RESUMEN
African swine fever, caused by the African swine fever virus (ASFV), is a viral hemorrhagic disease that affects domestic pigs and wild boars. ASFV infection causes extensive tissue damage, and the associated mechanism is poorly understood. Pyroptosis is characterized by the activation of inflammatory caspases and pore formation in the cellular plasma membrane, resulting in the release of inflammatory cytokines and cell damage. How ASFV infection regulates pyroptosis remains unclear. Here, using siRNA assay and overexpression methods, we report that ASFV infection regulated pyroptosis by cleaving the pyroptosis execution protein gasdermin A (GSDMA). ASFV infection activated caspase-3 and caspase-4, which specifically cleaved GSDMA at D75-P76 and D241-V242 to produce GSDMA into five fragments, including GSDMA-N1-75, GSDMA-N1-241, and GSDMA-N76-241 fragments at the N-terminal end of GSDMA. Only GSDMA-N1-241, which was produced in the late stage of ASFV infection, triggered pyroptosis and inhibited ASFV replication. The fragments, GSDMA-N1-75 and GSDMA-N76-241, lose the ability to induce pyroptosis. Overall ASFV infection differentially regulates pyroptosis by GSDMA in the indicated phase, which may be conducive to its own replication. Our findings reveal a novel molecular mechanism for the regulation of pyroptosis.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Caspasa 3 , Caspasas Iniciadoras , Piroptosis , Virus de la Fiebre Porcina Africana/metabolismo , Animales , Fiebre Porcina Africana/metabolismo , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/patología , Porcinos , Caspasa 3/metabolismo , Caspasa 3/genética , Caspasas Iniciadoras/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de Unión a Fosfato/metabolismo , Células HEK293 , Replicación ViralRESUMEN
The present investigation focuses on examining the clinical, histopathological, and ultrastructural changes that occurred in pig, during an outbreak of African swine fever (ASF) in 2022 in Assam, India. The disease initially manifested as a per-acute case with high mortality but without any evident clinical signs. Subsequently, some animals exhibited an acute form of the disease characterized by high fever (104-106 °F), anorexia, vomiting, respiratory distress, and bleeding from the anal and nasal orifices. During acute African swine fever virus (ASFV) infections, elevated levels of pro-inflammatory IL-1α, IL-1ß, IL-6, TNF, CCL2, CCL5, and CXCL10 were detected in the palatine tonsil, lymph nodes, spleen, and kidney using qPCR assay. These molecular changes were associated with haemorrhages, edemas, and lymphoid depletion. Postmortem examinations revealed prominent features such as splenomegaly with haemorrhages, haemorrhagic lymphadenitis, severe petechial haemorrhage in the kidney, pneumonia in the lungs, and necrotic palatine tonsil. Histopathological analysis demonstrated lymphocyte depletion in lymphoid organs, multi-organ haemorrhages, and interstitial pneumonia in the lungs. Scanning electron microscopy (SEM) further confirmed lymphocyte depletion in lymphoid organs through lymphocyte apoptosis and kidney damage with distorted tubules due to red blood cell destruction. Transmission electron microscopy reaffirmed lymphocyte apoptosis by observing chromatin condensation and nucleus margination in lymphocytes of lymphoid organs. These findings provide comprehensive insights into the clinical, histopathological, and ultrastructural aspects of ASF outbreak in pigs. Understanding the pathological changes associated with ASF can contribute to improved diagnosis, prevention, and control measures for this highly contagious and economically devastating viral disease.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Porcinos , Animales , Fiebre Porcina Africana/epidemiología , Fiebre Porcina Africana/patología , Linfocitos , Brotes de Enfermedades , Hemorragia , Sus scrofaRESUMEN
African swine fever (ASF) has brought excellent barriers to swine production in China and the world. Studies have shown that extracellular vesicles mediate the RNA and protein spread of pathogenic microorganisms and RNA and proteins. After infection by pathogenic microorganisms causes significant differences in the proteins contained within extracellular vesicles. Based on the above studies, the extracellular vesicles were extracted from ASF virus (ASFV)-infected swine plasma. And qPCR, western blot, and confocal experiment were carried out. The research shows that extracted extracellular vesicles significantly promote the replication of ASFV in susceptible and non-susceptible cells Proteomics analysis of the extracellular vesicle proteins revealed that ASFV infection could cause significant differences in the protein profile. This study demonstrates that extracellular vesicles play a critical role in ASFV replication and transmission and cause significant differences in the protein profile encapsulated in extracellular vesicles.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Vesículas Extracelulares , Fiebre Porcina Africana/metabolismo , Fiebre Porcina Africana/patología , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/metabolismo , Animales , Vesículas Extracelulares/metabolismo , Proteómica , Porcinos , Replicación ViralRESUMEN
The type I interferon (IFN-I) signaling pathway is an important part of the innate immune response and plays a vital role in controlling and eliminating pathogens. African swine fever virus (ASFV) encodes various proteins to evade the host's natural immunity. However, the molecular mechanism by which the ASFV-encoded proteins inhibit interferon production remains poorly understood. In the present study, ASFV MGF360-11L inhibited cGAS, STING, TBK1, IKKε, IRF7 and IRF3-5D mediated activation of the IFN-ß and ISRE promoters, accompanied by decreases in IFN-ß, ISG15 and ISG56 mRNA expression. ASFV MGF360-11L interacted with TBK1 and IRF7, degrading TBK1 and IRF7 through the cysteine, ubiquitin-proteasome and autophagy pathways. Moreover, ASFV MGF360-11L also inhibited the phosphorylation of TBK1 and IRF3 stimulated by cGAS-STING overexpression. Truncation mutation analysis revealed that aa 167-353 of ASFV MGF360-11L could inhibit cGAS-STING-mediated activation of the IFN-ß and ISRE promoters. Finally, the results indicated that ASFV MGF360-11L plays a significant role in inhibiting IL-1ß, IL-6 and IFN-ß production in PAM cells (PAMs) infected with ASFV. In short, these results demonstrated that ASFV MGF360-11L was involved in regulating IFN-I expression by negatively regulating the cGAS signaling pathway. In summary, this study preliminarily clarified the molecular mechanism by which the ASFV MGF360-11L protein antagonizes IFN-I-mediated antiviral activity, which will help to provide new strategies for the treatment and prevention of ASF.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Interferón Tipo I , Enfermedades de los Porcinos , Fiebre Porcina Africana/patología , Fiebre Porcina Africana/virología , Virus de la Fiebre Porcina Africana/metabolismo , Animales , Interferón Tipo I/genética , Interferón beta , Interferones/inmunología , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Transducción de Señal , Porcinos , Enfermedades de los Porcinos/patologíaRESUMEN
African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus that causes African swine fever, a lethal hemorrhagic disease that currently threatens the pig industry. Recent studies have identified the viral structural proteins of infectious ASFV particles. However, the functional roles of several ASFV structural proteins remain largely unknown. Here, we characterized the function of the ASFV structural protein H240R (pH240R) in virus morphogenesis. pH240R was identified as a capsid protein by using immunoelectron microscopy and interacted with the major capsid protein p72 by pulldown assays. Using a recombinant ASFV, ASFV-ΔH240R, with the H240R gene deleted from the wild-type ASFV (ASFV-WT) genome, we revealed that the infectious progeny virus titers were reduced by approximately 2.0 logs compared with those of ASFV-WT. Furthermore, we demonstrated that the growth defect was due to the generation of noninfectious particles with a higher particle-to-infectious titer ratio in ASFV-ΔH240R-infected primary porcine alveolar macrophages (PAMs) than in those infected with ASFV-WT. Importantly, we found that pH240R did not affect virus-cell binding, endocytosis, or egress but did affect ASFV assembly; noninfectious virions containing large aberrant tubular and bilobulate structures comprised nearly 98% of all virions observed in ASFV-ΔH240R-infected PAMs by electron microscopy. Notably, we demonstrated that ASFV-ΔH240R infection induced high-level expression of inflammatory cytokines in PAMs. Collectively, we show for the first time that pH240R is essential for ASFV icosahedral capsid formation and infectious particle production. Also, these results highlight the importance of pH240R in ASFV morphogenesis and provide a novel target for the development of ASF vaccines and antivirals. IMPORTANCE African swine fever is a lethal hemorrhagic disease of global concern that is caused by African swine fever virus (ASFV). Despite extensive research, there exist relevant gaps in knowledge of the fundamental biology of the viral life cycle. In this study, we identified pH240R as a capsid protein that interacts with the major capsid protein p72. Furthermore, we showed that pH240R was required for the efficient production of infectious progeny virions as indicated by the H240R-deleted ASFV mutant (ASFV-ΔH240R). More specifically, pH240R directs the morphogenesis of ASFV toward the icosahedral capsid in the process of assembly. In addition, ASFV-ΔH240R infection induced high-level expression of inflammatory cytokines in primary porcine alveolar macrophages. Our results elucidate the role of pH240R in the process of ASFV assembly, which may instruct future research on effective vaccines or antiviral strategies.
Asunto(s)
Virus de la Fiebre Porcina Africana/fisiología , Fiebre Porcina Africana/genética , Fiebre Porcina Africana/metabolismo , Proteínas de la Cápside/genética , Citocinas/metabolismo , Macrófagos/metabolismo , Eliminación de Secuencia , Fiebre Porcina Africana/patología , Virus de la Fiebre Porcina Africana/ultraestructura , Secuencia de Aminoácidos , Animales , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Citocinas/genética , Susceptibilidad a Enfermedades/inmunología , Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica , Genoma Viral , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Macrófagos/inmunología , Porcinos , Virión/ultraestructura , Internalización del Virus , Replicación ViralRESUMEN
African swine fever (ASF) is a severe hemorrhagic disease in swine characterized by massive lymphocyte depletion and cell death, with apoptosis and necrosis in infected lymphoid tissues. However, the molecular mechanism regarding ASFV-induced cell death remains largely unknown. In this study, 94 ASFV-encoded proteins were screened to determine the viral proteins involved in cell death in vitro, and pE199L showed the most significant effect. Ectopic expression of pE199L in porcine cells (CRL-2843) and human cells (HEK293T and HeLa cells) induced cell death remarkably, showing obvious shrinking, blistering, apoptotic bodies, and nuclear DNA fragments. Meanwhile, cell death was markedly alleviated when the expression of pE199L was knocked down during ASFV infection. Additionally, the expression of pE199L caused a loss of mitochondrial membrane potential, release of cytochrome C, and caspase-9 and -3/7 activation, indicating that the mitochondrial apoptotic pathway was involved in pE199L-induced apoptosis. Further investigations showed that pE199L interacted with several anti-apoptotic BCL-2 subfamily members (such as BCL-XL, MCL-1, BCL-W, and BCL-2A1) and competed with BAK for BCL-XL, which promoted BAK and BAX activation. Taken together, ASFV pE199L induces the mitochondrial-dependent apoptosis, which may provide clues for a comprehensive understanding of ASFV pathogenesis.
Asunto(s)
Virus de la Fiebre Porcina Africana , Apoptosis , Mitocondrias , Proteínas Virales , Animales , Humanos , Fiebre Porcina Africana/patología , Fiebre Porcina Africana/virología , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/crecimiento & desarrollo , Virus de la Fiebre Porcina Africana/metabolismo , Virus de la Fiebre Porcina Africana/patogenicidad , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína bcl-X/metabolismo , Caspasas/metabolismo , Línea Celular , Citocromos c/metabolismo , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Porcinos , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
African swine fever (ASF) is a severe hemorrhagic infectious disease in pigs caused by African swine fever virus (ASFV), leading to devastating economic losses in epidemic regions. Its control currently depends on thorough culling and clearance of the diseased and surrounding suspected pigs. An ASF vaccine has been extensively explored for years worldwide, especially in hog-intensive areas where it is highly desired, but it is still unavailable for numerous reasons. Here, we report another ASF vaccine candidate, named SY18ΔI226R, bearing a deletion of the I226R gene with a replacement of an enhanced green fluorescent protein (eGFP) expression cassette at the right end of the viral genome. This deletion results in the complete loss of virulence of SY18 as the gene-deleted strain does not cause any clinical symptoms in all pigs inoculated with a dosage of either 104.0 or 107.0 50% tissue culture infective doses (TCID50). Apparent viremia with a gradual decline was monitored, while virus shedding was detected only occasionally in oral or anal swabs. ASFV-specific antibody appeared at 9 days postinoculation. After intramuscular challenge with its parental strain ASFV SY18 at 21 days postinoculation, all the challenged pigs survived, without obvious febrile or abnormal clinical signs. No viral DNA could be detected upon the dissection of any tissue when viremia disappeared. These results indicated that SY18ΔI226R is safe in swine and elicits robust immunity to virulent ASFV infection. IMPORTANCE Outbreaks of African swine fever have resulted in devastating losses to the swine industry worldwide, but there is currently no commercial vaccine available. Although several vaccine candidates have been reported, none has been approved for use for several reasons, especially ones concerning biosafety. Here, we identified a new undescribed functional gene, I226R. When deleted from the ASFV genome, the virus completely loses its virulence in swine. Importantly, pigs infected with this gene-deleted virus were resistant to infection by intramuscular challenge with 102.5 or 104.0 TCID50 of its virulent parental virus. Furthermore, the nucleic acid of the gene-deleted virus and its virulent parental virus was rarely detected from oral or anal swabs. Viruses could not be detected in any tissues after necropsy when viremia became negative, indicating that robust immunity was achieved. Therefore, SY18ΔI226R is a novel, ideal, and efficacious vaccine candidate for genotype II ASF.
Asunto(s)
Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/inmunología , Eliminación de Gen , Genoma Viral , Fiebre Porcina Africana/patología , Fiebre Porcina Africana/prevención & control , Animales , ADN Viral , Genes Virales/genética , Genotipo , Análisis de Secuencia , Porcinos , Vacunas Virales/inmunología , Viremia/genética , Virulencia/genéticaRESUMEN
African swine fever virus (ASFV) is a large DNA virus that is highly contagious and pathogenic in domestic pigs with a mortality rate up to 100%. However, how ASFV suppresses JAK-STAT1 signaling to evade the immune response remains unclear. In this study, we found that the ASFV-encoded protein MGF-505-7R inhibited proinflammatory IFN-γ-mediated JAK-STAT1 signaling. Mechanistically, MGF-505-7R was found to interact with JAK1 and JAK2 and mediate their degradation. Further study indicated that MGF-505-7R promoted degradation of JAK1 and JAK2 by upregulating the E3 ubiquitin ligase RNF125 expression and inhibiting expression of Hes5, respectively. Consistently, MGF-505-7R-deficient ASFV induced high levels of IRF1 expression and displayed compromised replication both in primary porcine alveolar macrophages and pigs compared with wild-type ASFV. Furthermore, MGF-505-7R deficiency attenuated the virulence of the ASFV and pathogenesis of ASF in pigs. These findings suggest that the JAK-STAT1 axis mediates the innate immune response to the ASFV and that MGF-505-7R plays a critical role in the virulence of the ASFV and pathogenesis of ASF by antagonizing this axis. Thus, we conclude that deletion of MGF-505-7R may serve as a strategy to develop attenuated vaccines against the ASFV.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Janus Quinasa 1 , Janus Quinasa 2 , Sistema de Señalización de MAP Quinasas , Macrófagos Alveolares , Proteínas Virales , Factores de Virulencia , Fiebre Porcina Africana/genética , Fiebre Porcina Africana/metabolismo , Fiebre Porcina Africana/patología , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/metabolismo , Virus de la Fiebre Porcina Africana/patogenicidad , Animales , Línea Celular , Humanos , Janus Quinasa 1/genética , Janus Quinasa 1/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Porcinos , Proteínas Virales/genética , Proteínas Virales/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
African swine fever (ASF) is currently causing an epizootic, affecting pigs throughout Eurasia, and causing significant economic losses in the swine industry. ASF is caused by African swine fever virus (ASFV) that consists of a large dsDNA genome that encodes for more than 160 genes; few of these genes have been studied in detail. ASFV contains four multi-gene family (MGF) groups of genes that have been implicated in regulating the immune response and host specificity; however, the individual roles of most of these genes have not been well studied. Here, we describe the evaluation of the previously uncharacterized ASFV MGF110-1L open reading frame (ORF) using a deletion mutant of the ASFV currently circulating throughout Eurasia. The recombinant ASFV lacking the MGF110-1L gene (ASFV-G-ΔMGF110-1L) demonstrated in vitro that the MGF110-1L gene is non-essential, since ASFV-G-ΔMGF110-1L had similar replication kinetics in primary swine macrophage cell cultures when compared to parental highly virulent field isolate Georgia2007 (ASFV-G). Experimental infection of domestic pigs with ASFV-G-ΔMGF110-1L produced a clinical disease similar to that caused by the parental ASFV-G, confirming that deletion of the MGF110-1L gene from the ASFV genome does not affect viral virulence.
Asunto(s)
Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/patogenicidad , Fiebre Porcina Africana/patología , Sistemas de Lectura Abierta/genética , Factores de Virulencia/genética , Replicación Viral/genética , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases , Células Cultivadas , Eliminación de Gen , Regulación Viral de la Expresión Génica/genética , Genoma Viral/genética , Georgia (República) , Macrófagos/virología , Alineación de Secuencia , Sus scrofa , Porcinos/virología , Enfermedades de los Porcinos/virología , Virulencia/genéticaRESUMEN
African swine fever (ASF) has evolved from an exotic animal disease to a threat to global pig production. An important avenue for the wide-spread transmission of animal diseases is their dissemination through boar semen used for artificial insemination. In this context, we investigated the role of male reproductive organs in the transmission of ASF. Mature domestic boars and adolescent wild boars, inoculated with different ASF virus strains, were investigated by means of virological and pathological methods. Additionally, electron microscopy was employed to investigate in vitro inoculated sperm. The viral genome, antigens and the infectious virus could be found in all gonadal tissues and accessory sex glands. The viral antigen and viral mRNAs were mainly found in mononuclear cells of the respective tissues. However, some other cell types, including Leydig, endothelial and stromal cells, were also found positive. Using RNAScope, p72 mRNA could be found in scattered halo cells of the epididymal duct epithelium, which could point to the disruption of the barrier. No direct infection of spermatozoa was observed by immunohistochemistry, or electron microscopy. Taken together, our results strengthen the assumption that ASFV can be transmitted via boar semen. Future studies are needed to explore the excretion dynamics and transmission efficiency.
Asunto(s)
Virus de la Fiebre Porcina Africana/aislamiento & purificación , Fiebre Porcina Africana/transmisión , Fiebre Porcina Africana/virología , Genitales Masculinos/virología , Fiebre Porcina Africana/patología , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/fisiología , Animales , Glándulas Bulbouretrales/patología , Glándulas Bulbouretrales/virología , ADN Viral/análisis , Epidídimo/patología , Epidídimo/virología , Genitales Masculinos/patología , Leucocitos Mononucleares/virología , Masculino , Próstata/patología , Próstata/virología , ARN Mensajero/análisis , ARN Viral/análisis , Espermatozoides/ultraestructura , Espermatozoides/virología , Sus scrofa , Porcinos , Testículo/patología , Testículo/virología , Replicación ViralRESUMEN
Background: The first confirmed case of African swine fever (ASF) in Vietnam was reported officially in February 2019. To date, ASF virus (ASFV) have been detected in 63/63 provinces in Vietnam. Currently, real-time polymerase chain reaction (PCR) is considered to be a powerful tool for viral detection in field samples, including ASFV. However, some recent reports have suggested that mismatches in primer and probe binding regions may directly affect real-time PCR qualification, leading a false-negative result. Aim: This study aims to further examine a conflicting result obtained from two OIE recommended methods, conventional PCR and real-time PCR, for ASFV detection. Methods: Two ASF suspected pigs from different provinces in the north of Vietnam were selected for this study based on clinical signs and postmortem lesions. The different results obtained by OIE-recommended conventional PCR and real-time PCR were further analyzed by the Sanger sequencing method and virus isolation in combination with hemadsorption (HAD) test using porcine alveolar macrophages cells. Results: The results showed that when the primer sequence matched perfectly with the sequences of field isolates, a mutation in probe binding region was found, indicating that a single mismatch in the probe binding site may cause a false-negative result by real-time PCR in detecting ASFV in clinical samples in Vietnam. An agreement between conventional PCR, using PPA1/PPA2 primers and two golden standard methods, virus isolation in combination with HAD assay, and sequencing method was observed in this study. Conclusion: A single mismatch in the probe binding site caused a failse-negative result by realtime PCR method in field diagnosis of ASFV. The needs consideration when selecting the appropriate molecular diagnostic methods is based on the current databases of ASFV sequences, particularly for epidemiological surveillance of ASF.
Asunto(s)
Virus de la Fiebre Porcina Africana/aislamiento & purificación , Fiebre Porcina Africana/diagnóstico , Fiebre Porcina Africana/patología , Fiebre Porcina Africana/virología , Virus de la Fiebre Porcina Africana/genética , Animales , Reacciones Falso Negativas , Macrófagos Alveolares/virología , Técnicas de Diagnóstico Molecular/veterinaria , Reacción en Cadena de la Polimerasa/veterinaria , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Porcinos , VietnamRESUMEN
African swine fever virus (ASFV) is the causative agent of the African swine fever (ASF) epizootic currently affecting pigs throughout Eurasia, causing significant economic losses in the swine industry. The virus genome encodes for more than 160 genes, of which only a few have been studied in detail. Here we describe the previously uncharacterized ASFV open reading frame (ORF) C962R, a gene encoding for a putative NTPase. RNA transcription studies using infected swine macrophages demonstrate that the C962R gene is translated as a late virus protein. A recombinant ASFV lacking the C962R gene (ASFV-G-ΔC962R) demonstrates in vivo that the C962R gene is non-essential, since ASFV-G-ΔC962R has similar replication kinetics in primary swine macrophage cell cultures when compared to parental highly virulent field isolate Georgia2007 (ASFV-G). Experimental infection of domestic pigs with ASFV-G-ΔC962R produced a clinical disease similar to that caused by the parental ASFV-G, confirming that deletion of the C962R gene from the ASFV genome does not impact virulence.
Asunto(s)
Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/patogenicidad , Fiebre Porcina Africana/patología , Nucleósido-Trifosfatasa/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , Eliminación de Gen , Genoma Viral/genética , Macrófagos/virología , Sistemas de Lectura Abierta/genética , Alineación de Secuencia , Porcinos , Enfermedades de los Porcinos/virología , Proteínas Virales/genética , Factores de Virulencia/genética , Replicación Viral/genéticaRESUMEN
Following short immunization protocols, naturally attenuated African swine fever virus (ASFV) isolate OURT88/3 and deletion mutant BeninΔMGF have previously been shown to induce high percentages of protection in domestic pigs against challenge with virulent virus. The results obtained in the present study show that a single intramuscular immunization of domestic pigs with OURT88/3 or BeninΔMGF followed by a challenge with the virulent Benin 97/1 isolate at day 130 postimmunization did not trigger the mechanisms necessary to generate immunological memory able to induce long-term protection against disease. All pigs developed acute forms of acute swine fever (ASF). Gamma interferon-producing cells peaked at day 24 postimmunization, declining thereafter. Surprisingly, the levels of regulatory T cells (Tregs) and interleukin-10 (IL-10) were elevated at the end of the experiment, suggesting that regulatory components of the immune system may inhibit effective protection.IMPORTANCE The duration of immunity for any vaccine candidate is crucial. In the case of African swine fever virus vaccine candidates, this issue has received little attention. Attenuated viruses have proven protective following short immunization protocols in which pigs were challenged a few weeks after the first immunization. Here, the duration of immunity and the immune responses induced over a duration of 130 days were studied during prechallenge and after challenge of pigs immunized with the naturally attenuated isolate OURT88/3 and an attenuated gene-deleted isolate, BeninΔMGF. After a single intramuscular immunization of domestic pigs with the OURT88/3 isolate or BeninΔMGF virus, animals were not protected against challenge with the virulent Benin 97/1 ASFV genotype I isolate at day 130 postimmunization. The levels of regulatory T cells and IL-10 were elevated at the end of the experiment, suggesting that regulatory components of the immune system may inhibit effective protection.
Asunto(s)
Virus de la Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/inmunología , Interleucina-10/inmunología , Linfocitos T Reguladores/inmunología , Vacunas Virales/inmunología , Fiebre Porcina Africana/patología , Fiebre Porcina Africana/prevención & control , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Animales , Porcinos , Linfocitos T Reguladores/patología , Vacunas Atenuadas/inmunologíaRESUMEN
African swine fever (ASF) is one of the most important and complex viral diseases in domestic pigs and wild boar. Over the last decade, the disease has spread to several European and Asian countries and is now one of the major threats to profitable pig production worldwide. One of the more recently affected western countries is Belgium. To date, only wild boar are affected in a rather defined area in the Luxembourg region close to France, Luxembourg and Germany. While detailed sequence analyses were recently performed, biological characterization was still pending. Here, we report on the experimental inoculation of four sub-adult wild boar to further characterize the virus and its distribution in different tissues. After oronasal inoculation with the virus strain 'Belgium 2018/1', all animals developed an acute and severe disease course with typical pathomorphological and histopathological lesions. Organs and blood samples were positive in qPCR, haemadsorption test and antigen lateral flow devices (LFD). Virus and viral genome were also detected in genitals and accessory sex glands of two boars. There were no antibodies detectable in commercial antibody ELISAs, antibody LFDs and indirect immunoperoxidase tests. Thus, the genotype II ASF virus isolate 'Belgium 2018/1' showed a highly virulent phenotype in European wild boar similar to parental viruses like Armenia 2007 and other previously characterized ASFV strains. The study also provided a large set of well-characterized sample materials for test validation and assay harmonization.
Asunto(s)
Virus de la Fiebre Porcina Africana/patogenicidad , Fiebre Porcina Africana/virología , Sus scrofa/virología , Enfermedades de los Porcinos/virología , Fiebre Porcina Africana/patología , Virus de la Fiebre Porcina Africana/inmunología , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Animales , Animales Salvajes/virología , Anticuerpos Antivirales/sangre , Bélgica , Ensayo de Inmunoadsorción Enzimática/veterinaria , Genoma Viral , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Porcinos , Enfermedades de los Porcinos/patología , Virulencia/fisiologíaRESUMEN
: African swine fever is a devastating hemorrhagic infectious disease, which affects domestic and wild swines (Susscrofa) of all breeds and ages, with a high lethality of up to 90-100% in naïve animals. The causative agent, African swine fever virus (ASFV), is a large and complex double-stranded DNA arbovirus which is currently spreading worldwide, with serious socioeconomic consequences. There is no treatment or effective vaccine commercially available, and most of the current research is focused on attenuated viral models, with limited success so far. Thus, new strategies are under investigation. Extracellular vesicles (EVs) have proven to be a promising new vaccination platform for veterinary diseases in situations in which conventional approaches have not been completely successful. Here, serum extracellular vesicles from infected pigs using two different ASFV viruses (OURT 88/3 and Benin ΔMGF), corresponding to a naturally attenuated virus and a deletion mutant, respectively, were characterized in order to determine possible differences in the content of swine and viral proteins in EV-enriched fractions. Firstly, EVs were characterized by their CD5, CD63, CD81 and CD163 surface expression. Secondly, ASFV proteins were detected on the surface of EVs from ASFV-infected pig serum. Finally, proteomic analysis revealed few specific proteins from ASFV in the EVs, but 942 swine proteins were detected in all EV preparations (negative controls, and OURT 88/3 and Benin ΔMGF-infected preparations). However, in samples from OURT 88/3-infected animals, only a small number of proteins were differentially identified compared to control uninfected animals. Fifty-six swine proteins (Group Benin) and seven proteins (Group OURT 88/3) were differentially detected on EVs when compared to the EV control group. Most of these were related to coagulation cascades. The results presented here could contribute to a better understanding of ASFV pathogenesis and immune/protective responses in the host.
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Virus de la Fiebre Porcina Africana/patogenicidad , Fiebre Porcina Africana/virología , Vesículas Extracelulares/metabolismo , Fiebre Porcina Africana/metabolismo , Fiebre Porcina Africana/patología , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/metabolismo , Animales , Antígenos de Diferenciación/metabolismo , Vesículas Extracelulares/virología , Femenino , Mutación , Proteómica , Sus scrofa , Porcinos , Carga Viral , Proteínas Virales/metabolismoRESUMEN
The continuing spread of African swine fever (ASF) outside Africa in Europe, the Russian Federation, China and most recently to Mongolia and Vietnam, has heightened awareness of the threat posed by this devastating disease to the global pig industry and food security. In this review we summarise what we know about the African swine fever virus (ASFV), the disease it causes, how it spreads and the current global situation. We discuss current control methods in domestic and wild pigs and prospects for development of vaccines and other tools for control.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , África , Fiebre Porcina Africana/tratamiento farmacológico , Fiebre Porcina Africana/patología , Fiebre Porcina Africana/prevención & control , Fiebre Porcina Africana/transmisión , Virus de la Fiebre Porcina Africana/inmunología , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/patogenicidad , Virus de la Fiebre Porcina Africana/ultraestructura , Animales , Antivirales/uso terapéutico , Asia , China , Brotes de Enfermedades , Europa (Continente) , Ornithodoros/virología , Federación de Rusia , Sus scrofa/virología , Porcinos , Enfermedades por Picaduras de Garrapatas/transmisión , Vacunas ViralesRESUMEN
African swine fever (ASF) entered China in August 2018 and rapidly spread across the entire country, severely threatening the Chinese domestic pig population, which accounts for more than 50% of the pig population worldwide. In this study, an ASFV isolate, Pig/Heilongjiang/2018 (Pig/HLJ/18), was isolated in primary porcine alveolar macrophages (PAMs) from a pig sample from an ASF outbreak farm. The isolate was characterized by using the haemadsorption (HAD) test, Western blotting and immunofluorescence, and electronic microscopy. Phylogenetic analysis of the viral p72 gene revealed that Pig/HLJ/18 belongs to Genotype II. Infectious titres of virus propagated in primary PAMs and pig marrow macrophages were as high as 107.2 HAD50/ml. Specific-pathogen-free pigs intramuscularly inoculated with different virus dosages at 103.5-106.5 HAD50 showed acute disease with fever and haemorrhagic signs. The incubation periods were 3-5 days for virus-inoculated pigs and 9 days for contact pigs. All virus-inoculated pigs died between 6-9 days post-inoculation (p.i.), and the contact pigs died between 13-14 days post-contact (p.c.). Viremia started on day 2 p.i. in inoculated pigs and on day 9 p.c. in contact pigs. Viral genomic DNA started to be detected from oral and rectal swab samples on 2-5 days p.i. in virus-inoculated pigs, and 6-10 days p.c. in contact pigs. These results indicate that Pig/HLJ/18 is highly virulent and transmissible in domestic pigs. Our study demonstrates the threat of ASFV and emphasizes the need to control and eradicate ASF in China.
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Virus de la Fiebre Porcina Africana/crecimiento & desarrollo , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Fiebre Porcina Africana/patología , Fiebre Porcina Africana/virología , Replicación Viral , Virus de la Fiebre Porcina Africana/clasificación , Virus de la Fiebre Porcina Africana/genética , Animales , Sangre/virología , China , Genotipo , Macrófagos Alveolares/virología , Boca/virología , Filogenia , Recto/virología , Análisis de Supervivencia , Sus scrofa , Porcinos , Factores de TiempoRESUMEN
There is significant evidence that pathology of the microcirculation occurs in African swine fever (ASF); however, the mechanisms by which it develops are largely unknown. In the present experimental infection study, we show that an increase in vascular permeability in the initial stages of acute ASF is dependent on viraemia and elevation of the concentration of serum nitric oxide (NO). Macrophages activated by ASF virus (ASFV) are stimulated to produce NO and simultaneously to sensitize the endothelial cells through the action of vascular endothelial growth factor Β (VEGFΒ), which is followed by an increase in VEGF-mediated endothelial permeability. In the later stages of disease, the endothelial cells undergo DNA proliferation, which may additionally provoke capillary leakage, point haemorrhages and migration of blood cells into tissues. The possible mechanism of a shift in the cell cycle from the G1 to S and G2 stages could be a direct effect of ASFV. The terminal stages of disease are characterized by triggering of compensatory mechanisms such as stimulation of the synthesis of stromal cell-derived factor-1.
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Fiebre Porcina Africana/patología , Quimiocina CXCL12/sangre , Endotelio Vascular/patología , Óxido Nítrico/sangre , Factor A de Crecimiento Endotelial Vascular/sangre , Fiebre Porcina Africana/metabolismo , Animales , Ciclo Celular/fisiología , Proliferación Celular/fisiología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Endotelio Vascular/metabolismo , PorcinosRESUMEN
BACKGROUND: Georgia is a country in the Caucasus region with a traditional backyard and highly variable pig farming system. The practices of such sectors have seldom been described and analyzed to better understand their implication in the introduction and spread of infectious pig diseases. Moreover, the Georgian pig sector was badly hit by an epidemic of African swine fever in 2007 that quickly spread throughout the region. MATERIALS AND METHODS: We interviewed 487 pig farmers and 116 butchers using closed questionnaires on socioeconomic issues related to pig production, husbandry practices, biosecurity, marketing and movements, and disease awareness. Surveys were conducted in four regions of Georgia and descriptive statistics were computed. Factorial analyses of mixed data and hierarchical clustering on principal components were applied to study the relationship among collected variables for both farmers and butchers. RESULTS: Results show that pig farming in Georgia is a non-professional sector, highly heterogeneous by region, characterized by smallholdings of few animals, with low inputs, outdated technologies, and poor biosecurity, which all translates into low outputs and productivity. The hierarchical clustering on principal components confirmed that there are five major production and husbandry strategies, which match the four regions where the study was conducted. CONCLUSIONS: Our results are the first step to quantify biosecurity gaps and risky behaviours, develop risk profiles, and identify critical control points across the market chain where to implement mitigation measures. This study provides the baseline information needed to design realistic and sustainable prevention, surveillance and control strategies.
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Agricultores/psicología , Enfermedades de los Porcinos/transmisión , Fiebre Porcina Africana/epidemiología , Fiebre Porcina Africana/patología , Fiebre Porcina Africana/transmisión , Crianza de Animales Domésticos , Animales , Cruzamiento , Georgia (República)/epidemiología , Entrevistas como Asunto , Análisis Multivariante , Estaciones del Año , Factores Socioeconómicos , Encuestas y Cuestionarios , Porcinos , Enfermedades de los Porcinos/epidemiología , Enfermedades de los Porcinos/patologíaRESUMEN
African swine fever (ASF) in wild boar emerged in Estonia for the first time in September 2014. The first affected region was located in the South of Estonia close to the border with Latvia. It was considered to be epidemiologically connected to the outbreaks in the North of Latvia. About two weeks later, cases were detected in the North of Estonia, close to the Russian border. In the present study, we aimed to investigate the epidemiological courses of the disease in the South and in the North of Estonia. Potential associations between risk factors and the laboratory test results for ASF were examined. A hierarchical Bayesian space-time model was used to analyze the temporal trend of the ASF seroprevalence in the two areas. Young wild boar were statistically significant more likely to be ASF-positive by both, serology and virus detection, than older animals. A statistically significant difference between the two areas in the temporal course of the seroprevalence was found. While the seroprevalence clearly increased in the South, it remained relatively constant in the North. These findings led to the hypothesis that ASF might have been introduced earlier into the North of Estonia then into the South of the country.