RESUMEN
BACKGROUND: African swine fever (ASF) is a viral disease that affects pigs and wild boars providing economic burden in swine industry. METHODS AND RESULTS: In this study, we investigated the effect of deleting the ASFV multigene family 110 (MGF110) fragment (1 L-5-6 L) on apoptosis modulation and the expression of proinflammatory cytokines. Gene expression in swine peripheral blood macrophages infected with either the parental "Volgograd/14c" strain or the gene-deleted "Volgograd/D(1L-5-6L) MGF110" strain was analyzed. Caspase-3 activity was 1.15 times higher in macrophages infected with the parental ASFV strain compared to the gene-deleted strain. Gene expression analysis of Caspase-3 (Cas-3), Interferon-A (IFN-A), Tumor Necrosis Factor A (TNF-A), B-cell Lymphoma-2 (Bcl-2), Nuclear Factor Kappa B (NF-kB), Interleukin-12 (IL-12), and Heat Shock Protein-70 (HSP-70) using RT-qPCR at various time points after infection revealed significant differences in expression profiles between the strains. The peak expression of cytokines (except NF-kB) occurred at 24 h post-infection with the "Volgograd/D(1L-5-6L) MGF110" strain. In samples infected with the ASFV "Volgograd/14c" strain, the most intense expression was observed at 72 and 96 h, except for Bcl-2 and NF-kB, which peaked at 6 h post-infection. The cytokine expression trend for the "Volgograd/D(1L-5-6L) MGF110" strain was more stable with higher expression values. CONCLUSION: The expression trend for the parental strain increased over time, reaching maximum values at 72 and 96 h post-infection, but the overall expression level was lower than that of the gene-deleted strain. These findings suggest that deleting the multigene family 110 members (1 L-5-6 L) contributes to ASFV attenuation without affecting virus replication kinetics.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Citocinas , Macrófagos , Familia de Multigenes , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/patogenicidad , Animales , Porcinos , Citocinas/metabolismo , Citocinas/genética , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/genética , Fiebre Porcina Africana/metabolismo , Macrófagos/metabolismo , Macrófagos/virología , Apoptosis/genética , FN-kappa B/metabolismo , FN-kappa B/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Regulación de la Expresión GénicaRESUMEN
OBJECTIVE: This study validates a direct multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay which was previously established for enabling rapid and simultaneous detection of African swine fever (ASF) virus (ASFV) and classical swine fever virus. The assay eliminates the need for viral nucleic acid purification using a buffer system for crude extraction and an impurity-tolerant enzyme. However, the assay had not yet been validated using field samples of ASFV-infected pigs. Therefore, to address this gap, we tested 101 samples collected from pigs in Vietnam during 2018 and 2021 for validation. RESULTS: The rRT-PCR assay demonstrated a diagnostic sensitivity of 98.8% and a specificity of 100%. Remarkably, crude samples yielded results comparable to those of purified samples, indicating the feasibility of using crude samples without compromising accuracy in ASFV detection. Our findings emphasize the effectiveness of the rRT-PCR assay for the prompt and accurate diagnosis of both swine fever viruses, which is essential for effective disease prevention and control in swine populations.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Animales , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Porcinos , Vietnam , Fiebre Porcina Africana/diagnóstico , Fiebre Porcina Africana/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normasRESUMEN
Rapid and reliable detection method for African swine fever virus (ASFV) is proposed by surface-enhanced Raman spectroscopy (SERS). The ASFV target DNA can be specifically captured by sandwich hybridization between nanomagnetic beads and a SERS probe. Experimental results show that the significant Raman signal of the SERS probe with gold nanoparticles and a molecular reporter DTNB (5,5'-dimercapto-bis (2-nitrobenzoic acid)) can be adopted for detecting the hybridization chain reaction of ASFV DNA. The advantage of the SERS sandwich hybridization assay is the large response range from the single molecule level to 108 copies per mL, which not only can overcome the tedious time required for the amplification reaction but also provides a comparative method to polymerase chain reaction. Furthermore, real samples of African swine fever virus were detected from different subjects of swine fever virus including porcine reproductive respiratory syndrome virus and Japanese encephalitis virus. The proposed biosensor method can rapidly detect ASFV correctly within 15 min as a simple, convenient, low-cost detection approach. The biosensor can be used as a platform for the determination in biological, food, and environmental analytical fields.
Asunto(s)
Virus de la Fiebre Porcina Africana , Oro , Nanopartículas del Metal , Hibridación de Ácido Nucleico , Espectrometría Raman , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/genética , Espectrometría Raman/métodos , Nanopartículas del Metal/química , Animales , Oro/química , Técnicas Biosensibles/métodos , Porcinos , ADN Viral/análisis , ADN Viral/genética , Límite de Detección , Fiebre Porcina Africana/diagnóstico , Fiebre Porcina Africana/virologíaRESUMEN
African swine fever (ASF) is a highly contagious and severe hemorrhagic transboundary swine viral disease with up to a 100% mortality rate, which leads to a tremendous socio-economic loss worldwide. The lack of safe and efficacious ASF vaccines is the greatest challenge in the prevention and control of ASF. In this study, we generated a safe and effective live-attenuated virus (LAV) vaccine candidate VNUA-ASFV-LAVL3 by serially passaging a virulent genotype II strain (VNUA-ASFV-L2) in an immortalized porcine alveolar macrophage cell line (3D4/21, 50 passages). VNUA-ASFV-LAVL3 lost its hemadsorption ability but maintained comparable growth kinetics in 3D4/21 cells to that of the parental strain. Notably, it exhibited significant attenuation of virulence in pigs across different doses (103, 104, and 105 TCID50). All vaccinated pigs remained healthy with no clinical signs of African swine fever virus (ASFV) infection throughout the 28-day observation period of immunization. VNUA-ASFV-LAVL3 was efficiently cleared from the blood at 14-17 days post-infection, even at the highest dose (105 TCID50). Importantly, the attenuation observed in vivo did not compromise the ability of VNUA-ASFV-LAVL3 to induce protective immunity. Vaccination with VNUA-ASFV-LAVL3 elicited robust humoral and cellular immune responses in pigs, achieving 100% protection against a lethal wild-type ASFV (genotype II) challenge at all tested doses (103, 104, and 105 TCID50). Furthermore, a single vaccination (104 TCID50) provided protection for up to 2 months. These findings suggest that VNUA-ASFV-LAVL3 can be utilized as a promising safe and efficacious LAV candidate against the contemporary pandemic genotype II ASFV.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Genotipo , Vacunas Atenuadas , Vacunas Virales , Animales , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/inmunología , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/genética , Vacunas Atenuadas/administración & dosificación , Porcinos , Fiebre Porcina Africana/prevención & control , Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/virología , Vacunas Virales/inmunología , Vacunas Virales/genética , Vacunas Virales/administración & dosificación , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Línea Celular , Virulencia , Vacunación/veterinariaRESUMEN
The African swine fever virus (ASFV) is a large enveloped DNA virus that causes a highly pathogenic hemorrhagic disease in both domestic pigs and wild boars. The ASFV genome contains a double-stranded DNA encoding more than 150 proteins. The ASFV possesses only one protease, pS273R, which is important for virion assembly and host immune evasion. Therefore, the specific monoclonal antibody (mAb) against pS273R is useful for ASFV research. Here, we generated two specific anti-pS273R mAbs named 2F3 and 3C2, both of which were successfully applied for ELISA, Western blotting, and immunofluorescence assays. Further, we showed that both 2F3 and 3C2 mAbs recognize a new epitope of N terminal 1-25 amino acids of pS273R protein, which is highly conserved across different ASFV strains including all genotype I and II strains. Based on the recognized epitope, an indirect ELISA was established and was effective in detecting antibodies during ASFV infection. To conclude, the specific pS273R mAbs and corresponding epitope identified will strongly promote ASFV serological diagnosis and vaccine research.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Anticuerpos Monoclonales , Epítopos , Virus de la Fiebre Porcina Africana/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Porcinos , Ratones , Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/virología , Anticuerpos Antivirales/inmunología , Ratones Endogámicos BALB C , Proteínas Virales/inmunología , Péptido Hidrolasas/inmunología , Péptido Hidrolasas/metabolismo , Antígenos Virales/inmunología , Ensayo de Inmunoadsorción Enzimática , Mapeo EpitopoRESUMEN
African swine fever (ASF) has become a global pandemic due to inadequate prevention and control measures, posing a significant threat to the swine industry. Despite the approval of a single vaccine in Vietnam, no antiviral drugs against the ASF virus (ASFV) are currently available. Aloperine (ALO), a quinolizidine alkaloid extracted from the seeds and leaves of bitter beans, exhibits various biological functions, including anti-inflammatory, anti-cancer, and antiviral activities. In this study, we found that ALO could inhibit ASFV replication in MA-104, PK-15, 3D4/21, and WSL cells in a dose-dependent manner without cytotoxicity at 100 µM. Furthermore, it was verified that ALO acted on the co- and post-infection stages of ASFV by time-of-addition assay, and inhibited viral internalization rather than directly inactivating the virus. Notably, RT-qPCR analysis indicated that ALO did not exert anti-inflammatory activity during ASFV infection. Additionally, gene ontology (GO) and KEGG pathway enrichment analyses of transcriptomic data revealed that ALO could inhibit ASFV replication via the PRLR/JAK2 signaling pathway. Together, these findings suggest that ALO effectively inhibits ASFV replication in vitro and provides a potential new target for developing anti-ASFV drugs.
Asunto(s)
Virus de la Fiebre Porcina Africana , Antivirales , Janus Quinasa 2 , Piperidinas , Quinolizidinas , Transducción de Señal , Replicación Viral , Janus Quinasa 2/metabolismo , Animales , Transducción de Señal/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Quinolizidinas/farmacología , Porcinos , Piperidinas/farmacología , Virus de la Fiebre Porcina Africana/efectos de los fármacos , Virus de la Fiebre Porcina Africana/metabolismo , Antivirales/farmacología , Línea Celular , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/metabolismoRESUMEN
African swine fever (ASF) is a deadly hemorrhagic disease of domestic and wild swine that was first described in the early 20th century after the introduction of European pigs to Kenya. The etiological agent, the African swine fever virus (ASFV), is a large DNA virus within the Asfarviridae family that is broadly categorized epidemiologically into genotypes based on the nucleotide sequence of B646L, the gene encoding the major capsid protein p72. ASF outbreaks in Africa have been linked historically to 25 genotypes by p72 nucleotide analysis and, recently, to 6 genotypes by amino acid comparison, whereas global outbreaks of ASF outside of Africa have only been linked to 2 genotypes: genotype I, which led to an outbreak in Europe during the 1960s that later spread to South America, and genotype II, responsible for the current pandemic that began in Georgia in 2007 and has since spread to Europe, Asia, and Hispaniola. Here, we present an analysis of the genome of ASFV Spencer, an isolate that was collected in 1951 near Johannesburg, South Africa. While nucleotide analysis of Spencer indicates the p72 coding sequence is unique, differentiating from the closest reference by five nucleotides, the predicted amino acid sequence indicates that it is 100% homologous to contemporary genotype 1. Full genome analysis reveals it is more similar to Mkuzi1979 and encodes genes that share similarity with either genotype 1 or genotype 2 outbreak strains.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Brotes de Enfermedades , Genoma Viral , Genotipo , Filogenia , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/clasificación , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/epidemiología , Animales , Brotes de Enfermedades/veterinaria , Porcinos , Sudáfrica/epidemiología , Proteínas de la Cápside/genética , Análisis de Secuencia de ADN , Historia del Siglo XXRESUMEN
The first report of African swine fever virus (ASFV) genotype II in Italy in 2022 marked the beginning of a significant invasion in at least eight Italian regions with different infection clusters. In this study, we used the multi-gene approach to investigate the epidemiological associations between ASFV strains causing cases and outbreaks in wild boar and pigs in Italy from January 2022 to the end of 2023. Our results confirm that all the tested ASFV-positive Italian samples belonged to genotype II and show high homology with genotype II ASFV sequences previously collected in Eurasian countries. Molecular characterization revealed the presence of four genetic groups in Italy. The majority of African swine fever (ASF) samples analyzed in the current study (72%) belonged to genetic group 3, which was the most representative in Europe. The results also provide evidence of the prevalence of genetic group 19 (15.9%). In addition, we identified new putative genetic groups, genetic group 25 (9.1%) and genetic group 26 (3.0%), which have never been described before. This is the first detailed report on the molecular characterization of more than 130 ASFV strains circulating in Italy.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Genotipo , Filogenia , Sus scrofa , Fiebre Porcina Africana/epidemiología , Fiebre Porcina Africana/virología , Animales , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/clasificación , Italia/epidemiología , Porcinos , Sus scrofa/virología , Brotes de Enfermedades , Epidemias , Variación GenéticaRESUMEN
The spread of the African swine fever virus (ASF virus) genotype ii in the Eurasian region has been very successful and often inexplicable. The virus spreads rapidly and persists in areas with wild boar populations, but areas without feral pig populations are also affected. The virus has shown the ability to survive for a long time in the environment without a population of susceptible hosts, both pigs and Ornithodoros soft ticks. Published data indicated that ASF viruses persist significantly longer in an environment with some freshwater snails (especially Pomacea bridgesii, Tarebia granifera, Asolene spixii, Melanoides tuberculate, and Physa fontinalis), compared to freshwater without snails. Data obtained in this study suggest that gastropods theoretically can be the hosts of the ASF virus. Also, we have proven the possibility of long-term existence of an infectious virus when infected in vitro.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Animales , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/fisiología , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Porcinos , Fiebre Porcina Africana/virología , Gastrópodos/virología , Ornithodoros/virologíaRESUMEN
The emergence and spread of the African swine fever virus (ASFV) posed a significant threat to the global swine breeding industry, calling for innovative approaches benefiting viral containment and control. A recent study (Z. Zheng, L. Xu, H. Dou, Y. Zhou, X., et al., Microbiol Spectr 12: e02164-23, 2024, https://doi.org/10.1128/spectrum.02164-23) established a multiplexed CRISPR-Cas system targeting the genome of ASFV and tested the consequent antiviral activity both in vitro and in vivo. Application of this system showed a significant reduction of viral replication in vitro, while the germline-edited pigs expressing this system exhibited normal growth with continuous guide RNA expression. Although no survival advantage was observed upon ASFV challenge compared with nonengineered pigs, this marks the first attempt of germline editing to pursue ASFV resistance and paves the way for future disease-resistant animal breeding approaches utilizing CRISPR-Cas technology.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Sistemas CRISPR-Cas , Edición Génica , Animales , Virus de la Fiebre Porcina Africana/genética , Porcinos , Fiebre Porcina Africana/virología , Edición Génica/métodos , Replicación Viral/genética , Genoma Viral/genética , Resistencia a la Enfermedad/genéticaRESUMEN
African swine fever virus (ASFV) infection causes a frequently fatal disease in domestic swine that has affected more than 50 countries worldwide since 2021, with a major impact on animal welfare and economy. The development of effective vaccines or antivirals against this disease are urgently required for its effective control. Live detection of viral replication has been used as a tool for the screening and characterization of antiviral compounds in other dsDNA genome containing viruses. Here, we have adapted the ANCHOR fluorescent DNA labelling system to ASFV by constructing and characterizing a novel recombinant virus. We show that this virus is viable and effectively tags viral DNA replication sites, which can be detected and quantified in real time. Further, we have used high content cell microscopy to test the antiviral activity of bisbenzimide compounds and show that Hoechst 33342 has specific anti-ASFV activity. We expect this novel tool to be useful both in the further study of ASFV replication as in the screening of new specific antiviral compounds.
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Virus de la Fiebre Porcina Africana , Antivirales , Bencimidazoles , ADN Viral , Replicación Viral , Virus de la Fiebre Porcina Africana/efectos de los fármacos , Virus de la Fiebre Porcina Africana/fisiología , Virus de la Fiebre Porcina Africana/genética , Replicación Viral/efectos de los fármacos , Animales , Antivirales/farmacología , Bencimidazoles/farmacología , Porcinos , ADN Viral/genética , Replicación del ADN/efectos de los fármacos , Fiebre Porcina Africana/virología , Chlorocebus aethiops , Coloración y Etiquetado/métodos , Células Vero , Línea CelularRESUMEN
Wild boar population dynamics promote the increase in numbers and distribution of the species in Eurasia, leading to a rise in the interaction with human activities, as well as generating problems with the management of certain infectious diseases, most notably African swine fever (ASF). ASF virus possesses high stability in several contaminated pork and pork products that can be a source of indirect transmission to susceptible hosts habituated to anthropogenic food waste. This transmission route is a concerning threat for the dispersion of the disease, primarily into unaffected areas given the worldwide widespread distribution of the disease and the increase of wild boar contact with humans. Thus, in this study, a straightforward tool to assess the relative risk of wild boar natural populations potentially consuming food waste is presented using synthetic data. Three risk groups were defined related to urban areas, travel, and leisure. The surrounding quality of habitat of wild boar was used to obtain the relative risk of wild boar potentially consuming anthropogenic food waste. To assign the relative risk to the corresponding risk unit, we also included the population for the urban areas group, and traffic volume for the travel risk group. The leisure group had higher scaled risk scores, followed by the urban areas group. Higher risk was found in the edges of the study area where more natural landscapes are found. The implications of this risk are discussed focusing on the context of ASF transmission. The outputs can help prioritize decision-making in terms of the improvement of preventive measures against the habituation of wild boar to anthropogenic food waste and ASFV introduction in a given study area.
Asunto(s)
Fiebre Porcina Africana , Sus scrofa , Animales , Fiebre Porcina Africana/transmisión , Fiebre Porcina Africana/epidemiología , Fiebre Porcina Africana/virología , Porcinos , Sus scrofa/virología , Humanos , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/patogenicidad , Alimento Perdido y DesperdiciadoRESUMEN
The African swine fever virus (ASFV) is an ancient, structurally complex, double-stranded DNA virus that causes African swine fever. Since its discovery in Kenya and Africa in 1921, no effective vaccine or antiviral strategy has been developed. Therefore, the selection of more suitable vaccines or antiviral targets is the top priority to solve the African swine fever virus problem. B125R, one of the virulence genes of ASFV, encodes a non-structural protein (pB125R), which is important in ASFV infection. However, the epitope of pB125R is not well characterized at present. We observed that pB125R is specifically recognized by inactivated ASFV-positive sera, suggesting that it has the potential to act as a protective antigen against ASFV infection. Elucidation of the antigenic epitope within pB125R could facilitate the development of an epitope-based vaccine targeting ASFV. In this study, two strains of monoclonal antibodies (mAbs) against pB125R were produced by using the B cell hybridoma technique, named 9G11 and 15A9. The antigenic epitope recognized by mAb 9G11 was precisely located by using a series of truncated ASFV pB125R. The 52DPLASQRDIYY62 (epitope on ASFV pB125R) was the smallest epitope recognized by mAb 9G11 and this epitope was highly conserved among different strains. The key amino acid sites were identified as D52, Q57, R58, and Y62 by the single-point mutation of 11 amino acids of the epitope by alanine scanning. In addition, the immunological effects of the epitope (pB125R-DY) against 9G11 were evaluated in mice, and the results showed that both full-length pB125R and the epitope pB125R-DY could induce effective humoral and cellular immune responses in mice. The mAbs obtained in this study reacted with the eukaryotic-expressed antigen proteins and the PAM cell samples infected with ASFV, indicating that the mAb can be used as a good tool for the detection of ASFV antigen infection. The B cell epitopes identified in this study provide a fundamental basis for the research and development of epitope-based vaccines against ASFV.
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Virus de la Fiebre Porcina Africana , Anticuerpos Monoclonales , Anticuerpos Antivirales , Epítopos de Linfocito B , Animales , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/genética , Virus de la Fiebre Porcina Africana/inmunología , Virus de la Fiebre Porcina Africana/genética , Anticuerpos Monoclonales/inmunología , Ratones , Anticuerpos Antivirales/inmunología , Ratones Endogámicos BALB C , Porcinos , Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/virología , Virulencia , Mapeo Epitopo , FemeninoRESUMEN
African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), a highly infectious and lethal disease of domesticated swine. Outbreaks of ASF have been mostly restricted to the continent of Africa. The outbreaks that have occurred outside of Africa were controlled by extensive depopulation of the domesticated pig population. However, in 2007, an outbreak occurred in the country of Georgia, where ASFV infected wild pigs and quickly spread across eastern Europe. Since the reintroduction of ASF into Europe, variants of the current pandemic strain, ASFV Georgia 2007/01 (ASFV-G), which is classified as Genotype 2 based on p72 sequencing, have been reported in countries within western Europe, Asia, and the island of Hispaniola. Additionally, isolates collected in 2020 confirmed the presence of variants of ASFV-G in Nigeria. Recently, we reported similar variants of ASFV-G collected from domestic pigs suspected of dying of ASF in Ghana in 2022. Here, we retroactively report, based on full-length sequencing, that similar variants were present in Ghana in 2021. The SNP analysis revealed derivatives of ASFV with distinct genetic markers. Furthermore, we identified three full-length ASFV genomes as Genotype 1, indicating that there were two genotypes circulating in proximity during the 2021 ASF outbreaks in Ghana.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Brotes de Enfermedades , Genoma Viral , Genotipo , Filogenia , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/clasificación , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Animales , Fiebre Porcina Africana/epidemiología , Fiebre Porcina Africana/virología , Ghana/epidemiología , Porcinos , Brotes de Enfermedades/veterinaria , Estudios Retrospectivos , Variación GenéticaRESUMEN
Obtaining a complete good-quality sequence and annotation for the long double-stranded DNA genome of the African swine fever virus (ASFV) from next-generation sequencing (NGS) technology has proven difficult, despite the increasing availability of reference genome sequences and the increasing affordability of NGS. A gap analysis conducted by the global African swine fever research alliance (GARA) partners identified that a standardized, automatic pipeline for NGS analysis was urgently needed, particularly for new outbreak strains. Whilst there are several diagnostic and research labs worldwide that collect isolates of the ASFV from outbreaks, many do not have the capability to analyze, annotate, and format NGS data from outbreaks for submission to NCBI, and some publicly available ASFV genomes have missing or incorrect annotations. We developed an automated, standardized pipeline for the analysis of NGS reads that directly provides users with assemblies and annotations formatted for their submission to NCBI. This pipeline is freely available on GitHub and has been tested through the GARA partners by examining two previously sequenced ASFV genomes; this study also aimed to assess the accuracy and limitations of two strategies present within the pipeline: reference-based (Illumina reads) and de novo assembly (Illumina and Nanopore reads) strategies.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Anotación de Secuencia Molecular , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/clasificación , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Animales , Porcinos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Fiebre Porcina Africana/virología , Análisis de Secuencia de ADN/métodos , Biología Computacional/métodosRESUMEN
African swine fever (ASF) continues to spread in Africa, Europe, Asia and the island of Hispaniola, increasing the need to develop more streamlined and highly efficient surveillance and diagnostic capabilities. One way to achieve this is by further optimization of already established standard operating procedures to remove bottlenecks for high-throughput screening. Real-time polymerase chain reaction (real-time PCR) is the most sensitive and specific assay available for the early detection of the ASF virus (ASFV) genome, but it requires high-quality nucleic acid extracted from the samples. Whole blood from live pigs and spleen tissue from dead pigs are the preferred samples for real-time PCR. Whole blood can be used as is in nucleic acid extractions, but spleen tissues require an additional homogenization step. In this study, we compared the homogenates and swabs prepared from 52 spleen samples collected from pigs experimentally inoculated with highly and moderately virulent ASF virus strains. The results show that not only are the spleen swabs more sensitive when executed with a low-cell-count nucleic acid extraction procedure followed by real-time PCR assays but they also increase the ability to isolate ASFV from positive spleen samples. Swabbing is a convenient, simpler and less time-consuming alternative to tissue homogenization. Hence, we recommend spleen swabs over tissue homogenates for high-throughput detection of ASFV by real-time PCR.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Bazo , Animales , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/genética , Fiebre Porcina Africana/diagnóstico , Fiebre Porcina Africana/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Porcinos , Bazo/virología , Ensayos Analíticos de Alto Rendimiento/métodosRESUMEN
African swine fever (ASF) is a rapidly fatal viral haemorrhagic fever in Chinese domestic pigs. Although very high mortality is observed in pig farms after an ASF outbreak, clinically healthy and antibody-positive pigs are found in those farms, and viral detection is rare from these pigs. The ability of pigs to resist ASF viral infection may be modulated by host genetic variations. However, the genetic basis of the resistance of domestic pigs against ASF remains unclear. We generated a comprehensive set of structural variations (SVs) in a Chinese indigenous Xiang pig with ASF-resistant (Xiang-R) and ASF-susceptible (Xiang-S) phenotypes using whole-genome resequencing method. A total of 53,589 nonredundant SVs were identified, with an average of 25,656 SVs per individual in the Xiang pig genome, including insertion, deletion, inversion and duplication variations. The Xiang-R group harboured more SVs than the Xiang-S group. The F-statistics (FST) was carried out to reveal genetic differences between two populations using the resequencing data at each SV locus. We identified 2,414 population-stratified SVs and annotated 1,152 Ensembl genes (including 986 protein-coding genes), in which 1,326 SVs might disturb the structure and expression of the Ensembl genes. Those protein-coding genes were mainly enriched in the Wnt, Hippo, and calcium signalling pathways. Other important pathways associated with the ASF viral infection were also identified, such as the endocytosis, apoptosis, focal adhesion, Fc gamma R-mediated phagocytosis, junction, NOD-like receptor, PI3K-Akt, and c-type lectin receptor signalling pathways. Finally, we identified 135 candidate adaptive genes overlapping 166 SVs that were involved in the virus entry and virus-host cell interactions. The fact that some of population-stratified SVs regions detected as selective sweep signals gave another support for the genetic variations affecting pig resistance against ASF. The research indicates that SVs play an important role in the evolutionary processes of Xiang pig adaptation to ASF infection.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Animales , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/genética , Porcinos , Virus de la Fiebre Porcina Africana/genética , Resistencia a la Enfermedad/genética , Variación Genética , Genoma/genética , Secuenciación Completa del Genoma , Variación Estructural del Genoma , China , Sus scrofaRESUMEN
African swine fever (ASF) is a highly contagious and fatal viral disease that has caused huge economic losses to the pig and related industries worldwide. At present, rapid, accurate, and sensitive laboratory detection technologies are important means of preventing and controlling ASF. However, because attenuated strains of African swine fever virus (ASFV) are constantly emerging, an ASFV antibody could be used more effectively to investigate the virus and control the disease on pig farms. The isolation of ASFV-specific antibodies is also essential for the diagnosis of ASF. Therefore, in this study, we developed two chemiluminescence immunoassays (CLIAs) to detect antibodies directed against ASFV p72: a traditional plate-type blocking CLIA (p72-CLIA) and an automatic tubular competitive CLIA based on magnetic particles (p72-MPCLIA). We compared the diagnostic performance of these two methods to provide a feasible new method for the effective prevention and control of ASF and the purification of ASFV. The cut-off value, diagnostic sensitivity (Dsn), and diagnostic specificity (Dsp) of p72-CLIA were 40%, 100%, and 99.6%, respectively, in known background serum, whereas those of p72-MPCLIA were 36%, 100%, and 99.6%, respectively. Thus, both methods show good Dsn, Dsp, and repeatability. However, when analytical sensitivity was evaluated, p72-MPCLIA was more sensitive than p72-CLIA or a commercial enzyme-linked immunosorbent assay. More importantly, p72-MPCLIA reduced the detection time to 15 min and allowed fully automated detection. In summary, p72-MPCLIA showed superior diagnostic performance and offered a new tool for detecting ASFV infections in the future. KEY POINTS: ⢠Two chemiluminescence immunoassay (plate-type CLIA and tubular CLIA) methods based on p72 monoclonal antibody (mAb) were developed to detect ASFV antibody. ⢠Both methods show good diagnostic performance (Dsn (100%), Dsp (99.6%), and good repeatability), and p72-MPCLIA detects antibodies against ASFV p72 with high efficiency in just 15 min.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Anticuerpos Antivirales , Mediciones Luminiscentes , Sensibilidad y Especificidad , Virus de la Fiebre Porcina Africana/inmunología , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Animales , Fiebre Porcina Africana/diagnóstico , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/inmunología , Porcinos , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Inmunoensayo/métodos , Mediciones Luminiscentes/métodosRESUMEN
African swine fever virus (ASFV) is the causal agent of African swine fever (ASF), which is contagious and highly lethal to domestic pigs and wild boars. The genome of ASFV encodes many proteins important for ASFV life cycle. The functional importance of topoisomerase AsfvTopII has been confirmed by in vivo and in vitro assays, but the structure of AsfvTopII is poorly studied. Here, we report four AsfvTopII complex structures. The ATPase domain structures reveal the detailed basis for ATP binding and hydrolysis, which is shared by AsfvTopII and eukaryotic TopIIs. The DNA-bound structures show that AsfvTopII follows conserved mechanism in G-DNA binding and cleavage. Besides G-DNA, a T-DNA fragment is also captured in one AsfvTopII structure. Mutagenesis and in vitro assays confirm that Pro852 and the T-DNA-binding residue Tyr744 are important for the function of AsfvTopII. Our study not only advances the understanding on the biological function of AsfvTopII, but also provides a solid basis for the development of AsfvTopII-specific inhibitors.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Proteínas Virales , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/enzimología , Animales , Porcinos , Fiebre Porcina Africana/virología , Proteínas Virales/metabolismo , Proteínas Virales/genética , Proteínas Virales/química , Adenosina Trifosfato/metabolismo , Modelos Moleculares , Unión Proteica , ADN Viral/genética , ADN Viral/metabolismo , Cristalografía por Rayos XRESUMEN
African swine fever virus (ASFV) severely threatens the global economy and food security. ASFV encodes >150 genes, but the functions of most of them have yet to be characterized in detail. Here we explored the function of the ASFV CP312R gene and found that CP312R plays an essential role in ASFV replication. Knockout of the CP312R gene terminated viral replication and CP312R knockdown substantially suppressed ASFV infection in vitro. Furthermore, we resolved the crystal structure of pCP312R to 2.3 Å resolution and found that pCP312R has the potential to bind nucleic acids. LC-MS analysis and co-immunoprecipitation assay revealed that pCP312R interacts with RPS27A, a component of the 40S ribosomal subunit. Confocal microscopy showed the interaction between pCP312R and RPS27A leaded to a modification in the subcellular localization of this host protein, which suppresses host protein translation. Renilla-Glo luciferase assay and Ribopuromycylation analysis evidenced that knockout of RPS27A completely aborted the shutoff activity of pCP312R, and trans-complementation of RPS27A recovered pCP312R shutoff activity in RPS27A-knockout cells. Our findings shed light on the function of ASFV CP312R gene in virus infection, which triggers inhibition of host protein synthesis.