Attenuation of very virulent infectious bursal disease virus and comparison of full sequences of virulent and attenuated strains.
ABSTRACT A very virulent strain of infectious bursal disease virus (IBDVks) was isolated from the bursae of Fabricius of IBDV-affected broiler chickens. Following 43 serial passages in specific pathogen-free embryonated eggs, an attenuated strain was established (IBDVmb). Dosages of IBDVmb in the range 10(2) to 10(4) embryo infective dose of 50% were found to be safe and protective for commercial chicks. Chickens vaccinated with live vaccine containing IBDVmb responded with precipitating and type-specific neutralizing antibodies, and were immune to subsequent challenge with a very virulent IBDV. IBDVmb has been used as an attenuated vaccine throughout the world since 1993. A comparison of the full sequences of the virulent and attenuated strains (IBDVks and IBDVmb, respectively) revealed seven nucleotides that were different, four of them leading to changes in the amino-acid sequence. Comparison of the protein sequence of these strains and published sequences of very virulent and attenuated phenotypes lead us to suggest that the novel difference responsible for virulence of the Israeli strains are: residue 272 (VP2, very conserved site) and residue 527 (VP4), both in segment A, and in segment B (VP1) residues 96 and 161 (both conserved). Our study strengthens the possibility that more than one protein is involved in IBDV attenuation. In all reports, including ours, virulence was reduced without affecting antigenicity of the neutralizing epitopes in VP2. This could have practical implications for attenuated-vaccine development.
- SourceAvailable from: Vikram N Vakharia[show abstract] [hide abstract]
ABSTRACT: We have developed a system for generation of infectious bursal disease virus (IBDV), a segmented double-stranded RNA virus of the Birnaviridae family, with the use of synthetic transcripts derived from cloned cDNA. Independent full-length cDNA clones were constructed that contained the entire coding and noncoding regions of RNA segments A and B of two distinguishable IBDV strains of serotype I. Segment A encodes all of the structural (VP2, VP4, and VP3) and nonstructural (VP5) proteins, whereas segment B encodes the RNA-dependent RNA polymerase (VP1). Synthetic RNAs of both segments were produced by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of Vero cells with combined plus-sense transcripts of both segments generated infectious virus as early as 36 hr after transfection. The infectivity and specificity of the recovered chimeric virus was ascertained by the appearance of cytopathic effect in chicken embryo cells, by immunofluorescence staining of infected Vero cells with rabbit anti-IBDV serum, and by nucleotide sequence analysis of the recovered virus, respectively. In addition, transfectant viruses containing genetically tagged sequences in either segment A or segment B of IBDV were generated to confirm the feasibility of this system. The development of a reverse genetics system for double-stranded RNA viruses will greatly facilitate studies of the regulation of viral gene expression, pathogenesis, and design of a new generation of live vaccines.Proceedings of the National Academy of Sciences 11/1996; 93(20):11131-6. · 9.74 Impact Factor
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ABSTRACT: Infectious bursal disease viruses (IBDVs), belonging to the Birnaviridae family, cause severe immunodeficiency in young chickens by destroying the precursors of antibody-producing B cells in the bursa of Fabricius (BF). Different pathotypes of IBDVs, including cell culture-adapted viruses, differ markedly in virulence, which is characterized by mortality and bursal damage. To study the molecular determinants of virulence in IBDV, the genomic segments A and B of GLS bursa-derived (GLSBD) and tissue culture-adapted (GLSTC) viruses were cloned and sequenced. Comparison of the deduced amino acid sequences of segments A and B revealed only two amino acid substitutions at positions 87 (Q --> R) and 261 (P --> L) in segment B, and at positions 253 (Q --> H) and 284 (A --> T) in segment A; the latter of which has been shown to be involved in tissue culture adaptation and attenuation of the virus. To study the function of VP1 protein encoded by segment B, reassortant viruses between tissue culture-adapted strains, GLSTC and D78, and GLSBD were recovered using the reverse genetics system. The recombinant virus rGLSBDB containing segment B of GLSBD was able to replicate in Vero and chicken embryo fibroblast (CEF) cells but exhibited delayed replication kinetics. To evaluate the characteristics of these viruses in vivo, 3-week-old chickens were given equal doses of parental viruses or reassortant viruses by ocular inoculation. The pathological lesions and viral antigen distribution in BF were analyzed at 1, 2, or 3 days postinfection. Parental GLSBD and the recovered rGLSBDB viruses propagate most efficiently in the BF and cause severe bursal lesions, whereas the tissue culture-adapted GLSTC virus replicates less efficiently and induces mild bursal lesions at 3 days postinfection. Taken together, our results demonstrate that the VP1 protein of IBDV is involved in the efficiency of viral replication and modulates the virulence in vivo.Virology 12/2004; 330(1):62-73. · 3.37 Impact Factor
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ABSTRACT: The structural protein VP2 of infectious bursal disease virus (IBDV) spontaneously forms a dodecahedral T=1 subviral particle (SVP), and is a primary immunogen of the virus. In this study, the structure of IBDV SVP was determined in a cubic crystal and refined to 2.6A resolution. It contains 20 independent VP2 subunits in a crystallographic asymmetric unit. Each subunit is folded mainly into a shell domain and a protrusion domain, both with the Swiss-roll topology, plus a small helical base domain. Three VP2 subunits constitute a tight trimer, which is the building block of IBDV (sub)viral particles. The structure revealed a calcium ion bound to three pairs of symmetry-related Asp31 and Asp174 to stabilize the VP2 trimer. Our results of treatment of SVP with EGTA, a Ca(2+)-chelating reagent, indicated that the metal-ion may be important not only in maintaining highly stable quaternary structure but also in regulating the swelling and dissociation of the icosahedral particles. A Ca(2+)-dependent assembly pathway was thus proposed, which involves further interactions between the trimers. The 20 independent subunits showed conformational variations, with the surface loops of the protrusion domain being the most diverse. These loops are targets of the neutralizing antibodies. Several common interactions between the surface loops were clearly observed, suggesting a possible major conformation of the immunogenic epitopes.Journal of Structural Biology 08/2006; 155(1):74-86. · 3.36 Impact Factor