RESUMO
Infectious bursal disease (IBD) is an acute and fatal immunosuppressive disease caused by infectious bursal disease virus (IBDV). As an obligate intracellular parasite, IBDV infection is strictly regulated by host factors. Knowledge on the antiviral activity and possible mechanism of host factors might provide the theoretical basis for the prevention and control of IBD. In this study, RNA-sequencing results indicated that many host factors were induced by IBDV infection, among which the expression levels of OASL (2´,5´-oligadenylate synthetase-like protein) was significantly upregulated. OASL overexpression significantly inhibited IBDV replication, whereas OASL knockdown promoted IBDV replication. Interestingly, the antiviral ability of OASL was independent of its canonical enzymatic activity, i.e., OASL targeted viral protein VP2 for degradation, depending on the autophagy receptor p62/SQSTM1 in the autophagy pathway. Additionally, the 316 lysine (K) of VP2 was the key site for autophagy degradation, and its replacement with arginine disrupted VP2 degradation induced by OASL and enhanced IBDV replication. Importantly, our results for the first time indicate a unique and potent defense mechanism of OASL against double-stranded RNA virus by interaction with viral proteins, which leads to their degradation. IMPORTANCE: OASL (2´,5´-oligadenylate synthetase-like protein) exhibits broad-spectrum antiviral effects against single-stranded RNA viruses in mammals, potentially serving as a promising target for novel antiviral strategies. However, its role in inhibiting the replication of double-stranded RNA viruses (dsRNA viruses), such as infectious bursal disease virus (IBDV), in avian species remains unclear. Our findings indicated a unique and potent defense mechanism of OASL against dsRNA viruses. It has been previously shown in mammals that OASL inhibits virus replication through increasing interferon production. The groundbreaking aspect of our study is the finding that OASL has the ability to interact with IBDV viral protein VP2 and target it for degradation and thus exerts its antiviral effect. Our results reveal the interaction between avian natural antiviral immune response and IBDV infection. Our study not only enhances our understanding of bird defenses against viral infections but can also inform strategies for poultry disease management.
Assuntos
2',5'-Oligoadenilato Sintetase , Autofagia , Infecções por Birnaviridae , Galinhas , Vírus da Doença Infecciosa da Bursa , Proteínas Estruturais Virais , Replicação Viral , Vírus da Doença Infecciosa da Bursa/fisiologia , Animais , Infecções por Birnaviridae/virologia , Infecções por Birnaviridae/metabolismo , Proteínas Estruturais Virais/metabolismo , Proteínas Estruturais Virais/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , 2',5'-Oligoadenilato Sintetase/genética , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/metabolismo , Interações Hospedeiro-Patógeno , Células HEK293 , Humanos , Linhagem CelularRESUMO
IMPORTANCE: The Avibirnavirus infectious bursal disease virus is still an important agent which largely threatens global poultry farming industry economics. VP3 is a multifunctional scaffold structural protein that is involved in virus morphogenesis and the regulation of diverse cellular signaling pathways. However, little is known about the roles of VP3 phosphorylation during the IBDV life cycle. In this study, we determined that IBDV infection induced the upregulation of Cdc7 expression and phosphorylated the VP3 Ser13 site to promote viral replication. Moreover, we confirmed that the negative charge addition of phosphoserine on VP3 at the S13 site was essential for IBDV proliferation. This study provides novel insight into the molecular mechanisms of VP3 phosphorylation-mediated regulation of IBDV replication.
Assuntos
Avibirnavirus , Proteínas de Ciclo Celular , Galinhas , Vírus da Doença Infecciosa da Bursa , Proteínas Serina-Treonina Quinases , Proteínas Estruturais Virais , Replicação Viral , Animais , Avibirnavirus/química , Avibirnavirus/crescimento & desenvolvimento , Avibirnavirus/metabolismo , Infecções por Birnaviridae/enzimologia , Infecções por Birnaviridae/metabolismo , Infecções por Birnaviridae/veterinária , Infecções por Birnaviridae/virologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Galinhas/virologia , Vírus da Doença Infecciosa da Bursa/química , Vírus da Doença Infecciosa da Bursa/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/metabolismoRESUMO
The infectious bursal disease (IBD) is an acute immunosuppressive viral disease that significantly affects the economics of the poultry industry. The IBD virus (IBDV) was known to infect B lymphocytes and activate macrophage and T lymphocytes, but there are limited studies on the impact of IBDV infection on chicken intraepithelial lymphocyte natural killer (IEL-NK) cells. This study employed an mRNA sequencing approach to investigate the early regulation of gene expression patterns in chicken IEL-NK cells after infection with very virulent IBDV strain UPM0081. A total of 12,141 genes were expressed in uninfected chicken IEL-NK cells, and most of the genes with high expression were involved in the metabolic pathway, whereas most of the low expressed genes were involved in the cytokine-cytokine receptor pathway. A total of 1,266 genes were differentially expressed (DE) at 3 day-post-infection (dpi), and these DE genes were involved in inflammation, antiviral response and interferon stimulation. The innate immune response was activated as several genes involved in inflammation, antiviral response and recruitment of NK cells to the infected area were up-regulated. This is the first study to examine the whole transcriptome profile of chicken NK cells towards IBDV infection and provides better insight into the early immune response of chicken NK cells.
Assuntos
Infecções por Birnaviridae/veterinária , Galinhas/virologia , Vírus da Doença Infecciosa da Bursa , Células Matadoras Naturais/metabolismo , Doenças das Aves Domésticas/virologia , Animais , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/metabolismo , Infecções por Birnaviridae/virologia , Quimiocinas/metabolismo , Galinhas/imunologia , Galinhas/metabolismo , Citocinas/metabolismo , Perfilação da Expressão Gênica/veterinária , Regulação Viral da Expressão Gênica , Vírus da Doença Infecciosa da Bursa/patogenicidade , Interferons/metabolismo , Redes e Vias Metabólicas , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/metabolismo , Análise de Sequência de RNA/veterinária , Transcriptoma , Carga ViralRESUMO
Layer chickens were artificially challenged with infectious bursal disease virus (IBDV), and the kinetics of IFN-λ and antiviral genes in the bursa were explored using quantitative real-time PCR. Data showed that after the chickens were infected with IBDV, the virus load in the bursa of the Fabricius peaked at 96 h and gradually decreased. The relative mRNA expression levels of IFN-λ and antiviral genes (zinc-finger antiviral protein [ZAP], interferon alpha-inducible protein 6 [IFI6], laboratory of genetics and physiology 2 [LGP2], virus inhibitory protein [Viperin], and Mx) of the infected group dramatically increased at 24-168 h compared with those of the negative-infected group. Furthermore, the ZAP mRNA expression peaked at 24 h (3.97-fold). The Viperin mRNA transcript level was highest at 48 h (384.60-fold). The mRNA expression levels of IFI6 (96.31-fold), LGP2 (18.29-fold), and Mx (88.85-fold) peaked at 72 h, and that of IFN-λ was most remarkable at 96 h (2978.81-fold). Furthermore, the ZAP change rule was significantly positively correlated with the change rule of the IBDV load. The mRNA expression levels of IFN-λ and antiviral genes (ZAP, IFI6, LGP2, Viperin, and Mx) increased as the virus expression increased and then decreased. These results further corroborated that the IBDV infection seriously interfered with the chicken's innate immune response.
Assuntos
Antivirais/metabolismo , Infecções por Birnaviridae/metabolismo , Expressão Gênica , Vírus da Doença Infecciosa da Bursa/fisiologia , Interferons/metabolismo , Animais , Infecções por Birnaviridae/virologia , Bolsa de Fabricius/virologia , Galinhas/imunologia , Imunidade Inata , Interferons/genética , Proteínas Mitocondriais/metabolismo , Doenças das Aves Domésticas/virologia , RNA Helicases/metabolismo , Proteínas Repressoras/metabolismoRESUMO
MicroRNAs are small noncoding RNAs playing an important role in host response to pathogenic infection. Here we show that IBDV infection induced the demethylation of the pre-miR-27 promoter and upregulated gga-miR-27b-3p expression. We found that ectopic expression of miR-27b-3p in DF-1 cells enhanced the expression of chicken IFN-ß, IRF3 and NF-κB, via directly targeting cellular suppressors of cytokine signaling 3 and 6 (SOCS3 and 6), inhibiting IBDV replication in host cells, while inhibition of endogenous miR-27b-3p by its inhibitors suppressed the expression of IFN-ß, IRF3 and NF-κB, enhancing SOCS3 and 6 expressions and facilitating IBDV replication. Furthermore, transfection of DF-1 cells with miR-27b-3p markedly increased phosphorylation of STAT1 on Tyr701 in cells post chIFN-γ treatment. On the contrary, inhibition of endogenous miR-27b-3p reduced phosphorylation of STAT1 on Tyr701 in cells with chIFN-γ treatment. These findings indicate that gga-miR-27b-3p serves as an inducible antiviral mediator in host response to IBDV infection.
Assuntos
Infecções por Birnaviridae , Vírus da Doença Infecciosa da Bursa , MicroRNAs/fisiologia , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina , Animais , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/metabolismo , Linhagem Celular , Galinhas , Citocinas/metabolismo , Interações entre Hospedeiro e Microrganismos , Vírus da Doença Infecciosa da Bursa/fisiologia , Interferon Tipo I/metabolismo , Replicação ViralRESUMO
Infectious bursal disease virus (IBDV) infection triggers the induction of type I IFN, which is mediated by melanoma differentiation-associated protein 5 recognition of the viral genomic double-stranded RNA (dsRNA). However, the mechanism of IBDV overcoming the type I IFN antiviral response remains poorly characterized. Here, we show that IBDV genomic dsRNA selectively binds to the host cellular RNA binding protein Staufen1 (STAU1) in vitro and in vivo. The viral dsRNA binding region was mapped to the N-terminal moiety of STAU1 (residues 1-468). Down-regulation of STAU1 impaired IBDV replication and enhanced IFN-ß transcription in response to IBDV infection, while having little effect on the viral attachment to the host cells and cellular entry. Conversely, overexpression of STAU1 but not the IBDV dsRNA-binding deficient STAU1 mutant (469-702) led to a suppression of IBDV dsRNA-induced IFN-ß promoter activity. Moreover, we found that the binding of STAU1 to IBDV dsRNA decreased the association of melanoma differentiation-associated protein 5 but not VP3 with the IBDV dsRNA in vitro. Finally, we showed that STAU1 and VP3 suppressed IFN-ß gene transcription in response to IBDV infection in an additive manner. Collectively, these findings provide a novel insight into the evasive strategies used by IBDV to escape the host IFN antiviral response.-Ye, C., Yu, Z., Xiong, Y., Wang, Y., Ruan, Y., Guo, Y., Chen, M., Luan, S., Zhang, E., Liu, H. STAU1 binds to IBDV genomic double-stranded RNA and promotes viral replication via attenuation of MDA5-dependent ß interferon induction.
Assuntos
Infecções por Birnaviridae/virologia , Proteínas do Citoesqueleto/metabolismo , Vírus da Doença Infecciosa da Bursa/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferon beta/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral , Animais , Antivirais/metabolismo , Infecções por Birnaviridae/genética , Infecções por Birnaviridae/metabolismo , Galinhas , Proteínas do Citoesqueleto/genética , Genômica , Células HEK293 , Células HeLa , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Helicase IFIH1 Induzida por Interferon/genética , Interferon beta/genética , RNA de Cadeia Dupla/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genéticaRESUMO
Infectious bursa disease virus (IBDV) pathogenesis is characterized by increased numbers of T cells and decreased numbers of B cells in the bursa. Currently, little is about the key factor that affects T migration into bursa. In humans, CC chemokine ligand 19 (CCL19) recruits monocytes and neutrophils and is usually involved in various inflammatory disorders. The aim of this study was to assess the roles of CCL19 in driving peripheral blood cells infiltration into bursa of Fabricius of chickens infected with IBDV. Bursal samples were collected from chickens of the infection group and the control group on day 1, 3, 5, and 7 post infection (dpi) with IBDV. The mRNA or protein levels of ccl19 and ccr7 genes in bursae were determined by real-time PCR and immunohistochemistry (IHC) methods. Moreover, an in vitro chemotaxis assay was performed to evaluate the chemotaxis ability of CCL19 and bursal total protein. The results have displayed that the mRNA levels of ccl19 were significantly increased on 1, 3, 5, and 7 dpi in the infection group. The highest value amounted to 73.4-fold of the control group. Also, the mRNA levels of CCR7, the receptor of CCL19, began to increase on 3 dpi and reached to the highest value of 206.3-fold on 5 dpi after IBDV infection. Then the gene expression of CCR7 in bursae of the infection group returned to the normal level. IHC results of CCL19 protein level accorded with the mRNA levels of CCL19, with the highest value on 5 dpi. Then, in vitro chemotaxis test demonstrated that the total bursal protein had the ability of recruiting peripheral white blood cells (PWBC) and the migration percentage was a little higher than that of the blank control with only basal medium (P < 0.05). Taken together, these data suggest that CCL19 acts as a chicken PWBC chemotactic factor and facilitate the infiltration of PWBC (especially T cells) into the bursae after IBDV infection.
Assuntos
Proteínas Aviárias/genética , Infecções por Birnaviridae/veterinária , Bolsa de Fabricius/metabolismo , Quimiocina CCL19/genética , Fatores Quimiotáticos/fisiologia , Doenças das Aves Domésticas/metabolismo , Linfócitos T/imunologia , Animais , Proteínas Aviárias/metabolismo , Infecções por Birnaviridae/metabolismo , Infecções por Birnaviridae/virologia , Quimiocina CCL19/metabolismo , Vírus da Doença Infecciosa da Bursa/fisiologia , Monócitos/metabolismo , Neutrófilos/metabolismo , Doenças das Aves Domésticas/virologiaRESUMO
Infectious bursal disease virus (IBDV) is a very important small RNA virus in the family of Birnaviridae, which can cause severe immunosuppressive effects and pathological damages in young chickens. It can replicate in bursal lymphocytes and impede the growth and development of B cells, finally causing bursal lymphocytes apoptosis. Previous results have shown that protocatechuic acid (PCA) as an important phenolic compound could effectively improve the survival rate of chickens infected with IBDV. The current study aimed to explore how PCA influenced the pathogenesis of IBDV, especially lymphocyte apoptosis in the process of IBDV infection. The results showed that PCA could effectively alleviate bursal pathological changes at the early stage of IBDV invasion. Moreover, bursal lymphocyte apoptosis for tissue section samples was largely elevated by PCA by using the terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method while the bursal lymphocyte apoptosis ratio was also increased by PCA by flow cytometry in the early stage of IBDV infection in vivo. Meanwhile, PCA could promote non-lymphocyte apoptosis in vitro. Further study displayed that the potential mechanisms mainly relied on regulation of the expressions of pro-apoptotic protein Bax and anti-apoptotic Bcl-2, thus speeding up the process of IBDV-infected cell apoptosis and preventing virus infection. Meanwhile, the results displayed that the PI3K/Akt and NF kappa B signal pathways might play an important role in promoting cell apoptosis after IBDV infection. Overall, PCA as a potent antiviral drug precursor is expected to be applied in the poultry industry as a substitute for clinical antiviral application.
Assuntos
Apoptose/efeitos dos fármacos , Infecções por Birnaviridae/tratamento farmacológico , Hidroxibenzoatos/administração & dosagem , Vírus da Doença Infecciosa da Bursa/fisiologia , Doenças das Aves Domésticas/tratamento farmacológico , Animais , Infecções por Birnaviridae/metabolismo , Infecções por Birnaviridae/fisiopatologia , Infecções por Birnaviridae/virologia , Bolsa de Fabricius/citologia , Bolsa de Fabricius/efeitos dos fármacos , Bolsa de Fabricius/metabolismo , Bolsa de Fabricius/virologia , Galinhas , Feminino , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Masculino , NF-kappa B/genética , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/fisiopatologia , Doenças das Aves Domésticas/virologiaRESUMO
Infectious bursal disease virus (IBDV), a nonenveloped, double-stranded RNA (dsRNA) virus with a T=13 icosahedral capsid, has a virion assembly strategy that initiates with a precursor particle based on an internal scaffold shell similar to that of tailed double-stranded DNA (dsDNA) viruses. In IBDV-infected cells, the assembly pathway results mainly in mature virions that package four dsRNA segments, although minor viral populations ranging from zero to three dsRNA segments also form. We used cryo-electron microscopy (cryo-EM), cryo-electron tomography, and atomic force microscopy to characterize these IBDV populations. The VP3 protein was found to act as a scaffold protein by building an irregular, â¼40-Å-thick internal shell without icosahedral symmetry, which facilitates formation of a precursor particle, the procapsid. Analysis of IBDV procapsid mechanical properties indicated a VP3 layer beneath the icosahedral shell, which increased the effective capsid thickness. Whereas scaffolding proteins are discharged in tailed dsDNA viruses, VP3 is a multifunctional protein. In mature virions, VP3 is bound to the dsRNA genome, which is organized as ribonucleoprotein complexes. IBDV is an amalgam of dsRNA viral ancestors and traits from dsDNA and single-stranded RNA (ssRNA) viruses.IMPORTANCE Structural analyses highlight the constraint of virus evolution to a limited number of capsid protein folds and assembly strategies that result in a functional virion. We report the cryo-EM and cryo-electron tomography structures and the results of atomic force microscopy studies of the infectious bursal disease virus (IBDV), a double-stranded RNA virus with an icosahedral capsid. We found evidence of a new inner shell that might act as an internal scaffold during IBDV assembly. The use of an internal scaffold is reminiscent of tailed dsDNA viruses, which constitute the most successful self-replicating system on Earth. The IBDV scaffold protein is multifunctional and, after capsid maturation, is genome bound to form ribonucleoprotein complexes. IBDV encompasses numerous functional and structural characteristics of RNA and DNA viruses; we suggest that IBDV is a modern descendant of ancestral viruses and comprises different features of current viral lineages.
Assuntos
Infecções por Birnaviridae/virologia , Genoma Viral , Vírus da Doença Infecciosa da Bursa/fisiologia , RNA de Cadeia Dupla/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Estruturais Virais/metabolismo , Montagem de Vírus , Animais , Infecções por Birnaviridae/genética , Infecções por Birnaviridae/metabolismo , Capsídeo/fisiologia , Capsídeo/ultraestrutura , Células Cultivadas , Coturnix/virologia , Microscopia Crioeletrônica , Vírus da Doença Infecciosa da Bursa/ultraestrutura , Células Musculares/virologia , Proteínas de Ligação a RNA/genética , Proteínas Estruturais Virais/genética , VírionRESUMO
Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is responsible for a devastating immunosuppressive disease affecting juvenile domestic chickens. IBDV particles are naked icosahedrons enclosing a bipartite double-stranded RNA genome harboring three open reading frames (ORF). One of these ORFs codes for VP5, a non-structural polypeptide dispensable for virus replication in tissue culture but essential for IBDV pathogenesis. Using two previously described recombinant viruses, whose genomes differ in a single nucleotide, expressing or not the VP5 polypeptide, we have analyzed the role of this polypeptide during the IBDV replication process. Here, we show that VP5 is not involved in house-keeping steps of the virus replication cycle; i.e. genome transcription/replication, protein translation and virus assembly. Although infection with the VP5 expressing and non-expressing viruses rendered similar intracellular infective progeny yields, striking differences were detected on the ability of their progenies to exiting infected cells. Experimental data shows that the bulk of the VP5-expressing virus progeny efficiently egresses infected cells during the early phase of the infection, when viral metabolism is peaking and virus-induced cell death rates are as yet minimal, as determined by qPCR, radioactive protein labeling and quantitative real-time cell death analyses. In contrast, the release of the VP5-deficient virus progeny is significantly abridged and associated to cell death. Taken together, data presented in this report show that IBDV uses a previously undescribed VP5-dependent non-lytic egress mechanism significantly enhancing the virus dissemination speed. Ultrastructural analyses revealed that newly assembled IBDV virions associate to a vesicular network apparently facilitating their trafficking from virus assembly factories to the extracellular milieu, and that this association requires the expression of the VP5 polypeptide.
Assuntos
Infecções por Birnaviridae/virologia , Vírus da Doença Infecciosa da Bursa/patogenicidade , Proteínas não Estruturais Virais/metabolismo , Vírion/metabolismo , Liberação de Vírus/fisiologia , Replicação Viral , Animais , Infecções por Birnaviridae/metabolismo , Células Cultivadas , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Músculo Esquelético/virologia , Mioblastos/citologia , Mioblastos/metabolismo , Mioblastos/virologia , CodornizRESUMO
While the entry of infectious bursal disease virus (IBDV) is initiated by the binding of the virus to the two major receptors integrin and HSP90, the signaling events after receptor binding and how they contribute to virus entry remain elusive. We show here that IBDV activates c-Src by inducing the phosphorylation of the Y416 residue in c-Src both in DF-1 chicken fibroblasts and in vivo in the bursa of Fabricius from specific-pathogen-free (SPF) chickens. Importantly, inactivated IBDV fails to stimulate c-Src Y416 phosphorylation, and a very virulent IBDV strain induces a much higher level of c-Src Y416 phosphorylation than does an attenuated strain. Inhibition of c-Src activation by an Src kinase inhibitor or expression of a c-Src dominant negative mutant results in a significant decrease in the internalization of IBDV but has little effect on virus adhesion. Furthermore, short hairpin RNA (shRNA) downregulation of integrin, either the α4 or ß1 subunit, but not HSP90 remarkably attenuates IBDV-induced c-Src Y416 phosphorylation, resulting in a decrease in IBDV internalization but not virus adhesion. Moreover, interestingly, inhibition of either c-Src downstream of the phosphatidylinositol 3-kinase (PI3K)/Akt-RhoA signaling cascade or actin rearrangement leads to a significant decrease in IBDV internalization irrespective of the IBDV-induced high levels of c-Src phosphorylation. Cumulatively, our results suggest a novel feed-forward model whereby IBDV activates c-Src for benefiting its cell entry via an integrin-mediated pathway by the activation of downstream PI3K/Akt-RhoA signaling and cytoskeleton actin rearrangement. IMPORTANCE: While IBDV-caused immunosuppression is highly related to viral invasion, the molecular basis of the cellular entry of IBDV remains elusive. In this study, we demonstrate that IBDV activates c-Src by inducing the phosphorylation of the Y416 residue in c-Src to promote virus internalization but not virus adhesion. The ability to induce the level of c-Src Y416 phosphorylation correlates with the pathogenicity of an IBDV strain. IBDV-induced c-Src Y416 activation is α4ß1 integrin but not HSP90 dependent and involves the activation of the downstream PI3K/Akt-RhoA GTPase-actin rearrangement cascade. Thus, our findings provide new insights into the IBDV infection process and the potential for c-Src as a candidate target for the development of IBDV therapeutic drugs.
Assuntos
Infecções por Birnaviridae/metabolismo , Infecções por Birnaviridae/virologia , Vírus da Doença Infecciosa da Bursa/fisiologia , Integrina alfa4beta1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Internalização do Vírus , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Actinas/metabolismo , Animais , Proteína Tirosina Quinase CSK , Linhagem Celular , Embrião de Galinha , Galinhas , Fibroblastos , Proteínas de Choque Térmico HSP90/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Fosforilação , Transdução de Sinais , Ligação ViralRESUMO
Histamine is an endogenous nitrogenous compound with extensive effects on immunologic cells and involved in many physiological functions. The current aim was to determine histamine levels in embryonic liver and its association with the pathogenicity of a very virulent infectious bursal disease virus (vvIBDV) isolate serially passaged in chicken embryos. A vvIBDV isolate and the passaged viruses were inoculated into SPF embryonated chicken eggs (0.2 ml per egg) via the chorioallantoic membrane. Embryonic livers were collected at 24, 48, 72, 96, and 120 h post-inoculation and histamine contents were quantified by fluorescence spectrophotometry analyses. Results showed that the histamine content in embryonic livers infected with the original vvIBDV isolate and the early passaged viruses significantly increased 48 h post-inoculation, as compared with the adapted IBDV isolate (p<0.01) and controls (p<0.01), with the concentration peaking from 72 h to 96 h. Most of the infected chicken embryos died from 48 h to 96 h post-inoculation. Moreover, the histamine content in dead embryos was markedly increased compared with live embryos (p<0.05), peaking at 72 h post-inoculation (p<0.01). There was an association between histamine content in embryonic livers and an elevation in histidine decarboxylase activity. Taken together, our results suggest that an excess of histamine correlates with inflammatory responses during vvIBDV infection. This study provides an incremental step in the understanding of the pathogenesis of vvIBDV.
Assuntos
Embrião de Galinha/metabolismo , Embrião de Galinha/virologia , Histamina/metabolismo , Vírus da Doença Infecciosa da Bursa/patogenicidade , Fígado/metabolismo , Fígado/virologia , Animais , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/metabolismo , Infecções por Birnaviridae/veterinária , Embrião de Galinha/imunologia , Galinhas , Histidina Descarboxilase/metabolismo , Vírus da Doença Infecciosa da Bursa/genética , Vírus da Doença Infecciosa da Bursa/imunologia , Mediadores da Inflamação/metabolismo , Fígado/imunologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/virologia , RNA Viral/genética , RNA Viral/metabolismo , Carga Viral , VirulênciaRESUMO
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-ß 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.
Assuntos
Infecções por Birnaviridae/metabolismo , Infecções por Birnaviridae/veterinária , Vírus da Doença Infecciosa da Bursa/fisiologia , Doenças das Aves Domésticas/metabolismo , Proteínas/metabolismo , Proteômica/métodos , Animais , Linhagem Celular , Galinhas , Cromatografia Líquida/métodos , Citoplasma/metabolismo , Citoplasma/virologia , Interações Hospedeiro-Patógeno , Vírus da Doença Infecciosa da Bursa/isolamento & purificação , Proteínas/análise , Transdução de Sinais , Espectrometria de Massas em Tandem/métodos , Proteínas Estruturais Virais/análise , Proteínas Estruturais Virais/metabolismoRESUMO
BACKGROUND: Very virulent infectious bursal disease virus (vvIBDV) induces immunosuppression and inflammation in young birds, which subsequently leads to high mortality. In addition, infectious bursal disease (IBD) is one of the leading causes of vaccine failure on farms. Therefore, understanding the immunopathogenesis of IBDV in both the spleen and the bursae could help effective vaccine development. However, previous studies only profiled the differential expression of a limited number of cytokines, in either the spleen or the bursae of Fabricius of IBDV-infected chickens. Thus, this study aims to evaluate the in vitro and in vivo immunoregulatory effects of vvIBDV infection on macrophage-like cells, spleen and bursae of Fabricius. RESULTS: The viral load was increased during the progression of the in vitro infection in the HD11 macrophage cell line and in vivo, but no significant difference was observed between the spleen and the bursae tissue. vvIBDV infection induced the expression of pro-inflammatory and Th1 cytokines, and chemokines from HD11 cells in a time- and dosage-dependent manner. Furthermore, alterations in the lymphocyte populations, cytokine and chemokine expression, were observed in the vvIBDV-infected spleens and bursae. A drastic rise was detected in numbers of macrophages and pro-inflammatory cytokine expression in the spleen, as early as 2 days post-infection (dpi). On 4 dpi, macrophage and T lymphocyte infiltration, associated with the peak expression of pro-inflammatory cytokines in the bursae tissues of infected chickens were observed. The majority of the significantly regulated pro-inflammatory cytokines and chemokines, in vvIBDV-infected spleens and bursae, were also detected in vvIBDV-infected HD11 cells. This cellular infiltration subsequently resulted in a sharp rise in nitric oxide (NO) and lipid peroxidation levels. CONCLUSION: This study suggests that macrophage may play an important role in regulating the early expression of pro-inflammatory cytokines, first in the spleen and then in the bursae, the latter tissue undergoing macrophage infiltration at 4 dpi.
Assuntos
Infecções por Birnaviridae/veterinária , Bolsa de Fabricius/metabolismo , Galinhas , Citocinas/metabolismo , Vírus da Doença Infecciosa da Bursa/patogenicidade , Doenças das Aves Domésticas/virologia , Animais , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/metabolismo , Infecções por Birnaviridae/virologia , Linhagem Celular , Citocinas/genética , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/metabolismo , RNA Viral/metabolismo , Organismos Livres de Patógenos Específicos , Baço/metabolismo , Carga Viral , VirulênciaRESUMO
The mechanisms by which viruses modulate the immune system include changes in host genomic methylation. 5-hydroxymethylcytosine (5hmC) is the catalytic product of the Tet (Ten-11 translocation) family of enzymes and may serve as an intermediate of DNA demethylation. Recent reports suggest that 5hmC may confer consequences on cellular events including the pathogenesis of disease; in order to explore this possibility further we investigated both 5-methylcytosine (5mC) and 5hmC levels in healthy and diseased chicken bursas of Fabricius. We discovered that embryonic B-cells have high 5mC content while 5hmC decreases during bursa development. We propose that a high 5mC level protects from the mutagenic activity of the B-cell antibody diversifying enzyme activation induced deaminase (AID). In support of this view, AID mRNA increases significantly within the developing bursa from embryonic to post hatch stages while mRNAs that encode Tet family members 1 and 2 reduce over the same period. Moreover, our data revealed that infectious bursal disease virus (IBDV) disrupts this genomic methylation pattern causing a global increase in 5hmC levels in a mechanism that may involve increased Tet 1 and 2 mRNAs. To our knowledge this is the first time that a viral infection has been observed to cause global increases in genomic 5hmC within infected host tissues, underlining a mechanism that may involve the induction of B-cell genomic instability and cell death to facilitate viral egress.
Assuntos
5-Metilcitosina/metabolismo , Infecções por Birnaviridae/veterinária , Galinhas , Citosina/análogos & derivados , Metilação de DNA , Genoma , Doenças das Aves Domésticas/imunologia , Animais , Linfócitos B/fisiologia , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/metabolismo , Infecções por Birnaviridae/virologia , Bolsa de Fabricius/imunologia , Bolsa de Fabricius/metabolismo , Citosina/metabolismo , Vírus da Doença Infecciosa da Bursa/fisiologia , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/virologiaRESUMO
Infectious pancreatic necrosis virus (IPNV) causes high incidence of disease in salmonids during the first period after SW transfer. During this period as well as during periods of stress, cortisol levels increase and indications of a relationship between IPNV susceptibility and cortisol have been suggested. The intestine is an entry route and a target tissue for IPNV displaying severe enteritis and sloughing of the mucosa in infected fish. The mechanisms behind effects of the virus on the intestinal tissue and the impact of cortisol on the effect remain unclear. In the present study, Atlantic salmon post smolts treated with or without slow release cortisol implants were subjected to a cohabitant IPNV challenge. Analysis of genes and proteins related to the innate and acquired immune responses against virus was performed 6 days post-challenge using qPCR and immunohistochemistry. An increased mRNA expression of anti-viral cytokine interferon type I was observed in the proximal intestine and head kidney as a response to the viral challenge and this effect was suppressed by cortisol. No effect was seen in the distal intestine. T-cell marker CD3 as well as MHC-I in both intestinal regions and in the head kidney was down regulated at the mRNA level. Number of CD8α lymphocytes decreased in the proximal intestine in response to cortisol. On the other hand, mRNA expression of Mx and IL-1ß increased in the proximal intestine and head kidney in IPNV challenged fish in the presence of cortisol suggesting that the immune activation shifts in timing and response pathway during simulated stress. The present study clearly demonstrates that IPNV infection results in a differentiated epithelial immune response in the different intestinal regions of the Atlantic salmon. It also reveals that the epithelial immune response differs from the systemic, but that both are modulated by the stress hormone cortisol.
Assuntos
Infecções por Birnaviridae/veterinária , Doenças dos Peixes/imunologia , Hidrocortisona/farmacologia , Imunidade nas Mucosas/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Salmo salar/imunologia , Animais , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/metabolismo , Doenças dos Peixes/metabolismo , Vírus da Necrose Pancreática Infecciosa , Interferon Tipo I/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/imunologia , Rim/efeitos dos fármacos , Rim/imunologia , Rim/metabolismoRESUMO
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/imunologiaRESUMO
Previously we have identified a series of cellular miRNA molecules up- or down-regulated in infectious bursal disease virus (IBDV) infected chicken embryo fibroblasts and Bursa of Fabricius with gene microarray analysis. Here we studied in detail a relatively well studied miRNA, gga-miR-21, for better understanding miRNAs involvement in IBDV-host interactions. Chicken pri-gga-miRNA-21 and a control miRNA Caenorhabditis elegans pri-cel-lin-4 gene were cloned into a lentiviral vector, respectively. The resulting recombinant lentiviruses were used to infect chicken fibroblast cell line DF-1, and two stable cell lines, DF-miR-21 (overexpressing gga-miR-21) and DF-lin-4 (overexpressing cel-lin-4), were selected. Replication of IBDV in DF-miR-21, DF-lin-4 and DF-1 cells were compared and molecular mechanism of IBDV replication alteration was explored using bioinformatics, reporter gene system, qRT-PCR and Western blot analysis. IBDV replication was markedly lower in DF-miR-21 than in DF-lin-4 or DF-1 cells. A gga-miR-21 target sequence was identified within IBDV VP1 gene (1713-1734bp). Fusion of a 520nt long partial IBDV VP1 gene containing the target with a luciferase gene resulted in significantly lower transient luciferase activity in DF-miR-21 cells as compared to that in DF-lin-4 or DF-1 cell. Following IBDV infection of the cell lines, VP1 protein level in DF-miR-21 cells was dramatically lower than that in DF-lin-4 or DF-1 cells but VP1 mRNA level was not different. The finding indicated that gga-miR-21 could suppress IBDV replication through down regulating IBDV VP1 expression at translational level.
Assuntos
Infecções por Birnaviridae/veterinária , Fibroblastos/virologia , Vírus da Doença Infecciosa da Bursa/fisiologia , MicroRNAs/genética , Doenças das Aves Domésticas/genética , Biossíntese de Proteínas , Proteínas Estruturais Virais/genética , Replicação Viral , Animais , Infecções por Birnaviridae/genética , Infecções por Birnaviridae/metabolismo , Infecções por Birnaviridae/virologia , Embrião de Galinha , Galinhas , Regulação para Baixo , Fibroblastos/metabolismo , Vírus da Doença Infecciosa da Bursa/genética , MicroRNAs/metabolismo , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/virologia , Proteínas Estruturais Virais/metabolismoRESUMO
This study investigates the occurrence and distribution pattern of infectious pancreatic necrosis virus (IPNV) within the pancreas, liver, kidney and spleen of naturally infected cultured rainbow trout, Oncorhynchus mykiss (Walbaum), using immunohistochemistry (IHC). A nested PCR was also employed to confirm the presence of the virus in the pooled tissues of the specimens. All the examined tissues except spleen were immunohistochemically positive for IPNV, but staining intensity and distribution pattern varied. The kidney tubules had the most intense and widespread staining by IHC, indicating a specific tissue tropism at least for this particular serotype. The nucleotide sequence had the greatest identity with the Sp serotype confirming the presence of the nucleic acid of IPNV in the pooled tissues. Based on the present findings, it could be concluded that the absence of lesions consistent with infectious pancreatic necrosis (IPN) disease in the H&E-stained sections cannot rule out the presence of the IPNV, and the use of an alternative rapid confirmatory method such as IHC with formalin-fixed, paraffin-embedded tissue sections is helpful for the final diagnosis of IPN in rainbow trout.
Assuntos
Infecções por Birnaviridae/veterinária , Doenças dos Peixes/virologia , Vírus da Necrose Pancreática Infecciosa/genética , Oncorhynchus mykiss , Animais , Infecções por Birnaviridae/diagnóstico , Infecções por Birnaviridae/metabolismo , Infecções por Birnaviridae/virologia , Doenças dos Peixes/diagnóstico , Doenças dos Peixes/metabolismo , Imuno-Histoquímica , Vírus da Necrose Pancreática Infecciosa/classificação , Vírus da Necrose Pancreática Infecciosa/isolamento & purificação , Vírus da Necrose Pancreática Infecciosa/fisiologia , Oncorhynchus mykiss/crescimento & desenvolvimento , Reação em Cadeia da Polimerase/veterinária , Distribuição TecidualRESUMO
The present work describes the generation of a cell line from newly hatched Atlantic cod (Gadus morhua) larvae (ACL cells). Primary cultures were initiated by explant outgrowth from partially minced tissues and subcultured cells were exposed to UV radiation. After a substantial period of growth lag, cells started to proliferate and different growth conditions were tested to establish the cell line. At present, the ACL cell line has been subcultured for more than 100 passages. ACL cells had a polygonal shape and the morphology appeared homogenous with epithelial-like cells. Cell growth was dependent on the presence of foetal bovine serum and cells proliferated in a wide temperature range with optimal growth at 15 °C. By exposure to a viral dsRNA mimic (poly I:C) the cells expressed high levels of a repertoire of genes comprising both inflammatory mediators and interferon stimulated genes. Infection studies with two different viruses showed that infectious pancreatic necrosis virus (IPNV) propagated efficiently, and induced low level expression of genes of both pathways before the cells rapidly died. No productive infection was obtained with nervous necrosis virus (NNV), but a transient increase in the viral RNA level, followed by a high increase in expression of selected ISGs, suggests that the virus enters the cells but is unable to complete its replication cycle. To our knowledge, ACL cells are at the moment the only existing cell line from Atlantic cod. Our results demonstrate that ACL cells can be a useful research tool for further exploration of host-pathogen interactions and it is believed that this cell line will serve as a valuable tool also for studies within other research areas.