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1.
Microb Pathog ; 135: 103632, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31325569

RESUMO

Infectious bursal disease virus (IBDV) is the etiological agent of a highly contagious and immunosuppressive disease that affects domestic chickens. Toll-like receptors (TLRs), a kind of pattern recognition receptors, help the host to detect invading pathogens. To date, few systematic studies have been reported about the expression changes of TLR in chickens infected with pathogens. In the present study, layer chickens were infected with IBDV and the expression of chicken TLRs (chTLRs) was assayed by quantitative real-time PCR. The results showed that the expression of chTLR1a, 1b, 2a, 3, 4 and 15 was upregulated in the bursa of chickens infected with IBDV compared with noninfected chickens, while chTLR2b, 5, 7 and 21 expression was downregulated. Correlation analysis showed that chTLR3 expressions was directly associated with IBDV VP2 mRNA expression in bursa. These results suggested that different TLRs have different responses to the same viral infection. Some TLRs were activated early on, some later, and some were suppressed. This is the first study to report on the response of all chTLRs to one virus. This provids a valuable overview of the expression pattern of chTLRs when chickens are challenged by pathogens.


Assuntos
Infecções por Birnaviridae/imunologia , Galinhas/virologia , Vírus da Doença Infecciosa da Bursa/metabolismo , Doenças das Aves Domésticas/imunologia , Receptores Toll-Like/metabolismo , Animais , Infecções por Birnaviridae/genética , Infecções por Birnaviridae/virologia , Regulação Viral da Expressão Gênica , Imunidade Inata , Vírus da Doença Infecciosa da Bursa/genética , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Receptores Toll-Like/genética , Proteínas Estruturais Virais/metabolismo
2.
J Virol ; 93(10)2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30842328

RESUMO

SUMOylation is a posttranslational modification that has crucial roles in diverse cellular biological pathways and in various viral life cycles. In this study, we found that the VP1 protein, the RNA-dependent RNA polymerase of avibirnavirus infectious bursal disease virus (IBDV), regulates virus replication by SUMOylation during infection. Our data demonstrated that the polymerase VP1 is efficiently modified by small ubiquitin-like modifier 1 (SUMO1) in avibirnavirus-infected cell lines. Mutation analysis showed that residues 404I and 406I within SUMO interaction motif 3 of VP1 constitute the critical site for SUMO1 modification. Protein stability assays showed that SUMO1 modification enhanced significantly the stability of polymerase VP1 by inhibiting K48-linked ubiquitination. A reverse genetic approach showed that only IBDV with I404C/T and I406C/F mutations of VP1 could be rescued successfully with decreased replication ability. Our data demonstrated that SUMO1 modification is essential to sustain the stability of polymerase VP1 during IBDV replication and provides a potential target for designing antiviral drugs targeting IBDV.IMPORTANCE SUMOylation is an extensively discussed posttranslational modification in diverse cellular biological pathways. However, there is limited understanding about SUMOylation of viral proteins of IBDV during infection. In the present study, we revealed a SUMO1 modification of VP1 protein, the RNA-dependent RNA polymerase of avibirnavirus infectious bursal disease virus (IBDV). The required site of VP1 SUMOylation comprised residues 404I and 406I of SUMO interaction motif 3, which was essential for maintaining its stability by inhibiting K48-linked ubiquitination. We also showed that IBDV with SUMOylation-deficient VP1 had decreased replication ability. These data demonstrated that the SUMOylation of IBDV VP1 played an important role in maintaining IBDV replication.


Assuntos
Vírus da Doença Infecciosa da Bursa/metabolismo , Proteína SUMO-1/metabolismo , Proteínas Estruturais Virais/metabolismo , Avibirnavirus/metabolismo , Avibirnavirus/patogenicidade , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Vírus da Doença Infecciosa da Bursa/patogenicidade , Vírus da Doença Infecciosa da Bursa/fisiologia , Processamento de Proteína Pós-Traducional , RNA Replicase/genética , Proteína SUMO-1/fisiologia , Sumoilação , Ubiquitinação , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/genética , Replicação Viral/fisiologia
4.
Virus Res ; 247: 55-60, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29427596

RESUMO

Infectious Bursal Disease (IBD) is an acute, highly contagious and immunosuppressive disease of young chicken. The causative virus (IBDV) is a bi-segmented, double-stranded RNA virus. The virus encodes five major proteins, viral protein (VP) 1-5. VPs 1-3 have been characterized crystallographically. Albeit a rise in the number of studies reporting successful heterologous expression of VP5 in recent times, challenging the notion that rapid death of host cells overexpressing VP5 disallows obtaining sufficiently pure preparations of the protein for crystallographic studies, the structure of VP5 remains unknown and its function controversial. Our study describes the first 3D model of IBD VP5 obtained through an elaborate computational workflow. Based on the results of the study, IBD VP5 can be predicted to be a structural analog of the leucine-rich repeat (LRR) family of proteins. Functional implications arising from structural similarity of VP5 with host Toll-like receptor (Tlr) 3 also satisfy the previously reported opposing roles of the protein in first abolishing and later inducing host-cell apoptosis.


Assuntos
Vírus da Doença Infecciosa da Bursa/química , Receptor 3 Toll-Like/química , Proteínas não Estruturais Virais/química , Animais , Galinhas , Expressão Gênica , Vírus da Doença Infecciosa da Bursa/genética , Vírus da Doença Infecciosa da Bursa/isolamento & purificação , Vírus da Doença Infecciosa da Bursa/metabolismo , Simulação de Dinâmica Molecular , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
5.
Microb Pathog ; 107: 122-128, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28351707

RESUMO

Both CCR5 and CXCR4 are important chemokine receptors and take vital role in migration, development and distribution of T cells, however, whether they will influence the process of T cell infiltration into bursa of Fabricius during infectious bursal disease virus (IBDV) infection is unclear. In the current study, CCR5 and CXCR4 antagonists, Maraviroc and AMD3100, were administrated into chickens inoculated with IBDV, and the gene levels of IBDV VP2, CCR5, CXCR4 and related cytokines were determined by real-time PCR. The results showed that large number of T cells began to migrate into the bursae on Day 3 post infection with IBDV and the mRNA of chemokine receptors CCR5 and CXCR4 began to increase on Day 1. Moreover, antagonist treatments have increased the VP2, CCR5 and CXCR4 gene transcriptions and influenced on the gene levels of IL-2, IL-6, IL-8, IFN-γ, TGF-ß4, MHC-I and MDA5. In conclusion, the chemokine receptors CCR5 and CXCR4 might influence virus replication during IBDV infection and further study would focus on the interaction between chemokine receptors and their ligands.


Assuntos
Vírus da Doença Infecciosa da Bursa/metabolismo , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Receptores de Quimiocinas/metabolismo , Replicação Viral/fisiologia , Animais , Bolsa de Fabricius/virologia , Antagonistas dos Receptores CCR5 , Movimento Celular , Galinhas , Cicloexanos/antagonistas & inibidores , Citocinas/genética , Compostos Heterocíclicos/antagonistas & inibidores , Vírus da Doença Infecciosa da Bursa/genética , Interferon gama/genética , Interleucina-2/genética , Interleucina-6/genética , Interleucina-8/genética , Maraviroc , Doenças das Aves Domésticas/virologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Receptores CCR5/genética , Receptores CXCR4/genética , Linfócitos T/metabolismo , Linfócitos T/virologia , Transcrição Genética , Fator de Crescimento Transformador beta/genética , Triazóis/antagonistas & inibidores , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo
6.
Virus Res ; 232: 77-79, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28189698

RESUMO

Green fluorescent protein (GFP) has been successfully incorporated into the viral-like particles of infectious bursal disease virus (IBDV) with a linker at the C-terminus of VP3 in a baculovirus system. However, when the same locus in segment A was used to express GFP by a reverse genetic (RG) system, no viable GFP-expressing IBDV was recovered. To elucidate the underlying mechanism, cDNA construct of segment A with only the linker sequence (9 amino acids) was applied to generate RG IBDV virus (rIBDV). Similarly, no rIBDV was recovered. Moreover, when the incubation after transfection was extended, wildtype rIBDV without the linker was recovered suggesting a free C-terminus of VP3 might be necessary for IBDV replication. On the other hand, rIBDV could be recovered when additional sequence (up to 40 nucleotides) were inserted at the 3' noncoding region (NCR) adjacent to the stop codon of VP3, suggesting that the burden of the linker sequence was not in the stretched genome size but the disruption of the VP3 function. Finally, when the stop codon of VP3 was deleted in segment A to extend the translation into the 3' NCR without introducing additional genomic sequence, no rIBDV was recovered. Our data suggest that a free VP3 C-terminus is essential for IBDV replication.


Assuntos
Regulação Viral da Expressão Gênica , Vírus da Doença Infecciosa da Bursa/genética , Genética Reversa/métodos , Proteínas Estruturais Virais/genética , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Linhagem Celular Transformada , Galinhas , Clonagem Molecular , Fibroblastos/virologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Vírus da Doença Infecciosa da Bursa/metabolismo , Domínios Proteicos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Transfecção , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/metabolismo , Replicação Viral
7.
Parasit Vectors ; 9: 463, 2016 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-27553200

RESUMO

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.


Assuntos
Antígenos Virais/imunologia , Infecções por Birnaviridae/veterinária , Galinhas/imunologia , Eimeria tenella/virologia , Vírus da Doença Infecciosa da Bursa/metabolismo , Proteínas Estruturais Virais/metabolismo , Animais , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/prevenção & controle , Infecções por Birnaviridae/virologia , Regulação Viral da Expressão Gênica , Vírus da Doença Infecciosa da Bursa/imunologia , Vírus da Doença Infecciosa da Bursa/patogenicidade , Organismos Geneticamente Modificados , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/virologia , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologia , Vacinas Virais/imunologia , Virulência
8.
Genet Mol Res ; 15(2)2016 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-27323008

RESUMO

In this study, the immune response induced by a mixture of polysaccharide and nucleic acid extracted from Bacillus Calmette-Guerin (BCG) was evaluated in chickens inoculated with infectious bursal disease virus (IBDV) vaccine. After the mixture was injected intramuscularly at a dose of 0.075, 0.15 or 0.3 mg·kg(-1)·day(-1) for 3 days, the 14-day-old chickens were inoculated with the attenuated IBDV vaccine via intranasal and ocular routes. The relative weight of bursa of Fabricius (BF) and thymus, the serum IBD antibody titer, the CD4+/CD8+ ratio, and the concentrations of IFN-γ, IL-2 and IL-6 in peripheral blood were investigated on days 5, 15 and 25. The IBD antibody titer in BCG-treated groups was higher than in the negative control and only IBD-vaccinated chickens, indicating that the mixture of BCG can significantly enhance chicken humoral response. CD4+/CD8+ and the secretions of IFN-γ, IL-2 and IL-6 were also clearly increased compared with that in the negative control and IBD-vaccinated chickens, indicating that the mixture can also enhance the cell-mediated immune response. The results also showed that the relative weights of BF and thymus increased after chickens were inoculated with BCG, indicating that the BCG mixture can clearly enhance the immunity of IBD-vaccine and can be expected to be viewed as a candidate for a new type of immune adjuvant.


Assuntos
Vacina BCG/imunologia , Infecções por Birnaviridae/veterinária , Vírus da Doença Infecciosa da Bursa/imunologia , Ácidos Nucleicos/imunologia , Polissacarídeos/imunologia , Doenças das Aves Domésticas/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Antivirais/sangue , Vacina BCG/química , Vacina BCG/farmacologia , Infecções por Birnaviridae/imunologia , Galinhas/imunologia , Vírus da Doença Infecciosa da Bursa/metabolismo , Masculino , Ácidos Nucleicos/isolamento & purificação , Ácidos Nucleicos/farmacologia , Polissacarídeos/isolamento & purificação , Polissacarídeos/farmacologia , Doenças das Aves Domésticas/terapia , Distribuição Aleatória , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/farmacologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/farmacologia , Vacinas Virais/imunologia , Vacinas Virais/farmacologia
9.
Sci Rep ; 5: 14794, 2015 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-26440769

RESUMO

Unlike other viral protease, Avibirnavirus infectious bursal disease virus (IBDV)-encoded viral protease VP4 forms unusual intracellular tubule-like structures during viral infection. However, the formation mechanism and potential biological functions of intracellular VP4 tubules remain largely elusive. Here, we show that VP4 can assemble into tubules in diverse IBDV-infected cells. Dynamic analysis show that VP4 initiates the assembly at early stage of IBDV infection, and gradually assembles into larger size of fibrils within the cytoplasm and nucleus. Intracellular assembly of VP4 doesn't involve the host cytoskeleton, other IBDV-encoded viral proteins or vital subcellular organelles. Interestingly, the last C-terminal hydrophobic and amyloidogenic stretch (238)YHLAMA(243) with two "aggregation-prone" alanine residues was found to be essential for its intracellular self-assembly. The assembled VP4 fibrils show significantly low solubility, subsequently, the deposition of highly assembled VP4 structures ultimately deformed the host cytoskeleton and nucleus, which was potentially associated with IBDV lytic infection. Importantly, the assembly of VP4 significantly reduced the cytotoxicity of protease activity in host cells which potentially prevent the premature cell death and facilitate viral replication. This study provides novel insights into the formation mechanism and biological functions of the Avibirnavirus protease-related fibrils.


Assuntos
Avibirnavirus/metabolismo , Interações Hospedeiro-Patógeno , Serina Endopeptidases/metabolismo , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/metabolismo , Animais , Avibirnavirus/patogenicidade , Embrião de Galinha , Chlorocebus aethiops , Citoesqueleto/metabolismo , Células HEK293/virologia , Humanos , Vírus da Doença Infecciosa da Bursa/metabolismo , Vírus da Doença Infecciosa da Bursa/patogenicidade , Peptídeos/química , Peptídeos/metabolismo , Serina Endopeptidases/química , Solubilidade , Células Vero/virologia , Proteínas Virais/química , Proteínas Estruturais Virais/química
10.
Virol J ; 12: 177, 2015 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-26502988

RESUMO

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.


Assuntos
Infecções por Birnaviridae/veterinária , Vírus da Doença Infecciosa da Bursa/imunologia , Doenças das Aves Domésticas/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Baculoviridae/genética , Infecções por Birnaviridae/patologia , Infecções por Birnaviridae/prevenção & controle , Western Blotting , Bolsa de Fabricius/patologia , Linhagem Celular , Galinhas , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Vírus da Doença Infecciosa da Bursa/genética , Vírus da Doença Infecciosa da Bursa/metabolismo , Insetos , Microscopia Eletrônica de Transmissão , Poliproteínas , Doenças das Aves Domésticas/patologia , Multimerização Proteica , Resultado do Tratamento , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/ultraestrutura , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Virossomos/genética , Virossomos/metabolismo , Virossomos/ultraestrutura
11.
PLoS One ; 10(6): e0128828, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26046798

RESUMO

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.


Assuntos
Fibroblastos/virologia , Regulação Viral da Expressão Gênica , Fosfoproteínas/genética , Precursores de Proteínas/genética , Proteínas Estruturais Virais/genética , Motivos de Aminoácidos , Animais , Anticorpos Monoclonais/química , Linhagem Celular , Galinhas , Citoesqueleto/virologia , Mapeamento de Epitopos , Células HEK293 , Humanos , Vírus da Doença Infecciosa da Bursa/genética , Vírus da Doença Infecciosa da Bursa/metabolismo , Ponto Isoelétrico , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfoproteínas/metabolismo , Fosforilação , Precursores de Proteínas/metabolismo , Proteólise , Proteínas Estruturais Virais/metabolismo
12.
PLoS One ; 10(4): e0123470, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25886023

RESUMO

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.


Assuntos
Fusão Celular , Vírus da Doença Infecciosa da Bursa/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Humanos , Vírus da Doença Infecciosa da Bursa/fisiologia , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Proteínas não Estruturais Virais/química , Ensaio de Placa Viral
13.
Anal Chim Acta ; 853: 682-688, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25467518

RESUMO

In this study, we show a significantly reduced assay time and a greatly increased bead recovery for a commercial Luminex-based multiplex diagnostic immunoassay by performing all liquid handling steps of the assay protocol in a non-contact acoustic trapping platform. The Luminex assay is designed for detecting antibodies in poultry serum for infectious bursal disease virus, infectious bronchitis virus, Newcastle disease virus and avian reovirus. Here, we show proof-of-concept of a microfluidic system capable of being fully automated and handling samples in a parallel format with a miniature physical footprint where the affinity beads are retained in a non-contact levitated mode in a glass capillary throughout the assay protocol. The different steps are: incubation with the serum sample, secondary antibodies and fluorescent reporters and finally washing to remove any non-specifically bound species. A Luminex 200 instrument was used for the readout. The flow rates applied to the capillary during the initial trapping event and the wash steps were optimised for maximum bead recovery, resulting in a bead recovery of 75% for the complete assay. This can be compared to a bead recovery of approximately 30% when an automatic wash station was used when the assay was performed in the conventional manual format. The time for the incubation steps for a single assay was reduced by more than 50%, without affecting assay performance, since intermediate wash steps became redundant in the continuously perfused bead trapping capillary. We analyzed seven samples, in triplicates, and we can show that the readout of the assay performed in the acoustic trap compared 100% to the control ELISAs (positive or negative readout) and resulted in comparable S/P values as the conventional manual protocol. As the acoustic trapping does not require the particles to have magnetic properties, a greater degree of freedom in selecting microparticles can be provided. In extension, this can provide an opportunity to develop cheaper and more effective microparticles.


Assuntos
Antígenos Virais/análise , Ensaio de Imunoadsorção Enzimática/métodos , Animais , Anticorpos/imunologia , Antígenos Virais/imunologia , Ensaio de Imunoadsorção Enzimática/instrumentação , Vírus da Bronquite Infecciosa/metabolismo , Vírus da Doença Infecciosa da Bursa/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Vírus da Doença de Newcastle/metabolismo , Orthoreovirus Aviário/metabolismo , Infecções por Vírus de RNA/diagnóstico , Infecções por Vírus de RNA/virologia , Sonicação
14.
Biochim Biophys Acta ; 1844(7): 1173-82, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24732578

RESUMO

VP2 protein is the primary host-protective immunogen of infectious bursal disease virus (IBDV). His249 and His253 are two surface histidine residues in IBDV subviral particles (SVP), which is formed by twenty VP2 trimers when the VP2 protein of a local isolate is expressed. Here, a systemic study was performed to investigate His249 or/and His253 on self-assembly, cell attachment and immunogenicity of SVP. Point-mutagenesis of either or both histidine residues to alanine did not affect self-assembly of the SVP, but the SVP lost its Ni-NTA binding affinity when the His253 was mutated. Indirect immunofluorescence assays and inhibitory experiments also showed that His253 is essential for SVP to attach onto the DF-1 cells and to inhibit IBDV infection of DF-1 cells. Finally, enzyme-linked immunosorbent assays and chicken protection assays demonstrated that SVP with a mutation of His253 to alanine induced comparable neutralizing antibody titers in chickens as the wild-type SVP did. It was concluded that VP2's His253, a site not significant for the overall immunogenicity induced by SVP, is crucial for the binding affinity of SVP to Ni-NTA and the attachment of an IBDV host cell line. This is the first paper to decipher the role of His253 played in receptor interaction and immunogenicity.


Assuntos
Cromatografia de Afinidade , Vírus da Doença Infecciosa da Bursa/metabolismo , Níquel/metabolismo , Proteínas Estruturais Virais/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/metabolismo , Infecções por Birnaviridae/prevenção & controle , Galinhas , Imunofluorescência , Histidina/genética , Vírus da Doença Infecciosa da Bursa/genética , Vírus da Doença Infecciosa da Bursa/imunologia , Mutação/genética , Níquel/química , Conformação Proteica , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologia
15.
Biotechnol Lett ; 36(5): 1029-35, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24563296

RESUMO

Infectious bursal disease is an economically important disease that affects chickens worldwide. Here, a recombinant single chain variable fragment (scFv) antibody library derived from chickens immunized with VP2 protein of infectious bursal disease virus (IBDV) was constructed. The library was subjected to three rounds of screening by flow cytometry against VP2 protein through a bacteria display technology, resulting in the enrichment of scFv. Three scFv clones with different fluorescence intensity were obtained by random colony pick up. The isolated scFv antibodies were expressed and purified. Relative affinity assay showed the three clones had different sensitivity to VP2, in accordance with fluorescence activity cell sorting analysis. The potential use of the selected IBDV-specific scFv antibodies was demonstrated by the successful application of the isolated antibodies in western blotting assay and ELISA.


Assuntos
Vírus da Doença Infecciosa da Bursa/imunologia , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/isolamento & purificação , Afinidade de Anticorpos/imunologia , Técnicas de Química Combinatória , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Citometria de Fluxo , Humanos , Vírus da Doença Infecciosa da Bursa/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo
16.
PLoS One ; 9(1): e87790, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24498196

RESUMO

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.


Assuntos
Vírus da Doença Infecciosa da Bursa/genética , Vírus de RNA/genética , Ribonucleoproteínas/genética , Animais , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Vírus da Doença Infecciosa da Bursa/metabolismo , RNA de Cadeia Dupla/genética , RNA Viral/genética , Ribonucleoproteínas/metabolismo , Vírion/genética , Vírion/metabolismo , Montagem de Vírus/genética
17.
PLoS One ; 8(6): e65999, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23805195

RESUMO

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.


Assuntos
Vírus da Doença Infecciosa da Bursa/genética , Proteínas Estruturais Virais/metabolismo , Sequência de Aminoácidos , Animais , Teorema de Bayes , Infecções por Birnaviridae/virologia , Galinhas , Cuba , Bases de Dados Genéticas , Evolução Molecular , Vírus da Doença Infecciosa da Bursa/classificação , Vírus da Doença Infecciosa da Bursa/isolamento & purificação , Vírus da Doença Infecciosa da Bursa/metabolismo , Filogenia , Filogeografia , Doenças das Aves Domésticas/virologia , RNA Viral/isolamento & purificação , RNA Viral/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genética
18.
Prev Vet Med ; 111(1-2): 181-5, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23639492

RESUMO

To identify risk factors associated with infectious bursal disease (IBD) in commercial chickens in Bangladesh, we conducted a matched case-control study on 32 commercial farms affected with IBD and 96 IBD unaffected farms taking flock size (1000 vs. >1000 birds) and location of the farms (whether it is in rural or urban area) as matching variables. Epidemiological data from case and control farms were collected through the use of a pretested questionnaire, and analyzed by matched-pair analysis and multivariable conditional logistic regression. In the multivariable analysis, the variables 'receive visitors on the farm premises' (odds ratio [OR]=4.49; 95% confidence interval [CI]=1.50-13.42; p=0.007), 'purchase live poultry from the open market' (OR=7.59; 95% CI=2.69-21.45; p=<0.001), 'workers live outside the farms premises' (OR=5.89; 95% CI=1.87-18.60; p=0.002) and 'access of vendor vehicles on the farm premises' (OR=5.19; 95% CI=1.39-19.31; p=0.014) were identified as risk factors for IBD in commercial chicken farms. The proper management of the risk factors along with knowledge of the disease could help to reduce the incidence of IBDV infection in commercial chickens in Bangladesh.


Assuntos
Infecções por Birnaviridae/veterinária , Galinhas , Vírus da Doença Infecciosa da Bursa/isolamento & purificação , Doenças das Aves Domésticas/epidemiologia , Animais , Bangladesh/epidemiologia , Infecções por Birnaviridae/epidemiologia , Infecções por Birnaviridae/microbiologia , Estudos de Casos e Controles , Feminino , Vírus da Doença Infecciosa da Bursa/genética , Vírus da Doença Infecciosa da Bursa/metabolismo , Modelos Logísticos , Masculino , Análise Multivariada , Razão de Chances , Doenças das Aves Domésticas/microbiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Fatores de Risco , Inquéritos e Questionários
19.
J S Afr Vet Assoc ; 84(1): E1-4, 2013 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-25687581

RESUMO

Nucleotide sequences of the VP2 hypervariable region (VP2-HVR) of 10 infectious bursal disease viruses detected in indigenous and exotic chickens in Zambia from 2004 to 2005 were determined. Phylogenetic analysis showed that the viruses diverged into two genotypes and belonged to the African very virulent types (VV1 and VV2). In the phylogenetic tree, strains in one genotype clustered in a distinct group and were closely related to some strains isolated in western Africa (VV1), with nucleotide similarities of 95.7%- 96.5%. Strains in the other genotype were clustered within the eastern African VV type (VV2), with nucleotide similarities of 97.3%- 98.5%. Both genotypes were distributed in the southern parts of Zambia and had a unique conserved amino acid substitution at 300 (E→A) in addition to the putative virulence marker at positions 222(A), 242(I), 256(I), 294(I) and 299(S). These findings represent the first documentation of the existence of the African VV-IBDV variants in both indigenous and exotic chickens in Zambia.


Assuntos
Infecções por Birnaviridae/veterinária , Proteínas do Capsídeo/genética , Vírus da Doença Infecciosa da Bursa/genética , Epidemiologia Molecular , Doenças das Aves Domésticas/epidemiologia , Animais , Infecções por Birnaviridae/epidemiologia , Infecções por Birnaviridae/genética , Infecções por Birnaviridae/virologia , Proteínas do Capsídeo/metabolismo , Galinhas , Genótipo , Vírus da Doença Infecciosa da Bursa/metabolismo , Dados de Sequência Molecular , Filogenia , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , Análise de Sequência de RNA/veterinária , Zâmbia/epidemiologia
20.
Trop Anim Health Prod ; 45(5): 1107-12, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23212841

RESUMO

Infectious bursal disease virus (IBDV) is a double-stranded RNA virus that causes immunosuppressive disease in young chickens. Thousands of cases of IBDV infection are reported each year in South China, and these infections can result in considerable economic losses to the poultry industry. To monitor variations of the virus during the outbreaks, 30 IBDVs were identified from vaccinated chicken flocks from nine provinces in South China in 2011. VP2 fragments from different virus strains were sequenced and analyzed by comparison with the published sequences of IBDV strains from China and around the world. Phylogenetic analysis of hypervariable regions of the VP2 (vVP2) gene showed that 29 of the isolates were very virulent (vv) IBDVs, and were closely related to vvIBDV strains from Europe and Asia. Alignment analysis of the deduced amino acid (aa) sequences of vVP2 showed the 29 vv isolates had high uniformity, indicated low variability and slow evolution of the virus. The non-vvIBDV isolate JX2-11 was associated with higher than expected mortality, and had high deduced aa sequence similarity (99.2 %) with the attenuated vaccine strain B87 (BJ). The present study has demonstrated the continued circulation of IBDV strains in South China, and emphasizes the importance of reinforcing IBDV surveillance.


Assuntos
Infecções por Birnaviridae/veterinária , Galinhas , Vírus da Doença Infecciosa da Bursa/genética , Doenças das Aves Domésticas/epidemiologia , Proteínas Estruturais Virais/genética , Substituição de Aminoácidos , Animais , Infecções por Birnaviridae/epidemiologia , Infecções por Birnaviridae/virologia , China/epidemiologia , Vírus da Doença Infecciosa da Bursa/isolamento & purificação , Vírus da Doença Infecciosa da Bursa/metabolismo , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase/veterinária , Doenças das Aves Domésticas/virologia , Prevalência , Análise de Sequência de DNA/veterinária , Análise de Sequência de Proteína/veterinária , Homologia de Sequência de Aminoácidos , Proteínas Estruturais Virais/metabolismo
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