RESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) is a severe disease with substantial economic consequences for the swine industry. The DEAD-box helicase 3 (DDX3X) is an RNA helicase that plays a crucial role in regulating RNA metabolism, immunological response, and even RNA virus infection. However, it is unclear whether it contributes to PRRSV infection. Recent studies have found that the expression of DDX3X considerably increases in Marc-145 cells when infected with live PRRSV strains Ch-1R and SD16; however, it was observed that inactivated viruses did not lead to any changes. By using the RK-33 inhibitor or DDX3X-specific siRNAs to reduce DDX3X expression, there was a significant decrease in the production of PRRSV progenies. In contrast, the overexpression of DDX3X in host cells substantially increased the proliferation of PRRSV. A combination of transcriptomics and metabolomics investigations revealed that in PRRSV-infected cells, DDX3X gene silencing severely affected biological processes such as ferroptosis, the FoxO signalling pathway, and glutathione metabolism. The subsequent transmission electron microscopy (TEM) imaging displayed the typical ferroptosis features in PRRSV-infected cells, such as mitochondrial shrinkage, reduction or disappearance of mitochondrial cristae, and cytoplasmic membrane rupture. Conversely, the mitochondrial morphology was unchanged in DDX3X-inhibited cells. Furthermore, silencing of the DDX3X gene changed the expression of ferroptosis-related genes and inhibited the virus proliferation, while the drug-induced ferroptosis inversely promoted PRRSV replication. In summary, these results present an updated perspective of how PRRSV infection uses DDX3X for self-replication, potentially leading to ferroptosis via various mechanisms that promote PRRSV replication.
Assuntos
RNA Helicases DEAD-box , Ferroptose , Vírus da Síndrome Respiratória e Reprodutiva Suína , Replicação Viral , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Animais , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Ferroptose/fisiologia , Suínos , Síndrome Respiratória e Reprodutiva Suína/virologia , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Linhagem CelularRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) induces a poor innate immune response following infection. This study evaluates the effects of transforming growth factor beta 1 (TGFß1) up-regulated by PRRSV on gene expressions of co-stimulatory molecules, type I interferon (IFN), type I IFN-regulated genes (IRGs), pattern recognition receptors, and pro-inflammatory cytokines in PRRSV-inoculated monocyte-derived macrophages (MDMs). Phosphorothioate-modified antisense oligodeoxynucleotides (AS ODNs) specific to various regions of porcine TGFß1 mRNA were synthesized, and those specific to the AUG region efficiently knockdown TGFß1 mRNA expression and protein translation. Transfection of TGFßAS ODNs in MDMs inoculated with either classical PRRSV-2 (cPRRSV-2) or highly pathogenic PRRSV-2 (HP-PRRSV-2) significantly reduced TGFß1 mRNA expression and significantly increased mRNA expressions of CD80, CD86, IFNß, IRGs (i.e. IFN regulatory factor 3 (IRF3), IRF7, myxovirus resistance 1, osteopontin, and stimulator of IFN genes), Toll-like receptor 3, and tumor necrosis factor-alpha. Transfection of TGFßAS ODNs in MDMs inoculated with HP-PRRSV-2 also significantly increased mRNA expressions of IFNα, IFNγ, and 2'-5'-oligoadenylate synthetase 1. The quantity of PRRSV-2 RNA copy numbers was significantly reduced in MDMs transfected with TGFßAS ODNs as compared to untransfected MDMs. Recombinant porcine TGFß1 (rTGFß1) and recombinant porcine IFNα (rIFNα) sustained and reduced the yields of PRRSV-2 RNA copy numbers in PRRSV-2 inoculated MDMs, respectively. These findings demonstrate a strategy of PRRSV for innate immune suppression via an induction of TGFß expression. These findings also suggest TGFß as a potential parameter that future PRRSV vaccine and vaccine adjuvant candidates should take into consideration.
Assuntos
Citocinas , Interferon Tipo I , Macrófagos , Vírus da Síndrome Respiratória e Reprodutiva Suína , Animais , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Suínos , Interferon Tipo I/metabolismo , Citocinas/genética , Citocinas/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Macrófagos/imunologia , Macrófagos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Técnicas de Silenciamento de Genes , Imunidade InataRESUMO
Seven novel porcine parvoviruses (PPV2 to PPV8) have been discovered in the last two decades. The last one reported was PPV8 in China in 2022, which was proposed to be a member of the genus Protoparvovirus. Here, we report the first detection of PPV8 outside China - in two provinces from Colombia. Six out of 146 (4.1%) pigs showing porcine respiratory disease (PRD) tested positive for PPV8. Sequencing and phylogenetic analysis of two Colombian PPV8 isolates (GenBank database accession numbers PP335559 and PP335560) showed them to be members of the genus Protoparvovirus. Furthermore, PPV8 was detected in coinfections with porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV), which are associated with PRD.
Assuntos
Infecções por Parvoviridae , Parvovirus Suíno , Filogenia , Doenças dos Suínos , Animais , Suínos , Colômbia/epidemiologia , Parvovirus Suíno/genética , Parvovirus Suíno/isolamento & purificação , Parvovirus Suíno/classificação , Infecções por Parvoviridae/veterinária , Infecções por Parvoviridae/virologia , Infecções por Parvoviridae/epidemiologia , Doenças dos Suínos/virologia , Doenças dos Suínos/epidemiologia , Coinfecção/virologia , Coinfecção/veterinária , Coinfecção/epidemiologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Vírus da Síndrome Respiratória e Reprodutiva Suína/classificação , Síndrome Respiratória e Reprodutiva Suína/virologia , Síndrome Respiratória e Reprodutiva Suína/epidemiologiaRESUMO
Reports of Parainfluenza virus 5 (PIV5) epidemics have been on a global upward trend, with an expanding host range across various animals. In 2020, we isolated a PIV5 strain from a PRRSV-positive serum sample. This strain was named GX2020. Genetic analysis revealed that GX2020 belongs to group A, represented by the AGS strain isolated from a human in the USA. Comparisons of amino acid identity in the coding regions showed that GX2020 had the highest amino acid identity (99.6%) with the AGS strain. The emergence of PIV5 strains genetically similar to human strains in pigs highlights its zoonotic potential and underscores the need for enhanced PIV5 surveillance in the future.
Assuntos
Vírus da Parainfluenza 5 , Filogenia , Síndrome Respiratória e Reprodutiva Suína , Animais , Suínos , Síndrome Respiratória e Reprodutiva Suína/virologia , Síndrome Respiratória e Reprodutiva Suína/epidemiologia , China/epidemiologia , Humanos , Vírus da Parainfluenza 5/genética , Vírus da Parainfluenza 5/isolamento & purificação , Vírus da Parainfluenza 5/classificação , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/classificação , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Genoma Viral , Infecções por Rubulavirus/virologia , Infecções por Rubulavirus/veterinária , Infecções por Rubulavirus/epidemiologiaRESUMO
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.
Assuntos
Ácido Araquidônico , Glutamina , Inflamação , Vírus da Síndrome Respiratória e Reprodutiva Suína , Quercetina , Quercetina/farmacologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , Animais , Linhagem Celular , Inflamação/virologia , Inflamação/tratamento farmacológico , Glutamina/metabolismo , Glutamina/farmacologia , Ácido Araquidônico/metabolismo , Suínos , Síndrome Respiratória e Reprodutiva Suína/virologia , Síndrome Respiratória e Reprodutiva Suína/tratamento farmacológico , Chlorocebus aethiopsRESUMO
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.
Assuntos
Alcaloides , Antivirais , Ácido Glicirrízico , Matrinas , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Quinolizinas , Replicação Viral , Animais , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , Alcaloides/farmacologia , Quinolizinas/farmacologia , Quinolizinas/uso terapêutico , Suínos , Antivirais/farmacologia , Antivirais/uso terapêutico , Ácido Glicirrízico/farmacologia , Ácido Glicirrízico/uso terapêutico , Síndrome Respiratória e Reprodutiva Suína/tratamento farmacológico , Síndrome Respiratória e Reprodutiva Suína/virologia , Síndrome Respiratória e Reprodutiva Suína/prevenção & controle , Replicação Viral/efeitos dos fármacos , Citocinas/metabolismo , Análise de SobrevidaRESUMO
One of the most significant diseases in the swine business, porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory problems in piglets and reproductive failure in sows. The PRRSV nucleocapsid (N) protein is essential for the virus' assembly, replication, and immune evasion. Stages in the viral replication cycle can be impacted by interactions between the PRRSV nucleocapsid protein and the host protein components. Therefore, it is of great significance to explore the interaction between the PRRSV nucleocapsid protein and the host. Nevertheless, no information has been published on the network of interactions between the nucleocapsid protein and the host proteins in primary porcine alveolar macrophages (PAMs). In this study, 349 host proteins interacting with nucleocapsid protein were screened in the PRRSV-infected PAMs through a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics approach. Bioinformatics analysis, which included gene ontology annotation, Kyoto Encyclopedia of Genes and Genomes database enrichment, and a protein-protein interaction (PPI) network, revealed that the host proteins interacting with PRRSV-N may be involved in protein binding, DNA transcription, metabolism, and innate immune responses. This study confirmed the interaction between the nucleocapsid protein and the natural immune-related proteins. Ultimately, our findings suggest that the nucleocapsid protein plays a pivotal role in facilitating immune evasion during a PRRSV infection. This study contributes to enhancing our understanding of the role played by the nucleocapsid protein in viral pathogenesis and virus-host interaction, thereby offering novel insights for the prevention and control of PRRS as well as the development of vaccines.
Assuntos
Interações Hospedeiro-Patógeno , Macrófagos Alveolares , Proteínas do Nucleocapsídeo , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Mapas de Interação de Proteínas , Proteômica , Espectrometria de Massas em Tandem , Animais , Suínos , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virologia , Proteômica/métodos , Proteínas do Nucleocapsídeo/metabolismo , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Síndrome Respiratória e Reprodutiva Suína/virologia , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida , Biologia Computacional/métodos , Ontologia GenéticaRESUMO
Porcine reproductive and respiratory syndrome (PRRS), which poses substantial threats to the global pig industry, is primarily characterized by interstitial pneumonia. Cluster of differentiation 163 (CD163) is the essential receptor for PRRSV infection. Metalloproteinase-mediated cleavage of CD163 leads to the shedding of soluble CD163 (sCD163), thereby inhibiting PRRSV proliferation. However, the exact cleavage site in CD163 and the potential role of sCD163 in inflammatory responses during PRRSV infection remain unclear. Herein, we found that PRRSV infection increased sCD163 levels, as demonstrated in primary alveolar macrophages (PAMs), immortalized PAM (IPAM) cell lines, and sera from PRRSV-infected piglets. With LC-MS/MS, Arg-1041/Ser-1042 was identified as the cleavage site in porcine CD163, and an IPAM cell line with precise mutation at the cleavage site was constructed. Using the precisely mutated IPAM cells, we found that exogenous addition of sCD163 protein promoted inflammatory responses, while mutation at the CD163 cleavage site suppressed inflammatory responses. Consistently, inhibition of sCD163 using its neutralizing antibodies reduced PRRSV infection-triggered inflammatory responses. Importantly, sCD163 promoted cell polarization from M2 to M1 phenotype, which in turn facilitated inflammatory responses. Taken together, our findings identify sCD163 as a novel proinflammatory mediator and provide valuable insights into the mechanisms underlying the induction of inflammatory responses by PRRSV infection.
Assuntos
Antígenos CD , Antígenos de Diferenciação Mielomonocítica , Inflamação , Macrófagos Alveolares , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Receptores de Superfície Celular , Animais , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/imunologia , Antígenos de Diferenciação Mielomonocítica/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Suínos , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos CD/imunologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Macrófagos Alveolares/virologia , Macrófagos Alveolares/imunologia , Inflamação/virologia , Linhagem CelularRESUMO
Metabolism in host cells can be modulated after viral infection, favoring viral survival or clearance. Here, we report that lipid droplet (LD) synthesis in host cells can be modulated by yin yang 1 (YY1) after porcine reproductive and respiratory syndrome virus (PRRSV) infection, resulting in active antiviral activity. As a ubiquitously distributed transcription factor, there was increased expression of YY1 upon PRRSV infection both in vitro and in vivo. YY1 silencing promoted the replication of PRRSV, whereas YY1 overexpression inhibited PRRSV replication. PRRSV infection led to a marked increase in LDs, while YY1 knockout inhibited LD synthesis, and YY1 overexpression enhanced LD accumulation, indicating that YY1 reprograms PRRSV infection-induced intracellular LD synthesis. We also showed that the viral components do not colocalize with LDs during PRRSV infection, and the effect of exogenously induced LD synthesis on PRRSV replication is nearly lethal. Moreover, we demonstrated that YY1 affects the synthesis of LDs by regulating the expression of lipid metabolism genes. YY1 negatively regulates the expression of fatty acid synthase (FASN) to weaken the fatty acid synthesis pathway and positively regulates the expression of peroxisome proliferator-activated receptor gamma (PPARγ) to promote the synthesis of LDs, thus inhibiting PRRSV replication. These novel findings indicate that YY1 plays a crucial role in regulating PRRSV replication by reprogramming LD synthesis. Therefore, our study provides a novel mechanism of host resistance to PRRSV and suggests potential new antiviral strategies against PRRSV infection.IMPORTANCEPorcine reproductive and respiratory virus (PRRSV) has caused incalculable economic damage to the global pig industry since it was first discovered in the 1980s. However, conventional vaccines do not provide satisfactory protection. It is well known that viruses are parasitic pathogens, and the completion of their replication life cycle is highly dependent on host cells. A better understanding of host resistance to PRRSV infection is essential for developing safe and effective strategies to control PRRSV. Here, we report a crucial host antiviral molecule, yin yang 1 (YY1), which is induced to be expressed upon PRRSV infection and subsequently inhibits virus replication by reprogramming lipid droplet (LD) synthesis through transcriptional regulation. Our work provides a novel antiviral mechanism against PRRSV infection and suggests that targeting YY1 could be a new strategy for controlling PRRSV.
Assuntos
Gotículas Lipídicas , Vírus da Síndrome Respiratória e Reprodutiva Suína , Replicação Viral , Fator de Transcrição YY1 , Fator de Transcrição YY1/metabolismo , Fator de Transcrição YY1/genética , Animais , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Suínos , Gotículas Lipídicas/metabolismo , Síndrome Respiratória e Reprodutiva Suína/virologia , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Síndrome Respiratória e Reprodutiva Suína/genética , Linhagem Celular , Metabolismo dos Lipídeos , Interações Hospedeiro-PatógenoRESUMO
Porcine reproductive and respiratory syndrome (PRRS) is a globally prevalent contagious disease caused by the positive-strand RNA PRRS virus (PRRSV), resulting in substantial economic losses in the swine industry. Modifying the CD163 SRCR5 domain, either through deletion or substitution, can eff1ectively confer resistance to PRRSV infection in pigs. However, large fragment modifications in pigs inevitably raise concerns about potential adverse effects on growth performance. Reducing the impact of genetic modifications on normal physiological functions is a promising direction for developing PRRSV-resistant pigs. In the current study, we identified a specific functional amino acid in CD163 that influences PRRSV proliferation. Viral infection experiments conducted on Marc145 and PK-15 CD163 cells illustrated that the mE535G or corresponding pE529G mutations markedly inhibited highly pathogenic PRRSV (HP-PRRSV) proliferation by preventing viral binding and entry. Furthermore, individual viral challenge tests revealed that pigs with the E529G mutation had viral loads two orders of magnitude lower than wild-type (WT) pigs, confirming effective resistance to HP-PRRSV. Examination of the physiological indicators and scavenger function of CD163 verified no significant differences between the WT and E529G pigs. These findings suggest that E529G pigs can be used for breeding PRRSV-resistant pigs, providing novel insights into controlling future PRRSV outbreaks.
Assuntos
Antígenos CD , Antígenos de Diferenciação Mielomonocítica , Mutação Puntual , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Receptores de Superfície Celular , Animais , Suínos , Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Animais Geneticamente Modificados/genética , Linhagem CelularRESUMO
Porcine reproductive and respiratory syndrome (PRRS) is the most economically significant disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). Type I interferon (IFN) induces a large number of interferon-stimulated genes (ISGs) expression to inhibit PRRSV infection. To survive in the host, PRRSV has evolved multiple strategies to antagonize host innate immune response. Previous studies have reported that PRRSV N protein decreases the expression of TRIM25 and TRIM25-mediated RIG-I ubiquitination to suppress IFN-ß production. However, whether other PRRSV proteins inhibit the antiviral function of TRIM25 is less well understood. In this study, we first found that PRRSV NSP1α decreased ISGylation of TRIM25. Meanwhile, NSP1α significantly suppressed TRIM25-mediated IFN-ß production to promote PRRSV replication. Further studies demonstrated that PRRSV NSP1α reduced the protein level of TRIM25 in proteasome system but did not regulate the transcription level of TRIM25. In addition, the function of NSP1α in TRIM25 degradation did not rely on its papain-like cysteine protease activity. Taken together, PRRSV NSP1α antagonizes the antiviral response of TRIM25 by mediating TRIM25 degradation to promote PRRSV replication. Our data identify TRIM25 as a natural target of PRRSV NSP1α and reveal a novel mechanism that PRRSV induces TRIM25 degradation and inhibits host antiviral immune response.
Assuntos
Imunidade Inata , Vírus da Síndrome Respiratória e Reprodutiva Suína , Complexo de Endopeptidases do Proteassoma , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Proteínas não Estruturais Virais , Replicação Viral , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Animais , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Suínos , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Interferon beta/genética , Interferon beta/metabolismo , Interferon beta/imunologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Linhagem Celular , Ubiquitinação , Humanos , Células HEK293 , Interações Hospedeiro-Patógeno/imunologiaRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly contagious disease that threatens the global swine industry. Recent studies have focused on the damage that PRRSV causes to the reproductive system of male pigs, although pathological research is lacking. Therefore, we examined the pathogenic mechanisms in male piglets infected with PRRSV. Gross and histopathological changes indicated that PRRSV affected the entire reproductive system, as confirmed via immunohistochemical analysis. PRRSV infected Sertoli cells and spermatogonia. To test the new hypothesis that PRRSV infection in piglets impairs blood - testis barrier (BTB) development, we investigated the pathology of PRRSV damage in the BTB. PRRSV infection significantly decreased the quantity and proliferative capacity of Sertoli cells constituting the BTB. Zonula occludens-1 and ß-catenin were downregulated in cell - cell junctions. Transcriptome analysis revealed that several crucial genes and signalling pathways involved in the growth and development of Leydig cells, Sertoli cells, and tight junctions in the testes were downregulated. Apoptosis, necroptosis, inflammatory, and oxidative stress-related pathways were activated, whereas hormone secretion-related pathways were inhibited. Many Sertoli cells and spermatogonia underwent apoptosis during early differentiation. Infected piglets exhibited disrupted androgen secretion, leading to significantly reduced testosterone and anti-Müllerian hormone levels. A cytokine storm occurred, notably upregulating cytokines such as tumour necrosis factor-α and interleukin-6. Markers of oxidative-stress damage (i.e. H2O2, malondialdehyde, and glutathione) were upregulated, whereas antioxidant-enzyme activities (i.e. superoxide dismutase, total antioxidant capacity, and catalase) were downregulated. Our results demonstrated that PRRSV infected multiple organs in the male reproductive system, which impaired growth in the BTB.
Assuntos
Barreira Hematotesticular , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Células de Sertoli , Testículo , Animais , Masculino , Suínos , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Síndrome Respiratória e Reprodutiva Suína/patologia , Células de Sertoli/virologia , Células de Sertoli/metabolismo , Barreira Hematotesticular/virologia , Testículo/virologia , Testículo/patologia , Espermatogônias/virologia , Apoptose , Células Intersticiais do Testículo/virologia , Citocinas/metabolismo , Testosterona/sangue , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genéticaRESUMO
Recombination is a pervasive phenomenon in RNA viruses and an important strategy for accelerating the evolution of RNA virus populations. Recombination in the porcine reproductive and respiratory syndrome virus (PRRSV) was first reported in 1999, and many case reports have been published in recent years. In this review, all the existing reports on PRRSV recombination events were collected, and the genotypes, parental strains, and locations of the recombination breakpoints have been summarized and analyzed. The results showed that the recombination pattern constantly changes; whether inter- or intra-lineage recombination, the recombination hotspots vary in different recombination patterns. The virulence of recombinant PRRSVs was higher than that of the parental strains, and the emergence of virulence reversion was caused by recombination after using MLV vaccines. This could be attributed to the enhanced adaptability of recombinant PRRSV for entry and replication, facilitating their rapid propagation. The aim of this paper was to identify common features of recombinant PRRSV strains, reduce the recombination risk, and provide a foundation for future research into the mechanism of PRRSV recombination.
Assuntos
Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Recombinação Genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/classificação , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Animais , Suínos , Síndrome Respiratória e Reprodutiva Suína/virologia , Genótipo , Virulência , Genoma Viral , Replicação Viral , FilogeniaRESUMO
Since it was first reported in 2013, the NADC30-like PRRSV has been epidemic in China. Hubei Province is known as China's key hog-exporting region. To understand the prevalence and genetic variation of PRRSV, herein, we detected and analyzed 317 lung tissue samples from pigs with respiratory disease in Hubei Province, and demonstrated that the NADC30-like strain was the second-most predominant strain during 2017-2018, following the highly pathogenic PRRSV (HP-PRRSV). Additionally, we isolated a new NADC30-like PRRSV strain, named CHN-HB-2018, which could be stably passaged in Marc-145 cells. Genetic characterization analysis showed that compared with the NADC30 strain, the CHN-HB-2018 strain had several amino acid variations in glycoprotein (GP) 3, GP5, and nonstructural protein 2 (NSP2). Moreover, the CHN-HB-2018 strain showed a unique 5-amino acid (aa) deletion in NSP2, which has not previously been reported. Gene recombination analysis identified the CHN-HB-2018 strain as a potentially recombinant PRRSV of the NADC30-like strain and HP-PRRSV. Animal experiments indicated that the CHN-HB-2018 strain has a mild pathogenicity, with no mortality and only mild fever observed in piglets. This study contributes to defining the evolutionary characteristics of PRRSV and its molecular epidemiology in Hubei Province, and provides a potential candidate strain for PRRSV vaccine development.
Assuntos
Filogenia , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Vírus da Síndrome Respiratória e Reprodutiva Suína/classificação , Animais , Suínos , Síndrome Respiratória e Reprodutiva Suína/virologia , Síndrome Respiratória e Reprodutiva Suína/epidemiologia , China/epidemiologia , Virulência , Genoma Viral , Recombinação Genética , Variação Genética , Pulmão/virologia , Pulmão/patologiaRESUMO
BACKGROUND: Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) infection during late gestation substantially lowers fetal viability and survival. In a previous genome-wide association study, a single nucleotide polymorphism on chromosome 7 was significantly associated with probability of fetuses being viable in response to maternal PRRSV-2 infection at 21 days post maternal inoculation. The iodothyronine deiodinase 2 (DIO2) gene, located ~ 14 Kilobase downstream of this SNP, was selected as a priority candidate related to fetal susceptibility following maternal PRRSV-2 infection. Our objectives were to identify mutation(s) within the porcine DIO2 gene and to determine if they were associated with fetal outcomes after PRRSV-2 challenge. Sequencing of the DIO2, genotyping identified variants, and association of DIO2 genotypes with fetal phenotypes including DIO2 mRNA levels, viability, survival, viral loads, cortisol and thyroid hormone levels, and growth measurements were conducted. RESULTS: A missense variant (p.Asn91Ser) was identified in the parental populations from two independent PRRSV-2 challenge trials. This variant was further genotyped to determine association with fetal PRRS outcomes. DIO2 mRNA levels in fetal heart and kidney differed by the genotypes of Asn91Ser substitution with significantly greater DIO2 mRNA expression in heterozygotes compared with wild-type homozygotes (P < 0.001 for heart, P = 0.002 for kidney). While Asn91Ser did not significantly alter fetal viability and growth measurements, interaction effects of the variant with fetal sex or trial were identified for fetal viability or crown rump length, respectively. However, this mutation was not related to dysregulation of the hypothalamic-pituitary-adrenal and thyroid axis, indicated by no differences in circulating cortisol, T4, and T3 levels in fetuses of the opposing genotypes following PRRSV-2 infection. CONCLUSIONS: The present study suggests that a complex relationship among DIO2 genotype, DIO2 expression, fetal sex, and fetal viability may exist during the course of fetal PRRSV infection. Our study also proposes the increase in cortisol levels, indicative of fetal stress response, may lead to fetal complications, such as fetal compromise, fetal death, or premature farrowing, during PRRSV infection.
Assuntos
Iodeto Peroxidase , Mutação de Sentido Incorreto , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Animais , Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/virologia , Feminino , Suínos , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Gravidez , Iodotironina Desiodinase Tipo II , Genótipo , Feto/virologiaRESUMO
This study was designed to investigate the different sequential order of infection for porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV). Thirty-six pigs were randomly assigned to six different treatment groups. The first (hereafter referred to as PRRSV-PCV2) group was inoculated with PRRSV first followed by PCV2d. The second (hereafter referred to as PCV2+PRRSV) group was co-infected with both viruses at the same timepoint (42 days of age). The third (hereafter referred to as PCV2-PRRSV) group was inoculated with PCV2d first followed by PRRSV. A fourth group was only inoculated with PCV2d at 42 days of age, while a fifth group was only inoculated with PRRSV at the same timepoint. The sixth group served as a negative control group. The most important observation discovered that PRRSV only had a potentiation effect on PCV2 in both PRRS-PCV2 and PCV2+PRRSV groups. Both PRRSV-PCV2 and PCV2+PRRSV groups experienced a significant reduction in growth performance compared with control pigs. In addition, PRRSV-PCV2 and PCV2+PRRSV groups exhibited a greater severity in their clinical signs, and/or had higher PCV2 blood and lymphoid viral loads that resulted in a stronger severity of lymphoid lesions compared with PCV2-PRRSV group. Serum TNF-α levels were significantly higher in both PRRS-PCV2 and PCV2+PRRSV groups compared with those in PCV2-PRRS, PCV2, and PRRSV groups. The results of this study demonstrated that divergent clinical outcomes are dependent on the sequential infection order of PCV2 and PRRSV.
Assuntos
Infecções por Circoviridae , Circovirus , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Animais , Suínos , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Circovirus/fisiologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Infecções por Circoviridae/virologia , Infecções por Circoviridae/veterinária , Coinfecção/virologia , Coinfecção/veterinária , Carga Viral , Doenças dos Suínos/virologiaRESUMO
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.
Assuntos
Antivirais , Ensaios de Triagem em Larga Escala , Nitrocompostos , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Tiazóis , Animais , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , Nitrocompostos/farmacologia , Suínos , Antivirais/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Síndrome Respiratória e Reprodutiva Suína/tratamento farmacológico , Síndrome Respiratória e Reprodutiva Suína/virologia , Tiazóis/farmacologia , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Viremia/tratamento farmacológico , Viremia/virologiaRESUMO
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.
Assuntos
Antivirais , Citocinas , Flavonoides , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Replicação Viral , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , Animais , Flavonoides/farmacologia , Antivirais/farmacologia , Suínos , Replicação Viral/efeitos dos fármacos , Citocinas/metabolismo , Síndrome Respiratória e Reprodutiva Suína/virologia , Síndrome Respiratória e Reprodutiva Suína/tratamento farmacológico , Antígenos CD/metabolismo , Receptores de Superfície Celular/metabolismo , Linhagem Celular , Antígenos de Diferenciação MielomonocíticaRESUMO
Porcine reproductive and respiratory syndrome (PRRS) is one of the most detrimental contagious swine ailments worldwide. Currently, no effective drugs are available for its treatment. Targeting the structural and non-structural proteins (NSP) of the type 2 PRRS virus (PRRSV-2) with small interfering RNA (siRNA) is an effective approach to inhibit PRRSV replication. NSP4, which is highly conserved and possesses 3â¯C-like serine protease activity (3CLSP), can cleave PRRSV self-proteins, thereby contributing to viral replication. To investigate the mechanism by which NSP4 regulates PRRSV-2 replication and screen for effective siRNA inhibitors of PRRSV-2 replication, the recombinant plasmid pEGFP-C1-NSP4 was constructed, and a control siRNA pair and two siRNA pairs targeting the PRRSV-2 NSP4 gene (shRNA-ctr, shRNA-150, and shRNA-536) were synthesized and cloned into the pSilencer4.1-CMV vector. After 24â¯h of incubation, Marc-145 cells were transfected with recombinant plasmids, and subsequently infected with different PRRSV-2 (XH-GD, ZQ-GD, GDr180, and JXA1-R). Subsequently, the effects of NSP4 overexpression, shRNA on PRRSV-2 replication were evaluated by assessing cytopathic effects (CPE), TCID50, quantitative real-time PCR (qPCR), immunofluorescence assays (IFA), and Western blotting. The data from these CPE, TCID50, qPCR, and IFA experiments revealed that NSP4 overexpression significantly enhanced PRRSV-2 replication and shRNA targeting NSP4 can inhibit PRRSV-2 replication in Marc-145 cells, indicating that shRNA could serve as candidate molecules for fundamental research on PRRSV-2.
Assuntos
Vírus da Síndrome Respiratória e Reprodutiva Suína , RNA Interferente Pequeno , Proteínas não Estruturais Virais , Replicação Viral , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Animais , Suínos , Linhagem Celular , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Síndrome Respiratória e Reprodutiva Suína/virologiaRESUMO
Autophagy is an important biological process in host defense against viral infection. However, many viruses have evolved various strategies to disrupt the host antiviral system. Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus with a large economic impact on the swine industry. At present, studies on the escape mechanism of PRRSV in the autophagy process, especially through chaperone-mediated autophagy (CMA), are limited. This study confirmed that PRRSV glycoprotein 5 (GP5) could disrupt the formation of the GFAP-LAMP2A complex by inhibiting the MTORC2/PHLPP1/GFAP pathway, promoting the dissociation of the pGFAP-EF1α complex, and blocking the K63-linked polyubiquitination of LAMP2A to inhibit the activity of CMA. Further research demonstrated that CMA plays an anti-PRRSV role by antagonizing nonstructural protein 11 (NSP11)-mediated inhibition of type I interferon (IFN-I) signaling. Taken together, these results indicate that PRRSV GP5 inhibits the antiviral effect of CMA by targeting LAMP2A. This research provides new insight into the escape mechanism of immunosuppressive viruses in CMA. IMPORTANCE: Viruses have evolved sophisticated mechanisms to manipulate autophagy to evade degradation and immune responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus that causes enormous economic losses in the swine industry. However, the mechanism by which PRRSV manipulates autophagy to defend against host antiviral effects remains unclear. In this study, we found that PRRSV GP5 interacts with LAMP2A and disrupts the formation of the GFAP-LAMP2A complex, thus inhibiting the activity of CMA and subsequently enhancing the inhibitory effect of the NSP11-mediated IFN-I signaling pathway, ultimately facilitating PRRSV replication. Our study revealed a novel mechanism by which PRRSV escapes host antiviral effects through CMA, providing a potential host target, LAMP2A, for developing antiviral drugs and contributing to understanding the escape mechanism of immunosuppressive viruses.