RESUMEN
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.
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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 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.
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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/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.
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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.
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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
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.
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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
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 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
A low-cost, handheld centrifugal microfluidic system for multiplexed visual detection based on recombinase polymerase amplification (RPA) was developed. A concise centrifugal microfluidic chip featuring four reaction units was developed to run multiplexed RPA amplification in parallel. Additionally, a significantly shrunk-size and cost-effective handheld companion device was developed, incorporating heating, optical, rotation, and sensing modules, to perform multiplexed amplification and visual detection. After one-time sample loading, the metered sample was equally distributed into four separate reactors with high-speed centrifugation. Non-contact heating was adopted for isothermal amplification. A tiny DC motor on top of the chip was used to drive steel beads inside reactors for active mixing. Another small DC motor, which was controlled by an elaborate locking strategy based on magnetic sensing, was adopted for centrifugation and positioning. Visual fluorescence detection was optimized from different sides, including material, surface properties, excitation light, and optical filters. With fluorescence intensity-based visual detection, the detection results could be directly observed through the eyes or with a smartphone. As a proof of concept, the handheld device could detect multiple targets, e.g., different genes of African swine fever virus (ASFV) with the comparable LOD (limit of detection) of 75 copies/test compared to the tube-based RPA.
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Técnicas de Amplificación de Ácido Nucleico , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Técnicas de Amplificación de Ácido Nucleico/métodos , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/genética , Dispositivos Laboratorio en un Chip , Límite de Detección , Centrifugación/instrumentación , Animales , Teléfono Inteligente , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/economíaRESUMEN
African swine fever (ASF) is a highly contagious and severe hemorrhagic disease caused by the African swine fever virus (ASFV). The continuous spread of ASFV affects the safety of the global meat supply; therefore, the establishment of sensitive and specific detection methods for ASFV has become an important hot spot in food safety. Herein, we developed a flexible magnetoelastic (ME) biosensor based on PDMS/FeSiB/QDs composite films for the detection of ASFV P72 protein. Based on the high luminescence performance of CsPbBr3 quantum dots and the excellent magnetoelastic effect of FeSiB, flexible ME biosensors convert stress signals generated by antibody-antigen-specific binding into optical and electromagnetic signals. The nanostructures covalently linked by quantum dots and PDMS provide biomodification sites for ASFV P72 antibodies, simplifying the functionalization modification process compared to the case of conventional biosensors. The deformation of the PDMS film is amplified, and the conversion of surface stress signals to electrical signals is enhanced by exposing the biosensor to a uniform magnetic field. The experimental results proved that the flexible ME biosensor has a wide linear range of 10 ng mL-1-100 µg mL-1, and the detection limit is as low as 0.079 ng mL-1. Moreover, the flexible ME biosensor also shows good stability, sensitivity and specificity, confirming the potential for early disease screening.
Asunto(s)
Virus de la Fiebre Porcina Africana , Técnicas Biosensibles , Dimetilpolisiloxanos , Puntos Cuánticos , Técnicas Biosensibles/métodos , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Dimetilpolisiloxanos/química , Puntos Cuánticos/química , Animales , Proteínas Virales/química , Porcinos , Elasticidad , Límite de DetecciónRESUMEN
African swine fever virus (ASFV) has spread through many countries and regions worldwide, causing significant losses. Timely detection of ASFV-infected pigs is crucial for disease control. In this study, we assessed the performance of two pen-side tests: a portable real-time PCR (qPCR) test for detecting viral genomic DNA and a lateral flow immunoassay (LFIA) for detecting viral antigens. To determine the time from infection to the earliest detection, 10 ASFV-seronegative pigs were inoculated intramuscularly with 104.0 hemadsorption dose 50 of a highly virulent ASFV strain. Whole blood and oral swab samples were alternately collected from each group of five pigs daily until all succumbed to the infection. Samples were promptly subjected to the two pen-side tests upon collection, and a subset was transported to a veterinary diagnostic laboratory for analysis using a reference qPCR assay. Viral genomic DNA was consistently detected by the reference qPCR assay in all blood samples from 2 days postinfection (dpi), preceding the onset of clinical signs, and in oral swabs from 4 dpi onwards. The portable qPCR test demonstrated comparable performance to the reference qPCR assay for both whole blood and oral swab samples. The LFIA exhibited 100% specificity when testing with whole blood samples but showed reduced sensitivity, particularly for blood samples collected early or late after infection. The antigen test did not perform well with oral swabs.
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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 , Animales , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/genética , Fiebre Porcina Africana/diagnóstico , Fiebre Porcina Africana/virología , Porcinos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , ADN Viral/genética , Inmunoensayo/métodos , Antígenos Virales/análisisRESUMEN
African Swine Fever (ASF) is caused by a DNA virus (AFSV) maintained and transmitted by the Argasid ticks. The re-emergence of the disease in Africa coupled with its rapid spread globally is a threat to the pig industry, food security and livelihoods. The ecology and epidemiology of the ASFV sylvatic cycle, especially in the face of changing land use and land cover, further compounds the menace and impacts of this disease in Kenya. The study aimed to determine the occurrence and distribution of ASFV seroprevalence in warthog populations, the tick vectors and extent of tick infestation of warthog burrows, and the genotypes of ASFV in soft ticks in Kenya. Warthogs from different parts of Kenya were captured and venous blood was centrifuged to harvest sera. Warthog burrows were examined for their conditions and to extract ticks. Sera were analyzed for antibodies against ASFV using a commercial ELISA kit coated with p32 ASFV recombinant protein. Ticks were pooled, DNA extracted and the p72 gene of the ASFV was amplified by qPCR and conventional PCR. The overall seroprevalence of ASFV in warthogs was 87.5 %. A total of 228 warthog burrows were examined and 2154 argasid ticks were extracted from the burrows. Tick pools from Kigio Farm and Lewa Wildlife Conservancies were ASFV-positive by qPCR and conventional PCR. ASFV was further confirmed by the Twist Comprehensive Viral Research Panel (TCVRP), which also identified the argasid ticks as Ornithodoros porcinus. The ticks were infected with virus genotype IX, and their occurrence overlaps with regions of previous ASF outbreaks in domestic pigs. Further, Viruses that could be tick endosymbionts/commensals or due to bloodmeal were detected in ticks by TCVRP; Porcine type-C oncovirus; Pandoravirus neocaledonia; Choristoneura fumiferana granulovirus; Enterobacteria phage p7; Leporid herpesvirus 4 isolate; 5; Human Lymphotropic virus; Human herpesvirus 5. In conclusion, our results suggest that infected Ornithodoros spp. seems to have a rich virome, which has not been explored but could be exploited to inform ASF control in Kenya. Further, the ecology of Ornithodoros spp. and burrow-use dynamics are complex and more studies are needed to understand these dynamics, specifically in the spread of ASFV at the interface of wild and domestic pigs. Further, our results provide evidence of genotype IX ASFV sylvatic cycle which through O. porcinus tick transmission has resulted in high exposure of adult common warthogs. Finally, the co-circulation of ASFV genotype IX in the same location with past ASF outbreaks in domestic pigs and presently in ticks brings to focus the role of the interface and ticks on virus transmission to pigs and warthogs.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Anticuerpos Antivirales , Animales , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/fisiología , Fiebre Porcina Africana/epidemiología , Fiebre Porcina Africana/transmisión , Fiebre Porcina Africana/virología , Kenia/epidemiología , Porcinos , Estudios Seroepidemiológicos , Anticuerpos Antivirales/sangre , Genotipo , Infestaciones por Garrapatas/epidemiología , Infestaciones por Garrapatas/veterinaria , Vectores Arácnidos/virologíaRESUMEN
INTRODUCTION: The rapid spread of African swine fever in the Kaliningrad region makes it necessary to use the methods of molecular epidemiology to determine the dynamics and direction of ASF spread in this region of Russia. The aim of the study was to determine single nucleotide polymorphisms within molecular markers K145R, O174L and MGF 505-5R of ASFVs isolated in Kaliningrad region and to study the circulating of the pathogen in European countries by subgenotyping and spatio-temporal clustering analysis. MATERIALS AND METHODS: Blood samples from living domestic pigs and organs from dead domestic pigs and wild boars, collected in the Kaliningrad region between 2017 and 2022 were used. Virus isolation was carried out in porcine bone-marrow primary cell culture. Amplicons of genome markers were amplified by PCR with electrophoretic detection and subsequent extraction of fragments from agarose gel. Sequencing was performed using the Sanger method. RESULTS: The circulation of two genetic clusters of ASFV isolates on the territory of the Kaliningrad has been established: epidemic (K145R-III, MGF 505-5R-II, O174L-I - 94.3% of the studied isolates) and sporadic (K145R-II, MGF 505-5R-II, O174L-I - 5.7%). CONCLUSION: The broaden molecular genetic surveillance of ASFV isolates based on sequencing of genome markers is necessary in the countries of the Eurasian continent to perform a more detailed analysis of ASF spread between countries and within regions.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Genoma Viral , Animales , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/clasificación , Porcinos , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/epidemiología , Federación de Rusia/epidemiología , Filogenia , Polimorfismo de Nucleótido Simple , Marcadores Genéticos , Sus scrofa/virología , Análisis Espacio-TemporalRESUMEN
Owing to the lack of effective vaccines, current control measures and eradication strategies for the African swine fever virus (ASFV) rely on early detection and stringent stamping-out procedures. In the present study, we developed two independent isothermal amplification assays, namely, loop-mediated isothermal amplification (LAMP) and polymerase spiral reaction (PSR), for quick visualization of the ASFV genome in clinical samples. Additionally, a quantitative real-time PCR (qRT-PCR)-based hydrolysis probe assay was developed for comparative assessment of sensitivity with the developed isothermal assays. The analytical sensitivity of the LAMP, PSR, and qRT-PCR was found to be 2.64 ×105 copies/µL, 2.64 ×102 copies/µL, and 2.64 ×101 copies/µL, respectively. A total of 165 clinical samples was tested using the developed visual assays. The relative accuracy, relative specificity, and relative diagnostic sensitivity for LAMP vs PSR were found to be 95.37% vs 102.48%, 97.46% vs 101.36%, and 73.33% vs 113.33%, respectively.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Técnicas de Amplificación de Ácido Nucleico , Sensibilidad y Especificidad , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Animales , Técnicas de Amplificación de Ácido Nucleico/métodos , Porcinos , Fiebre Porcina Africana/diagnóstico , Fiebre Porcina Africana/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Técnicas de Diagnóstico Molecular/métodos , Genoma Viral/genéticaRESUMEN
African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a disease with detrimental effects on the health, welfare, and production of domestic and wild pigs. The ASF laboratory confirmation is based on the analysis of blood, serum and organ samples. However, testing these samples could not be always convenient, economically feasible or possible. This study describes the validation process of a PCR-based assay targeting a portion of p72 gene, used for the molecular detection of ASFV, from meat juice samples obtained from pigs succumbed to ASFV. More specifically, we investigated the capability of a real-time PCR assay to detect ASFV DNA in meat juices obtained from the diaphragmatic muscle along with the correspondent spleens of 55 ASFV-positive pigs and wild boars sampled from confirmed outbreaks in Romania and from 73 ASFV-negative and regularly slaughtered healthy pigs collected in the Abruzzo region (Italy). The test was able to detect viral DNA in both types of samples, with lower Ct values in spleens (mean=21.11, median=20.61) than meat juices (mean=23.08, median=22.40). However, distributions of Ct values were strongly correlated each other (R2= 0.83, P<0.001). Considering the distribution of the observed Ct values in the 55 positive meat juice samples, a 1:10 dilution would be able to detect 90 % of positive samples, whereas a 1:100 dilution would reduce the detectability to 78 % of more contaminated samples. As meat juice could be obtained easily from muscles and considering the potential use of this test on pooled samples, it could represent a tool to aid the investigation of ASFV spread.
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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 , Animales , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Porcinos , Fiebre Porcina Africana/diagnóstico , Fiebre Porcina Africana/virología , Rumanía , Italia , ADN Viral/genética , ADN Viral/aislamiento & purificación , Carne de Cerdo/virología , Bazo/virología , Proteínas de la CápsideRESUMEN
BACKGROUND: African swine fever (ASF) is a highly contagious and severe haemorrhagic disease of Suidae, with mortalities that approach 100 percent. Several studies suggested the potential implication of non-biting dipterans in the spread of ASFV in pig farms due to the identification of the ASFV DNA. However, to our knowledge, no study has evaluated the viral DNA load in non-biting dipterans collected in outbreak farms and no risk factors have been analysed. In this context, our study aimed to analyse the risk factors associated with the presence of non-biting dipterans collected from ASF outbreaks in relation to the presence and load of viral DNA. METHODS: Backyard farms (BF), type A farms (TAF), and commercial farms (CF), were targeted for sampling in 2020. In 2021, no BF were sampled. Each farm was sampled only once. The identification of the collected flies to family, genus, or species level was performed based on morphological characteristics using specific keys and descriptions. Pools were made prior to DNA extraction. All extracted DNA was tested for the presence of the ASFV using a real-time PCR protocol. For this study, we considered every sample with a CT value of 40 as positive. The statistical analysis was performed using Epi Info 7 software (CDC, USA). RESULTS: All collected non-biting flies belonged to five families: Calliphoridae, Sarcophagidae, Fanniidae, Drosophilidae, and Muscidae. Of the 361 pools, 201 were positive for the presence of ASFV DNA. The obtained CT values of the positive samples ranged from 21.54 to 39.63, with a median value of 33.59 and a mean value of 33.56. Significantly lower CT values (corresponding to higher viral DNA load) were obtained in Sarcophagidae, with a mean value of 32.56; a significantly higher number of positive pools were noticed in August, mean value = 33.12. CONCLUSIONS: Our study brings compelling evidence of the presence of the most common synanthropic flies near domestic pig farms carrying ASFV DNA, highlighting the importance of strengthening the biosecurity measures and protocols for prevention of the insect life cycle and distribution.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , ADN Viral , Dípteros , Brotes de Enfermedades , Granjas , Animales , 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/epidemiología , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/transmisión , Porcinos , Brotes de Enfermedades/veterinaria , ADN Viral/genética , Rumanía/epidemiología , Dípteros/virología , Dípteros/clasificación , Dípteros/genética , Insectos Vectores/virología , Insectos Vectores/clasificaciónRESUMEN
Since the confirmation of African swine fever (ASF) in South Korea in 2019, its spread, predominantly in wild boars, has been a significant concern. A key factor in this situation is the lack of identification of risk factors by surveillance bias. The unique orography, characterized by high mountains, complicates search efforts, leading to overlooked or delayed case detection and posing risks to the swine industry. Additionally, shared rivers with neighboring country present a continual threat of virus entry. This study employs geospatial analysis and statistical methods to 1) identify areas at high risk of ASF occurrence but possibly under-surveilled, and 2) indicate strategic surveillance points for monitoring the risk of ASF virus entry through water bodies and basin influences. Pearson's rho test indicated that elevation (rho = -0.908, p-value < 0.001) and distance from roads (rho = -0.979, p-value < 0.001) may have a significant impact on limiting surveillance activities. A map of potential under-surveilled areas was created considering these results and was validated by a chi-square goodness-of-fit test (X-square = 208.03, df = 1, p-value < 0.001). The strong negative correlation (rho = -0.997, p-value <0.001) between ASF-positive wild boars and distance from water sources emphasizes that areas surrounding rivers are one of the priority areas for monitoring. The subsequent hydrological analyses provided important points for monitoring the risk of virus entry via water from the neighboring country. This research aims to facilitate early detection and prevent further spread of ASF.
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Fiebre Porcina Africana , Fiebre Porcina Africana/epidemiología , Fiebre Porcina Africana/virología , Animales , Porcinos , República de Corea/epidemiología , Animales Salvajes/virología , Sus scrofa/virología , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/patogenicidad , Monitoreo Epidemiológico/veterinariaRESUMEN
The present study focuses on the pathological and molecular characterization of African swine fever virus (ASFV) associated with an outbreak in wild boars in two national parks in southern India in 2022-2023. Significant mortality was observed among free-ranging wild boars at Bandipur National Park, Karnataka, and Mudumalai National Park, Tamil Nadu. Extensive combing operations were undertaken in both national parks, spanning an area of around 100 km2, originating from the reported epicenter, to estimate the mortality rate. Recovered carcasses were pathologically examined, and ASFV isolates was genetically characterized. Our findings suggested spillover infection of ASFV from nearby domestic pigs, and the virus was equally pathogenic in wild boars and domestic pigs. ASFV intrusion was reported in the Northeastern region of the country, which borders China and Myanmar, whereas the current outbreak is very distantly located, in southern India. Molecular data will help in tracing the spread of the virus in the country.