Detection Of African Swine Fever Virus Research Articles

Field Verification of an African Swine Fever Virus Loop-Mediated Isothermal Amplification (LAMP) Assay During an Outbreak in Timor-Leste.

Recent outbreaks of African swine fever virus (ASFV) have seen the movement of this virus into multiple new regions with devastating impact. Many of these outbreaks are occurring in remote, or resource-limited areas, that do not have access to molecular laboratories. Loop-mediated isothermal amplification (LAMP) is a rapid point of care test that can overcome a range of inhibitors. We outline further development of a real-time ASFV LAMP, including field verification during an outbreak in Timor-Leste. To increase field applicability, the extraction step was removed and an internal amplification control (IAC) was implemented. Assay performance was assessed in six different sample matrices and verified for a range of clinical samples. A LAMP detection limit of 400 copies/rxn was determined based on synthetic positive control spikes. A colourmetric LAMP assay was also assessed on serum samples. Comparison of the LAMP assay to a quantitative polymerase chain reaction (qPCR) was performed on clinical ASFV samples, using both serum and oral/rectal swabs, with a substantial level of agreement observed. The further verification of the ASFV LAMP assay, removal of extraction step, implementation of an IAC and the assessment of a range of sample matrix, further support the use of this assay for rapid in-field detection of ASFV.

Direct colorimetric LAMP assay for rapid detection of African swine fever virus: A validation study during an outbreak in Vietnam

African swine fever (ASF) is a highly infectious viral disease with high mortality. The most recent ASF outbreak in Vietnam began in 2019, posing a threat to spread to the neighbouring Asian countries. Without a commercial vaccine or efficient chemotherapeutics, rapid diagnosis and necessary biosecurity procedures are required to control the disease. While the diagnostic method of ASF recommended by the World Organization of Animal Health is real-time PCR, the ideal diagnosis procedure including master mix setup, template extraction and a high-cost qPCR equipment for many samples being tested simultaneously is not portable. In this study, a colorimetric loop-mediated isothermal amplification (LAMP) assay was modified and evaluated for ASF virus detection using crude serum samples collected from domestic pigs in Vietnam during the 2019 outbreak. The LAMP results can be readily visualized to the naked eye within 30min without the requirement of DNA extraction and sophisticated equipment. The sensitivity, specificity and limit of detection of direct colorimetric LAMP assay were comparable to a commercial diagnostic real-time PCR kit. Results strongly indicate that the adapted colorimetric LAMP assay has a remarkable potential for the in-field diagnosis of ASF.

Development a multiplex RT-PCR assay for simultaneous detection of African swine fever virus, classical swine fever virus and atypical porcine pestivirus

African swine fever virus (ASFV), classical swine fever virus (CSFV) and atypical porcine pestivirus (APPV) have caused considerable financial losses to the pig industry worldwide, and it is critical to achieve early and accurate diagnosis of these viruses to control the diseases induced by them. In this study, three pairs of specific primers were designed based on the highly conserved genome regions of these viruses, and a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay for ASFV, CSFV and APPV was established after various reaction conditions were optimized. The mRT-PCR assay consisted of two steps, that is, reverse transcription (RT) and mPCR. The assay was highly specific, sensitive, and reproducible for ASFV, CSFV and APPV without cross-reaction with other swine pathogens. The sensitivity of this assay, which used purified plasmid constructs containing specific viral target fragments as templates, was 6.34 × 102 copies/μL for ASFV and 6.34 × 101 copies/μL for both CSFV and APPV. A total of 384 clinical samples from piglets suspected to be infected in Guangxi Province, Southern China, during 2018−2019 were analyzed by the established mRT-PCR method. The results showed that the positive rates of ASFV, CSFV and APPV were 43.75 %, 13.28 % and 4.17 %, respectively, and the coinfection rates of ASFV/CSFV, ASFV/APPV and CSFV/APPV were 5.47 %, 1.83 % and 1.30 %, respectively. To understand the epidemiological characteristics of APPV, the newly discovered virus, in Guangxi Province, the clinical samples from APPV-positive animals were selected randomly for amplification and sequencing, and the complete genomic sequences of four APPV strains were obtained. Phylogenetic analysis demonstrated that APPV strains from Guangxi Province had a high degree of genetic diversity. This study provides an important tool for rapid detection and accurate diagnosis of ASFV, CSFV and APPV.

Multiplex and visual detection of African Swine Fever Virus (ASFV) based on Hive-Chip and direct loop-mediated isothermal amplification

African swine fever is caused by African swine fever virus (ASFV), and has a mortality rate approaching 100%. It has already caused tremendous economy lost around the world. Without effective vaccine, rapid and accurate on-site detection plays an indispensable role in controlling outbreaks. Herein, by combining Hive-Chip and direct loop-mediated isothermal amplification (LAMP), we establish a multiplex and visual detection platform. LAMP primers targeting five ASFV genes (B646L, B962L, C717R, D1133L, and G1340L) were designed and pre-fixed in Hive-Chip. On-chip LAMP showed the limits of detection (LOD) of ASFV synthetic DNAs and mock samples are 30 and 50 copies per microliter, respectively, and there is no cross-reaction among the target genes. The overall performance of our platform is comparable to that of the commercial kits. From sample preparation to results readout, the entire process takes less than 70 min. Multiplex detection of real samples of ASFV and other swine viruses further demonstrates the high sensitivity and specificity of Hive-Chip. Overall, our platform provides a promising option for on-site, fast and accurate detection of ASFV.

Development and clinical application of a novel CRISPR-Cas12a based assay for the detection of African swine fever virus

BackgroundAs no treatment or effective vaccine for African swine fever virus (ASFV) is currently available, a rapid, highly sensitive diagnostic is urgently needed to curb the spread of ASFV.ResultsHere we designed a novel CRISPR-Cas12a based assay for ASFV detection. To detect different ASFV genotypes, 19 crRNAs were designed to target the conserved p72 gene in ASFV, and several crRNAs with high activity were identified that could be used as alternatives in the event of new ASFV variants. The results showed that the sensitivity of the CRISPR-Cas12a based assay is about ten times higher than either the commercial quantitative PCR (qPCR) kit or the OIE-recommended qPCR. CRISPR-Cas12a based assay could also detect ASFV specifically without cross-reactivity with other important viruses in pigs and various virus genotypes. We also found that longer incubation times increased the detection limits, which could be applied to improve assay outcomes in the detection of weakly positive samples and new ASFV variants. In addition, both the CRISPR-Cas12a based assay and commercial qPCR showed very good consistency.ConclusionsIn summary, the CRISPR-Cas12a based assay offers a feasible approach and a new diagnostic technique for the diagnosis of ASFV, particularly in resource-poor settings.

Towards a Sampling Rationale for African Swine Fever Virus Detection in Pork Products.

African swine fever (ASF) is a highly lethal disease of pigs caused by the ASF virus (ASFV), which presents a serious threat to global food security. The movement of contaminated pork products has previously been postulated as contributing to the introduction of ASF into new areas. To evaluate the performance of ASFV detection systems in multi-component pork products, we spiked sausage meat with four different ASFV-containing materials (ASFV cell culture, pork loin, meat juice and bone marrow). DNA was extracted using two manual systems (MagMAX CORE, Qiagen) and one automated (MagMAX CORE) one, and three qPCR assays (VetMAX, King, UPL) were used. The performance of the DNA extraction systems was as follows; automated MagMAX > manual MagMAX > manual Qiagen. The commercial VetMAX qPCR assay yielded significantly lower CT values (p < 0.001), showing greater sensitivity than the World Organization for Animal Health (OIE)-prescribed assays (King, UPL). Detection probability was the highest for matrices contaminated with bone marrow compared with pork loin or meat juice. An estimated minimum sample size of one 1-g sample is sufficient to detect ASFV in a homogenous pork product if bone marrow from infected pigs comprises 1 part in 10,000. We demonstrated that existing ASFV detection systems are appropriate for use in a food-testing capacity, which can provide an additional control measure for ASF.

Rapid and semi-quantitative colorimetric loop-mediated isothermal amplification detection of ASFV via HSV color model transformation.

African Swine Fever (ASF) is a highly contagious and lethal viral disease of swine, the presence of which in groups of pigs leads to enormous economic losses in the farming industry. However, vaccines and drugs to treat ASF have yet to be developed. To control the spread of the African Swine Fever Virus (ASFV), a diagnostic method that can be applied rapidly and can detect the disease during the early stages of infection is urgently needed. In this study, we demonstrate a rapid and easy-to-use ASFV detection method that combines loop-mediated isothermal amplification (LAMP) and image processing with the hue-saturation-value (HSV) color model. This method was validated through use of synthetic ASFV DNA. The method shows high sensitivity, as it detects as few as 10 copies per reaction within 20min. The speed and sensitivity of this newly developed assay are superior to those reported in previous studies. In addition, through HSV color space transformation, the colorimetric result of this LAMP assay can be used for a semi-quantitative analysis for ASFV (ranging from 108 to 101 copies per reaction) and improve the discern to low concentration samples from a negative control. These results show that the combination of ASFV-LAMP assay and HSV color space transformation may accelerate the screening process of pigs for ASFV infection. Overall, this study provides a rapid, sensitive, early-stage, on-site diagnosis of ASFV infection and has potential to be applied to other infectious diseases.

Application of CRISPR-Cas12a Enhanced Fluorescence Assay Coupled with Nucleic Acid Amplification for the Sensitive Detection of African Swine Fever Virus.

African swine fever (ASF) is one of the most severe diseases of pigs. In this study, a CRISPR-Cas12a (also known as Cpf1) system coupled with nucleic acid amplification was optimized for the detection of ASF virus (ASFV). Two novel single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters were developed to increase the brightness of the fluorescent signal for the visualization of nucleic acid detection. The CRISPR-Cas12a system was used to simultaneously cleave the polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP) amplicons and the newly developed ssDNA-FQ reporter, resulting in fluorescence that could be easily detected in multiple platforms, especially on cheap and portable blue or UV light transilluminators. This specific cleavage with fluorescence reveals the presence of the amplicon and confirms its identity, thereby preventing false-positive test results from nonspecific amplicons. This method is also uninterfered by the presence of large amounts of irrelevant background DNA and displays no cross-reactivity with other porcine DNA or RNA viruses. When coupled with LAMP, the Cas12a platform can detect a plasmid containing p72 with as few as 2 copies/μL reaction. Our results indicate that the CRISPR-Cas12a enhanced fluorescence assay coupled with nucleic acid amplification is robust, convenient, specific, confirmatory, affordable, and potentially adaptable for ASF diagnosis.

A visual on‐site method for African swine fever virus detection in raw pig tissues

AbstractThe spread of African swine fever virus (ASFV) leads to high mortality in domestic pigs, and has caused huge losses in the global pork industry. Therefore, the rapid detection of ASFV is essential for the prevention and control of the epidemic. Herein, a denaturation bubble‐mediated strand exchange amplification (SEA) with colorimetric detection strategy for the rapid detection of ASFV was established and the results were visible with the naked eyes. The entire detection process based on isothermal amplification was performed on a portable metal bath only. And the whole process including rapid extraction and visual detection was finished within 50 min. The limit of detection of this method reached to 104 copies/μl of plasmid containing p72 gene. And the ASFV was successfully detected in eight different tissue and seven types of pig feed which revealed a strong anti‐interference ability in the presence of complex matrix. Through comparing 94 field positive and negative ASFV samples, excellent performance of SEA assay with 92.86% sensitivity and 100% specificity relative to PCR was observed. The developed method could be used for on‐site detection of ASFV with simplicity, rapidity, and visuality, especially suitable for pork industry food safety and ASFV control in resource‐limited areas.

Identification of a Continuously Stable and Commercially Available Cell Line for the Identification of Infectious African Swine Fever Virus in Clinical Samples.

African swine fever virus (ASFV) is causing outbreaks both in domestic pigs and wild boar in Europe and Asia. In 2018, the largest pig producing country, China, reported its first outbreak of African swine fever (ASF). Since then, the disease has quickly spread to all provinces in China and to other countries in southeast Asia, and most recently to India. Outbreaks of the disease occur in Europe as far west as Poland, and one isolated outbreak has been reported in Belgium. The current outbreak strain is highly contagious and can cause a high degree of lethality in domestic pigs, leading to widespread and costly losses to the industry. Currently, detection of infectious ASFV in field clinical samples requires accessibility to primary swine macrophage cultures, which are infrequently available in most regional veterinary diagnostic laboratories. Here, we report the identification of a commercially available cell line, MA-104, as a suitable substrate for virus isolation of African swine fever virus.

Clinical Validation of Two Recombinase-Based Isothermal Amplification Assays (RPA/RAA) for the Rapid Detection of African Swine Fever Virus.

African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating infectious disease of domestic pigs and wild boars, and has tremendous negative socioeconomic impact on the swine industry and food security worldwide. It is characterized as a notifiable disease by World Organisation for Animal Health (OIE). No effective vaccine or treatment against ASF has so far been available. Early detection and rapid diagnosis are of potential significance to control the spread of ASF. Recombinase-based isothermal amplification assay, recombinase polymerase amplification (RPA) developed by TwistDx (Cambridge, United Kingdom) or recombinase-aided amplification (RAA) by Qitian (Wuxi, China), is becoming a molecular tool for the rapid, specific, and cost-effective identification of multiple pathogens. In this study, we aim to investigate if RPA/RAA can be a potential candidate for on-site, rapid and primary detection of ASFV. A panel of 152 clinical samples previously well-characterized by OIE-recommended qPCR was enrolled in this study, including 20 weak positive (Ct value ≥ 30) samples. This panel was consisted of different types, such as EDTA-blood, spleen, lung, lymph node, kidney, tonsil, liver, brain. We evaluated two recombinase-based isothermal amplification assays, RPA or RAA, by targeting the ASFV B646L gene (p72), and validated the clinical performance in comparison with OIE real-time PCR. Our result showed that the analytical sensitivity of RPA and RAA was as 93.4 and 53.6 copies per reaction, respectively at 95% probability in 16 min, at 39°C. They were universally specific for all 24 genotypes of ASFV and no cross reaction to other pathogens including Classical swine fever virus (CSV), Foot-and-mouth disease virus (FMDV), Pseudorabies virus, Porcine circovirus 2 (PCV2), Porcine Reproductive and respiratory syndrome virus (PPRSV). The results on detection of various kinds of clinical samples indicated an excellent diagnostic agreement between RPA, RAA and OIE real-time PCR method, with the kappa value of 0.960 and 0.973, respectively. Compared to real-time PCR, the specificity of both RPA and RAA was 100% (94.40% ∼ 100%, 95% CI), while the sensitivity was 96.59% (90.36% ∼ 99.29%, 95% CI) and 97.73% (92.03% ∼ 99.72%, 95% CI), respectively. Our data demonstrate that the developed recombinase-based amplification assay (RPA/RAA), promisingly equipped with field-deployable instruments, offers a sensitive and specific platform for the rapid and reliable detection of ASFV, especially in the resource-limited settings for the purpose of screening and surveillance of ASF.

Stability of African swine fever virus on heat-treated field crops.

African swine fever (ASF) is an infectious disease of pigs and represents a massive threat to animal health and the pig industry worldwide. The ASF virus (ASFV) is efficiently transmitted via blood and meat from infected animals and can be highly stable in the environment. There is therefore great concern about the potential role of contaminated raw materials used for feed or bedding in the spread of ASFV. Especially crops and derived products originating from areas with ASF in wild boar and thus with high environmental ASFV contamination may be a risk for virus introduction into domestic pig herds. However, little is known about the stability of ASFV on contaminated crops and possible inactivation methods. In this study, we tested the effect of drying and heat treatment on the inactivation of ASFV on six different types of field crops, namely wheat, barley, rye, triticale, corn, and peas, contaminated with infectious blood. Samples were analysed for the presence of viral DNA and infectious virus after 2hr drying at room temperature or after drying and 1hr exposure to moderate heat at a specific temperature between 40°C and 75°C. ASFV genome was detected in all samples by real-time polymerase chain reaction (PCR), including samples that had been dried for 2hr and incubated for 1hr at 75°C. On the other hand, no infectious virus could be detected after 2hr drying using virus isolation in porcine macrophages in combination with the detection of ASFV by the haemadsorption test (HAT). We therefore conclude that the risk of ASFV transmission via contaminated crops is most likely low, if they are incubated for at least 2hr minimum at room temperature. Nonetheless, to minimize the risk of transmission as much as possible crops from ASF-affected zones should not be used for pig feed.

A recombinase polymerase amplification combined with lateral flow dipstick for rapid and specific detection of African swine fever virus

African swine fever (ASF) is an acute, hemorrhagic, highly contagious disease caused by African swine fever virus (ASFV) infection of domestic pigs and wild boars, showing mortality rates up to 100 %. There are no effective vaccines or antiviral drugs available for ASFV. Therefore, disease control is mainly based on animal slaughtering and the enforcement of strict sanitary measures. In order to establish a rapid, sensitive and simple method for on-site detection of ASFV, a recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD) was developed using a pair of specific primers and probe. Using recombinant plasmid pMD19-T-K205R DNA as a template, the RPA-LFD detection could be accomplished in 10 min at a temperature of 36℃-44℃. More specific than PCR and more rapid and simpler than real-time PCR, RPA-LFD has the same detection limit of 1 × 102 copies/reaction as real-time PCR, also with no cross-reaction with other viral strains. A convenient and rapid ASFV RPA-LFD detection method was developed, which will provide an efficient method for investigating epidemiology of ASFV infection.

Rapid on-site detection of African swine fever virus using polymerase chain reaction with a lateral flow strip

African swine fever (ASF) is a highly contagious disease caused by African swine fever virus (ASFV) in domestic pigs and wild boars, which results in huge economic losses to the pig industry. Because of its extremely infectious capacity, there is an urgent need to establish a convenient and low-cost diagnostic method for rapid and on-site detection of the virus to timely implement the control measures. In this study, a polymerase chain reaction (PCR) in combination with lateral flow strip (LFS), targeting the major structural protein p72 gene, was established for rapid detection of ASFV. The PCR-LFS assay displayed no cross-reactivity to other swine viruses. The sensitivity of this assay was 15 copies/reaction of ASFV plasmid template. In clinical diagnoses of 300 plasmid-spiked samples, the detection rate of the diagnosis of ASFV by PCR-LFS assay, OIE-validated real-time PCR assay, and commercial kit reached 36.33% (109/300), 26.67% (80/300) and 37.33% (112/300) respectively. The coincidence rate of PCR-LFS assay and OIE-validated real-time PCR assay was 89.67% (269/300), with a kappa value of 0.763 (p < 0.001) and that of PCR-LFS and commercial kit was 97.67% (293/300), with a kappa value of 0.950 (p < 0.001). The entire procedure, from specimen processing to result reporting, can be completed within 1 hr. Our results demonstrated that the established PCR-LFS assay is suitable for on-site detection of ASFV.

Evaluation of seven commercial African swine fever virus detection kits and three Taq polymerases on 300 well-characterized field samples

African swine fever virus (ASFV) is a complex double stranded DNA virus, responsible for a highly infectious and fatal disease in pigs and boars and for important deterioration of animal welfare. Over the last decade, the disease spread to several European and Asian countries causing unprecedented dramatic economic losses in pig industry. In the absence of a vaccine, affected countries rely on trustful diagnostic tests and adapted testing policies to set up control programs to fight against the disease. In this study, we evaluated the sensitivity and specificity of seven commercially available ASFV real-time PCR detection kits and three Taq polymerases on 300 well-characterized wild boar samples collected in Belgium during the 2018–2019 outbreak. This study confirms that all commercial kits and two Taq polymerases are suitable for ASFV detection in diagnostic laboratories. Furthermore, the use of endogenous controls is emphasized when testing field samples harvested on carcasses in an advanced stage of decomposition, in order to avoid false negative results.

Rapid detection of African swine fever virus using Cas12a-based portable paper diagnostics

African swine fever virus (ASFV) is a dsDNA virus responsible for a severe, highly contagious, and lethal disease affecting both domestic and wild pigs. ASFV has brought enormous economic loss to a number of countries, and effective vaccine and therapy are still lacking. Therefore, a rapid, sensitive, and field-deployable detection of ASFV is important for disease surveillance and control. Herein, we developed a Cas12a-mediated portable paper assay to rapidly and precisely detect ASFV. We identified a robust set of crRNAs that recognized the highly conserved region of essential ASFV genes. The Cas12a-mediated detection assay showed low tolerance for mismatch mutations, and no cross-reactivity against other common swine pathogens. We further developed a paper-based assay to allow instrument-free detection of ASFV. Specifically, we applied gold nanoparticle–antibody conjugate to engineer homemade strips and combined it with Cas12a-mediated ASFV detection. This portable paper, instrument-free diagnostics, faithfully detected ASFV in swine samples, showing comparable sensitivity to the traditionally instrument-dependent qPCR method. Taking together, we developed a highly sensitive, instant, and economic Cas12a-mediated paper diagnostics of ASFV, with a great application potential for monitoring ASFV in the field.

Evaluation of different methods for preventing the African swine fever virus

African swine fever (ASF) is a contagious disease of key importance that occurs in wild boar population and domestic pigs. To date, there is no vaccine or treatment available, hence disease surveillance and stamping out in case of an outbreak is the only way to control it. A duplex ASF-IC real-time PCR assay was optimised using field samples obtained in Lithuania and results of singleplex and duplex real-time PCR assays allowing the identification of the ASF virus were compared. The specificity and sensitivity of ASF virus detection was validated using a reference sample of positive and negative virus nucleic acid and ASF virus positive and negative samples obtained from animals in Lithuania. Results prove that duplex ASF-IC real-time PCR assay provides a rapid, sensitive, and reliable molecular tool for ASF virus detection in wild boar population and domestic pigs. In general, it was found that duplex ASF-IC real-time PCR assay proves to be faster and more sensitive than singleplex real-time PCR and therefore has a high potential to be applied in routine analysis.

CRISPR/Cas12a technology combined with immunochromatographic strips for portable detection of African swine fever virus

African swine fever virus (ASFV), the aetiological agent of African swine fever (ASF), causes lethal haemorrhagic fever in domestic pigs with high mortality and morbidity and has devastating consequences on the global swine industry. On-site rapid and sensitive detection of ASFV is key to the timely implementation of control. In this study, we developed a rapid, sensitive and instrument-free ASFV detection method based on CRISPR/Cas12a technology and lateral flow detection (named CRISPR/Cas12a-LFD). The limit of detection of CRISPR/Cas12a-LFD is 20 copies of ASFV genomic DNA per reaction, and the detection process can be completed in an hour. The assay showed no cross-reactivity with other swine DNA viruses, and has 100% agreement with real-time PCR detection of ASFV in 149 clinical samples. Overall, the CRISPR/Cas12a-LFD method provides a novel alternative for the portable, simple, sensitive, and specific detection of ASFV and may contribute to the prevention and control of ASF outbreaks.

High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system

Here we report the development of a high throughput, all-solution phase, and isothermal detection system for African Swine Fever Virus (ASFV). CRISPR-Cas12a programmed with a CRISPR RNA (crRNA) is used to detect ASFV target DNA. Upon ASFV DNA binding, the Cas12a/crRNA/ASFV DNA complex becomes activated and degrades a fluorescent single stranded DNA (ssDNA) reporter present in the assay. We combine this powerful CRISPR-Cas assay with a fluorescence-based point-of-care (POC) system for rapid and accurate virus detection. Without nucleic acid amplification, a detection limit of 1 pM is achieved within 2 h. In addition, the ternary Cas12a/crRNA/ASFV DNA complex is highly stable at physiological temperature and continues to cleave the ssDNA reporter even after 24 h of incubation, resulting in an improved detection limit of 100 fM. We show that this system is very specific and can differentiate nucleic acid targets with closely matched sequences. The high sensitivity and selectivity of our system enables the detection of ASFV in femtomolar range. Importantly, this system features a disposable cartridge and a sensitive custom designed fluorometer, enabling compact and simple ASFV detection, intended for low resource settings.

African swine fever in Benin and prevalence of the disease in Southern Benin: A retrospective study (2014-2018).

Objective:This study aimed to assess the prevalence and risk factors of African swine fever (ASF) disease in Benin.Materials and methods:A retrospective study was conducted on 70 pig farms from the Departement of Atlantique and Ouémé and also by using the data available from the Directorate of Livestock on the spread of ASF in Benin from 2014 to 2018. The prevalence of ASF was assessed with 106 nasal swabs from apparently healthy domestic pigs and 15 organ samples from dead ASF-suspected pigs. ASF virus detection was carried out by conventional polymerase chain reaction using Qiagen Kit for DNA extraction. Data recorded were processed with SAS software (2006).Results:It appears that ASF is an endemic disease in Benin with the Department of Ouémé as the hotspot of dissemination of the virus in the country. The losses due to ASF recorded from 2014 to 2018 are evaluated to 884,850,000 CFA Franc by estimating the average cost of a pig at 25,000 FCFA. A prevalence of 1.89% (CI at 95%, 0.71–3.49) was recorded for live animals with a positive result in organs from all dead pigs suspected of ASF. Breeding practices related to the sharing of breeding males, scavenging pigs, and non-compliance with biosecurity measures were the risk factors identified.Conclusion:The present study sheds light on the areas prone to the ASF virus in Benin. Moreover, the cross-sectional data recorded on the prevalence of ASF will help to better rule on the spread of the disease. It would be interesting for the Beninese Republic to increase its efforts for ASF control.