RESUMEN
The Porcine reproductive and respiratory syndrome (PRRS) causes severe financial losses to the global swine industry. Due to continuous virus evolution, the protection against the PRRS provided by current vaccines is limited. In order to find new antiviral strategies, this study investigated the antiviral potential of antimicrobial peptides (AMPs) against PRRSV. Given the diversity of PRRSV strains and the limited effectiveness of existing vaccines in controlling PRRSV, this study evaluated the inhibitory effects of KLAK, Cecropin B, Piscidin1, and Caerin1.1 on 3 strains of PRRSV (lineage 5 classical strain, lineage 8 highly pathogenic strain, and lineage 1 NADC30-like strain). Caerin1.1 exhibited significant dose-dependent antiviral activity, with an effective concentration (EC50) of 7.5⯵M. Caerin1.1 effectively inhibited PRRSV replication when added before or in early infection but showed reduced effectiveness when added in late infection, indicating its potential involvement in targeting early transcription mechanisms of viral RNA polymerase and significantly upregulating cytokine gene expression. In the NADC30 strain-based animal infection model, Caerin1.1 treatment significantly reduced lung viral loads and inflammation in the lungs of PRRSV-infected pigs, with a mortality rate of 0â¯% (0/5) in the treated group compared to 66.67â¯% (4/6) in the untreated group, indicating a reduction in the mortality rate. Additionally, compared with the untreated group, the Caerin1.1-treated group showed significant improvements, such as lighter fever, more daily weight gain, less clinical symptoms, less viral load in blood, and less virus oral shedding (P < 0.05). These findings reveal the potential of antimicrobial peptides as PRRSV therapeutic agents and suggest that Caerin1.1 is a promising candidate for a novel anti-PRRSV drug.
Asunto(s)
Péptidos Antimicrobianos , Antivirales , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Carga Viral , Replicación Viral , Animales , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Porcinos , Síndrome Respiratorio y de la Reproducción Porcina/tratamiento farmacológico , Síndrome Respiratorio y de la Reproducción Porcina/virología , Antivirales/farmacología , Replicación Viral/efectos de los fármacos , Péptidos Antimicrobianos/farmacología , Carga Viral/efectos de los fármacos , Pulmón/virología , Pulmón/efectos de los fármacos , Péptidos Catiónicos Antimicrobianos/farmacología , Línea CelularRESUMEN
Porcine reproductive and respiratory syndrome (PRRS) is endemic worldwide, seriously affecting the development of the pig industry, but vaccines have limited protective effects against PRRSV transmission. The aim of this study was to identify potential anti-PRRSV drugs. We examined the cytotoxicity of seven compounds formulated based on the mass ratio of glycyrrhizic acid to matrine and calculated their inhibition rates against PRRSV in vitro. The results showed that the seven compounds all had direct killing and therapeutic effects on PRRSV, and the compounds inhibited PRRSV replication in a time- and dose-dependent manner. The compound with the strongest anti-PRRSV effect was selected for subsequent in vivo experiments. Pigs were divided into a control group and a medication group for the in vivo evaluation. The results showed that pigs treated with the 4:1 compound had 100% morbidity after PRRSV challenge, and the mortality rate reached 75% on the 8th day of the virus challenge. These results suggest that this compound has no practical anti-PRRSV effect in vivo and can actually accelerate the death of infected pigs. Next, we further analyzed the pigs that exhibited semiprotective effects following vaccination with the compound to determine whether the compound can synergize with the vaccine in vivo. The results indicated that pigs treated with the compound had higher mortality rates and more severe clinical reactions after PRRSV infection (p < 0.05). The levels of proinflammatory cytokines (IL-6, IL-8, IL-1ß, IFN-γ, and TNF-α) were significantly greater in the compound-treated pigs than in the positive control-treated pigs (p < 0.05), and there was no synergistic enhancement with the live attenuated PRRSV vaccine (p < 0.05). The compound enhanced the inflammatory response, prompted the body to produce excessive levels of inflammatory cytokines and caused body damage, preventing a therapeutic effect. In conclusion, the present study revealed that the in vitro effectiveness of these agents does not indicate that they are effective in vivo or useful for developing anti-PRRSV drugs. Our findings also showed that, to identify effective anti-PRRSV drugs, comprehensive drug screening is needed, for compounds with solid anti-inflammatory effects both in vitro and in vivo. Our study may aid in the development of new anti-PRRSV drugs.
Asunto(s)
Alcaloides , Antivirales , Ácido Glicirrínico , Matrinas , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Quinolizinas , Replicación Viral , Animales , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Alcaloides/farmacología , Quinolizinas/farmacología , Quinolizinas/uso terapéutico , Porcinos , Antivirales/farmacología , Antivirales/uso terapéutico , Ácido Glicirrínico/farmacología , Ácido Glicirrínico/uso terapéutico , Síndrome Respiratorio y de la Reproducción Porcina/tratamiento farmacológico , Síndrome Respiratorio y de la Reproducción Porcina/virología , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Replicación Viral/efectos de los fármacos , Citocinas/metabolismo , Análisis de SupervivenciaRESUMEN
BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a prevalent swine pathogen, which has caused adverse impact on the global swine industry for almost 30 years. However, due to the immune suppression caused by the virus and the genetic diversity in PRRSV, no virus-targeting broad neutralizing strategy has been successfully developed yet. Antiviral peptide and nanobody have attracted extensive attention with the ease in production and the efficacy in practice. In this study, four new fusion proteins named nanobody peptide conjugates (NPCs) were developed by combining PRRSV specific non-neutralizing nanobodies with CD163-derived peptides targeting the receptor binding domain (RBD) of PRRSV proteins. RESULTS: Four NPCs were successfully constructed using two nanobodies against PRRSV N and nsp9 individually, recombining with two antiviral peptides 4H7 or 8H2 from porcine CD163 respectively. All four NPCs demonstrated specific capability of binding to PRRSV and broad inhibitory effect against various lineages of PRRSV in a dose-dependent manner. NPCs interfere with the binding of the RBD of PRRSV proteins to CD163 in the PRRSV pre-attachment stage by CD163 epitope peptides in the assistance of Nb components. NPCs also suppress viral replication during the stage of post-attachment, and the inhibitory effects depend on the antiviral functions of Nb parts in NPCs, including the interference in long viral RNA synthesis, NF-κB and IFN-ß activation. Moreover, an interaction was predicted between aa K31 and T32 sites of neutralizing domain 4H7 of NPC-N/nsp9-4H7 and the motif 171NLRLTG176 of PRRSV GP2a. The motif 28SSS30 of neutralizing domain 8H2 of NPC-N/nsp9-8H2 could also form hydrogens to bind with the motif 152NAFLP156 of PRRSV GP3. The study provides valuable insights into the structural characteristics and potential functional implications of the RBD of PRRSV proteins. Finally, as indicated in a mouse model, NPC intranasally inoculated in vivo for 12-24 h sustains the significant neutralizing activity against PRRSV. These findings inspire the potential of NPC as a preventive measure to reduce the transmission risk in the host population against respiratory infectious agents like PRRSV. CONCLUSION: The aim of the current study was to develop a peptide based bioactive compound to neutralize various PRRSV strains. The new antiviral NPC (nanobody peptide conjugate) consists of a specific nanobody targeting the viral protein and a neutralizing CD163 epitope peptide for virus blocking and provides significant antiviral activity. The study will greatly promote the antiviral drug R&D against PRRSV and enlighten a new strategy against other viral diseases.
Asunto(s)
Anticuerpos Neutralizantes , Antígenos CD , Antígenos de Diferenciación Mielomonocítica , Péptidos , Virus del Síndrome Respiratorio y Reproductivo Porcino , Receptores de Superficie Celular , Anticuerpos de Dominio Único , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Animales , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/química , Porcinos , Antígenos de Diferenciación Mielomonocítica/inmunología , Antígenos de Diferenciación Mielomonocítica/metabolismo , Receptores de Superficie Celular/inmunología , Antígenos CD/inmunología , Antígenos CD/metabolismo , Anticuerpos Neutralizantes/inmunología , Péptidos/química , Péptidos/farmacología , Péptidos/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Ratones , Replicación Viral/efectos de los fármacos , Línea CelularRESUMEN
BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes severe inflammatory response, respiratory disease and sow reproductive failure. Quercetin is among the widely occurring polypheno found abundantly in nature. Quercetin has anti-inflammatory, anti-oxidative and anti-viral properties. OBJECTIVES: This study aimed to explore the effect and mechanism of quercetin on PRRSV-induced inflammation in MARC-145 cells. METHODS: Observing the cytopathic effect and measurements of inflammatory markers in MARC-145 cells collectively demonstrate that quercetin elicits a curative effect on PRRSV-induced inflammation. Liquid chromatography-mass spectrometry was further used for a non-targeted metabolic analysis of the role of quercetin in the metabolic regulation of PRRSV inflammation in MARC-145 cells. RESULTS: It was shown that quercetin attenuated PRRSV-induced cytopathy in MARC-145 cells. Quercetin treatment inhibited PRRSV replication in MARC-145 cells in a dose-dependent manner. We also found that quercetin inhibited PRRSV-induced mRNA expression and secretion levels of tumour necrosis factor-α, interleukin 1ß and interleukin 6. Metabolomics analysis revealed that quercetin ameliorated PRRSV-induced inflammation. Pathway analysis results revealed that PRRSV-induced pathways including arachidonic acid metabolism, linoleic acid, glycerophospholipid and alanine, aspartate and glutamate metabolism were suppressed by quercetin. Moreover, we confirmed that quercetin inhibited the activation of NF-κB/p65 pathway, probably by attenuating PLA2, ALOX and COX mRNA expression. CONCLUSIONS: These results provide a crucial insight into the molecular mechanism of quercetin in alleviating PRRSV-induced inflammation.
Asunto(s)
Ácido Araquidónico , Glutamina , Inflamación , Virus del Síndrome Respiratorio y Reproductivo Porcino , Quercetina , Quercetina/farmacología , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Animales , Línea Celular , Inflamación/virología , Inflamación/tratamiento farmacológico , Glutamina/metabolismo , Glutamina/farmacología , Ácido Araquidónico/metabolismo , Porcinos , Síndrome Respiratorio y de la Reproducción Porcina/virología , Síndrome Respiratorio y de la Reproducción Porcina/tratamiento farmacológico , Chlorocebus aethiopsRESUMEN
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) poses a major threat to the global swine industry, yet effective prevention and control measures remain elusive. This study unveils Nitazoxanide (NTZ) as a potent inhibitor of PRRSV both in vitro and in vivo. Through High-Throughput Screening techniques, 16 potential anti-PRRSV compounds are identified from a library comprising FDA-approved and pharmacopeial drugs. We show that NTZ displays strong efficacy in reducing PRRSV proliferation and transmission in a swine model, alleviating viremia and lung damage. Additionally, Tizoxanide (TIZ), the primary metabolite of NTZ, has been identified as a facilitator of NMRAL1 dimerization. This finding potentially sheds light on the underlying mechanism contributing to TIZ's role in augmenting the sensitivity of the IFN-ß pathway. These results indicate the promising potential of NTZ as a repurposed therapeutic agent for Porcine Reproductive and Respiratory Syndrome (PRRS). Additionally, they provide valuable insights into the antiviral mechanisms underlying NTZ's effectiveness.
Asunto(s)
Antivirales , Ensayos Analíticos de Alto Rendimiento , Nitrocompuestos , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Tiazoles , Animales , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Nitrocompuestos/farmacología , Porcinos , Antivirales/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Síndrome Respiratorio y de la Reproducción Porcina/tratamiento farmacológico , Síndrome Respiratorio y de la Reproducción Porcina/virología , Tiazoles/farmacología , Replicación Viral/efectos de los fármacos , Línea Celular , Viremia/tratamiento farmacológico , Viremia/virologíaRESUMEN
Porcine reproductive and respiratory syndrome (PRRS) is a major challenge for the global swine industry, causing huge economic losses worldwide. To date, there are no effective measures to prevent and control the spread of PRRS virus (PRRSV). Baicalin (BA) is a natural flavonoid with various pharmacological effects, including antiviral, anti-inflammatory, antioxidant and immunomodulatory. Here, we demonstrate that BA exhibits potent anti-PRRSV activity in vitro, BA concentrations in the range of 5-20 µg/mL significantly inhibited PRRSV infection in a dose-dependent manner and were independent of PRRSV strain. Mechanistically, BA inhibited PRRSV replication by directly interacting with virions, thereby affecting multiple stages of the virus life cycle. Meanwhile, the preventive effect of BA on PRRSV could be realized by inhibiting CD151 and CD163 expression. Furthermore, BA reduced the PRRSV-induced expression of PAMs cytokines (IFN-α, IL-6, IL-8, and TNF-α), suggesting that BA-induced antiviral cytokines may help BA inhibit PRRSV infection. Taken together, BA can be used as an inhibitor of PRRSV infection in vitro, which provides a theoretical basis for the clinical application of BA and the prevention and control of PRRSV infection, which is worthy of further in vivo studies in swine.
Asunto(s)
Antivirales , Citocinas , Flavonoides , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Replicación Viral , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Animales , Flavonoides/farmacología , Antivirales/farmacología , Porcinos , Replicación Viral/efectos de los fármacos , Citocinas/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virología , Síndrome Respiratorio y de la Reproducción Porcina/tratamiento farmacológico , Antígenos CD/metabolismo , Receptores de Superficie Celular/metabolismo , Línea Celular , Antígenos de Diferenciación MielomonocíticaRESUMEN
BACKGROUND: Traditional Chinese medicine (TCM) has been garnering ever-increasing worldwide attention as the herbal extracts and formulas prove to have potency against disease. Fuzhengjiedu San (FZJDS), has been extensively used to treat viral diseases in pigs, but its bioactive components and therapeutic mechanisms remain unclear. METHODS: In this study, we conducted an integrative approach of network pharmacology and experimental study to elucidate the mechanisms underlying FZJDS's action in treating porcine reproductive and respiratory syndrome virus (PRRSV). We constructed PPI network and screened the core targets according to their degree of value. GO and KEGG enrichment analyses were also carried out to identify relevant pathways. Lastly, qRT-PCR, flow cytometry and western blotting were used to determine the effects of FZJDS on core gene expression in PRRSV-infected monkey kidney (MARC-145) cells to further expand the results of network pharmacological analysis. RESULTS: Network pharmacology data revealed that quercetin, kaempferol, and luteolin were the main active compounds of FZJDS. The phosphatidylinositol-3-kinase (PI3K)/Akt pathway was deemed the cellular target as it has been shown to participate most in PRRSV replication and other PRRSV-related functions. Analysis by qRT-PCR and western blotting demonstrated that FZJDS significantly reduced the expression of P65, JNK, TLR4, N protein, Bax and IĸBa in MARC-145 cells, and increased the expression of Bcl-2, consistent with network pharmacology results. This study provides that FZJDS has significant antiviral activity through its effects on the PI3K/AKT signaling pathway. CONCLUSION: We conclude that FZJDS is a promising candidate herbal formulation for treating PRRSV and deserves further investigation.
Asunto(s)
Medicamentos Herbarios Chinos , Fosfatidilinositol 3-Quinasas , Virus del Síndrome Respiratorio y Reproductivo Porcino , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Línea Celular , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Quempferoles/farmacología , Luteolina/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/tratamiento farmacológico , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quercetina/farmacología , Quercetina/análogos & derivados , Transducción de Señal/efectos de los fármacos , Porcinos , Replicación Viral/efectos de los fármacosRESUMEN
Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), has caused substantial economic losses to the global swine industry due to the lack of effective commercial vaccines and drugs. There is an urgent need to develop alternative strategies for PRRS prevention and control, such as antiviral drugs. In this study, we identified ursonic acid (UNA), a natural pentacyclic triterpenoid from medicinal herbs, as a novel drug with anti-PRRSV activity in vitro. Mechanistically, a time-of-addition assay revealed that UNA inhibited PRRSV replication when it was added before, at the same time as, and after PRRSV infection was induced. Compound target prediction and molecular docking analysis suggested that UNA interacts with the active pocket of PTPN1, which was further confirmed by a target protein interference assay and phosphatase activity assay. Furthermore, UNA inhibited PRRSV replication by targeting PTPN1, which inhibited IFN-ß production. In addition, UNA displayed antiviral activity against porcine epidemic diarrhoea virus (PEDV) and Seneca virus A (SVA) replication in vitro. These findings will be helpful for developing novel prophylactic and therapeutic agents against PRRS and other swine virus infections.
Asunto(s)
Antivirales , Inmunidad Innata , Virus del Síndrome Respiratorio y Reproductivo Porcino , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Triterpenos , Replicación Viral , Animales , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Antivirales/farmacología , Porcinos , Triterpenos/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Plantas Medicinales/química , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/virologíaRESUMEN
The aim of this study was to investigate the in vitro and in vivo antiviral effects of CLEVir-X, against porcine reproductive and respiratory syndrome virus (PRRSV). CLEVir-X is a nucleoside analogue and a dialdehyde form of xanthosine. CLEVir-X demonstrated antiviral action during the in vitro portion of this experiment with its inosine monophosphate dehydrogenase (IMPDH) inhibition against PRRSV. The anti-PRRSV effect of CLEVir-X was recovered through supplementation with guanosine. This suggests that PRRSV replication may be regulated through IMPDH and its guanosine biosynthetic pathway. CLEVir-X treatment in cultures resulted in mutation frequency increase of up to 7.8-fold within the viral genomes (e.g. ORF6) compared to their parallel, untreated cultures. The incorporation of CLEVir-X into the viral genome causes lethal mutagenesis and subsequent decrease in specific infectivity. During the in vivo antiviral experiment, 21-day-old pigs began oral administration of 5 mL of phosphate buffered saline containing CLEVir-X (with purity of 68 % and dosage of 40 mg/kg body weight). This treatment was provided twice daily at 9:00AM and 5:00PM for 14 days. Pigs were simultaneously intranasally inoculated with PRRSV at the beginning of CLEVir-X treatment (21 days of age). Several beneficial effects from the oral administration of CLEVir-X were observed including reduction of body temperature, alleviation of respiratory clinical signs, decreased PRRSV load in both blood and lung tissues, and mitigation of lung interstitial pneumonia lesions. The results of the present study demonstrated that CLEVir-X has mutagenic and nonmutagenic modes of antiviral action against PRRSV based on both in vitro and in vivo antiviral experiments.
Asunto(s)
Antivirales , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Animales , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Porcinos , Antivirales/farmacología , Síndrome Respiratorio y de la Reproducción Porcina/tratamiento farmacológico , Síndrome Respiratorio y de la Reproducción Porcina/virología , Replicación Viral/efectos de los fármacos , Pulmón/virología , Pulmón/efectos de los fármacos , Pulmón/patología , Carga Viral/efectos de los fármacos , Administración OralRESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the pork industry worldwide. Currently, vaccine strategies provide limited protection against PRRSV transmission, and no effective drug is commercially available. Therefore, there is an urgent need to develop novel antiviral strategies to prevent PRRSV pandemics. This study showed that artesunate (AS), one of the antimalarial drugs, potently suppressed PRRSV replication in Marc-145 cells and ex vivo primary porcine alveolar macrophages (PAMs) at micromolar concentrations. Furthermore, we demonstrated that this suppression was closely associated with AS-activated AMPK (energy homeostasis) and Nrf2/HO-1 (inflammation) signaling pathways. AS treatment promoted p-AMPK, Nrf2, and HO-1 expression and, thus, inhibited PRRSV replication in Marc-145 and PAM cells in a time- and dose-dependent manner. These effects of AS were reversed when the AMPK or HO-1 gene was silenced by short interfering RNA. In addition, we demonstrated that AMPK works upstream of Nrf2/HO-1, as its activation by AS is AMPK dependent. Adenosine phosphate analysis showed that AS activates AMPK via improving the AMP/ADP-to-ATP ratio rather than direct interaction with AMPK. Altogether, our findings indicate that AS is a promising novel therapeutic for controlling PRRSV and that its anti-PRRSV mechanism, which involves the functional link between energy homeostasis and inflammation suppression pathways, may provide opportunities for developing novel antiviral agents. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) infections have continuously threatened the pork industry worldwide. Vaccination strategies provide very limited protection against PRRSV infection, and no effective drug is commercially available. We show that artesunate (AS), one of the antimalarial drugs, is a potent inhibitor against PRRSV replication in Marc-145 cells and ex vivo primary porcine alveolar macrophages (PAMs). Furthermore, we demonstrate that AS inhibits PRRSV replication via activation of AMPK-dependent Nrf2/HO-1 signaling pathways, revealing a novel link between energy homeostasis (AMPK) and inflammation suppression (Nrf2/HO-1) during viral infection. Therefore, we believe that AS may be a promising novel therapeutics for controlling PRRSV, and its anti-PRRSV mechanism may provide a strategy to develop novel antiviral agents.
Asunto(s)
Antimaláricos/farmacología , Artesunato/farmacología , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Transducción de Señal/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Antimaláricos/química , Artesunato/química , Línea Celular , Susceptibilidad a Enfermedades , Hemo-Oxigenasa 1/metabolismo , Interacciones Huésped-Patógeno , Modelos Biológicos , Factor 2 Relacionado con NF-E2/metabolismo , PorcinosRESUMEN
Owing to several limitations of porcine reproductive and respiratory syndrome virus (PRRSV) control procedures, the importance of antiviral agents is increasing; however, limited studies have been done on the development of anti-PRRSV agents. Herein, we explored the antiviral effect and mechanism of rottlerin against PRRSV. We demonstrated that treatment of rottlerin at an early stage of PRRSV infection significantly inhibited the viral replication. PRRSV infection induced protein kinase C-δ phosphorylation, which was specifically downregulated by rottlerin. The treatment of rottlerin led to disrupting the PRRSV entry pathway by blocking endocytosis of the virions. Further, to evaluate the anti-PRRSV effect of the rottlerin in vivo, we administrated rottlerin loaded liposome to pigs infected with PRRSV LMY or FL12 strain. The treatment of rottlerin-liposome reduced the blood viral load, interstitial pneumonia and clinical scores compared to untreated pigs. These results provide an evidence of anti-PRRSV effect of rottlerin in vitro via inhibiting PRRSV internalization and in vivo, all of which strongly suggest the applicability of rottlerin as a potential PRRSV prophylactic treatment.
Asunto(s)
Acetofenonas/farmacología , Antivirales/farmacología , Benzopiranos/farmacología , Endocitosis/efectos de los fármacos , Síndrome Respiratorio y de la Reproducción Porcina/tratamiento farmacológico , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Animales , Línea Celular , Enfermedades Pulmonares Intersticiales/prevención & control , Síndrome Respiratorio y de la Reproducción Porcina/patología , Porcinos , Carga Viral/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe reproductive failure in sows and respiratory diseases in growing and finishing pigs and results in great economic losses to the swine industry. Although vaccines are available, PRRSV remains a major threat to the pig farms. Thus, there is an urgent need to develop antiviral drugs to compensate for vaccines. In this study, we report that Aloe extract (Ae) can strongly inhibit PRRSV in Marc-145 cells and porcine alveolar macrophages lines (iPAMs) in vitro. Furthermore, we identified a novel anti-PRRSV molecule, Emodin, from Ae by high-performance liquid chromatography (HPLC). Emodin exerted its inhibitory effect through targeting the whole stages of PRRSV infectious cycle. Moreover, we also found that Emodin can inactivate PRRSV particles directly. Notably, we confirmed that Emodin was able to significantly induce Toll-like receptor 3 (TLR3) (p < 0.01), IFN-α (p < 0.05) and IFN-ß expression in iPAMs, indicating that induction of antiviral agents via TLR3 activation by Emodin might contribute to its anti-PRRSV effect. These findings imply that the Emodin from Aloe could hamper the proliferation of PRRSV in vitro and might constitute a new approach for treating PRRSV infection.
Asunto(s)
Aloe/química , Antivirales/farmacología , Emodina/farmacología , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Receptor Toll-Like 3/genética , Animales , Línea Celular , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/virología , Síndrome Respiratorio y de la Reproducción Porcina , Porcinos , Receptor Toll-Like 3/inmunología , Replicación Viral/efectos de los fármacosRESUMEN
Porcine reproductive and respiratory syndrome (PRRS) is a viral infectious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV) and is devastating the swine industry. MARC-145 cells, an African green monkey kidney cell line, are sensitive to PRRSV-2, and are often used for in vitro studies on PRRSV-2. Preliminary research has shown that glycyrrhizin, an important active component extracted from traditional Chinese medicinal licorice, significantly inhibits the proliferation of PRRSV-2 in MARC-145 cells; however, the in-depth molecular mechanism remains unclear. By determining the cell growth cycle, this study found that PRRSV-2 infection first increased the content of G1-phase MARC-145 cells and then decreased the content of G1-phase cells. Moreover, glycyrrhizin affected the role of PRRSV-2 in regulating the cell cycle. Furthermore, PRRSV-2 had the highest proliferation titer in G0/G1-phase MARC-145 cells, and glycyrrhizin reduced the content of PRRSV-2 in synchronized MARC-145 cells. According to the results of ATPase detection, PRRSV-2 infection weakened the Na+/K+-ATPase and Ca2+/Mg2+-ATPase activities in MARC-145 cells, while glycyrrhizin significantly enhanced their activities in PRRSV-2-infected MARC-145 cells. The above results provide theoretical support toward clarifying the mechanism by which glycyrrhizin inhibits the proliferation of PRRSV-2 in MARC-145 cells. Moreover, these results offer references for the development and use of glycyrrhizin and the clinical treatment of PRRSV-2 infection.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Antivirales/farmacología , Ácido Glicirrínico/farmacología , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Animales , Línea Celular , Chlorocebus aethiops , Riñón , Síndrome Respiratorio y de la Reproducción Porcina/tratamiento farmacológico , Virus del Síndrome Respiratorio y Reproductivo Porcino/enzimología , Virus del Síndrome Respiratorio y Reproductivo Porcino/crecimiento & desarrollo , PorcinosRESUMEN
The objective of the present work was to evaluate the efficacy of a novel antigen carrier using mannosylated gelatin nanoparticles with entrapped inactivated porcine reproductive and respiratory syndrome virus (PRRSV) in inducing T cell mediated immunity in vitro. Gelatin nanoparticles (GNP) were modified with mannose to form mannosylated gelatin nanoparticles (MnGNP), which can efficiently and specifically target monocyte derived dendritic cells (MoDCs). The inactivated PRRSV was encapsulated in the MnGNP and GNP, referred to as MnGNP-PRRSV and GNP-PRRSV, respectively. All these prepared nanometer particles were characterized for size, surface charge, drug encapsulation efficiency, and drug release. The efficacy of MnGNP in targeting MoDCs was investigated, as well as the subsequent MoDCs maturation and T cell mediated cytotoxicity. The developed MnGNP-PRRSV particle was characterized with a nanometric size of 302.67⯱â¯3.2â¯nm, surface charge of 23.81⯱â¯1.26â¯mV, and PRRSV encapsulation efficiency of 63.2⯱â¯1.85 %. The maximum uptake of MnGNP in MoDCs in vitro was 15.5 times higher than GNP with a shorter reaction time that peaked 4â¯h earlier. The uptake of MnGNP-PRRSV induced maturation of MoDCs and significantly enhanced expression of SWC-3a, CD80, CD1, SLA I, SLA II on MoDCs, compared to PRRSV (p < 0.001). The cytokine secretion of IL-1ß, IL-6, IL-10, and IL-12 was also increased in MoDCs when treated with MnGNP-PRRSV, compared to PRRSV (p < 0.05). The matured MoDCs triggered T lymphocytes in autologous peripheral blood mononuclear cells (PBMCs) activation, proliferation, and differentiation into effector cytotoxic T lymphocyte, suggesting increased amount of activated T cells after MnGNP-PRRSV treatment. Additionally, the function of T cells to kill PRRSV infected cells was 83.98⯱â¯2.62 % when triggered by MnGNP-PRRSV, compared to 60⯱â¯4.7 % in PRRSV group (p < 0.001). These results indicate that MnGNP with entrapped inactivated PRRSV can effectively and specifically target dendritic cells for maturation and activation, and subsequently improve T cell activation, proliferation and function to kill PRRSV infected cells.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Gelatina/química , Gelatina/inmunología , Manosa/metabolismo , Nanopartículas/química , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Linfocitos T/inmunología , Animales , Citocinas/genética , Citocinas/inmunología , Células Dendríticas/inmunología , Células Dendríticas/virología , Gelatina/farmacología , Activación de Linfocitos , Manosa/química , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Transducción de Señal , Porcinos , Inactivación de VirusRESUMEN
This study evaluated the immunomodulatory effect of two types of phytochemicals, i.e. rutin and ß-carotene, and two types of vitamins, i.e. α-tocopherol and l-ascorbic acid on improving innate immune responses to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). Monocyte-derived macrophages (MDM) from eight PRRSV-seronegative pigs were inoculated with HP-PRRSV and subsequently stimulated with rutin, ß-carotene, α-tocopherol, and l-ascorbic acid in the absence or presence of either polyinosinic:polycytidylic acid or lipopolysaccharide. The mRNA expression levels of myxovirus resistance 1, interferon regulatory factor 3 (IRF3), IRF7, 2'-5'-oligoadenylatesynthetase 1, stimulator of interferon genes (STING), osteopontin (OPN), interferon alpha (IFNα), IFNß, IFNγ, interleukin-10 (IL-10), tumor necrosis factor alpha (TNFα), and transforming growth factor beta (TGFß) were evaluated by real-time PCR. Compared with control MDM, HP-PRRSV significantly suppressed mRNA expressions of all immune-related genes except IL-10 and TGFß. Compared with HP-PRRSV-inoculated MDM, stimulation with rutin, α-tocopherol, and l-ascorbic acid, but not ß-carotene significantly enhanced mRNA expression levels of IRF3, IRF7, STING, OPN, IFNα, IFNß, and IFNγ in HP-PRRSV-inoculated MDM. Stimulation with rutin also significantly reduced mRNA expression levels of TNFα and TGFß, whereas stimulation with ß-carotene and α-tocopherol significantly reduced TNFα mRNA expression in HP-PRRSV-inoculated MDM. Our findings demonstrate the potentials of rutin, α-tocopherol, and l-ascorbic acid in enhancing type I interferon-regulated genes and type I and II IFN expressions, and in reducing pro- and/or anti-inflammatory cytokine expressions in HP-PRRSV-inoculated MDM. Our findings suggest that rutin, α-tocopherol, and l-ascorbic acid may serve as effective immunomodulators for improving innate immune response to HP-PRRSV.
Asunto(s)
Citocinas/genética , Interferón Tipo I/genética , Interferón beta/genética , Macrófagos/efectos de los fármacos , Macrófagos/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Animales , Ácido Ascórbico/farmacología , Línea Celular , Citocinas/antagonistas & inhibidores , Citocinas/inmunología , Inmunidad Innata/efectos de los fármacos , Interferón Tipo I/inmunología , Interferón beta/inmunología , Macrófagos/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Rutina/farmacología , Porcinos , alfa-Tocoferol/farmacologíaRESUMEN
As a severe disease characterized by reproductive failure and respiratory distress, porcine reproductive and respiratory syndrome (PRRS) is one of the most leading threats to the swine industry worldwide. Highly evolving porcine reproductive and respiratory syndrome virus (PRRSV) strains with distinct genetic diversity make the current vaccination strategy much less cost-effective and thus urge alternative protective host directed therapeutic approaches. RACK1-PKC-NF-κB signalling axis was suggested as a potential therapeutic target for PRRS control, therefore we tested the inhibitory effect of PKC inhibitor dequalinium chloride (DECA) on the PRRSV infection in vitro. RT-qPCR, western blot, Co-IP and cytopathic effect (CPE) observations revealed that DECA suppressed PRRSV infection and protected Marc-145 cells and porcine alveolar macrophages (PAMs) from severe cytopathic effects, by repressing the PKCα expression, the interaction between RACK1 and PKCα, and subsequently the NF-κB activation. In conclusion, the data presented in this study shed more light on deeper understanding of the molecular pathogenesis upon PRRSV infection and more importantly suggested DECA as a potential promising drug candidate for PRRS control.
Asunto(s)
Decualinio/farmacología , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Proteína Quinasa C-alfa/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Animales , Línea Celular , Células Cultivadas , Efecto Citopatogénico Viral , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/virología , Transducción de Señal , PorcinosRESUMEN
BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a serious viral disease of swine. At present, there are vaccines for the control of PRRSV infection, but the effect is not satisfactory. The recombination of attenuated vaccines causes significant difficulties with the prevention and control of PRRSV. Type III interferons (IFNs), also called IFN-λs, were newly identified and showed potent antiviral activity within the mucosal surface and immune organs. RESULTS: Therefore, primary porcine alveolar macrophages (PAMs) were used for this investigation. To this end, we found that the replication of PRRSV in PAMs was significantly reduced after pre-treatment with IFN-λ3, and such inhibition was dose- and time-dependent. The plaque formation of PRRSV abrogated entirely, and virus yields were reduced by four orders of magnitude when the primary PAMs were treated with IFN-λ3 at 1000 ng/ml. In addition, IFN-λ3 in our study was able to induce the expression of interferon-stimulated genes 15 (ISG15), 2'-5'-oligoadenylate synthase 1 (OAS1), IFN-inducible transmembrane 3 (IFITM3), and myxoma resistance protein 1(Mx1) in primary PAMs. CONCLUSIONS: IFN-λ3 had antiviral activity against PRRSV and can stimulate the expression of pivotal interferon-stimulated genes (ISGs), i.e., ISG15, Mx1, OAS1, and IFITM3. So, IFN-λ3 may serve as a useful antiviral agent.
Asunto(s)
Interferones/farmacología , Macrófagos Alveolares/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Línea Celular , Chlorocebus aethiops , Síndrome Respiratorio y de la Reproducción Porcina , Porcinos , Interferón lambdaRESUMEN
TRIM26, a member of the tripartite motif (TRIM) family has been shown to be involved in modulation of innate antiviral response. However, the functional characteristics of porcine TRIM26 (porTRIM26) are unclear. In this study, we used a synthesized antigen peptide to generate a polyclonal antibody against porTRIM26 with which to study the expression and function of porTRIM26. We demonstrated that polyinosinic:polycytidylic acid (poly (I:C)) stimulation and viral infection (vesicular stomatitis (VSV) or porcine reproductive and respiratory syndrome virus (PRRSV)) induce expression of porTRIM26, whereas knock-down expression of porTRIM26 promotes interferon (IFN)- production after poly (I:C) stimulation and virus infection (VSV or PRRSV). The importance of the porTRIM26-mediated modulation of the antiviral response was also shown in VSV- or PRRSV-infected cells. In summary, these findings show that porTRIM26 has an inhibitory role in IFN- expression and the antiviral response.
Asunto(s)
Antivirales/farmacología , Interferón beta/antagonistas & inhibidores , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Proteínas de Motivos Tripartitos/metabolismo , Animales , Síndrome Respiratorio y de la Reproducción Porcina/tratamiento farmacológico , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Porcinos , Proteínas de Motivos Tripartitos/genéticaRESUMEN
The aim of this study was to test in vitro the ability of a mixture of citrus extract, maltodextrin, sodium chloride, lactic acid and citric acid (AuraShield L) to inhibit the virulence of infectious bronchitis, Newcastle disease, avian influenza, porcine reproductive and respiratory syndrome (PRRS) and bovine coronavirus viruses. Secondly, in vivo, we have investigated its efficacy against infectious bronchitis using a broiler infection model. In vitro, these antimicrobials had expressed antiviral activity against all five viruses through all phases of the infection process of the host cells. In vivo, the antimicrobial mixture reduced the virus load in the tracheal and lung tissue and significantly reduced the clinical signs of infection and the mortality rate in the experimental group E2 receiving AuraShield L. All these effects were accompanied by a significant reduction in the levels of pro-inflammatory cytokines and an increase in IgA levels and short chain fatty acids (SCFAs) in both trachea and lungs. Our study demonstrated that mixtures of natural antimicrobials, such AuraShield L, can prevent in vitro viral infection of cell cultures. Secondly, in vivo, the efficiency of vaccination was improved by preventing secondary viral infections through a mechanism involving significant increases in SCFA production and increased IgA levels. As a consequence the clinical signs of secondary infections were significantly reduced resulting in recovered production performance and lower mortality rates in the experimental group E2.
Asunto(s)
Antivirales/farmacología , Infecciones por Coronavirus/tratamiento farmacológico , Coronavirus Bovino/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Virus de la Bronquitis Infecciosa/efectos de los fármacos , Subtipo H9N2 del Virus de la Influenza A/efectos de los fármacos , Virus de la Enfermedad de Newcastle/efectos de los fármacos , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Enfermedades de las Aves de Corral/tratamiento farmacológico , Animales , Línea Celular , Embrión de Pollo , Pollos , Infecciones por Coronavirus/virología , Modelos Animales de Enfermedad , Células Epiteliales/virología , Humanos , Gripe Aviar/metabolismo , Gripe Aviar/virología , Gripe Humana/metabolismo , Gripe Humana/virología , Enfermedad de Newcastle/metabolismo , Enfermedad de Newcastle/virología , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virología , Enfermedades de las Aves de Corral/virología , PorcinosRESUMEN
Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV) infection, imposes enormous economic impact to the world pork industry. Currently there is no effective treatment to prevent PRRSV infection in swine. We report that the natural compound cryptotanshinone (Cpt) effectively inhibits the infection of various strains of PRRSV to porcine alveolar macrophages (PAMs), the primary cell target of PRRSV in vivo. Mechanistically, Cpt inhibits the activation of signal transducer and activator of transcription 3 (STAT3), and blocks the interleukin 10 (IL-10) stimulated as well as the basal level CD163 expression in PAMs. Cpt-treatment of PAMs is effective when applied either before or after PRRSV infection, with the combined pre- and post-PRRSV infection treatment resulting in the most significant, dose-dependent inhibition of PRRSV infection. Cpt inhibited both type I/II PRRSV infection in PAMs. Our study identified a new approach to prevent/treat PRRSV infection of pigs with natural compounds.