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The inflammatory response is an essential component of innate immunity to defense against pathogens. Infectious bursal disease (IBD) is the most important immunosuppressive disease in chickens and is caused by the infectious bursal disease virus (IBDV). Acute inflammation is a typical pathogenic process for IBD, however, the underlying mechanism is not clear. Here, we report that IBDV induces obvious inflammatory response in vivo and in vitro . Furthermore, viral VP2 is identified as an important inflammatory stimulus. It is observed that IBDV VP2 can activate NF-κB signaling pathway and then increase IL-1β production. In detail, IBDV VP2 interacts with myeloid differentiation primary response gene 88 (MyD88), potentiates the oligomerization of MyD88 and assembly of MyD88 complex, which is one important element leading to NF-κB signaling pathway activation and IL-1β production increase. More meaningfully, residues 253/284 of viral VP2 are significantly involved in IBDV-induced inflammatory response through modulating the interaction strength between VP2 and MyD88 and the following MyD88-NF-κB-IL-1β signaling pathway. This study reveals one molecular mechanism that trigger inflammation during IBDV infection, which is of great significance for a deeper understanding of the pathogenic mechanisms of IBDV.

ObjectivesTo develop a method for in vitro assembly of recombinant proteins expressed in E. coli into chimeric virus-like particles (cVLPs).ResultsA fusion protein (Bepi-Cap-A) between capsid protein (Cap) of PCV2b and B cell epitope (Bepi) of IBDV was expressed in E. Coli, and purified. For assembling them into cVLPs (Bepi-Cap-VLP), the Bepi-Cap-A was suspended in buffer C [0.03% (“%” stands for “v/v” unless otherwise indicated) polyethylene glycol, 0.4 M Tris, 10 mM β-mercaptoethanol, 5% glycerol, 0.02% (w/v) gellan gum, 0.1 M glycine, 0.03% Tween 80, 500 mM NaCl], and incubated. After centrifugation, the pellet was resuspended in buffer D [50 mM Na2HPO4, 50 mM NaH2PO4, 0.01% (w/v) gellan gum, 0.05 mM EDTA, 500 mM NaCl, 0.03% Tween 80, pH 6.5], and then dialyzed against dialysis buffer (50 mM Na2HPO4, 50 mM NaH2PO4, 500 mM NaCl, 0.03% Tween 80, pH 6.5). The procedure resulted in typical and immunogenic Bepi-Cap-VLP.ConclusionsThe data provide a method which is feasible for in vitro assembly of recombinant proteins into chimeric virus-like particles.

Infectious bursal disease is a highly contagious and acute viral disease caused by the infectious bursal disease virus (IBDV); it affects all major poultry producing areas of the world. The current study was designed to rigorously measure the global phylogeographic dynamics of IBDV strains to gain insight into viral population expansion as well as the emergence, spread and pattern of the geographical structure of very virulent IBDV (vvIBDV) strains. Sequences of the hyper-variable region of the VP2 (HVR-VP2) gene from IBDV strains isolated from diverse geographic locations were obtained from the GenBank database; Cuban sequences were obtained in the current work. All sequences were analysed by Bayesian phylogeographic analysis, implemented in the Bayesian Evolutionary Analysis Sampling Trees (BEAST), Bayesian Tip-association Significance testing (BaTS) and Spatial Phylogenetic Reconstruction of Evolutionary Dynamics (SPREAD) software packages. Selection pressure on the HVR-VP2 was also assessed. The phylogeographic association-trait analysis showed that viruses sampled from individual countries tend to cluster together, suggesting a geographic pattern for IBDV strains. Spatial analysis from this study revealed that strains carrying sequences that were linked to increased virulence of IBDV appeared in Iran in 1981 and spread to Western Europe (Belgium) in 1987, Africa (Egypt) around 1990, East Asia (China and Japan) in 1993, the Caribbean Region (Cuba) by 1995 and South America (Brazil) around 2000. Selection pressure analysis showed that several codons in the HVR-VP2 region were under purifying selection. To our knowledge, this work is the first study applying the Bayesian phylogeographic reconstruction approach to analyse the emergence and spread of vvIBDV strains worldwide.

Background Eimeria species are parasitic protozoa that cause coccidiosis, an intestinal disease commonly characterised by malabsorption, diarrhoea and haemorrhage that is particularly important in chickens. Vaccination against chicken coccidiosis is effective using wild-type or attenuated live parasite lines. The development of protocols to express foreign proteins in Eimeria species has opened up the possibility of using Eimeria live vaccines to deliver heterologous antigens and function as multivalent vaccine vectors that could protect chickens against a range of pathogens. Results In this study, genetic complementation was used to express immunoprotective virus antigens in Eimeria tenella . Infectious bursal disease virus (IBDV) causes Gumboro, an immunosuppressive disease that affects productivity and can interfere with the efficacy of poultry vaccination programmes. Infectious laryngotracheitis virus (ILTV) causes a highly transmissible respiratory disease for which strong cellular immunity and antibody responses are required for effective vaccination. Genes encoding the VP2 protein from a very virulent strain of IBDV ( vvVP2 ) and glycoprotein I from ILTV ( gI ) were cloned downstream of 5’ Et-Actin or 5’ Et-TIF promoter regions in plasmids that also contained a mCitrine fluorescent reporter cassette under control of the 5’ Et-MIC1 promoter. The plasmids were introduced by nucleofection into E. tenella sporozoites, which were then used to infect chickens. Progeny oocysts were sorted by FACS and passaged several times in vivo until the proportion of fluorescent parasites in each transgenic population reached ~20 % and the number of transgene copies per parasite genome decreased to < 10. All populations were found to transcribe and express the transgene and induced the generation of low titre, transgene-specific antibodies when used to immunise chickens. Conclusions E. tenella can express antigens of other poultry pathogens that are successfully recognised by the chicken immune system. Nonetheless, further work has to be done in order to improve the levels of expression for its future use as a multivalent vaccine vector.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a major avian pathogen responsible for an immunosuppressive disease affecting juvenile chickens. The IBDV genome is formed by two dsRNA segments. The largest one harbors two partially overlapping open reading frames encoding a non-structural polypeptide, known as VP5, and a large polyprotein, respectively. VP5 is non-essential for virus replication. However, it plays a major role in IBDV pathogenesis. VP5 accumulates at the plasma membrane (PM) of IBDV-infected cells. We have analyzed the mechanism underlying the VP5 PM targeting. Updated topological prediction algorithm servers fail to identify a transmembrane domain within the VP5 sequence. However, the VP5 polycationic C-terminal region, harboring three closely spaced patches formed by two or three consecutive basic amino acid residues (lysine or arginine), might account for its PM tropism. We have found that mutations, either C-terminal VP5 deletions or replacement of basic amino acids by alanine residues, that reduce the electropositive charge of the VP5 C-terminus abolish PM targeting. Lipid overlay assays performed with an affinity-purified Flag-tagged VP5 (FVP5) protein version show that this polypeptide binds several phosphoinositides (PIP), exhibiting a clear preference for monophosphate species. Experiments performed with FVP5 mutant proteins lacking the polycationic domain demonstrate that this region is essential for PIP binding. Data gathered with IBDV mutants expressing C-terminal deleted VP5 polypeptides generated by reverse genetics demonstrate that the VP5-PIP binding domain is required both for its PM targeting in infected cells, and for efficient virus dissemination. Data presented here lead us to hypothesize that IBDV might use a non-lytic VP5-dependent cell-to-cell spreading mechanism.

Infectious bursal disease virus (IBDV) is an avian pathogen responsible for an acute immunosuppressive disease that causes major losses to the poultry industry. Despite having a bipartite dsRNA genome, IBDV, as well as other members of the Birnaviridae family, possesses a single capsid layer formed by trimers of the VP2 capsid protein. The capsid encloses a ribonucleoprotein complex formed by the genome associated to the RNA-dependent RNA polymerase and the RNA-binding polypeptide VP3. A previous report evidenced that expression of the mature VP2 IBDV capsid polypeptide triggers a swift programmed cell death response in a wide variety of cell lines. The mechanism(s) underlying this effect remained unknown. Here, we show that VP2 expression in HeLa cells activates the double-stranded RNA (dsRNA)-dependent protein kinase (PKR), which in turn triggers the phosphorylation of the eukaryotic initiation factor 2α (eIF2α). This results in a strong blockade of protein synthesis and the activation of an apoptotic response which is efficiently blocked by coexpression of a dominant negative PKR polypeptide. Our results demonstrate that coexpression of the VP3 polypeptide precludes phosphorylation of both PKR and eIF2α and the onset of programmed cell death induced by VP2 expression. A mutation blocking the capacity of VP3 to bind dsRNA also abolishes its capacity to prevent PKR activation and apoptosis. Further experiments showed that VP3 functionally replaces the host-range vaccinia virus (VACV) E3 protein, thus allowing the E3 deficient VACV deletion mutant WRΔE3L to grow in non-permissive cell lines. According to results presented here, VP3 can be categorized along with other well characterized proteins such us VACV E3, avian reovirus sigmaA, and influenza virus NS1 as a virus-encoded dsRNA-binding polypeptide with antiapoptotic properties. Our results suggest that VP3 plays a central role in ensuring the viability of the IBDV replication cycle by preventing programmed cell death.

Birnaviruses are unconventional members of the icosahedral double-stranded (dsRNA) RNA virus group. The main differential birnavirus trait is the lack of the inner icosahedral transcriptional core, a ubiquitous structure conserved in all other icosahedral dsRNA viruses, that shelters the genome from cellular dsRNA sensors and provide the enzymatic machinery to produce and extrude mature messenger RNAs. In contrast, birnaviral particles enclose ribonucleoprotein (RNP) complexes formed by the genome segments, the dsRNA-binding VP3 polypeptide and the virus-encoded RNA polymerase (RdRp). The presence of RNPs suggests that the birnavirus replication program might exhibit significant differences with respect to those of prototypal dsRNA viruses. However, experimental evidences supporting this hypothesis are as yet scarce. Of particular relevance for the understanding of birnavirus replication is to determine whether RNPs act as intracellular capsid-independent transcriptional units. Our study was focused to answer this question using the infectious bursal disease virus (IBDV), the best characterized birnavirus, as model virus. Here, we describe the intracellular assembly of functional IBDV RNPs in the absence of the virus-encoded VP2 capsid polypeptide. Recombinant RNPs are generated upon coexpression of the IBDV VP1 and RdRp polypeptides and transfection of purified virus dsRNA. Presented data show that recombinant RNPs direct the expression of the IBDV polypeptide repertoire and the production of infectious virus in culture cells. Results described in this report constitute the first direct experimental evidence showing that birnaviral RNPs are intracellularly active in the absence of the virus capsid. This finding is consistent with presented data indicating that RNP formation precedes virus assembly in IBDV-infected cells, and supports the recently proposed IBDV replication model entailing the release of RNPs during the initial stages of the infection. Indeed, results presented here also support the previously proposed evolutionary connection between birnaviruses and positive-strand single-stranded RNA viruses.

Birnavirus-encoded viral protein 4 (VP4) utilizes a Ser/Lys catalytic dyad mechanism to process polyprotein. Here three phosphorylated amino acid residues Ser538, Tyr611 and Thr674 within the VP4 protein of the infectious bursal disease virus (IBDV), a member of the genus Avibirnavirus of the family Birnaviridae, were identified by mass spectrometry. Anti-VP4 monoclonal antibodies finely mapping to phosphorylated (p)Ser538 and the epitope motif 530PVVDGIL536 were generated and verified. Proteomic analysis showed that in IBDV-infected cells the VP4 was distributed mainly in the cytoskeletal fraction and existed with different isoelectric points and several phosphorylation modifications. Phosphorylation of VP4 did not influence the aggregation of VP4 molecules. The proteolytic activity analysis verified that the pTyr611 and pThr674 sites within VP4 are involved in the cleavage of viral intermediate precursor VP4-VP3. This study demonstrates that IBDV-encoded VP4 protein is a unique phosphoprotein and that phosphorylation of Tyr611 and Thr674 of VP4 affects its serine-protease activity.

Background Virus-like particle (VLP) technology is considered one of the most promising approaches in animal vaccines, due to the intrinsic immunogenic properties as well as high safety profile of VLPs. In this study, we developed a VLP vaccine against infectious bursal disease virus (IBDV), which causes morbidity and mortality in chickens, by expressing a baculovirus in insect cells. Methods To improve the self-proteolytic processing of precursor polyprotein (PP), we constructed a recombinant baculovirus transfer vector that co-expresses PP and the VP4 protease gene of IBDV. Results Expression and VLP assembly of recombinant proteins and antigenicity of the VLP were examined by Western blotting, ELISA, and transmission electron microscopy. In animal experiments, vaccination with the recombinant VLP induced strong and uniform humoral immunity and provided complete protection against challenge with very virulent (vv) IBDV in SPF chickens ( n  = 12). As determined by the bursa of Fabricius (BF)/body weight (B/BW) ratio, the protection against post-challenge bursal atrophy was significantly higher ( P  < 0.001) in VLP-vaccinated birds than in non-vaccinated controls. Conclusions Since the protective efficacy of the VLP vaccine was comparable to that of a commercially available inactivated vaccine, the recombinant VLP merits further investigation as an alternative means of protection against vvIBD.