Protein Vp1. The two minor structural Proteins, Vp2 and VP3, interact with the capsid. Here, the roles of Vp2 and VP3 were explored during the course of SV40 infection. Start codons of Vp2, VP3, or both Vp2 and VP3, were destroyed by site-directed mutagenesis, and mutant genomes were transfected into CV-1 cells. SV40DVp2 produced plaques and infectious virion particles with titres indistinguishable from wild-type. SV40DVP3 and SV40 DVp2/VP3 were defective in plaque formation and rendered no infectious particles. All three mutants showed normal nuclear localization of T-Ag and Vp1; they also showed packaging of SV40 DNA by nuclease digestion assays. Thus, VP3 is essential for formation of infectious SV40 particles, whereas Vp2 is not. One critical role of full-length VP3 appears to be in virus–cell interactions at post-packaging steps of a permissive infection.
Editte Gharakhanian - One of the best experts on this subject based on the ideXlab platform.
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The simian virus 40 minor structural Protein VP3, but not Vp2, is essential for infectious virion formation.
Journal of General Virology, 2003Co-Authors: Editte Gharakhanian, Luz Muñoz, Luz MayorcaAbstract:The SV40 capsid is composed of pentameric capsomeres of the major structural Protein Vp1. The two minor structural Proteins, Vp2 and VP3, interact with the capsid. Here, the roles of Vp2 and VP3 were explored during the course of SV40 infection. Start codons of Vp2, VP3, or both Vp2 and VP3, were destroyed by site-directed mutagenesis, and mutant genomes were transfected into CV-1 cells. SV40ΔVp2 produced plaques and infectious virion particles with titres indistinguishable from wild-type. SV40ΔVP3 and SV40 ΔVp2/VP3 were defective in plaque formation and rendered no infectious particles. All three mutants showed normal nuclear localization of T-Ag and Vp1; they also showed packaging of SV40 DNA by nuclease digestion assays. Thus, VP3 is essential for formation of infectious SV40 particles, whereas Vp2 is not. One critical role of full-length VP3 appears to be in virus–cell interactions at post-packaging steps of a permissive infection.
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The simian virus 40 minor structural Protein VP3, but not Vp2, is essential for infectious virion formation.
The Journal of general virology, 2003Co-Authors: Editte Gharakhanian, Luz Muñoz, Luz MayorcaAbstract:The SV40 capsid is composed of pentameric capsomeres of the major structural Protein Vp1. The two minor structural Proteins, Vp2 and VP3, interact with the capsid. Here, the roles of Vp2 and VP3 were explored during the course of SV40 infection. Start codons of Vp2, VP3, or both Vp2 and VP3, were destroyed by site-directed mutagenesis, and mutant genomes were transfected into CV-1 cells. SV40DeltaVp2 produced plaques and infectious virion particles with titres indistinguishable from wild-type. SV40DeltaVP3 and SV40 DeltaVp2/VP3 were defective in plaque formation and rendered no infectious particles. All three mutants showed normal nuclear localization of T-Ag and Vp1; they also showed packaging of SV40 DNA by nuclease digestion assays. Thus, VP3 is essential for formation of infectious SV40 particles, whereas Vp2 is not. One critical role of full-length VP3 appears to be in virus-cell interactions at post-packaging steps of a permissive infection.
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Short Communication The simian virus 40 minor structural Protein VP3, but not Vp2, is essential for infectious virion formation
2003Co-Authors: Editte Gharakhanian, Luz MayorcaAbstract:The SV40 capsid is composed of pentameric capsomeres of the major structural Protein Vp1. The two minor structural Proteins, Vp2 and VP3, interact with the capsid. Here, the roles of Vp2 and VP3 were explored during the course of SV40 infection. Start codons of Vp2, VP3, or both Vp2 and VP3, were destroyed by site-directed mutagenesis, and mutant genomes were transfected into CV-1 cells. SV40DVp2 produced plaques and infectious virion particles with titres indistinguishable from wild-type. SV40DVP3 and SV40 DVp2/VP3 were defective in plaque formation and rendered no infectious particles. All three mutants showed normal nuclear localization of T-Ag and Vp1; they also showed packaging of SV40 DNA by nuclease digestion assays. Thus, VP3 is essential for formation of infectious SV40 particles, whereas Vp2 is not. One critical role of full-length VP3 appears to be in virus–cell interactions at post-packaging steps of a permissive infection.
Nicholas Muzyczka - One of the best experts on this subject based on the ideXlab platform.
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Structure of Adeno-Associated Virus Type 4
Journal of virology, 2005Co-Authors: Eric Padron, Lakshmanan Govindasamy, John A. Chiorini, Valorie D. Bowman, Nikola Kaludov, Hazel C. Levy, Phillip Nick, Nicholas Muzyczka, Timothy S. BakerAbstract:Adeno-associated virus (AAV) is a member of the Parvoviridae, belonging to the Dependovirus genus. Currently, several distinct isolates of AAV are in development for use in human gene therapy applications due to their ability to transduce different target cells. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. The structure of AAV type 4 (AAV4), one of the most antigenically distinct serotypes, was determined to 13-A resolution by cryo-electron microscopy and image reconstruction. A pseudoatomic model was built for the AAV4 capsid by use of a structure-based sequence alignment of its major capsid Protein, VP3, with that of AAV2, to which AAV4 is 58% identical and constrained by its reconstructed density envelope. The model showed variations in the surface loops that may account for the differences in receptor binding and antigenicity between AAV2 and AAV4. The AAV4 capsid surface topology also shows an unpredicted structural similarity to that of Aleutian mink disease virus and human parvovirus B19, autonomous members of the genus, despite limited sequence homology.
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Structure of Adeno-Associated Virus Type 4
2004Co-Authors: Eric Padron, Lakshmanan Govindasamy, John A. Chiorini, Nikola Kaludov, Phillip Nick, Nicholas Muzyczka, Valorie Bowman, Hazel Levy, Timothy S. BakerAbstract:Adeno-associated virus (AAV) is a member of the Parvoviridae, belonging to the Dependovirus genus. Currently, several distinct isolates of AAV are in development for use in human gene therapy applications due to their ability to transduce different target cells. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. The structure of AAV type 4 (AAV4), one of the most antigenically distinct serotypes, was determined to 13-Å resolution by cryo-electron microscopy and image reconstruction. A pseudoatomic model was built for the AAV4 capsid by use of a structure-based sequence alignment of its major capsid Protein, VP3, with that of AAV2, to which AAV4 is 58 % identical and constrained by its reconstructed density envelope. The model showed variations in the surface loops that may account for the differences in receptor binding and antigenicity between AAV2 and AAV4. The AAV4 capsid surface topology also shows an unpredicted structural similarity to that of Aleutian mink disease virus and human parvovirus B19, autonomous members of the genus, despite limited sequence homology. Adeno-associated virus (AAV) is a member of the Parvoviridae family (45). AAV virions have a T�1 icosahedral capsid consisting of 60 copies of three related Proteins, VP1, VP2, and VP3, at an estimated ratio of 1:1:8, which surrounds a singlestrande
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mutational analysis of the adeno associated virus type 2 aav2 capsid gene and construction of aav2 vectors with altered tropism
Journal of Virology, 2000Co-Authors: Wu Xiao, Thomas J Conlon, Jeffrey A Hughes, Mavis Agbandjemckenna, Thomas W Ferkol, Terence R Flotte, Nicholas MuzyczkaAbstract:Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid Proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be β-barrel structures in the capsid Protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid Protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag insertions identified several other regions that were on the surface of the capsid. These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or coreceptor binding. Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues.
Polly Roy - One of the best experts on this subject based on the ideXlab platform.
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Assembly and Intracellular Localization of the Bluetongue Virus Core Protein VP3
Journal of virology, 2005Co-Authors: Alak Kanti Kar, Nao Iwatani, Polly RoyAbstract:The bluetongue virus (BTV) core Protein VP3 plays a crucial role in the virion assembly and replication process. Although the structure of the Protein is well characterized, much less is known about the intracellular processing and localization of the Protein in the infected host cell. In BTV-infected cells, newly synthesized viral core particles accumulate in specific locations within the host cell in structures known as virus inclusion bodies (VIBs), which are composed predominantly of the nonstructural Protein NS2. However, core Protein location in the absence of VIBs remains unclear. In this study, we examined VP3 location and degradation both in the absence of any other viral Protein and in the presence of NS2 or the VP3 natural associate Protein, VP7. To enable real-time tracking and processing of VP3 within the host cell, a fully functional enhanced green fluorescent Protein (EGFP)-VP3 chimera was synthesized, and distribution of the fusion Protein was monitored in different cell types using specific markers and inhibitors. In the absence of other BTV Proteins, EGFP-VP3 exhibited distinct cytoplasmic focus formation. Further evidence suggested that EGFP-VP3 was targeted to the proteasome of the host cells but was dispersed throughout the cytoplasm when MG132, a specific proteasome inhibitor, was added. However, the distribution of the chimeric EGFP-VP3 Protein was altered dramatically when the Protein was expressed in the presence of the BTV core Protein VP7, a normal partner of VP3 during BTV assembly. Interaction of EGFP-VP3 and VP7 and subsequent assembly of core-like particles was further examined by visualizing fluorescent particles and was confirmed by biochemical analysis and by electron microscopy. These data indicated the correct assembly of EGFP-VP3 subcores, suggesting that core formation could be monitored in real time. When EGFP-VP3 was expressed in BTV-infected BSR cells, the Protein was not associated with proteasomes but instead was distributed within the BTV inclusion bodies, where it colocalized with NS2. These findings expand our knowledge about VP3 localization and its fate within the host cell and illustrate the assembly capability of a VP3 molecule with a large amino-terminal extension. This also opens up the possibility of application as a delivery system.
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Mapping the assembly pathway of Bluetongue virus scaffolding Protein VP3.
Virology, 2004Co-Authors: Alak Kanti Kar, Mrinal K. Ghosh, Polly RoyAbstract:The structure of the Bluetongue virus (BTV) core and its outer layer VP7 has been solved by X-ray crystallography, but the assembly intermediates that lead to the inner scaffolding VP3 layer have not been defined. In this report, we addressed two key questions: (a) the role of VP3 amino terminus in core assembly and its interaction with the transcription complex (TC) components; and (b) the assembly intermediates involved in the construction of the VP3 shell. To do this, deletion mutants in the amino terminal and decamer-decamer interacting region of VP3 (DeltaDD) were generated, expressed in insect cells using baculovirus expression systems, and their ability to assemble into core-like particles (CLPs) and to incorporate the components of TC were investigated. Deletion of the N-terminal 5 (Delta5N) or 10 (Delta10N) amino acids did not affect the ability to assemble into CLPs in the presence of VP7 although the cores assembled using the 10 residue mutant (Delta10N) deletion were very unstable. Removal of five residues also did not effect incorporation of the internal VP1 RNA polymerase and VP4 mRNA capping enzyme Proteins of the TC. Removal of the VP3-VP3 interacting domain (DeltaDD) led to failure to assemble into CLPs yet retained interaction with VP1 and VP4. In solution, purified DeltaDD mutant Protein readily multimerized into dimers, pentamers, and decamers, suggesting that these oligomers are the authentic assembly intermediates of the subcore. However, unlike wild-type VP3 Protein, the dimerization domain-deleted assembly intermediates were found to have lost RNA binding ability. Our study emphasizes the requirement of the N-terminus of VP3 for binding and encapsidation of the TC components, and defines the role of the dimerization domain in subcore assembly and RNA binding.
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synthesis and characterization of chimeric particles between epizootic hemorrhagic disease virus and bluetongue virus functional domains are conserved on the VP3 Protein
Journal of Virology, 1991Co-Authors: Le H Blois, B Fayard, T Urakawa, Polly RoyAbstract: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.
