RESUMO
Porcine reproductive and respiratory syndrome (PRRS) is a serious global pig industry disease. Understanding the mechanism of viral replication and developing efficient antiviral strategies are necessary for combating with PRRS virus (PRRSV) infection. Recently, nanobody is considered to be a promising antiviral drug, especially for respiratory viruses. The present study evaluated two nanobodies against PRRSV nucleocapsid (N) protein (PRRSV-N-Nb1 and -Nb2) for their anti-PRRSV activity in vitro and in vivo. The results showed that intracellularly expressed PRRSV-N-Nb1 significantly inhibited PRRSV-2 replication in MARC-145 cells (approximately 100%). Then, the PRRSV-N-Nb1 fused with porcine IgG Fc (Nb1-pFc) as a delivering tag was produced and used to determine its effect on PRRSV-2 replication in porcine alveolar macrophages (PAMs) and pigs. The inhibition rate of Nb1-pFc against PRRSV-2 in PAMs could reach >90%, and it can also inhibit viral replication in vivo. Epitope mapping showed that the motif Serine 105 (S105) in PRRSV-2 N protein was the key amino acid binding to PRRSV-N-Nb1, which is also pivotal for the self-interaction of N protein via binding to Arginine 97. Moreover, viral particles were not successfully rescued when the S105 motif was mutated to Alanine (S105A). Attachment, entry, genome replication, release, docking model analysis, and blocking enzyme-linked immunosorbent assay (ELISA) indicated that the binding of PRRSV-N-Nb1 to N protein could block its self-binding, which prevents the viral replication of PRRSV. PRRSV-N-Nb1 may be a promising drug to counter PRRSV-2 infection. We also provided some new insights into the molecular basis of PRRSV N protein self-binding and assembly of viral particles.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) causes serious economic losses to the swine industry worldwide, and there are no highly effective strategies for prevention. Nanobodies are considered a promising novel approach for treating diseases because of their ease of production and low costing. Here, we showed that PRRSV-N-Nb1 against PRRSV-N protein significantly inhibited PRRSV-2 replication in vitro and in vivo. Furthermore, we demonstrated that the motif Serine 105 (S105) in PRRSV-N protein was the key amino acid to interact with PRRSV-N-Nb1 and bond to its motif R97, which is important for the self-binding of N protein. The PRRSV-N-Nb1 could block the self-interaction of N protein following viral assembly. These findings not only provide insights into the molecular basis of PRRSV N protein self-binding as a key factor for viral replication for the first time but also highlight a novel target for the development of anti-PRRSV replication drugs.
Assuntos
Proteínas do Nucleocapsídeo , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Anticorpos de Domínio Único , Animais , Aminoácidos , Linhagem Celular , Proteínas do Nucleocapsídeo/metabolismo , Síndrome Respiratória e Reprodutiva Suína/tratamento farmacológico , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Serina , Anticorpos de Domínio Único/farmacologia , Suínos , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Many proteins of African swine fever virus (ASFV, such as p72, p54, p30, CD2v, K205R) have been successfully expressed and characterized. However, there are few reports on the DP96R protein of ASFV, which is the virulence protein of ASFV and plays an important role in the process of host infection and invasion of ASFV. RESULTS: Firstly, the prokaryotic expression vector of DP96R gene was constructed, the prokaryotic system was used to induce the expression of DP96R protein, and monoclonal antibody was prepared by immunizing mice. Four monoclonal cells of DP96R protein were obtained by three ELISA screening and two sub-cloning; the titer of ascites antibody was up to 1:500,000, and the monoclonal antibody could specifically recognize DP96R protein. Finally, the subtypes of the four strains of monoclonal antibodies were identified and the minimum epitopes recognized by them were determined. CONCLUSION: Monoclonal antibody against ASFV DP96R protein was successfully prepared and identified, which lays a foundation for further exploration of the structure and function of DP96R protein and ASFV diagnostic technology.
Assuntos
Vírus da Febre Suína Africana , Anticorpos Monoclonais , Epitopos , Camundongos Endogâmicos BALB C , Proteínas Virais , Animais , Feminino , Camundongos , Febre Suína Africana/imunologia , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Suínos , Proteínas Virais/imunologiaRESUMO
Porcine reproductive and respiratory syndrome (PRRS) is caused by the PRRS virus (PRRSV), which has brought huge economic losses to the pork industry worldwide since its first discovery in the late 1980s in North America. To date, there are no effective commercial vaccines or therapeutic drugs available for controlling the spread of PRRSV. Due to their unique advantages of high affinity and high specificity, nanobodies (Nbs) have received increasing attention in the process of disease diagnosis and treatment. Trans-activator transcription (TAT) can serve as a vector to carry specific proteins into cells by passing through cell membranes. In our previous study, a specific Nb against the PRRSV nucleocapsid (N) protein was screened using phage display technology. For this study, we developed a novel recombinant protein constituting a TAT-conjugated Nb, which we call TAT-Nb1. The target cell entry efficiency of TAT-Nb1 and its effect on PRRSV infection and replication were then investigated. Our results indicate that TAT delivered Nb1 into Marc-145 cells and porcine alveolar macrophages (PAMs) in a dose- and time-dependent manner. Furthermore, TAT-Nb1 dose-dependently suppressed PRRSV infection and replication, where this antiviral effect was independent of PRRSV strain. Co-immunoprecipitation results revealed that Nb1 efficiently interacted with the N protein of PRRSV. Taken together, the presented results suggest that TAT-Nb1 can effectively suppress PRRSV replication, and it may be considered as a new anti-PRRSV candidate drug.
Assuntos
Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Animais , Suínos , Antivirais/farmacologia , Antivirais/metabolismo , Linhagem Celular , Replicação Viral , Síndrome Respiratória e Reprodutiva Suína/tratamento farmacológico , Proteínas do Nucleocapsídeo , Macrófagos Alveolares/metabolismoRESUMO
Porcine reproductive and respiratory syndrome (PRRS) is a highly infectious disease caused by PRRS virus (PRRSV) that causes great economic losses to the swine industry worldwide. PRRSV has been recognized to modulate the host antiviral interferon (IFN) response and downstream interferon-stimulated gene expression to intercept the antiviral effect of host cells. Guanylate-binding proteins (GBPs) are IFN-inducible GTPases that exert broad antiviral activity against several DNA and RNA viruses, of which GBP1 is considered to play a pivotal role. However, the role of GBP1 in PRRSV replication remains unknown. The present study showed that overexpression of GBP1 notably inhibited PRRSV infection, while the knockdown of endogenous GBP1 promoted PRRSV infection. The K51 and R48 residues of GBP1 were essential for the suppression of PRRSV replication. Furthermore, GBP1 abrogated PRRSV replication by disrupting normal fibrous actin structures, which was indispensable for effective PRRSV replication. By using a co-immunoprecipitation assay, we found that GBP1 interacted with the non-structural protein 4 (nsp4) protein of PRRSV, and this interaction was mapped to the N-terminal globular GTPase domain of GBP1 and amino acids 1-69 of nsp4. PRRSV infection significantly downregulated GBP1 protein expression in Marc-145 cells, and nsp4, a 3C-like serine proteinase, was responsible for GBP1 cleavage, and the cleaved site was located at glutamic acid 338 of GBP1. Additionally, the anti-PRRSV activity of GBP1 was antagonized by nsp4. Taken together, these findings expand our understanding of the sophisticated interaction between PRRSV and host cells, PRRSV pathogenesis and its mechanisms of evading the host immune response.
Assuntos
Cisteína Proteases , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Doenças dos Suínos , Animais , Antivirais , Linhagem Celular , Interações Hospedeiro-Patógeno , Interferons , Suínos , Replicação ViralRESUMO
African swine fever (ASF), which is caused by the ASF virus (ASFV), is a highly contagious hemorrhagic disease that causes high mortality to domestic porcine and wild boars and brings huge economic losses to world swine industry. Due to the lack of an effective vaccine, the control of ASF must depend on early, efficient, and cost-effective detection and strict control and elimination strategies. Traditional serological testing methods are generally associated with high testing costs, complex operations, and high technical requirements. As a promising alternative diagnostic tool to traditional antibodies, nanobodies (Nb) have the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity, although the system is dependent on the immunization of Bactrian camels to obtain the specific VHH library of the target protein. The application of Nbs in the detection of ASFV antibodies has not yet been reported yet. Using a phage display technology, one Nb against the ASFV p54 protein that exhibited high specificity and affinity, Nb8, was successfully screened. A HEK293T cell line stably expressing Nb8-horseradish peroxidase (HRP) fusion protein was established using the lentiviral expression system. Following the optimization of the reaction conditions, the Nb8-HRP fusion protein was successfully used to establish a competitive enzyme-linked immunosorbent assay (cELISA) to detect ASFV-specific antibodies in pig serum, for the first time. There was no cross-reaction with healthy pig serum, porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), and classical swine fever virus (CSFV) positive sera. The optimal cut-off value for the cELISA by ROC analysis was 52.5%. A total of 209 serum samples were tested using the developed cELISA and a commercial ELISA kit. The results showed that the relative specificity of the cELISA was 98.97%, and the relative sensitivity of the cELISA was 93.3%, with the percent agreement between the two ELISA methods being 98.56%. In conclusion, a specific, sensitive, and repeatable cELISA was successfully developed based on the Nb8 as a probe, providing a promising method for the detection of anti-ASFV antibodies in clinical pig serum. KEY POINTS: ⢠We successfully screened a specific, high affinity nanobody against ASFV p54 protein. ⢠We establish a method for continuous and stable expression of Nb-HRP fusion protein using a lentiviral packaging system. ⢠We establish a nanobody cELISA detection method that can monitor an ASF infection.
Assuntos
Febre Suína Africana , Vírus da Febre Suína Clássica , Anticorpos Anti-HIV/análise , Anticorpos de Domínio Único , Febre Suína Africana/epidemiologia , Animais , Anticorpos Antivirais , Ensaio de Imunoadsorção Enzimática/métodos , Células HEK293 , Peroxidase do Rábano Silvestre , Humanos , SuínosRESUMO
BACKGROUND: African swine fever virus (ASFV) is a highly contagious hemorrhagic disease and often lethal, which has significant economic consequences for the swine industry. Due to lacking of commercial vaccine, the prevention and control of ASF largely depend on early large-scale detection and screening. So far, the commercial ELISA kits have a long operation time and are expensive, making it difficult to achieve large-scale clinical applications. Nanobodies are single-domain antibodies produced by camelid animals, and have unique advantages such as smaller molecular weight, easy genetic engineering modification and low-costing of mass production, thus exhibiting good application prospects. RESULTS: The present study developed a new method for detection of ASFV specific antibodies using nanobody-horseradish peroxidase (Nb-HRP) fusion proteins as probe. By using camel immunization, phage library construction and phage display technology, five nanobodies against K205R protein were screened. Then, Nb-HRP fusion proteins were produced using genetic modification technology. Based on the Nb-HRP fusion protein as specific antibodies against K205R protein, a new type of cELISA was established to detect ASFV antibodies in pig serum. The cut-off value of the cELISA was 34.8%, and its sensitivity, specificity, and reproducibility were good. Furthermore, the developed cELISA exhibited 99.3% agreement rate with the commercial available ELISA kit (kappa value = 0.98). CONCLUSIONS: The developed cELISA method has the advantages of simple operation, rapid and low-costing, and can be used for monitoring of ASFV infection in pigs, thus providing a new method for the prevention and control of ASF.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Anticorpos de Domínio Único , Doenças dos Suínos , Vírus da Febre Suína Africana/genética , Animais , Camelus , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Peroxidase do Rábano Silvestre , Indicadores e Reagentes , Reprodutibilidade dos Testes , SuínosRESUMO
BACKGROUND: The prevalence of viral infectious diseases has become a serious threat to public safety, economic and social development. Vaccines have been served as the most effective platform to prevent virus transmission via the activation of host immune responses, while the low immunogenicity or safety, the high cost of production, storage, transport limit their effective clinical application. Therefore, there is a need to develop a promising strategy to improve the immunogenicity and safety of vaccines. METHODS: We developed a splenic-targeting biomimetic nanovaccine (NV) that can boost protective humoral and cellular immunity against african swine fever virus (ASFV) infection. The universal PLGA nanoparticles (CMR-PLGA/p54 NPs) coated with mannose and CpG (TLR9 agonist) co-modified red blood cell (RBC) membrane were prepared, which comprised a viral antigen (p54) and can be served as a versatile nanovaccine for elevating protective immunity. RESULTS: CMR-PLGA/p54 NVs could be effectively uptaken by BMDC and promoted BMDC maturation in vitro. After subcutaneous immunization, antigen could be effectively delivered to the splenic dendritic cells (DCs) due to the splenic homing ability of RBC and DC targeting capacity of mannose, which promoted antigen presentation and DCs maturation, and further elicited higher levels of cytokines secretion and specific IgG titers, CD4+ and CD8+ T cells activation and B maturation. Moreover, NVs demonstrated notable safety during the immunization period. CONCLUSIONS: This study demonstrates the high potential of CMR-PLGA NPs as vaccine delivery carriers to promote humoral and cellular immune responses, and it provides a promising strategy to develop safe and effective vaccines against viral infectious diseases.
Assuntos
Vírus da Febre Suína Africana , Viroses , Animais , Suínos , Biomimética , Manose , Linfócitos T CD8-PositivosRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) infection eliminates production of type I interferons (IFNs) in host cells, which triggers an antiviral immune response through the induction of downstream IFN-stimulated genes (ISGs), thus escaping the fate of host-mediated clearance. The IFN-induced transmembrane 3 (IFITM3) has recently been identified as an ISG and plays a pivotal role against enveloped RNA viruses by restricting cell entry. However, the role of IFITM3 in PRRSV replication is unknown. The present study demonstrated that overexpression of IFITM3 suppresses PRRSV replication, while silencing of endogenous IFITM3 prominently promoted PRRSV replication. Additionally, it was shown that IFITM3 undergoes S-palmitoylation and ubiquitination modification, and both posttranslational modifications contribute to the anti-PRRSV activity of IFITM3. Further study showed that PRRSV particles are transported into endosomes and then into lysosomes during the early stages of infection, and confocal microscopy results revealed that PRRSV particles are transported to IFITM3-positive cellular vesicles. By using a single virus particle fluorescent labeling technique, we confirmed that IFITM3 can restrict PRRSV membrane fusion by inducing accumulation of cholesterol in cellular vesicles. Additionally, we found that both endogenous and exogenous IFITM3 are incorporated into newly producing PRRS virions and diminish viral intrinsic infectivity. By using cell coculture systems, we found that IFITM3 effectively restricted PRRSV intercellular transmission, which may have been caused by disrupted membrane fusion and reduced viral infectivity. In conclusion, our results demonstrate, for the first time, that swine IFITM3 interferes with the life cycle of PRRSV, and possibly other enveloped arteritis viruses, at multiple steps.IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS), which is caused by PRRS virus (PRRSV), is of great economic significance to the swine industry. Due to the complicated immune escape mechanisms of PRRSV, there are no effective vaccines or therapeutic drugs currently available against PRRS. Identification of cellular factors and underlying mechanisms that establish an effective antiviral state against PRRSV can provide unique strategies for developing antiviral vaccines or drugs. As an interferon (IFN)-stimulated gene, the role of IFN-induced transmembrane 3 (IFITM3) in PRRSV infection has not been reported as of yet. In the present study, it was shown that IFITM3 can exert a potent anti-PRRSV effect, and PRRS virions are trafficked to IFITM3-containing cell vesicles, where viral membrane fusion is impaired by cholesterol accumulation that is induced by IFITM3. Additionally, both endogenous and exogenous IFITM3 are incorporated into newly assembled progeny virions, and this decreased their intrinsic infectivity.
Assuntos
Interferon Tipo I/metabolismo , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Proteínas Virais/metabolismo , Internalização do Vírus , Animais , Linhagem Celular , Colesterol/metabolismo , Endossomos/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Suínos , Vírion/metabolismo , Montagem de Vírus , Replicação ViralRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the pork industry worldwide each year. Our previous research demonstrated that heme oxygenase-1 (HO-1) can suppress PRRSV replication via an unknown molecular mechanism. In this study, inhibition of PRRSV replication was demonstrated to be mediated by carbon monoxide (CO), a downstream metabolite of HO-1. Using several approaches, we demonstrate that CO significantly inhibited PRRSV replication in both a PRRSV permissive cell line, MARC-145, and the predominant cell type targeted during in vivo PRRSV infection, porcine alveolar macrophages (PAMs). Our results showed that CO inhibited intercellular spread of PRRSV; however, it did not affect PRRSV entry into host cells. Furthermore, CO was found to suppress PRRSV replication via the activation of the cyclic GMP/protein kinase G (cGMP/PKG) signaling pathway. CO significantly inhibits PRRSV-induced NF-κB activation, a required step for PRRSV replication. Moreover, CO significantly reduced PRRSV-induced proinflammatory cytokine mRNA levels. In conclusion, the present study demonstrates that CO exerts its anti-PRRSV effect by activating the cellular cGMP/PKG signaling pathway and by negatively regulating cellular NF-κB signaling. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of PRRSV replication but also suggest potential new control measures for future PRRSV outbreaks. IMPORTANCE: PRRSV causes great economic losses each year to the swine industry worldwide. Carbon monoxide (CO), a metabolite of HO-1, has been shown to have antimicrobial and antiviral activities in infected cells. Our previous research demonstrated that HO-1 can suppress PRRSV replication. Here we show that endogenous CO produced through HO-1 catalysis mediates the antiviral effect of HO-1. CO inhibits PRRSV replication by activating the cellular cGMP/PKG signaling pathway and by negatively regulating cellular NF-κB signaling. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of PRRSV replication but also suggest potential new control measures for future PRRSV outbreaks.
Assuntos
Antivirais/farmacologia , Monóxido de Carbono/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/genética , Heme Oxigenase-1/genética , Macrófagos Alveolares/efeitos dos fármacos , NF-kappa B/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , Animais , Antivirais/metabolismo , Monóxido de Carbono/metabolismo , Linhagem Celular , Chlorocebus aethiops , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Regulação da Expressão Gênica , Heme Oxigenase-1/metabolismo , Interações Hospedeiro-Patógeno , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virologia , NF-kappa B/metabolismo , Compostos Organometálicos/metabolismo , Compostos Organometálicos/farmacologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Pirazinas/farmacologia , Pirróis/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Suínos , Internalização do Vírus , Replicação Viral/efeitos dos fármacosRESUMO
Bovine viral diarrhoea virus (BVDV) causes significant economic losses to the cattle industry worldwide. Previously, we demonstrated that heme oxygenase-1 (HO-1) can inhibit BVDV replication via an unknown molecular mechanism. To elucidate the mechanism involved, we assess whether the HO-1 downstream metabolites carbon monoxide (CO), biliverdin (BV) and iron affect BVDV replication. We treated Madin-Darby bovine kidney (MDBK) cells with an exogenous CO donor, CORM-2. We found that CORM-2 but not its inactive form (iCORM-2) inhibited BVDV replication in a dose-dependent and time duration-dependent manner, suggesting a CO-specific mediation of the CORM-2 antiviral effect. Direct incubation of BVDV with high-dose CORM-2 reduced virus titres, suggesting that CORM-2 attenuates BVDV growth by both physically inactivating virus particles in the extracellular environment and affecting intracellular BVDV replication, but mainly via an intracellular mechanism. Exogenous BV treatment, both post-infection and co-incubation with BVDV, inhibited BVDV replication in a dose-dependent manner, indicating that BV has potent antiviral activity against BVDV. Direct incubation of BVDV with BV had no significant effect on virus titres, indicating that BV is not virucidal and attenuates BVDV growth by affecting intracellular BVDV replication. Furthermore, BV was found to affect BVDV penetration but not attachment. However, increased iron via addition of FeCl3 did not interfere with BVDV replication. Collectively, the results of the present study demonstrate that the HO-1 metabolites BV and CO, but not iron, inhibit BVDV replication. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of BVDV replication but also suggest potential new control measures for future BVDV infection.
Assuntos
Antivirais/farmacologia , Biliverdina/farmacologia , Monóxido de Carbono/farmacologia , Vírus da Diarreia Viral Bovina/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos , Linhagem Celular , Cloretos/farmacologia , Vírus da Diarreia Viral Bovina/crescimento & desenvolvimento , Vírus da Diarreia Viral Bovina/metabolismo , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Compostos Férricos/farmacologia , Heme Oxigenase-1/metabolismo , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Internalização do Vírus/efeitos dos fármacosRESUMO
BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) could lead to pandemic diseases and huge financial losses to the swine industry worldwide. Curcumin, a natural compound, has been reported to serve as an entry inhibitor of hepatitis C virus, chikungunya virus and vesicular stomatitis virus. In this study, we investigated the potential effect of curcumin on early stages of PRRSV infection. RESULTS: Curcumin inhibited infection of Marc-145 cells and porcine alveolar macrophages (PAMs) by four different genotype 2 PRRSV strains, but had no effect on the levels of major PRRSV receptor proteins on Marc-145 cells and PAMs or on PRRSV binding to Marc-145 cells. However, curcumin did block two steps of the PRRSV infection process: virus internalization and virus-mediated cell fusion. CONCLUSIONS: Our results suggested that an inhibition of genotype 2 PRRSV infection by curcumin is virus strain-independent, and mainly inhibited by virus internalization and cell fusion mediated by virus. Collectively, these results demonstrate that curcumin holds promise as a new anti-PRRSV drug.
Assuntos
Antivirais/farmacologia , Curcumina/farmacologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Linhagem Celular , Curcumina/uso terapêutico , Genótipo , Macrófagos Alveolares , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Síndrome Respiratória e Reprodutiva Suína/tratamento farmacológico , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Receptores Virais/metabolismo , Suínos , Internalização do Vírus/efeitos dos fármacosRESUMO
UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important viruses affecting the swine industry worldwide. Our previous research showed that PRRSV downregulates the expression of heme oxygenase-1 (HO-1), a pivotal cytoprotective enzyme, postinfection and that overexpression of HO-1 inhibits PRRSV replication. MicroRNAs regulate gene expression at the posttranscriptional level and have recently been demonstrated to play vital roles in pathogen-host interactions. The present study sought to determine whether microRNAs modulate HO-1 expression and, by doing so, regulate PRRSV replication. Using bioinformatic prediction and experimental verification, we demonstrate that HO-1 expression is regulated by miR-24-3p. A direct interaction between miR-24-3p and HO-1 mRNA was confirmed using a number of approaches. Overexpression of miR-24-3p significantly decreased HO-1 mRNA and protein levels. PRRSV infection induced miR-24-3p expression to facilitate viral replication. The suppressive effect of HO-1 induction by protoporphyrin IX cobalt chloride (CoPP; a classical inducer of HO-1 expression) on PRRSV replication in MARC-145 cells and primary porcine alveolar macrophages could also be reversed by overexpression of miR-24-3p. Collectively, these results suggested that miR-24-3p promotes PRRSV replication through suppression of HO-1 expression, which not only provides new insights into virus-host interactions during PRRSV infection but also suggests potential new antiviral strategies against PRRSV infection. IMPORTANCE: MicroRNAs (miRNAs) play vital roles in viral infections by regulating the expression of viral or host genes at the posttranscriptional level. Heme oxygenase-1 (HO-1), a pivotal cytoprotective enzyme, has antiviral activity for a number of viruses, such as Ebola virus, hepatitis C virus, human immunodeficiency virus, and our focus, PRRSV, which causes great economic losses each year in the swine industry worldwide. Here, we show that PRRSV infection induces host miRNA miR-24-3p expression and that miR-24-3p regulates HO-1 expression through both mRNA degradation and translation repression. Suppression of HO-1 expression by miR-24-3p facilitates PRRSV replication. This work lends credibility to the hypothesis that an arterivirus can manipulate cellular miRNAs to enhance virus replication by regulating antiviral responses following viral infection. Therefore, our findings provide new insights into the pathogenesis of PRRSV.
Assuntos
Regulação da Expressão Gênica/genética , Heme Oxigenase-1/metabolismo , Interações Hospedeiro-Patógeno/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Replicação Viral/fisiologia , Análise de Variância , Animais , Western Blotting , Linhagem Celular , Chlorocebus aethiops , Biologia Computacional , Primers do DNA/genética , Citometria de Fluxo , Imunoprecipitação , Luciferases , Macaca mulatta , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Sus scrofa , Replicação Viral/genéticaRESUMO
The outbreak of African Swine Fever (ASF) has caused huge economic losses to the pig industry. There are no safe and effective vaccines or diagnostics available. The p30 protein serves as a key target for the detection of ASFV antibodies and is an essential antigenic protein for early serological diagnosis. Here, the p30 protein was purified after being expressed in E. coli and its immunogenicity was verified in sera from pigs naturally infected with ASFV. Furthermore, a monoclonal antibody (McAb) designated as McAb 1B4G2-4 (subtype IgG1/kappa-type) was produced and it was verified to specifically recognize the ASFV Pig/HLJ/2018/strain and eukaryotic recombinant ASFV p30 protein. The epitope identified by McAb 1B4G2-4, defining the unique B-cell epitope 164HNFIQTI170, was located using peptide scanning. Comparing amino acid (aa) sequence revealed that this epitope is conserved in all reference ASFV strains from different regions of China, including the highly pathogenic strain Georgia 2007/1 (NC_044959.2) that is widely distributed. It is also exposed to the surface of the p30 protein, suggesting that it could be an important B-cell epitope. Our study may serve as a basis for the development of serological diagnostic methods and subunit vaccines.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Suínos , Animais , Vírus da Febre Suína Africana/genética , Epitopos de Linfócito B/genética , Proteínas Virais/metabolismo , Anticorpos Monoclonais , Escherichia coli/metabolismo , Proteínas Recombinantes , Anticorpos AntiviraisRESUMO
The African swine fever virus (ASFV) continues to pose significant economic and pandemic risks. Consequently, discovering new, efficient vaccines is crucial. Messenger RNA (mRNA) vaccines have emerged as promising candidates, providing minimal risk of insertional mutagenesis, high safety profiles, effectiveness, rapid scalability in production, and cost-effectiveness. In this study, we have developed an ASF p30 mRNA vaccine candidate (mRNA/Man-LNP) employing mannose-modified lipid nanoparticles (LNPs). The mRNA/Man-LNP exhibited effective antigen presentation and facilitated dendritic cells (DCs) maturation. Notably, it elicited strong IgG titers and activated CD4+ and CD8+ T-cells in immunized mice, all while adhering to stringent biosafety standards. This investigation demonstrates that mRNA/Man-LNP can trigger both humoral and cellular immune responses, suggesting its potential as a potent and promising vaccine candidate for controlling African swine fever (ASF).
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Manose , Nanopartículas , Vacinas Virais , Animais , Nanopartículas/química , Vírus da Febre Suína Africana/imunologia , Vírus da Febre Suína Africana/genética , Febre Suína Africana/prevenção & controle , Febre Suína Africana/imunologia , Camundongos , Vacinas Virais/imunologia , Suínos , Manose/química , Células Dendríticas/imunologia , Lipídeos/química , Desenvolvimento de Vacinas , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Vacinas de mRNA , Feminino , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , LipossomosRESUMO
African swine fever virus (ASFV) poses a serious threat to domestic pigs and wild boars, which is responsible for substantial production and economic losses. A dominant ASFV specific linear B cell epitope that reacted with the convalescent serum was explored and identified with the help of immune informatics techniques. It is essential in understanding the host immunity and in developing diagnostic technical guidelines and vaccine design. The confirmation of dominant epitopes with a positive serological matrix is feasible. To improve the immunogenicity of the epitope, we designed the dominant epitope of CD2v in the form of 2 branch Multiple-Antigen peptide (MAPs-2), CD2v-MAPs-2. Notably, CD2v peptide can be taken up by dendritic cells (DCs) to activate T lymphocytes and induce highly effective valence antibodies in BALB/c mice. The specific CD8+ T cell response were observed. The dominant epitope peptide identified in this study was able to effectively activate humoral and cellular immunity in mice model.
Assuntos
Vírus da Febre Suína Africana , Camundongos , Suínos , Animais , Epitopos de Linfócito B , Proteínas Virais/metabolismo , Sus scrofa/metabolismoRESUMO
The African swine fever virus (ASFV) is a highly infectious viral pathogen that presents a major threat to the global pig industry. No effective vaccine is available for the virus. The p54 protein, a major structural component of ASFV, is involved in virus adsorption and entry to target cells and also plays a key role in ASFV vaccine development and disease prevention. Here, we generated species-specific monoclonal antibodies (mAbs), namely 7G10A7F7, 6E8G8E1, 6C3A6D12, and 8D10C12C8 (subtype IgG1/kappa type), against the ASFV p54 protein and characterized the specificity of these mAbs. Peptide scanning techniques were used to determine the epitopes that are recognized by the mAbs, which defined a new B-cell epitope, TMSAIENLR. Amino acid sequence comparison showed that this epitope is conserved among all reference ASFV strains from different regions of China, including the widely prevalent, highly pathogenic strain Georgia 2007/1 (NC_044959.2). This study reveals important signposts for the design and development of ASFV vaccines and also provides critical information for the functional studies of the p54 protein via deletion analysis.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Suínos , Animais , Epitopos de Linfócito B , Anticorpos Monoclonais , Proteínas ViraisRESUMO
The African swine fever virus (ASFV) causes high mortality in domestic pigs. ASFV encodes an important protein target for subunit vaccine development, CD2v, but its most effective immunological regions are not known. Herein, we generated a monoclonal antibody (mAb) named IF3 by immunizing mice against the intracellular region of the CD2v protein (CD2v-IR). 1F3 specifically recognized CD2v, which is expressed transiently in transfected Sf9 cells and also in inactivated ASFV-infected porcine alveolar macrophage (PAM) cells. The epitope recognized by 1F3 is 264EPSPREP270, which is highly conserved in ASFV genotypes. Immunization of mice with this epitope elicited an increased IgG response, including IgG1 and IgG2a subtypes, and also increased CD8+ T cells and cytokine expression. Overall, these results indicate that this epitope induces both humoral and cellular immune responses that may be used for ASFV-related subunit vaccine design and development.
RESUMO
African swine fever (ASF) is an acute, virulent, and highly fatal infectious disease caused by the African swine fever virus (ASFV). There is no effective vaccine or diagnostic method to prevent and control this disease currently, which highlights the significance of ASF early detection. In this study, we chose an early antigen and a late-expressed antigen to co-detect the target antibody, which not only helps in early detection but also improves accuracy and sensitivity. CP204L and B602L were successfully expressed as soluble proteins in an Escherichia coli vector system. By optimizing various conditions, a dual-antigen indirect ELISA for ASFV antibodies was established. The assay was non-cross-reactive with antibodies against the porcine reproductive and respiratory syndrome virus, classical swine fever virus, porcine circovirus type 2, and pseudorabies virus. The maximum serum dilution for detection of ASFV-positive sera was 1:1600. The intra-batch reproducibility coefficient of variation was <5% and the inter-batch reproducibility coefficient of variation was <10%. Compared with commercial kits, the dual-antigen indirect ELISA had good detection performance. In conclusion, we established a detection method with low cost, streamlined production process, and fewer instruments. It provides a new method for the serological diagnosis of ASF.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Animais , Suínos , Febre Suína Africana/diagnóstico , Reprodutibilidade dos Testes , Anticorpos , Ensaio de Imunoadsorção Enzimática , Escherichia coliRESUMO
African Swine Fever (ASF) is an acute and highly lethal disease in pigs caused by African Swine Fever Virus (ASFV). Viral proteins have been commonly used as antigenic targets for the development of ASF diagnostic methods. However, the prokaryotic expression of viral proteins has deficiencies such as instability, insolubility, and high cost in eukaryotic situations. This study screened and verified ASFV-encoded p72, p54, and p30 protein antigenic epitopes. Subsequently, a novel antigenic epitope-associated recombinant protein was designed based on an ideal structural protein and expressed in Escherichia coli (E. coli). Western blot analysis indicated that the recombinant protein could specifically react with the monoclonal antibody (mAb) of p72 and polyclonal antibodies of p54 and p30, respectively. Next, an ASF indirect ELISA (iELISA) method was established based on the recombinant protein, which has no specific reaction with sera of other important pig viral diseases. Meanwhile, it shows a sensitivity to detecting dilutions of ASF-positive reference serum up to 1:6400. The clinical sample detection results showed a high coincidence rate of 98 % with a commercial competition ELISA kit. In conclusion, we established a novel specific, and sensitive ASF serologic detection method that opens new avenues for ASF serodiagnostic method development.