Development of microsatellite markers for the soft tick Ornithodoros phacochoerus.

BACKGROUND: Soft ticks of the genus Ornithodoros are responsible for the maintenance and transmission of the African swine fever (ASF) virus in the sylvatic and domestic viral cycles in Southern Africa. They are also the main vectors of the Borrelia species causing relapsing fevers. Currently, no genetic markers are available for Afrotropical Ornithodoros ticks. As ASF spreads globally, such markers are needed to assess the role of ticks in the emergence of new outbreaks. The aim of this study is to design microsatellite markers that could be used for ticks of the Ornithodoros moubata complex, particularly Ornithodoros phacochoerus, to assess population structure and tick movements in ASF endemic areas. METHODS: A total of 151 markers were designed using the O. moubata and O. porcinus genomes after elimination of repeated sequences in the genomes. All designed markers were tested on O. phacochoerus and O. porcinus DNA to select the best markers. RESULTS: A total of 24 microsatellite markers were genotyped on two populations of O. phacochoerus and on individuals from four other Ornithodoros species. Nineteen markers were selected to be as robust as possible for population genetic studies on O. phacochoerus. CONCLUSIONS: The microsatellite markers developed here represent the first genetic tool to study nidicolous populations of O. phacochoerus.

Comparative assessment of two in-house-built isothermal assays for visual detection of African swine fever virus.

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.

Fangchinoline Inhibits African Swine Fever Virus Replication by Suppressing the AKT/mTOR/NF-κB Signaling Pathway in Porcine Alveolar Macrophages.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is one of the most important infectious diseases that cause high morbidity and mortality in pigs and substantial economic losses to the pork industry of affected countries due to the lack of effective vaccines. The need to develop alternative robust antiviral countermeasures, especially anti-ASFV agents, is of the utmost urgency. This study shows that fangchinoline (FAN), a bisbenzylisoquinoline alkaloid found in the roots of Stephania tetrandra of the family Menispermaceae, significantly inhibits ASFV replication in porcine alveolar macrophages (PAMs) at micromolar concentrations (IC50 = 1.66 µM). Mechanistically, the infection of ASFV triggers the AKT/mTOR/NF-κB signaling pathway. FAN significantly inhibits ASFV-induced activation of such pathways, thereby suppressing viral replication. Such a mechanism was confirmed using an AKT inhibitor MK2206 as it inhibited AKT phosphorylation and ASFV replication in PAMs. Altogether, the results suggest that the AKT/mTOR pathway could potentially serve as a treatment strategy for combating ASFV infection and that FAN could potentially emerge as an effective novel antiviral agent against ASFV infections and deserves further in vivo antiviral evaluations.

Pathogenicity and virulence of African swine fever virus.

African swine fever (ASF) is a devastating disease with a high impact on the pork industry worldwide. ASF virus (ASFV) is a very complex pathogen, the sole member of the family Asfaviridae, which induces a state of immune suppression in the host through infection of myeloid cells and apoptosis of lymphocytes. Moreover, haemorrhages are the other main pathogenic effect of ASFV infection in pigs, related to the infection of endothelial cells, as well as the activation and structural changes of this cell population by proinflammatory cytokine upregulation within bystander monocytes and macrophages. There are still many gaps in the knowledge of the role of proteins produced by the ASFV, which is related to the difficulty in producing a safe and effective vaccine to combat the disease, although few candidates have been approved for use in Southeast Asia in the past couple of years.

Geospatial analysis for strategic wildlife disease surveillance: African swine fever in South Korea (2019-2021).

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.

African swine fever virus DNA is present in non-biting flies collected from outbreak farms in Romania.

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.

An analysis of the social, cultural, and ecological factors that affect the implementation of biosecurity measures on smallholder commercial swine farms in Italy in the context of an emerging African Swine Fever outbreak.

African Swine Fever (ASF) is a contagious viral disease that infects wild and domesticated swine. In early 2022, the virus was found in wild boar in the Apennine mountains of mainland Italy.2 Since then, it has spread from wild boar to domesticated swine. To control the spread of ASF, an effective surveillance system and the implementation of strict biosecurity measures on farms are required yet are unevenly implemented across husbandry systems. Smallholder farms in particular are known to have low levels of biosecurity. In the Apennine mountains of Italy, small commercial farms have been found to have low levels of biosecurity despite being located in areas with high densities of wild boar, and, hence, being high-risk sites for potential ASF incursion and subsequent diffusion. To address the question as to why the level of biosecurity is low, interviews and participant observation were conducted with smallholder commercial farmers. The interviews identified the social, cultural, and ecological factors that affect the implementation of biosecurity measures in small commercial swine farms in the Apennines. Farmers expressed knowledge of priority biosecurity measures and an overall willingness to follow rules and regulations; however, the application of the measures in practice was uneven across farms. Economic, political, and ecological factors as well as farmer beliefs about biosecurity emerged as important factors affecting the implementation of biosecurity measures. These include economic constraints, challenges posed by the mountain environment, a shifting regulatory environment, and ideas about animal welfare. Other important factors include cultural factors such as the use of traditional agricultural methods and norms about customer access to animals, time constraints and the perceived hassle of implementing the measures, farmer age, farmer relationships with government officials and veterinarians, and the role of pigs in reducing farm waste. The study confirmed that wild boar are present in high numbers and in close proximity to smallholder commercial farms in the Apennines.

Transcriptome profiles of organ tissues from pigs experimentally infected with African swine fever virus in early phase of infection.

African swine fever, caused by African swine fever virus (ASFV), is a highly contagious and fatal disease that poses a significant threat to the global pig industry. The limited information on ASFV pathogenesis and ASFV-host interactions has recently prompted numerous transcriptomic studies. However, most of these studies have focused on elucidating the transcriptome profiles of ASFV-infected porcine alveolar macrophages in vitro. Here, we analyzed dynamic transcriptional patterns in vivo in nine organ tissues (spleen, submandibular lymph node, mesenteric lymph node, inguinal lymph node, tonsils, lungs, liver, kidneys, and heart) obtained from pigs in the early stages of ASFV infection (1 and 3 d after viremia). We observed rapid spread of ASFV to the spleen after viremia, followed by broad transmission to the liver and lungs and subsequently, the submandibular and inguinal lymph nodes. Profound variations in gene expression patterns were observed across all organs and at all time-points, providing an understanding of the distinct defence strategies employed by each organ against ASFV infection. All ASFV-infected organs exhibited a collaborative response, activating immune-associated genes such as S100A8, thereby triggering a pro-inflammatory cytokine storm and interferon activation. Functional analysis suggested that ASFV exploits the PI3K-Akt signalling pathway to evade the host immune system. Overall, our findings provide leads into the mechanisms underlying pathogenesis and host immune responses in different organs during the early stages of infection, which can guide further explorations, aid the development of efficacious antiviral strategies against ASFV, and identify valuable candidate gene targets for vaccine development.

Genetic Variations of African Swine Fever Virus: Major Challenges and Prospects.

African swine fever (ASF) is a contagious viral disease affecting pigs and wild boars. It typically presents as a hemorrhagic fever but can also manifest in various forms, ranging from acute to asymptomatic. ASF has spread extensively globally, significantly impacting the swine industry. The complex and highly variable character of the ASFV genome makes vaccine development and disease surveillance extremely difficult. The overall trend in ASFV evolution is towards decreased virulence and increased transmissibility. Factors such as gene mutation, viral recombination, and the strain-specificity of virulence-associated genes facilitate viral variations. This review deeply discusses the influence of these factors on viral immune evasion, pathogenicity, and the ensuing complexities encountered in vaccine development, disease detection, and surveillance. The ultimate goal of this review is to thoroughly explore the genetic evolution patterns and variation mechanisms of ASFV, providing a theoretical foundation for advancement in vaccine and diagnostic technologies.

The MGF300-2R Protein of African Swine Fever Virus Promotes IKKß Ubiquitination by Recruiting the E3 Ubiquitin Ligase TRIM21.

African swine fever (ASF) is an acute, hemorrhagic, highly contagious disease in pigs caused by African swine fever virus (ASFV). Our previous study identified that the ASFV MGF300-2R protein functions as a virulence factor and found that MGF300-2R degrades IKKß via selective autophagy. However, the E3 ubiquitin ligase responsible for IKKß ubiquitination during autophagic degradation still remains unknown. In order to solve this problem, we first pulled down 328 proteins interacting with MGF300-2R through immunoprecipitation-mass spectrometry. Next, we analyzed and confirmed the interaction between the E3 ubiquitin ligase TRIM21 and MGF300-2R and demonstrated the catalytic role of TRIM21 in IKKß ubiquitination. Finally, we indicated that the degradation of IKKß by MGF300-2R was dependent on TRIM21. In summary, our results indicate TRIM21 is the E3 ubiquitin ligase involved in the degradation of IKKß by MGF300-2R, thereby augmenting our understanding of the functions of MGF300-2R and offering insights into the rational design of live attenuated vaccines and antiviral strategies against ASF.

The evolutionary and genetic patterns of African swine fever virus.

African swine fever (ASF) is a serious animal disease, and has spread to Africa, Europe and Asia, causing massive economic losses. African swine fever virus (ASFV) is transmitted from a reservoir host (warthog) to domestic pigs via a sylvatic cycle (transmission between warthogs and soft ticks) and a domestic cycle (transmission between domestic pigs) and survives by expressing a variety of genes related to virus-host interactions. We evaluated differences in codon usage patterns among ASFV genotypes and clades and explored the common and specific evolutionary and genetic characteristics of ASFV sequences. We analysed the evolutionary relationships, nucleotide compositions, codon usage patterns, selection pressures (mutational pressure and natural selection) and viral adaptation to host codon usage based on the coding sequences (CDS) of key functional genes of ASFV. AT bias was detected in the six genes analysed, irrespective of clade. The AT bias of genes (A224L, A179L, EP153R) encoding proteins involved in interaction with host cells after infection was high; among them, the AT bias of EP153R was the greatest at 78.3%. A large number of overrepresented codons were identified in EP153R, whereas there were no overrepresented codons with a relative synonymous codon usage (RSCU) value of ≥3 in B646L. In most genes, the pattern of selection pressure was similar for each clade, but in EP153R, diverse patterns of selection pressure were captured within the same clade and genotype. As a result of evaluating host adaptation based on the codon adaptation index (CAI), for B646L, E183L, CP204L and A179L, the codon usage patterns in all sequences were more similar to tick than domestic pig or wild boar. However, EP153R showed the lowest average CAI value of 0.52 when selecting tick as a reference set. The genes analysed in this study showed different magnitudes of selection pressure at the clade and genotype levels, which is likely to be related to the function of the encoded proteins and may determine key evolutionary traits of viruses, such as the level of genetic variation and host range. The diversity of codon adaptations at the genetic level in ASFV may account for differences in translational selection in ASFV hosts and provides insight into viral host adaptation and co-evolution.

Identification of two novel B cell epitopes on E184L protein of African swine fever virus using monoclonal antibodies.

African swine fever virus (ASFV) is a large double-stranded DNA virus with a complex structural architecture and encodes more than 150 proteins, where many are with unknown functions. E184L has been reported as one of the immunogenic ASFV proteins that may contribute to ASFV pathogenesis and immune evasion. However, the antigenic epitopes of E184L are not yet characterized. In this study, recombinant E184L protein was expressed in prokaryotic expression system and four monoclonal antibodies (mAbs), designated as 1A10, 2D2, 3H6, and 4C10 were generated. All four mAbs reacted specifically with ASFV infected cells. To identify the epitopes of the mAbs, a series of overlapped peptides of E184L were designed and expressed as maltose binding fusion proteins. Accordingly, the expressed fusion proteins were probed with each E184L mAb separately by using Western blot. Following a fine mapping, the minimal linear epitope recognized by mAb 1A10 was identified as 119IQRQGFL125, and mAbs 2D2, 3H6, and 4C10 recognized a region located between 153DPTEFF158. Alignment of amino acids of E184L revealed that the two linear epitopes are highly conserved among different ASFV isolates. Furthermore, the potential application of the two epitopes in ASFV diagnosis was assessed through epitope-based ELISA using 24 ASFV positive and 18 negative pig serum and the method were able to distinguish positive and negative samples, indicating the two epitopes are dominant antigenic sites. To our knowledge, this is the first study to characterize the B cell epitopes of the antigenic E184L protein of ASFV, offering valuable tools for future research, as well as laying a foundation for serological diagnosis and epitope-based marker vaccine development.

High mortality in free-ranging wild boars associated with African swine fever virus in India.

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.

Characterization of an African swine fever virus outbreak in India and comparative analysis of immune genes in infected and surviving crossbreed vs. indigenous Doom pigs.

Since 2020, African swine fever (ASF) has affected all pig breeds in Northeast India except Doom pigs, a unique indigenous breed from Assam and the closest relatives of Indian wild pigs. ASF outbreaks result in significant economic losses for pig farmers in the region. Based on sequencing and phylogenetic analysis of the B646L (p72) gene, it has been determined that ASFV genotype II is responsible for outbreaks in this region. Recent studies have shown that MYD88, LDHB, and IFIT1, which are important genes of the immune system, are involved in the pathogenesis of ASFV. The differential expression patterns of these genes in surviving ASFV-infected and healthy Doom breed pigs were compared to healthy controls at different stages of infection. The ability of Doom pigs to withstand common pig diseases, along with their genetic resemblance to wild pigs, make them ideal candidates for studying tolerance to ASFV infection. In the present study, we investigated the natural resistance to ASF in Doom pigs from an endemic area in Northeast India. The results of this study provide important molecular insights into the regulation of ASFV tolerance genes.

A proposed update of African swine fever virus (genotype II) subgenotyping based on the central variable region (CVR) of Russian isolates.

African swine fever virus (ASFV) isolates are grouped and tracked through analysis of their central variable region (CVR) sequences. In this study, sequences of 70 ASFV isolates collected from different regions of Russia between 2018 and 2022 were analyzed. The analysis based on the CVR sequences indicated that the isolates belonged to three distinct groups. Group 1 shared 100% sequence identity to the isolate Georgia 2007/1. Group 5 had a C > A single-nucleotide polymorphism (SNP) at position 601, while group 13 is new and unique to the Far East of Russia, with five isolates from the Amur, Khabarovsk, and Primorsky regions. These findings demonstrate a new approach to phylogenomics and cladistics of ASFV isolates within genotype II on the basis of the CVR.

Development of novel monoclonal antibodies for blocking NF-κB activation induced by CD2v protein in African swine fever virus.

Background: CD2v, a critical outer envelope glycoprotein of the African swine fever virus (ASFV), plays a central role in the hemadsorption phenomenon during ASFV infection and is recognized as an essential immunoprotective protein. Monoclonal antibodies (mAbs) targeting CD2v have demonstrated promise in both diagnosing and combating African swine fever (ASF). The objective of this study was to develop specific monoclonal antibodies against CD2v. Methods: In this investigation, Recombinant CD2v was expressed in eukaryotic cells, and murine mAbs were generated through meticulous screening and hybridoma cloning. Various techniques, including indirect enzyme-linked immunosorbent assay (ELISA), western blotting, immunofluorescence assay (IFA), and bio-layer interferometry (BLI), were employed to characterize the mAbs. Epitope mapping was conducted using truncation mutants and epitope peptide mapping. Results: An optimal antibody pair for a highly sensitive sandwich ELISA was identified, and the antigenic structures recognized by the mAbs were elucidated. Two linear epitopes highly conserved in ASFV genotype II strains, particularly in Chinese endemic strains, were identified, along with a unique glycosylated epitope. Three mAbs, 2B25, 3G25, and 8G1, effectively blocked CD2v-induced NF-κB activation. Conclusions: This study provides valuable insights into the antigenic structure of ASFV CD2v. The mAbs obtained in this study hold great potential for use in the development of ASF diagnostic strategies, and the identified epitopes may contribute to vaccine development against ASFV.

In vitro phenotypic characterisation of two genotype I African swine fever viruses with genomic deletion isolated from Sardinian wild boars.

African swine fever virus (ASFV) causes a devastating disease affecting domestic and wild pigs. ASF was first introduced in Sardinia in 1978 and until 2019 only genotype I isolates were identified. A remarkable genetic stability of Sardinian ASFV isolates was described, nevertheless in 2019 two wild boar isolates with a sustained genomic deletion (4342 base pairs) were identified (7303WB/19, 7212WB/19). In this study, we therefore performed in vitro experiments with monocyte-derived macrophages (moMФ) to unravel the phenotypic characteristics of these deleted viruses. Both 7303WB/19 and 7212WB/19 presented a lower growth kinetic in moMФ compared to virulent Sardinian 26544/OG10, using either a high (1) or a low (0.01) multiplicity of infection (MOI). In addition, flow cytometric analysis showed that both 7303WB/19 and 7212WB/19 presented lower intracellular levels of both early and late ASFV proteins. We subsequently investigated whether deleted virus variants were previously circulating in wild boars in Sardinia. In the four years preceding the last genotype I isolation (February 2015-January 2019), other eight wild boar isolates were collected, all belonging to p72 genotype I, B602L subgroup X, but none of them presented a sustained genomic deletion. Overall, we observed the deleted virus isolates in Sardinia only in 2019, at the end of a strong eradication campaign, and our data suggest that it might possess an attenuated phenotype in vivo. A better understanding of ASFV evolution in endemic territories might contribute to development of effective control measures against ASF.

Novel estimation of African swine fever transmission parameters within smallholder villages in Lao P.D.R.

African Swine Fever (ASF) disease transmission parameters are crucial for making response and control decisions when faced with an outbreak, yet they are poorly quantified for smallholder and village contexts within Southeast Asia. Whilst disease-specific factors - such as latent and infectious periods - should remain reasonably consistent, host, environmental and management factors are likely to affect the rate of disease spread. These differences are investigated using Approximate Bayesian Computation with Sequential Monte-Carlo methods to provide disease parameter estimates in four naïve pig populations in villages of Lao People's Democratic Republic. The villages represent smallholder pig farmers of the Northern province of Oudomxay and the Southern province of Savannakhet, and the model utilised field mortality data to validate the transmission parameter estimates over the course of multiple model generations. The basic reproductive number between-pigs was estimated to range from 3.08 to 7.80, whilst the latent and infectious periods were consistent with those published in the literature for similar genotypes in the region (4.72 to 6.19 days and 2.63 to 5.50 days, respectively). These findings demonstrate that smallholder village pigs interact similarly to commercial pigs, however the spread of disease may occur slightly slower than in commercial study groups. Furthermore, the findings demonstrated that despite diversity across the study groups, the disease behaved in a consistent manner. This data can be used in disease control programs or for future modelling of ASF in smallholder contexts.

Infection of domestic pigs with a genotype II potent strain of ASFV causes cytokine storm and lymphocyte mass reduction.

The whole-genome sequence of an African swine fever virus (ASFV) strain (HuB/HH/2019) isolated from Hubei, China, was highly similar to that of the Georgia 2007/1 strain ASFV. After infection with strong strains, domestic pigs show typical symptoms of infection, including fever, depression, reddening of the skin, hemorrhagic swelling of various tissues, and dysfunction. The earliest detoxification occurred in pharyngeal swabs at 4 days post-infection. The viral load in the blood was extremely high, and ASFV was detected in multiple tissues, with the highest viral loads in the spleen and lungs. An imbalance between pro- and anti-inflammatory factors in the serum leads to an excessive inflammatory response in the body. Immune factor expression is suppressed without effectively eliciting an immune defense. Antibodies against p30 were not detected in acutely dead domestic pigs. Sequencing of the peripheral blood mononuclear cell transcriptome revealed elevated transcription of genes associated with immunity, defense, and stress. The massive reduction in lymphocyte counts in the blood collapses the body's immune system. An excessive inflammatory response with a massive reduction in the lymphocyte count may be an important cause of mortality in domestic pigs. These two reasons have inspired researchers to reduce excessive inflammatory responses and stimulate effective immune responses for future vaccine development.

Saccharomyces cerevisiae oral immunization in mice using multi-antigen of the African swine fever virus elicits a robust immune response.

African swine fever virus (ASFV) is one of the most complex viruses. ASFV is a serious threat to the global swine industry because no commercial vaccines against this virus are currently available except in Vietnam. Moreover, ASFV is highly stable in the environment and can survive in water, feed, and aerosols for a long time. ASFV is transmitted through the digestive and respiratory tract. Mucosal immunity is the first line of defense against ASFV. Saccharomyces cerevisiae (SC), which has been certified by the U.S. Food and Drug Administration and has a generally recognized as safe status in the food industry, was used for oral immunization in this study. ASFV antigens were effectively expressed in recombinant SC strains with high DNA copy numbers and stable growth though surface display technology and chromosome engineering (δ-integration). The recombinant SC strains containing eight ASFV antigens-KP177R, E183L, E199L, CP204L, E248R, EP402R, B602L, and B646L- induced strong humoral and mucosal immune responses in mice. There was no antigenic competition, and these antigens induced Th1 and Th2 cellular immune responses. Therefore, the oral immunization strategy using recombinant SC strains containing multiple ASFV antigens demonstrate potential for future testing in swine, including challenge studies to evaluate its efficacy as a vaccine against ASFV.