BackgroundInfectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultry industry. IBDV has a bi-segmented double-stranded RNA genome. Segments A and B encode the capsid, ribonucleoprotein and non-structural proteins, or the virus polymerase (RdRp), respectively. Since the late eighties, very virulent (vv) IBDV strains have emerged in Europe inducing up to 60% mortality. Although some progress has been made in understanding the molecular biology of IBDV, the molecular basis for the pathogenicity of vvIBDV is still not fully understood.Methodology, Principal FindingsStrain 88180 belongs to a lineage of pathogenic IBDV phylogenetically related to vvIBDV. By reverse genetics, we rescued a molecular clone (mc88180), as pathogenic as its parent strain. To study the molecular basis for 88180 pathogenicity, we constructed and characterized in vivo reassortant or mosaic recombinant viruses derived from the 88180 and the attenuated Cu-1 IBDV strains. The reassortant virus rescued from segments A of 88180 (A88) and B of Cu-1 (BCU1) was milder than mc88180 showing that segment B is involved in 88180 pathogenicity. Next, the exchange of different regions of BCU1 with their counterparts in B88 in association with A88 did not fully restore a virulence equivalent to mc88180. This demonstrated that several regions if not the whole B88 are essential for the in vivo pathogenicity of 88180.Conclusion, SignificanceThe present results show that different domains of the RdRp, are essential for the in vivo pathogenicity of IBDV, independently of the replication efficiency of the mosaic viruses.

The avian adenovirus CELO is a promising vector for gene transfer applications. In order to study this potentiality, we developed an improved method for construction of adenovirus vectors in cosmids that was used to engineer the CELO genome. For all the recombinant viruses constructed by this method, the ability to produce infectious particles and the stability of the genome were evaluated in a chicken hepatocarcinoma cell line (LMH cell line). Our aim was to develop a replication-competent vector for vaccination of chickens, so we first generated knockout point mutations into 16 of the 22 unassigned CELO open reading frames (ORFs) to determine if they were essential for virus replication. As the 16 independent mutant viruses replicated in our cellular system, we constructed CELO genomes with various deletions in the regions of these nonessential ORFs. An expression cassette coding for the enhanced green fluorescent protein (eGFP) was inserted in place of these deletions to easily follow expression of the transgene and propagation of the vector in cell monolayers. Height-distinct GFP-expressing CELO vectors were produced that were all replication competent in our system. We then retained the vector backbone with the largest deletion (i.e., 3.6 kb) for the construction of vectors carrying cDNA encoding infectious bursal disease virus proteins. These CELO vectors could be useful for vaccination in the chicken species.Members of the family Adenoviridae are nonenveloped viruses with double-stranded linear DNA genomes that are 25 to 45 kb in length (13). Human adenoviruses (type 2 and 5) have been for some time developed as gene delivery vectors for vaccination or gene therapy (2,9,17,24,35,36) and more recently, adenoviruses from various species (bovine, ovine, porcine, canine, and avian) have been studied for similar applications (11,16,23,25,28,29,30,32). Among avian adenoviruses, which include at least 10 serotypes (3,4,8,12,27,32), chicken embryo lethal orphan (CELO), which represents serotype 1, is the best characterized (6,7,19,20,22,23,26). The CELO virus genome is 43,804 bp long and has been completely sequenced (7), and its transcriptional organization has been established (26). The central region of the viral genome is strongly homologous with other adenoviruses: the lower strand encodes replication functions (DNA polymerase, DNA-binding protein, pTP), and the upper strand, which is transcribed under the control of a single major late promoter, encodes capsid structural proteins. On either side of this central part there are two regions encoding at least 22 unassigned open reading frames (ORFs) that have no sequence homology with the E1, E3, and E4 regions of mammalian adenoviruses (mastadenoviruses). Only 2 of these 22 genes have been studied: ORF8 encodes GAM-1 protein, which was identified as a functional homolog to human adenovirus E1B 19K protein (6), and ORF22 encodes a protein that interacts with the retinoblastoma protein, which is similar to human adenovirus E1A protein, and cooperates with GAM-1 ...

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Streptomyces coelicolor and Lemna minor were used as a model to study the modulation of bacterial gene expression during plant-streptomycete interactions. S. coelicolor was grown in minimal medium with and without L. minor fronds. Bacterial proteomes were analyzed by two-dimensional gel electrophoresis, and a comparison of the two culture conditions resulted in identification of 31 proteins that were induced or repressed by the presence of plant material. One-half of these proteins were identified by peptide mass fingerprinting by using matrix-assisted laser desorption ionization-time of flight mass spectrometry. The induced proteins were involved in energetic metabolism (glycolysis, pentose phosphate pathway, oxidative phosphorylation), protein synthesis, degradation of amino acids, alkenes, or cellulose, tellurite resistance, and growth under general physiological or oxidative stress conditions. The repressed proteins were proteins synthesized under starvation stress conditions. These results suggest that root exudates provide additional carbon sources to the bacteria and that physiological adaptations are required for efficient bacterial growth in the presence of plants.Plant rhizospheres harbor diverse communities of microorganisms. It is generally assumed that rhizosphere microbes use compounds released by the plant roots as their major nutrient sources. This ability is the nutritional basis of rhizosphere competence (32). Streptomycetes are gram-positive bacteria that are frequently isolated from terrestrial plant rhizospheres (3, 15), but Wohl and McArthur (49) also showed that streptomycetes are associated with freshwater plants. Until now, few research efforts have been dedicated to the study of plantstreptomycete interactions, and most previous efforts have involved the common scab-inducing streptomycetes and their host plants (29). As several Streptomyces species have been shown to be effective biocontrol agents for plant diseases (10), studies on the interactions between saprophytic streptomycetes and plants need to be documented more.We propose Streptomyces coelicolor and Lemna minor as an experimental model to study the modulation of bacterial gene expression during the interaction between a saprophytic streptomycete and a plant. Recent research in our laboratory has shown that L. minor, a small aquatic plant extensively used in bioremediation (38) and environmental studies (30), is colonized by various Streptomyces species (unpublished data). On the other hand, S. coelicolor is a common inhabitant of plant rhizospheres (11) that can be readily cocultivated in the presence of L. minor under sterile conditions. The fact that the S. coelicolor chromosome (6) has been fully sequenced is an important asset in proteomic studies.Proteomics has become an integral part of gene expression analysis. The functional complement of genetic information can be analyzed by a combination of two-dimensional gel electrophoresis for the separation of complex mixtures of proteins and mass spectrometry for identification...

Avian adenovirus CELO (chicken embryo lethal orphan virus, fowl adenovirus type 1) incorporates two different homotrimeric fiber proteins extending from the same penton base: a long fiber (designated fiber 1) and a short fiber (designated fiber 2). The short fibers extend straight outwards from the viral vertices, whilst the long fibers emerge at an angle. In contrast to the short fiber, which binds an unknown avian receptor and has been shown to be essential to the invasiveness of this virus, the long fiber appears to be unnecessary for infection in birds. Both fibers contain a short N-terminal virus-binding peptide, a slender shaft domain and a globular C-terminal head domain; the head domain, by analogy with human adenoviruses, is likely to be involved mainly in receptor binding. This study reports the high-resolution crystal structure of the head domain of the long fiber, solved using single isomorphous replacement (using anomalous signal) and refined against data at 1.6 Å (0.16 nm) resolution. The C-terminal globular head domain had an anti-parallel b-sandwich fold formed by two four-stranded b-sheets with the same overall topology as human adenovirus fiber heads. The presence in the sequence of characteristic repeats N-terminal to the head domain suggests that the shaft domain contains a triple b-spiral structure. Implications of the structure for the function and stability of the avian adenovirus long fiber protein are discussed; notably, the structure suggests a different mode of binding to the coxsackievirus and adenovirus receptor from that proposed for the human adenovirus fiber heads. INTRODUCTIONAvian adenovirus or chick embryo lethal orphan (CELO) virus (also known as fowl adenovirus type 1, species Fowl adenovirus A in the genus Adenovirus of the family Adenoviridae) is a large, non-enveloped, double-stranded (ds) DNA virus (Laver et al., 1971;McCracken & Adair, 1993). The CELO virus appears to be relatively benign and has not been associated with serious pathogenicity or economic losses (Cowen et al., 1978), nor does it give rise to any evident disease state when experimentally introduced into chickens. This apparent harmlessness has sparked interest in the possibility of using CELO virus in humans as a gene therapy vector (Kelleher & Vos, 1994;Stevenson et al., 2006) or chemotherapy vehicle (Logunov et al., 2004;Shashkova et al., 2005). Studies have also been undertaken to explore its potential as a vaccination vehicle in birds (Francois et al., 2004). The 44 kb dsDNA genome of the virus is contained within a single icosahedral capsid layer (Chiocca et al., 1996). As is the case for human adenoviruses (Fabry et al., 2005;Saban et al., 2006), the capsid vertices are comprised of penton proteins and the faces of hexon proteins, with further minor proteins incorporated as stabilizers. Each penton vertex contains a pentameric penton base and two trimeric fiber proteins, the short fiber (fiber 2) and the long fiber (fiber 1), with the short fiber emerging straight from the base and the long fiber at ...