RESUMO
Porcine parvovirus (PPV) is one of the most important pathogens that cause reproductive failure in pigs. However, the pathogenesis of PPV infection remains unclear. Proteomics is a powerful tool to understand the interaction between virus and host cells. In the present study, we analyzed the proteomics of PPV-infected PK-15 cells. A total of 32 and 345 proteins were differentially expressed at the early and replication stages, respectively. Subsequent gene ontology annotation and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed these differentially expressed proteins were significantly enriched in pathways including toll-like receptor signaling pathway, tumor necrosis factor signaling pathway, and viral carcinogenesis. The expression of poly (rC) binding protein 1 (PCBP1) was observed to decrease after PPV infection. Overexpressed or silenced PCBP1 expression inhibited or promoted PPV infection. Our studies established a foundation for further exploration of the multiplication mechanism of PPV. IMPORTANCE: Porcine parvovirus (PPV) is a cause of reproductive failure in the swine industry. Our knowledge of PPV remains limited, and there is no effective treatment for PPV infection. Proteomics of PPV-infected PK-15 cells was conducted to identify differentially expressed proteins at 6 hours post-infection (hpi) and 36 hpi. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that various pathways participate in PPV infection. Poly (rC) binding protein 1 was confirmed to inhibit PPV replication, which provided potential targets for anti-PPV infection. Our findings improve the understanding of PPV infection and pave the way for future research in this area.
Assuntos
Infecções por Parvoviridae , Parvovirus Suíno , Proteômica , Proteínas de Ligação a RNA , Doenças dos Suínos , Replicação Viral , Parvovirus Suíno/genética , Parvovirus Suíno/fisiologia , Animais , Suínos , Linhagem Celular , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Infecções por Parvoviridae/virologia , Infecções por Parvoviridae/metabolismo , Infecções por Parvoviridae/veterinária , Doenças dos Suínos/virologia , Doenças dos Suínos/metabolismo , Doenças dos Suínos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
(1) Background: Porcine Parvovirus (PPV) is a single-stranded DNA virus without envelope which causes great harm in relation to porcine reproductive disorders in clinic. Cluster of Differentiation 38 (CD38) is a transmembrane protein widely existing in mammals. Its various functions make it a very popular research object, including in the viral infection field. (2) Methods: Western blotting and an EdU Cell Proliferation Kit were used to evaluate the effect of CD38-deficient cells. Relative quantitative real-time RT-PCR was used to detect the transcription levels of cytokines after PPV infection. The renilla luciferase reporter gene assay was used to verify the activation function of CD38 on downstream factors. The fluorescence probe method was used to detect the level of intracellular reactive oxygen species (ROS). (3) Results: This study found that the loss of CD38 function inhibited the up-regulated state of Toll-like Receptor 9 (TLR9), Interferon-α (IFN-α), and Myxovirus Resistance 1 (Mx1) after PPV infection. The luminescence of the group transfected with both CD38 expression plasmid and TLR9 promoter renilla luciferase reporter plasmid was significantly up-regulated compared with the control, suggesting that CD38 may activate the promoter of TLR9. In addition, CD38 deficiency not only activated the transcription of Sirtuin-1 (SIRT1), but also inhibited ROS level and the transcription of NLR Family Pyrin Domain Containing 3 (NLRP3). (4) Conclusion: (i) CD38 may participate in the TLR9/IFN-α/Mx1 pathway by activating the expression of TLR9 after PPV infected PK-15 cells; (ii) CD38 may activate the NLRP3/CASP1 pathway by increasing ROS level; (iii) CD38 deficiency activates the expression of SIRT1 and can prevent the normal proliferation of PPV.
Assuntos
Infecções por Parvoviridae , Parvovirus Suíno , Animais , Inflamassomos/metabolismo , Luciferases de Renilla/metabolismo , Mamíferos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Infecções por Parvoviridae/veterinária , Parvovirus Suíno/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Sirtuína 1/metabolismo , Suínos , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismoRESUMO
Since 2001, strains of porcine parvovirus (PPV), designated 27a-like strains, were observed in Europe, suggesting a predominance of these viruses over older strains. The reasons for the obvious evolutionary advantage are unknown. Here, a series of mutants containing amino acid replacements found in the predominant field strains were generated in a PPV-NADL2 background, and their impact on replication efficiency and antibody binding activity was determined. Some amino acid substitutions observed in the 27a-like strains significantly increased viral fitness and decreased neutralization activity of serum samples raised against commercial vaccines and old virus strains (e.g., NADL2). These mutant viruses and a monoclonal antibody raised against a classical PPV strain defined a 27a-specific neutralizing epitope around amino acid 228 of the capsid protein VP2. Based on the analysis of the mutant viruses, it is hypothesized that the predominant factor for the global spread of the PPV-27a strain substitutions is an increased viral fitness of the 27a-like viruses, possibly supported by partial immune selection. This is reminiscent to the evolution of canine parvovirus and worldwide replacement of the original virus by the so-called new antigenic types. IMPORTANCE Porcine parvovirus is one of the most important causes of reproductive failure in swine. Recently, despite the continuous use of vaccines, "new" strains emerged, leading to the hypothesis that the emergence of new amino acid substitutions could be a viral adaptation to the immune response against the commercial vaccines. Our results indicate the amino acid substitutions observed in the 27a-like strains can modify viral fitness and antigenicity. However, an absolute immune escape was not evident.
Assuntos
Proteínas do Capsídeo/genética , Parvovirus Suíno/fisiologia , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Células Cultivadas , Epitopos/genética , Epitopos/imunologia , Modelos Moleculares , Testes de Neutralização , Parvovirus Suíno/genética , Parvovirus Suíno/imunologia , Suínos , Replicação ViralRESUMO
Porcine parvovirus (PPV) NS1, the major nonstructural protein of this virus, plays an important role in PPV replication. We show, for the first time, that NS1 dynamically shuttles between the nucleus and cytoplasm, although its subcellular localization is predominantly nuclear. NS1 contains two nuclear export signals (NESs) at amino acids 283 to 291 (designated NES2) and amino acids 602 to 608 (designated NES1). NES1 and NES2 are both functional and transferable NESs, and their nuclear export activity is blocked by leptomycin B (LMB), suggesting that the export of NS1 from the nucleus is dependent upon the chromosome region maintenance 1 (CRM1) pathway. Deletion and site-directed mutational analyses showed that NS1 contains a bipartite nuclear localization signal (NLS) at amino acids 256 to 274. Coimmunoprecipitation assays showed that NS1 interacts with importins α5 and α7 through its NLS. The overexpression of CRM1 and importins α5 and α7 significantly promoted PPV replication, whereas the inhibition of CRM1- and importin α/ß-mediated transport by specific inhibitors (LMB, importazole, and ivermectin) clearly blocked PPV replication. The mutant viruses with deletions of the NESs or NLS motif of NS1 by using reverse genetics could not be rescued, suggesting that the NESs and NLS are essential for PPV replication. Collectively, these findings suggest that NS1 shuttles between the nucleus and cytoplasm, mediated by its functional NESs and NLS, via the CRM1-dependent nuclear export pathway and the importin α/ß-mediated nuclear import pathway, and PPV proliferation was inhibited by blocking NS1 nuclear import or export. IMPORTANCE PPV replicates in the nucleus, and the nuclear envelope is a barrier to its entry into and egress from the nucleus. PPV NS1 is a nucleus-targeting protein that is important for viral DNA replication. Because the NS1 molecule is large (>50 kDa), it cannot pass through the nuclear pore complex by diffusion alone and requires specific transport receptors to permit its nucleocytoplasmic shuttling. In this study, the two functional NESs in the NS1 protein were identified, and their dependence on the CRM1 pathway for nuclear export was demonstrated. The nuclear import of NS1 utilizes importins α5 and α7 in the importin α/ß nuclear import pathway.
Assuntos
Interações Hospedeiro-Patógeno , Carioferinas/metabolismo , Infecções por Parvoviridae/veterinária , Parvovirus Suíno/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Doenças dos Suínos/metabolismo , Doenças dos Suínos/virologia , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Imunofluorescência , Humanos , Carioferinas/genética , Camundongos , Sinais de Exportação Nuclear/genética , Ligação Proteica , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/genética , Suínos , Proteínas não Estruturais Virais/genética , Replicação Viral , Proteína Exportina 1RESUMO
Porcine Parvovirus (PPV), a pathogen causing porcine reproductive disorders, encodes two capsid proteins (VP1 and VP2) and three nonstructural proteins (NS1, NS2 and SAT) in infected cells. The PPV NS2 mRNA is from NS1 mRNA after alternative splicing, yet the corresponding mechanism is unclear. In this study, we identified a PPV NS1 mRNA binding protein SYNCRIP, which belongs to the hnRNP family and has been identified to be involved in host pre-mRNA splicing by RNA-pulldown and mass spectrometry approaches. SYNCRIP was found to be significantly up-regulated by PPV infection in vivo and in vitro. We confirmed that it directly interacts with PPV NS1 mRNA and is co-localized at the cytoplasm in PPV-infected cells. Overexpression of SYNCRIP significantly reduced the NS1 mRNA and protein levels, whereas deletion of SYNCRIP significantly reduced NS2 mRNA and protein levels and the ratio of NS2 to NS1, and further impaired replication of the PPV. Furthermore, we found that SYNCRIP was able to bind the 3'-terminal site of NS1 mRNA to promote the cleavage of NS1 mRNA into NS2 mRNA. Taken together, the results presented here demonstrate that SYNCRIP is a critical molecule in the alternative splicing process of PPV mRNA, while revealing a novel function for this protein and providing a potential target of antiviral intervention for the control of porcine parvovirus disease.
Assuntos
DNA Viral/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas/genética , Infecções por Parvoviridae/veterinária , Parvovirus Suíno/fisiologia , RNA Mensageiro/genética , Doenças dos Suínos/genética , Proteínas não Estruturais Virais/genética , Processamento Alternativo , Animais , Replicação do DNA , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Infecções por Parvoviridae/genética , Infecções por Parvoviridae/metabolismo , Parvovirus Suíno/genética , RNA Mensageiro/metabolismo , Sus scrofa , Suínos , Doenças dos Suínos/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
Porcine parvovirus (PPV) is a major cause of reproductive failure in swine and has caused substantial losses throughout the world. Viral protein 2 (VP2) of PPV is a major structural protein that can self-assemble into virus-like particles (VLP) with hemagglutination (HA) activity. In order to identify the essential residues involved in the mechanism of capsid assembly and to further understand the function of HA, we analyzed a series of deletion mutants and site-directed mutations within the N-terminal of VP2 using the Escherichia coli system. Our results showed that deletion of the first 47 amino acids from the N-terminal of the VP2 protein did not affect capsid assembly, and further truncation to residue 48 Asparagine (Asn, N) caused detrimental effects. Site-directed mutagenesis experiments demonstrated that residue 47Asn reduced the assembly efficiency of PPV VLP, while residue 48Asn destroyed the stability, hemagglutination, and self-assembly characteristics of the PPV VP2 protein. Results from native PAGE inferred that macromolecular polymers were critical intermediates of the VP2 protein during the capsid assembly process. Site-directed mutation at 48Asn did not affect the ability of monomers to form into oligomers, but destroyed the ability of oligomers to assemble into macromolecular particles, influencing both capsid assembly and HA activity. Our findings provide valuable information on the mechanisms of PPV capsid assembly and the possibility of chimeric VLP vaccine development by replacing the first 47 amino acids at the N-terminal of the VP2 protein.
Assuntos
Antígenos Virais/genética , Asparagina/metabolismo , Proteínas do Capsídeo/genética , Capsídeo/fisiologia , Regulação Viral da Expressão Gênica , Parvovirus Suíno/genética , Parvovirus Suíno/fisiologia , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Antígenos Virais/metabolismo , Asparagina/análise , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Mutação , Suínos , Montagem de Vírus/genética , Montagem de Vírus/fisiologiaRESUMO
To protect the health of sows and gilts, significant investments are directed toward the development of vaccines against infectious agents that impact reproduction. We developed an intrauterine vaccine that can be delivered with semen during artificial insemination to induce mucosal immunity in the reproductive tract. An in vitro culture of uterine epithelial cells was used to select an adjuvant combination capable of recruiting antigen-presenting cells into the uterus. Adjuvant polyinosinic:polycytidylic acid (poly I:C), alone or in combination, induced expression of interferon gamma, tumor necrosis factor alpha, and select chemokines. A combination adjuvant consisting of poly I:C, host defense peptide and polyphosphazene (Triple Adjuvant; TriAdj), which previously was shown to induce robust mucosal and systemic humoral immunity when administered to the uterus in rabbits, was combined with boar semen to evaluate changes in localized gene expression and cellular recruitment, in vivo. Sows bred with semen plus TriAdj had decreased γδ T cells and monocytes in blood, however, no corresponding increase in the number of monocytes and macrophages was detected in the endometrium. Compared to sows bred with semen alone, sows bred with semen plus TriAdj showed increased CCL2 gene expression in the epithelial layer. These data suggest that the adjuvants may further augment a local immune response and, therefore, may be suitable for use in an intrauterine vaccine. When inactivated porcine parvovirus (PPV) formulated with the TriAdj was administered to the pig uterus during estrus along with semen, we observed induction of PPV antibodies in serum but only when the pigs were already primed with parenteral PPV vaccines. Recombinant protein vaccines and inactivated PPV vaccines administered to the pig uterus during breeding as a primary vaccine alone failed to induce significant humoral immunity. More trials need to be performed to clarify whether repeated intrauterine vaccination can trigger strong humoral immunity or whether the primary vaccine needs to be administered via a systemic route to promote a mucosal and systemic immune response.
Assuntos
Quimiocina CCL2/metabolismo , Endométrio/metabolismo , Células Epiteliais/fisiologia , Compostos Organofosforados/imunologia , Infecções por Parvoviridae/imunologia , Parvovirus Suíno/fisiologia , Poli I-C/imunologia , Sêmen/imunologia , Útero/imunologia , Vacinas/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Antivirais/sangue , Cruzamento , Células Cultivadas , Feminino , Inseminação Artificial , Polímeros , Reprodução , Suínos , Regulação para Cima , VacinaçãoRESUMO
BACKGROUND: Porcine parvovirus (PPV) is a single-stranded DNA virus that causes porcine reproductive failure. It is of critical importance to study PPV pathogenesis for the prevention and control of the disease. NS1, a PPV non-structural protein, is participated in viral DNA replication, transcriptional regulation, and cytotoxicity. Our previous research showed that PPV can activate nuclear factor kappa B (NF-κB) signaling pathway and then up-regulate the expression of interleukin (IL)-6. OBJECTIVES: Herein, the purpose of this study is to determine whether the non-structural protein NS1 of PPV also has the same function. METHODS: Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay, western blot, immunofluorescence assay and small interfering RNA (siRNA) were used. RESULTS: Our findings demonstrated that PPV NS1 protein can up-regulate the expression levels of IL-6 and tumor necrosis factor-alpha in a dose-dependent manner. Moreover, PPV NS1 protein was found to induce the phosphorylation of IκBα, then leading to the phosphorylation and nuclear translocation of NF-κB. In addition, the NS1 protein activated the upstream pathways of NF-κB. Meanwhile, TLR2-siRNA assay showed TLR2 plays an important role in the activation of NF-κB signaling pathway induced by PPV-NS1. CONCLUSIONS: These findings indicated that PPV NS1 protein induced the up-regulated of IL-6 expression through activating the TLR2 and NF-κB signaling pathways. In conclusion, these findings provide a new avenue to study the innate immune mechanism of PPV infection.
Assuntos
NF-kappa B/metabolismo , Infecções por Parvoviridae/veterinária , Parvovirus Suíno/fisiologia , Transdução de Sinais , Doenças dos Suínos/genética , Receptor 2 Toll-Like/metabolismo , Proteínas não Estruturais Virais/genética , Animais , Regulação da Expressão Gênica/imunologia , NF-kappa B/genética , Infecções por Parvoviridae/genética , Infecções por Parvoviridae/metabolismo , Parvovirus Suíno/genética , Suínos , Doenças dos Suínos/metabolismo , Receptor 2 Toll-Like/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Porcine parvovirus (PPV) is one of the most common and important virus causes of infectious infertility in swine throughout the world. Inactivated PPV vaccine is broadly used, however, there is no appropriate immunomodulatory adjuvant for enhancing present vaccines and developing new ones. Therefore, in this study, the water-soluble N-2-Hydroxypropyl trimethyl ammonium chloride chitosan (N-2-HACC) was synthesized, the adjuvant potential of chitosan derivative was evaluated in inactivated PPV vaccine. Twenty adult healthy sows were assigned to four groups and vaccinated with synthesized PPV/N-2-HACC, commercial inactivated vaccine, N-2-HACC adjuvant and PBS. After insemination, all sows were challenged with the homologous PPV-H strain. In vivo immunization showed that sows immunized with the PPV/N-2-HACC induced more long-lasting HI antibodies and strong immune responses. Importantly, immunization of PPV/N-2-HACC significantly protected sows from homologous PPV-H strain infection. However, immunization of PPV/N-2-HACC didn't change the level of IL-2, IL-4 and IFN-γ and the production of CD4+, CD8 + T lymphocyte. The results indicated that PPV/N-2-HACC protect PPV infection mainly through enhancing the humoral immunity rather than cellular immunity. In addition, the mummified fetuses were observed from the control groups, but neither of the two vaccine groups. In conclusion, these results suggest that N-2-HACC can be exploited as an effective adjuvant for vaccine development, and the PPV/N-2-HACC are potent immunization candidates against PPV infection.
Assuntos
Quitosana/análogos & derivados , Retardo do Crescimento Fetal/imunologia , Infecções por Parvoviridae/imunologia , Parvovirus Suíno/fisiologia , Complicações Infecciosas na Gravidez/imunologia , Vacinas de Produtos Inativados/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Antivirais/sangue , Quitosana/imunologia , Feminino , Imunidade Humoral , Imunogenicidade da Vacina , Gravidez , Solubilidade , Suínos , Vacinação , ÁguaRESUMO
Propolis from honeybee hives, which is a traditional Chinese medicine, is widely used in veterinary clinics. Many compounds have been identified and isolated from propolis. Ferulic acid (FA), one of the propolis components, previous studies have proven that it has antiviral effects. To study the mechanism of FA antiviral effects, experiments such as immunofluorescence, quantitative real-time PCR and immunoblotting were introduced. In porcine kidney (PK-15) cells, PPV infection induced the expression of the proapoptotic genes Bid, Bad, Bim and Bak, disrupted mitochondrial membrane potential, promoted mitochondria-mediated, caspase-dependent apoptotic signaling and induced apoptosis. Furthermore, the infected PK-15 cells had increased intracellular reactive oxygen species (ROS) generation. FA treatment, however, reversed these effects and increased cell viability. FA treatment also significantly decreased the PPV-induced expression of Bid, Cyt-c and Apaf-1, suggesting that ROS were involved in the activation of the mitochondria-mediated apoptosis pathway. This in vitro study showed that the antiviral activity of FA was probably associated with inhibiting the replication of PPV by blocking proapoptotic factors such as Bid, Bcl-2 and Mcl-1, and attenuating the mitochondria-mediated response by inhibiting the activation of the Bid-related signaling pathway. Pharmacological inhibitors inhibited PPV-induced apoptosis by blocking Bid, and also suppressed the expression of Caspase family proteins in ppv-induced apoptosis. Taken together, our results suggested that PPV induced PK-15 cell apoptosis via activation of Bid and Bid-related signaling pathways and that the mitochondria act as the mediators of these pathways. FA effectively and extensively attenuated this PPV action, and thus is a potential antiviral agent against PPV.
Assuntos
Antivirais/uso terapêutico , Ácidos Cumáricos/uso terapêutico , Rim/patologia , Infecções por Parvoviridae/tratamento farmacológico , Parvovirus Suíno/fisiologia , Animais , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Células Cultivadas , Ácidos Cumáricos/metabolismo , Medicina Tradicional Chinesa , Própole/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Suínos , Replicação Viral/efeitos dos fármacosRESUMO
Porcine parvovirus (PPV) is considered the main cause of reproductive disorders in pigs, which are summarized under the acronym SMEDI (stillbirth, mummification, embryonic death, and infertility). In this review the biology of the virus and its structure, pathogenic potential and strain variation, as well as the disease induced by the virus, are described. Known aspects of pathogenesis, diagnosis and prevention, particularly by vaccination, are summarized. Furthermore, in recent years 'new' parvoviruses (PPV2 to 7) have been described in pigs. They have been detected in pigs from various parts of the world and their association with clinical signs or disease will be discussed.
Assuntos
Infecções por Parvoviridae/veterinária , Parvovirus Suíno/fisiologia , Doenças dos Suínos/virologia , Animais , Doenças Transmissíveis Emergentes/veterinária , Desenvolvimento de Medicamentos , Genoma Viral , Genômica/métodos , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno , Técnicas de Diagnóstico Molecular , Parvovirus Suíno/classificação , Parvovirus Suíno/ultraestrutura , Filogenia , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/prevenção & controle , Tropismo Viral , Vacinas Virais/imunologiaRESUMO
Endoplasmic reticulum (ER) stress is associated with numerous mammalian diseases, especially viral diseases. Porcine parvovirus (PPV) is the causative agent of reproductive failure in swine. Here, we observed that the PPV infection of porcine kidney 15 and porcine testis cells resulted in the activation of ER stress sensors mediated by protein kinase R-like ER kinase (PERK), but not inositol-requiring enzyme 1 and activating transcription factor 6 (ATF6). ER stress activation obviously blocked PPV replication. Depletion of proteins, such as PERK, eukaryotic initiation factor 2, and ATF4, by small interfering RNA significantly enhanced PPV replication. Moreover, the pro-apoptotic factor C/EBP homologous protein was identified a key factor in the inhibition of PPV replication. These data demonstrate that PPV infection activates ER stress through the PERK signaling pathway and that ER stress inhibits further PPV replication by promoting apoptosis.
Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Infecções por Parvoviridae/virologia , Parvovirus Suíno/fisiologia , Transdução de Sinais , Replicação Viral , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Técnicas de Silenciamento de Genes , Infecções por Parvoviridae/metabolismo , Infecções por Parvoviridae/patologia , Parvovirus Suíno/metabolismo , Suínos , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/genéticaRESUMO
Autophagy plays important roles in the infection and pathogenesis of many viruses, yet the regulatory roles of autophagy in the process of porcine parvovirus (PPV) infection remain unclear. Herein, we show that PPV infection induces autophagy in porcine placental trophoblasts (PTCs). Induction of autophagy by rapamycin (RAPA) inhibited the occurrence of apoptotic cell death, yet promoted viral replication in PPV-infected cells; inhibition of autophagy by 3-MA or ATG5 knockdown increased cellular apoptosis and reduced PPV replication. Interestingly, we found that in the presence of caspase-inhibitor zVAD-fmk, PPV induces non-apoptotic cell death that was characterized by lysosomal damage and associated with autophagy. Induction of complete autophagy flux by RAPA markedly promoted PPV replication compared with incomplete autophagy induced by RAPA plus bafilomycin (RAPA/BAF) in the early phase of PPV infection (24 h.p.i.). Meanwhile, induction of complete autophagy with RAPA increased lysosomal damage and non-apoptotic cell death in the later phase of PPV infection. Therefore, our data suggest that autophagy can enhance PPV replication and promote the occurrence of lysosomal-damage-associated non-apoptotic cell death in PPV-infected porcine placental trophoblasts.
Assuntos
Apoptose , Autofagia , Infecções por Parvoviridae/veterinária , Parvovirus Suíno/fisiologia , Doenças dos Suínos/virologia , Trofoblastos/virologia , Replicação Viral , Animais , Autofagossomos/metabolismo , Biomarcadores , Linhagem Celular , Feminino , Lisossomos/metabolismo , Parvovirus Suíno/ultraestrutura , Placenta/metabolismo , Placenta/virologia , Gravidez , Suínos , Doenças dos Suínos/metabolismo , Trofoblastos/metabolismoRESUMO
Glutaraldehyde (GA), used medically as a disinfectant and as a crosslinker for haemoglobin (Hb)-based oxygen carriers (HBOCs), was investigated for its ability to inactivate viruses during the preparation of these artificial blood substitutes. Porcine parvovirus (PPV; a non-enveloped DNA virus) and porcine pseudorabies virus (PRV; an enveloped DNA virus) were used as the virus indicators. Upon treatment with 0.1 mM GA, the titer of PRV decreased from 9.62 log10 to 2.62 log10 within 0.5 h, whereas that of PPV decreased from 7.00 log10 to 2.30 log10 in 5 h. Following treatment with 1.0 mM GA, the titer of PRV decreased from 11.00 log10 to 1.97 log10 within 0.5 h, whereas that of PPV decreased from 7.50 log10 to 3.43 log10 in 4.5 h. During the polymerization of Hb with GA, the GA concentration decreased to 1.0 and 0.1 mM within 30 and 50 min, respectively, at a GA:Hb molar ratio of 10:1, whereas at a GA:Hb molar ratio of 30:1, GA decreased to those same concentrations in 1.5 and 2.5 h, respectively. This rapid decrease in GA concentration during its polymerization with Hb indicates that GA must be added into the Hb solution in a short time in order to get as high a initial concentration as possible. In this study, the GA can only inactivate PRV effectively, given that a longer time (4.5 h) was required for it to inactivate the PPV titer. This study therefore demonstrates that GA inactivates the enveloped DNA virus only during the preparation of HBOCs.
Assuntos
Glutaral/farmacologia , Hemoglobinas/metabolismo , Herpesvirus Suídeo 1/efeitos dos fármacos , Oxigênio/metabolismo , Parvovirus Suíno/efeitos dos fármacos , Inativação de Vírus/efeitos dos fármacos , Animais , Linhagem Celular , Herpesvirus Suídeo 1/fisiologia , Parvovirus Suíno/fisiologia , Polimerização , SegurançaRESUMO
Porcine parvovirus virus (PPV) is an animal virus that has caused high economic losses for the swine industry worldwide. Previous studies demonstrated that PPV infection induced significant production of interleukin 6 (IL-6) in vitro and in vivo. However, the inflammatory cytokines and specific signaling pathways induced during PPV infection remain largely unknown. In the present study, we analyzed the expression levels of IL-6 in PPV-infected porcine kidney 15 (PK-15) and the results showed that PPV infection induced the increase of IL-6 mRNA expression in a dose-dependent manner. We also detected the expression of toll-like receptor 9 (TLR9) signaling proteins in the mRNA expressing level, when compared with the control group, the TLR9 expression in mRNA level increased at 24h in PK-15 cells after PPV infection and reached the peak level at 48h. In addition, the transcript profile of nuclear factor kappa B (NF-κB) signal pathway relating genes (MyD88, IRAK1, TRAF6, TAK1α, IκBκB and NF-κB) expression levels increased at different times. Furthermore, to verify the IL-6 expression was specific with the TLR9 expression and then by activating the NF-κB signal pathway, TLR9 and NF-κB specific inhibitors were applied during PPV infection, separately, the result indicated that the expression of IL-6 was decreased after inhibitor treatment. Taken together, PPV infection significantly induced IL-6 expression and this induction depended on NF-κB activation and TLR9 signaling pathways in PK-15 cell.
Assuntos
Citocinas/metabolismo , Inflamação/metabolismo , NF-kappa B/metabolismo , Parvovirus Suíno/fisiologia , Receptor Toll-Like 9/metabolismo , Animais , Linhagem Celular , Citocinas/genética , Regulação da Expressão Gênica/imunologia , NF-kappa B/genética , Transdução de Sinais/fisiologia , Suínos , Receptor Toll-Like 9/genética , TranscriptomaRESUMO
The SAT protein (SATp) of porcine parvovirus (PPV) accumulates in the endoplasmic reticulum (ER), and SAT deletion induces the slow-spreading phenotype. The in vitro comparison of the wild-type Kresse strain and its SAT knockout (SAT-) mutant revealed that prolonged cell integrity and late viral release are responsible for the slower spreading of the SAT- virus. During PPV infection, regardless of the presence or absence of SATp, the expression of downstream ER stress response proteins (Xbp1 and CHOP) was induced. However, in the absence of SATp, significant differences in the quantity and the localization of CHOP were detected, suggesting a role of SATp in the induction of irreversible ER stress in infected cells. The involvement of the induction of irreversible ER stress in porcine testis (PT) cell necrosis and viral egress was confirmed by treatment of infected cells by ER stress-inducing chemicals (MG132, dithiothreitol, and thapsigargin), which accelerated the egress and spreading of both the wild-type and the SAT- viruses. UV stress induction had no beneficial effect on PPV infection, underscoring the specificity of ER stress pathways in the process. However, induction of CHOP and its nuclear translocation cannot alone be responsible for the biological effect of SAT, since nuclear CHOP could not complement the lack of SAT in a coexpression experiment.IMPORTANCE SATp is encoded by an alternative open reading frame of the PPV genome. Earlier we showed that SATp of the attenuated PPV NADL-2 strain accumulates in the ER and accelerates virus release and spreading. Our present work revealed that slow spreading is a general feature of SAT- PPVs and is the consequence of prolonged cell integrity. PPV infection induced ER stress in infected cells regardless of the presence of SATp, as demonstrated by the morphological changes of the ER and expression of the stress response proteins Xbp1 and CHOP. However, the presence of SATp made the ER stress more severe and accelerated cell death during infection, as shown by the higher rate of expression of CHOP and alteration of the localization of CHOP. The beneficial effect of irreversible ER stress on PPV spread was confirmed by treatment of infected cells with ER stress-inducing chemicals.
Assuntos
Estresse do Retículo Endoplasmático , Interações Hospedeiro-Patógeno , Parvovirus Suíno/fisiologia , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Liberação de Vírus , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Parvovirus Suíno/genética , Suínos , Proteínas Virais/genética , Fatores de Virulência/genéticaRESUMO
A SYBR Green-based real-time polymerase chain reaction (qPCR) was designed to detect Ungulate copiparvovirus 2, also known as porcine parvovirus 4 (PPV4). The test was applied to search for PPV4 DNAemia in sera from 1- to 4-month-old pigs displaying signs of postweaning multisystemic wasting syndrome (PMWS), as well as in sera from healthy swine at equivalent age and in sera from older healthy animals (>6 months old). High levels of PPV4 DNA were detected in PMWS-affected pigs. The mean viral DNA load in PMWS-affected pigs was 5.2 × 107 copies/mL, whereas in young healthy pigs it was 1.4 × 105 copies/mL (P ≤ 0.001). Although the copy numbers were lower in younger PMWS-affected individuals, this result sheds some light on the possible association between PPV4 viral load detection in this group and the immune impairment caused by PMWS.
Assuntos
Infecções por Parvoviridae/veterinária , Parvovirinae/isolamento & purificação , Síndrome Definhante Multissistêmico de Suínos Desmamados/virologia , Doenças dos Suínos/epidemiologia , Carga Viral/veterinária , Animais , DNA Viral/análise , Infecções por Parvoviridae/epidemiologia , Infecções por Parvoviridae/virologia , Parvovirus Suíno/fisiologia , Prevalência , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Suínos , Doenças dos Suínos/virologiaRESUMO
Pasteurization is regularly used to inactivate viruses for the safety of plasma derivatives. Influence of pasteurization at 60 °C for 10 h on α2-Macroglobulin activity and virus inactivation were studied. With 40% sugar as stabilizers more than 70% α2-Macroglobulin activity was reserved after pasteurization compared with 20% in control. Glucose presented a better activity protection effect than sucrose and maltose. By pasteurization without stabilizer the virus titers of pseudorabies virus, Sindbis virus, porcine parvovirus and encephalomyocarditis virus were reduced more than 5.88 log10, 7.50 log10, 4.88 log10, and 5.63 log10 respectively within 2 h. By pasteurization with 40% glucose vesicular stomatitis virus was inactivated more than 5.88 log10 within 1 h. Only 2.71 log10 reduction was achieved for encephalomyocarditis virus after 10 h. 40% glucose protected α2-M activity and viruses simultaneously from pasteurization. Other viral inactivation methods need to be incorporated to ensure viral safety of this manufacturing process of α2-Macroglobulin.
Assuntos
Proteínas Sanguíneas/metabolismo , Glucose/farmacologia , Temperatura Alta , Pasteurização/métodos , Inativação de Vírus/efeitos dos fármacos , alfa-Macroglobulinas/metabolismo , Animais , Linhagem Celular , Vírus da Encefalomiocardite/fisiologia , Herpesvirus Suídeo 1/fisiologia , Humanos , Parvovirus Suíno/fisiologia , Reprodutibilidade dos Testes , Sindbis virus/fisiologia , Suínos , Fatores de Tempo , Células Vero , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/fisiologiaRESUMO
BACKGROUND: Porcine parvovirus (PPV) infection primarily causes reproductive failure of pregnant swine and results in host cell death. Boars, as an important disseminator, shed PPV to sows via semen. PPV infects and numerously replicates in boar testicle, which results in damage of swine testicle in vivo. Reactive oxygen species (ROS), a mediator of cell apoptosis, play a crucial role in the mitochondria apoptotic pathway. However, whether PPV infection induces ST cells apoptosis and ROS accumulation is still unclear. METHODS: To determine the effects of PPV infection on the apoptosis, we detected morphological changes, DNA ladder, activities of caspases, and expression of PARP in PPV-infected ST cells. Moreover, aiming to investigate the effect of PPV infection on the mitochondrial apoptotic pathway and ROS accumulation, we detected the Δψm, apoptosis-related genes, and ROS. To investigate the role of ROS in the process of PPV-induced apoptosis, the ST cells were infected with PPV and treated with the ROS antioxidants. The ROS level was measured using Reactive Oxygen Species Assay Kit and the Δψm, expression level of Bcl-2, translocation of Bax, and redistribution of mitochondria cytochrome c were tested. RESULTS: In this study, we demonstrated that PPV infection could induce apoptosis that was characterized by morphological changes, DNA fragmentation and activation of caspases. Moreover, PPV infection suppressed Bcl-2 expression, enhanced Bax expression and translocation to mitochondria, decreased the mitochondrial transmembrane potential, and triggered the release of cytochrome c, which caused the subsequent activation of caspase-9 and caspase-3 and initiation of apoptosis. However, during the process of PPV-induced apoptosis, the protein levels of Fas and FasL were not affected. Further studies showed that PPV infection caused ROS accumulation. Inhibition of ROS could reduce mitochondrial transmembrane potential and could significantly block ST cells apoptosis via suppressing Bax translocation, cytochrome c and caspase-3 activation. CONCLUSIONS: All these results suggest that PPV-induced ROS accumulation mediates apoptosis in ST cells, which provided theoretical basis for the molecular pathogenesis of PPV infection.
Assuntos
Apoptose , Mitocôndrias/metabolismo , Parvovirus Suíno/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Animais , Caspase 3/metabolismo , Caspase 9/metabolismo , Células Cultivadas , Masculino , Potencial da Membrana Mitocondrial , Infecções por Parvoviridae/veterinária , Suínos , Doenças dos Suínos/metabolismo , Doenças dos Suínos/virologia , Testículo/citologia , Testículo/metabolismoRESUMO
Porcine parvovirus (PPV) infections can lead to significant losses to the swine industry by causing reproductive failure in pigs. Germacrone has been reported to efficiently suppress the replication of influenza virus. In this report, the antiviral activity of germacrone on PPV in swine testis (ST) cells was investigated. Here, we show for the first time that germacrone protects cells from PPV infection and suppresses the synthesis of viral mRNA and protein. Furthermore, we show that germacrone inhibits PPV replication at an early stage in a dose-dependent manner. These findings suggest that germacrone is a potential candidate for anti-PPV therapy.