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African swine fever virus (ASFV) is a large, icosahedral, double-stranded DNA virus in the Asfarviridae family and the causative agent of African swine fever (ASF). ASFV causes a hemorrhagic fever with high mortality rates in domestic and wild pigs. ASFV contains an open reading frame named EP152R, previous research has shown that EP152R is an essential gene for virus rescue in swine macrophages. However, the detailed functions of ASFV EP152R remain elusive. Herein, we demonstrate that EP152R, a membrane protein located in the endoplasmic reticulum (ER), induces ER stress and swelling, triggering the PERK/eIF2α pathway, and broadly inhibiting host protein synthesis in vitro. Additionally, EP152R strongly promotes immune evasion, reduces cell proliferation, and alters cellular metabolism. These results suggest that ASFV EP152R plays a critical role in the intracellular environment, facilitating viral replication. Furthermore, virus-level experiments have shown that the knockdown of EP152R or PERK inhibitors efficiently affects viral replication by decreasing viral gene expression. In summary, these findings reveal a series of novel functions of ASFV EP152R and have important implications for understanding host-pathogen interactions., (© 2024 Federation of American Societies for Experimental Biology.)

African swine fever (ASF) is an acute and severe infectious disease caused by the African Swine Fever Virus (ASFV). ASFV exhibits significant resistance and stability in the environment, which, coupled with its double-stranded DNA and large genome, predisposes it to contaminate laboratory samples. This characteristic can lead to false-positive results in swine farm settings even days after disinfection, as detectable through polymerase chain reaction (PCR) or real-time fluorescent quantitative PCR (qPCR) assays. To meet the demand for efficient clinical methods capable of discriminating between ASFV nucleic acid and ASFV virions, this study aims to ascertain the efficacy of the nuclease "BenzoNuclease" in distinguishing ASFV nucleic acid (ASFV-DNA) from ASFV virions. BenzoNuclease is a versatile nucleic acid enzyme with the capacity to degrade nearly all forms of DNA and RNA. Initially, this research established a highly sensitive general PCR detection method for ASFV. Subsequently, a positive control was constructed using the M13 bacteriophage to substitute for active ASFV, facilitating the development of an improved qPCR method. It is important to note that common disinfectants have the potential to deactivate BenzoNuclease. However, in an environment simulating actual production applications, residual disinfectants do not interfere with the enzymatic efficacy of BenzoNuclease, thus not affecting the detection capabilities of this method. Positive clinical samples from pig farms, upon testing with the improved method, revealed that three samples were positive, indicating the presence of viral particles, while the remaining samples were negative, indicating the presence of nucleic acids. This provides an additional new option for sample testing in pig farms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

African swine fever (ASF) is a contagious and fatal disease caused by the African swine fever virus (ASFV), which can infect pigs of all breeds and ages. Most infected pigs have poor prognosis, leading to substantial economic losses for the global pig industry. Therefore, it is imperative to develop a safe and efficient commercial vaccine against ASF. The development of ASF vaccine can be traced back to 1960. However, because of its large genome, numerous encoded proteins, and complex virus particle structure, currently, no effective commercial vaccine is available. Several strategies have been applied in vaccine design, some of which are potential candidates for vaccine development. This review provides a comprehensive analysis on the safety and effectiveness, suboptimal immunization effects at high doses, absence of standardized evaluation criteria, notable variations among strains of the same genotype, and the substantial impact of animal health on the protective efficacy against viral challenge. All the information will be helpful to the ASF vaccine development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The outbreak and spread of African swine fever virus (ASFV) have caused considerable economic losses to the pig industry worldwide. Currently, to promote the development of effective ASF vaccines, especially subunit vaccines, more antigenic protein targets are urgently needed. In this work, six transmembrane proteins (I329L, E146L, C257L, EP153R, I177L, and F165R) were expressed in mammalian cell lines and screened with pig anti-ASFV serum. It was found that the E146L protein was an immunodominant protein antigen among the six selected proteins. Moreover, the E146L protein induced antibody responses in all immunized pigs. To gain insight into the antigenic characteristics of the E146L protein, three monoclonal antibodies (mAbs; 12H12, 15G1, and 15H10) were generated by immunizing BALB/c mice with the purified E146L protein. The epitopes of the mAbs were further finely mapped through a peptide fusion protein expression strategy. Finally, the epitopes of the mAbs were identified as 48 PDESSIAYMRFRN 61 of the mAb 12H12, 138 TLTGLQRII 146 of the mAb 15G1, and 30 GWSPFKYSKGNT 41 of the mAb 15H10. Furthermore, the epitope of mAb 15H10 was validated as the immunodominant epitope with ASFV-infected pig sera. The chemically synthesized mAb 15H10 epitope peptide (EP1) exhibited the most extensive immunoreactivity with artificially or naturally ASFV-infected pig sera. The epitope 15H10 is located on the surface of the E146L protein and is highly conserved. These findings provide insight into the structure and function of the E146L protein of ASFV., Competing Interests: Declarations Animal ethics All the experiments were performed strictly in accordance with the guidelines of care and use of laboratory animals by the Ministry of Science and Technology of the People’s Republic of China. The mouse and pig experimental protocols were approved by the Harbin Veterinary Research Institute (approval number: mouse experiment, 220110-02-GR; pig experiment, 230414-01-SW). All animals used in this study were carefully fed, and the suffering of the animals was minimized. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

In 2018, African swine fever virus (ASFV) emerged in China, causing extremely serious economic losses to the domestic pig industry. Infection with ASFV can cause disseminated coagulation, leading to the consumption of platelets and coagulation factors and severe bleeding. However, the mechanism of virus-induced coagulation has yet to be established. In our study, ASFV downregulated the coagulation process, as detected by D-dimer (D2D) and Factor X (F10) expression in pigs challenged with ASFV HLJ/18. In vitro, ASFV infection increased Factor IX (F9) and Factor XII (F12) expression while downregulating F10 expression in porcine alveolar macrophages (PAMs). African swine fever virus induced both intrinsic and extrinsic coagulation cascades. In addition, several encoded proteins affect the expression of the crucial coagulation protein F10, and among the encoded proteins, p72 inhibits the activity and expression of F10. Proteomic analysis also revealed that p72 is involved in the coagulation cascade. p72 can interact with F10, and its inhibitory functional domains include amino acids 423-432 and amino acids 443-452. Finally, we found that F10 and p72 interact with tripartite motif-containing protein 28 (TRIM28). TRIM28 knockdown resulted in a decrease in F10 expression. Importantly, TRIM28 contributes to the reduction in F10 protein expression regulated by p72. Our findings revealed an inhibitory effect of the viral protein p72 on the ASFV infection-induced coagulation cascade and revealed a role of TRIM28 in reducing F10 expression, revealing a molecular mechanism of ASFV-associated coagulation., Competing Interests: Declarations Ethics approval and consent to participate All animal experiments were performed according to animal protocols approved by the Subcommittee on Research Animal Care at the HVRI (231008–02-GJ) and carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China. Competing interests The authors declare that they have no competing interests., (© 2024. The Author(s).)

Introduction: The lack of data on the whole-genome sequences of African swine fever virus (ASFV) variants circulating on the territory of the left bank of the Dnieper River complicates the understanding of the molecular evolution of the virus and the character of the epidemic process development in Russia and Ukraine. Understanding the genetic divergence and phylogenetic relatedness of isolates can largely adjust the strategy of general and specific prevention of the disease. The aim of the study - search and description of unique mutations (deletions/insertions/substitutions) in isolates collected from domestic pigs in Donetsk, Luhansk and Zaporozhye regions in 2023; determination of relatedness and level of homology with reference strains of ASFV genotype II; sub-genotyping and clustering of isolates based on whole-genome analysis., Materials and Methods: The samples used were a culture suspension of porcine bone marrow (PBM) cells containing ASFV isolates obtained from pathologic material from domestic pig carcasses. Genomic DNA was prepared by purification and concentration of virus followed by phenol-chloroform extraction of total nucleic acid. The high-throughput sequencing process was performed using MGI technology. Consensus sequences were assembled by mapping reads to the reference genome of strain Georgia 2007/1., Results: All isolates are assigned to genotype II, have a monophyletic origin, are phylogenetically close to the clusters «Europe» (4/5) and «Bryansk 2021» (1/5), and are divergent from the original parental genetic variants that make up the enlarged clades. In addition, numerous substitutions in the loci of the multigene family MGF 110 , 505 , and 360 , encoding virulence proteins, were detected in 4 isolates from Donetsk and Zaporozhye regions., Conclusion: The phylogeny of the genotype II ASFV, which originated from the reference strain Georgia 2007/1, is shown to be sufficient for isolate differentiation. The presented data are of theoretical and practical importance for domestic and international ASFV surveillance.

The African swine fever virus (ASFV) is spreading worldwide and causing huge economic losses to the global pig industry. The ASFV genome is 170-193 kb in length, contains approximately 150 open reading frames, and encodes more than 200 proteins, most of which have unknown functions. Owing to the unique viral structure, replication strategy, large number of genes of unknown function, and complicated pathogenesis, vaccine development research is challenging. Several naturally attenuated ASFV isolates have been extensively investigated and many genetically manipulated, gene-deleted, and cell-adapted ASFVs have been reported. Currently, live attenuated viruses prepared from weakly virulent strains are an efficient method to provide effective protection in vaccinated pigs; however, these have seldom been widely approved for vaccine use, except in Vietnam. Herein, we summarize the attenuated isolates or vaccine candidates for live vaccines derived from different sources, including naturally mutated, attenuated, cell-adapted, and genetically modified recombinant ASFVs. This will help to understand the gene function and immunogenicity of attenuated live ASFV, as well as the shortcomings of these viruses as vaccine candidates, and provide clues to prepare live, efficient, and safe vaccines for African swine fever.IMPORTANCEOutbreaks of African swine fever (ASF) have caused devastating losses to the global pig industry. Pigs immunized with ASFV attenuated virus can resist the lethal challenge of a strongly virulent virus. Here, we summarize the virulence of naturally mutated, cell-adapted, and genetically recombinant ASFV for pigs, and the protective effect after facing an attack challenge. We also analyze the advantages and disadvantages of ASFV attenuated viruses as vaccine candidates to provide clues for the preparation of efficient and safe live African swine fever vaccines., Competing Interests: The authors declare no conflict of interest.

African swine fever (ASF), a highly infectious and devastating disease affecting both domestic pigs and wild boars, owes its etiology to African swine fever virus (ASFV). ASFV encodes more than 165 proteins. However, novel immunogenic proteins remain unknown. This study aimed to determine the antigenicity of the F317L protein (pF317L) of ASFV. The results revealed that pF317L was able to react with convalescent pig sera, indicating that pF317L could be a candidate antigen. The antigenic potential of pF317L expressed by rHCLV-F317L, a recombinant virus in the backbone of C-strain (a lapinized live attenuated classical swine fever virus) was further investigated in rabbits and pigs. The results revealed that antibodies and cell-mediated immune responses against pF317L were induced in either rabbits or pigs inoculated with rHCLV-F317L. Importantly, anti-pF317L antibodies from rabbits or pigs immunized with rHCLV-F317L significantly inhibited ASFV replication in vitro. In conclusion, pF317L demonstrates favorable immunogenic properties, positioning it as a promising candidate for the development of protective antigens in the ongoing endeavor to formulate efficacious ASF vaccine strategies., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier B.V. All rights reserved.)

African swine fever (ASF) is an acute and devastating infectious disease that has caused significant economic losses to the global pig industry since it was first discovered and reported. African swine fever virus (ASFV) has a large genome encoding more than 160 proteins. The biological characteristics and functions of its various proteins still remain unclear; therefore, the efficacy of specific drugs and vaccines against ASFV remains limited. ASFV pCP312R is an important ASFV protein that exhibits good immunogenicity. In this study, five monoclonal antibodies (mAbs) targeting pCP312R were successfully prepared. Confocal microscopy observations showed that pCP312R was located in the viral factory at the late stage of ASFV infection, and was co-located with p30 and pK205R. These results suggested that pCP312R might be involved in ASFV assembly. Neutralization tests revealed that pCP312R mAb could not neutralize ASFV. Next, we identified the B cell epitopes of one of the most immunogenic mAbs and found a novel epitope of pCP312R, 72 TIPPSTDEEVIR 83 , which was conserved in different pCP312R strains. Overall, five ASFV pCP312R monoclonal antibodies were prepared, and the antigenic epitope of one strain was identified in this study, laying a foundation for further studies on ASFV pCP312R function and facilitating serological diagnosis vaccine development for ASFV., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To understand the clinicopathological forms of African swine fever (ASF) in wild boar, it is crucial to possess a basic knowledge of the biological characteristics of the currently circulating ASF virus isolates. The aim of this work is to establish an accurate and comprehensive histopathologic grading system to standardize the assessment of the ASF lesions in wild boar. The study evaluated the differences between animals infected with a high virulence genotype II isolate (Arm07) (HVI) through intramuscular (IM) (n = 6) and contact-infected (n = 12) routes, alongside those orally infected with a low virulence isolate (Lv17/WB/Riel) (LVI) (n = 6). The assessment included clinical (CS), macroscopic (MS), and histopathologic (HS) scores, as well as viral loads in blood and tissues by real-time quantitative polymerase chain reaction (qPCR). Tissues examined included skin, lymph nodes, bone marrow, palatine tonsil, lungs, spleen, liver, kidneys, thymus, heart, adrenal glands, pancreas, urinary bladder, brain, and gastrointestinal and reproductive tracts. The HVI group exhibited a 100% mortality rate with elevated CS, MS, and HS values. Animals infected by contact (CS = 12; MS = 58.5; HS = 112) and those intramuscularly infected (CS = 14.8; MS = 47; HS = 104) demonstrated similar values, indicating that the route of infection does not decisively influence the severity of clinical and pathological signs. The LVI group showed a 0% mortality rate, an inconspicuous clinical form, minimal lesions (CS = 0; MS = 12; HS = 29), and a lower viral load. Histopathologic evaluation has proven valuable in advancing our comprehension of ASF pathogenesis in wild boar and paves the groundwork for further research investigating protective mechanisms in vaccinated animals., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

African swine fever (ASF) is endemic to Uganda and causes annual outbreaks. Some pigs survive these outbreaks and remain asymptomatic but are African swine fever virus (ASFV) positive. The potential heritability and genetic disparities in disease susceptibility among Ugandan pigs are not fully understood. In a 12-year study, whole blood and tissue samples were collected from 212 pigs across 19 districts in Uganda. Polymerase chain reaction (PCR) assays were used to determine ASFV infection status and genotyping was completed using a commercial porcine array. The point prevalence of ASF was calculated for each district, and breed composition origins were quantified for the sampled pigs by implementing established ancestry analyses. Genome-wide associated studies (GWAS) were conducted using all available domestic swine samples (full study population; n = 206) as well as a reduced dataset (farm-level study population; n = 129). This study revealed a greater number of ASFV-positive pigs in border districts than in non-border districts, a high level of admixture among domestic pigs sampled from Ugandan smallholder farms, and 48 loci that were associated with ASFV infection status. The discovery of 48 significant SNPs and 28 putative candidate genes may imply the possibility of heritability for resistance to ASFV. However, additional investigations in ASFV-endemic regions are required to fully elucidate the heritability of ASFV susceptibility among surviving pigs in Uganda., (© 2024. The Author(s).)

The African swine fever virus (ASFV) causes severe disease in wild and domestic pigs, with high mortality rates, extensive spread, and significant economic losses globally. Despite ongoing efforts, an effective vaccine remains elusive. Therefore, effective diagnostic methods are needed to rapidly detect and prevent the further spread of ASF. This study assessed nine commercial kits based on real-time polymerase chain reaction (PCR) approved in the Republic of Korea using the synthesized ASFV plasmid, 20 food waste samples, and artificially spiked samples (ASSs). The kits were evaluated for their diagnostic sensitivity, specificity, cost per reaction, and reaction running time. In addition, the results were compared with those of the World Organization for Animal Health (WOAH) standard methods. Three commercial kits (VDx ® ASFV qPCR Kit, Palm PCR™ ASFV Fast PCR Kit, and PowerChek™ ASFV Real-time PCR Detection Kit Ver.1.0) demonstrated the highest sensitivity (100 ag/μL), cost-effectiveness (less than KRW 10,000), and shortest running time (less than 70 min). These kits are suitable for the monitoring, early diagnosis, and prevention of the spread of ASF. This is the first report on the performance comparison of ASFV diagnostic kits approved in the Republic of Korea, providing valuable information for selecting kits for testing with food waste samples.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a deadly, highly contagious disease in both domestic pigs and wild boar. With mortality up to 100%, the disease has been making a serious impact on the swine industry worldwide. Because no effective antiviral treatment has been observed, proactive prevention such as vaccination remains the key to controlling the outbreak. In the pursuit of expediting vaccine development, our current work has made the first report for heterologous production of the viral outer envelope glycoprotein CD2v extracellular domain (CD2v ED), a proposed promising vaccine antigen candidate in the "green" synthetic host Nicotiana benthamiana. Protein oligomerization strategies were implemented to increase the immunogenicity of the target antigen. Herein, the protein was expressed in oligomeric forms based on the C-terminally fused GCN4pII trimerization motif and GCN4pII_TP oligomerization motif. Quantitative western blot analysis showed significantly higher expression of trimeric CD2v ED_GCN4pII with a yield of about 12 mg/100 g of fresh weight, in comparison to oligomeric CD2v ED_GCN4pII_TP, revealing the former is the better choice for further studies. The results of purification and size determination by size exclusion chromatography (SEC) illustrated that CD2v ED_GCN4pII was successfully produced in stable oligomeric forms throughout the extraction, purification, and analysis process. Most importantly, purified CD2v ED_GCN4pII was demonstrated to induce both humoral and cellular immunity responses in mice to extents equivalent to those of the live attenuated vaccine ASFV-G-∆I177L, suggesting it as the potential subunit vaccine candidate for preventing ASFV., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

African Swine Fever Virus (ASFV), resulting in strain-dependent vascular pathology, leading to hemorrhagic fever, is an important pathogen in swine. The pathogenesis of ASFV is determined by the array and spatial distribution of susceptible cells within the host. In this study, the replication characteristics of ASFV genotype I E70 (G1-E70) and ASFV genotype II Belgium 2018/1 (G2-B18) in the environment of small veins were investigated in an established vein explant model. Immunofluorescence staining analysis revealed that perivenous macrophages (CD163 + cells) were widely distributed in the explant, with most of them (approximately 2-10 cells/0.03 mm 2 ) being present close to the vein (within a radius of 0-348 µm). Upon inoculation with G1-E70 and G2-B18, we observed an increase in the quantity of cells testing positive for viral antigens over time. G1-E70 replicated more efficiently than G2-B18 in the vein explants (7.6-fold for the ear explant at 72 hpi). The majority of ASFV + cells were CD163 + , indicating that macrophages are the primary target cells. Additional identification of cells infected with ASFV revealed the presence of vimentin + , CD14 + , and VWF + cells, demonstrating the cellular diversity and complexity associated with ASFV infection. By the use of this new vein explant model, the susceptibility of vascular and perivascular cells to an ASFV infection was identified. With this model, it will be possible now to conduct more functional analyses to get better insights into the pathogenesis of ASFV-induced hemorrhages.

African swine fever (ASF) is a devastating disease caused by African swine fever virus (ASFV) and leads to significant economic losses in the pig farming industry. Given the absence of an effective vaccine or treatment, the mortality rate of ASF is alarmingly close to 100%. Consequently, the ability to rapidly and accurately detect ASFV on site and promptly identify infected pigs is critical for controlling the spread of this pandemic. The dynamics of the ASF virus load and antibody response necessitate the adoption of various detection strategies at different stages of infection, a topic that has received limited attention to date. This review offers detailed guidance for choosing appropriate ASF diagnostic techniques tailored to the clinical manifestations observed from the acute to chronic phases, including asymptomatic cases. We comprehensively summarize and evaluate the latest advancements in ASFV detection methods, such as CRISPR-based diagnostics, biosensors, and microfluidics. Additionally, we address the challenges of false negatives or positives due to ASF variants or the use of injected live attenuated vaccines. This review provides an exhaustive list of diagnostic tests suitable for detecting each stage of symptoms and potential target genes for developing new detection methods. In conclusion, we highlight the current challenges and future directions in ASFV detection, underscoring the need for continued research and innovation in this field., (© 2024. The Author(s).)

African swine fever (ASF), caused by ASF virus (ASFV), is a highly infectious and severe hemorrhagic disease of pigs that causes major economic losses. Currently, no commercial vaccine is available and prevention and control of ASF relies mainly on early diagnosis. Here, a novel automated double antigen sandwich chemiluminescent immunoassay (DAgS-aCLIA) was developed to detect antibodies against ASFV p72 (p72-Ab). For this purpose, recombinant p72 trimer was produced, coupled to magnetic particles as carriers and labeled with acridinium ester as a signal trace. Finally, p72-Ab can be sensitively and rapidly measured on an automated chemiluminescent instrument. For quantitative analysis, a calibration curve was established with a laudable linearity range of 0.21 to 212.0 ng/mL (R 2 = 0.9910) and a lower detection limit of 0.15 ng/mL. For qualitative analysis, a cut-off value was set at 1.50 ng/mL with a diagnostic sensitivity of 100.00% and specificity of 98.33%. Furthermore, antibody response to an ASF gene-deleted vaccine candidate can be accurately quantified using this DAgS-aCLIA, as evidenced by early seroconversion as early as 7 days post-immunization and high antibody levels. Compared with available enzyme-linked immunosorbent assays, this DAgS-aCLIA demonstrated a wider linearity range of 4 to 16-fold, and excellent analytical sensitivity and agreement of over 95.60%. In conclusion, our proposed DAgS-aCLIA would be an effective tool to support ASF epidemiological surveillance.IMPORTANCEAfrican swine fever virus (ASFV) is highly contagious in wild boar and domestic pigs. There is currently no vaccine available for ASF, so serological testing is an important diagnostic tool. Traditional enzyme-linked immunosorbent assays provide only qualitative results and are time and resource consuming. This study will develop an automated chemiluminescent immunoassay (CLIA) that can quantitatively and qualitatively detect antibodies to ASFV p72, greatly reducing detection time and labour-intensive operation, and improving detection sensitivity and linearity range. This novel CLIA would serve as a reliable and convenient tool for ASF pandemic surveillance and vaccine development., Competing Interests: The authors declare no conflicts of interest.

African swine fever virus (ASFV) infection causes a frequently fatal disease in domestic swine that has affected more than 50 countries worldwide since 2021, with a major impact on animal welfare and economy. The development of effective vaccines or antivirals against this disease are urgently required for its effective control. Live detection of viral replication has been used as a tool for the screening and characterization of antiviral compounds in other dsDNA genome containing viruses. Here, we have adapted the ANCHOR fluorescent DNA labelling system to ASFV by constructing and characterizing a novel recombinant virus. We show that this virus is viable and effectively tags viral DNA replication sites, which can be detected and quantified in real time. Further, we have used high content cell microscopy to test the antiviral activity of bisbenzimide compounds and show that Hoechst 33342 has specific anti-ASFV activity. We expect this novel tool to be useful both in the further study of ASFV replication as in the screening of new specific antiviral compounds., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: SKG and FG are share-holders of NeoVirTech SAS. SKG is an employee of NeoVirTech SAS. No other conflict of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

African swine fever virus (ASFV) severely threatens the global economy and food security. ASFV encodes >150 genes, but the functions of most of them have yet to be characterized in detail. Here we explored the function of the ASFV CP312R gene and found that CP312R plays an essential role in ASFV replication. Knockout of the CP312R gene terminated viral replication and CP312R knockdown substantially suppressed ASFV infection in vitro. Furthermore, we resolved the crystal structure of pCP312R to 2.3 Å resolution and found that pCP312R has the potential to bind nucleic acids. LC-MS analysis and co-immunoprecipitation assay revealed that pCP312R interacts with RPS27A, a component of the 40S ribosomal subunit. Confocal microscopy showed the interaction between pCP312R and RPS27A leaded to a modification in the subcellular localization of this host protein, which suppresses host protein translation. Renilla-Glo luciferase assay and Ribopuromycylation analysis evidenced that knockout of RPS27A completely aborted the shutoff activity of pCP312R, and trans-complementation of RPS27A recovered pCP312R shutoff activity in RPS27A-knockout cells. Our findings shed light on the function of ASFV CP312R gene in virus infection, which triggers inhibition of host protein synthesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

African swine fever (ASF) is an acute, highly hemorrhagic viral disease in domestic pigs and wild boars. The disease is caused by African swine fever virus, a double stranded DNA virus of the Asfarviridae family. ASF can be classified into 25 different genotypes, based on a 478 bp fragment corresponding to the C-terminal sequence of the B646L gene, which is highly conserved among strains and encodes the major capsid protein p72. The C-terminal end of p72 has been used as a PCR target for quick diagnosis of ASF, and its characterization remains the first approach for epidemiological tracking and identification of the origin of ASF in outbreak investigations. Recently, a new classification of ASF, based on the complete sequence of p72, reduced the 25 genotypes into only six genotypes; therefore, it is necessary to have the capability to sequence the full-length B646L gene (p72) in a rapid manner for quick genotype characterization. Here, we evaluate the use of an amplicon approach targeting the whole B646L gene, coupled with nanopore sequencing in a multiplex format using Flongle flow cells, as an easy, low cost, and rapid method for the characterization and genotyping of ASF in real-time.

Introduction: African swine fever (ASF) is a contagious viral disease that affects pigs and wild boars, with a mortality rate of up to 100% in susceptible animals. The virus has been circulating in Europe and Asia since its introduction in 2007. Initially, all studied isolates were identified as genotype II, but in 2021 genotype I was reported in China. Later in 2023, the first recombinant virus of genotype I and II was identified in China, with an isolate dating back to 2021, this was followed by the detection of 6 recombinant isolates in Vietnam., Methods: In this study, an ASFV isolate from the Primorsky Region of Russia obtained from a domestic pig was analyzed by sequencing several genome markers as well as the full genome. Eight pigs were infected with the isolate to assess its virulence., Results: Virus replication in cell culture showed hemadsorption, while sequencing of genome markers clustered the isolate into both genotype I and genotype II. The whole-genome sequence showed that the Russian isolate shared a 99.99% identity with recombinant isolates described earlier in China. Experimental animals developed ASF disease after the introduction of a low dose of the virus (10 HAU50) and died within 7 days post-infection, presenting an acute form of the disease., Conclusion: This is the first report on recombinant ASFV in Russia's territory. The results once again confirm the transboundary nature of the disease, demonstrating the vulnerability of the global pig industry underscoring the need for developing new ASF vaccines effective against recombinant strains and emphasizing the importance of continuous molecular monitoring to detect emerging threats promptly., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

African swine fever virus (ASFV) is the causative agent of an often lethal disease in domestic pigs, African swine fever (ASF). ASF is currently a pandemic disease challenging pig production in Eurasia. While the ASFV genome encodes for over 160 proteins, the function of most of them are still not characterized. Among those ASF genes with unknown functions is the E111R gene. It has been recently reported that the deletion of the E111R gene from the genome of the virulent Chinese field isolate SY18 strain produced a reduction of virus virulence when pigs were inoculated at relatively low doses. Conversely, we report here that deletion of the ASFV gene E111R in the Georgia 2010 isolate does not alter the virulence of the parental virus in experimentally inoculated pigs. A recombinant virus lacking the E111R gene, ASFV-G-∆E111R was intramuscularly (IM) inoculated in domestic pigs at a dose of 10 2 HAD 50 of ASFV-G-∆E111R and compared with animals that received a similar dose of virulent ASFV-G. Both, animals inoculated with either the recombinant ASFV-G-∆E111R or the parental virus developed a fatal form of the disease and were euthanized around the 6th-7th day post-inoculation (dpi).

African swine fever virus (ASFV) is one of the most important causative agents of animal diseases and can cause highly fatal diseases in swine. ASFV DNA polymerase (DNAPol) is responsible for genome replication and highly conserved in all viral genotypes showing an ideal target for drug development. Here, we systematically determined the structures of ASFV DNAPol in apo, replicating and editing states. Structural analysis revealed that ASFV DNAPol had a classical right-handed structure and showed the highest similarity to the structure of human polymerase delta. Intriguingly, ASFV DNAPol has a much longer fingers subdomain, and the thumb and palm subdomain form a unique interaction that has never been seen. Mutagenesis work revealed that the loss of this unique interaction decreased the enzymatic activity. We also found that the β-hairpin of ASFV DNAPol is located below the template strand in the editing state, which is different from the editing structures of other known B family DNAPols with the β-hairpin above the template strand. It suggests that B family DNAPols have evolved two ways to facilitate the dsDNA unwinding during the transition from replicating into editing state. These findings figured out the working mechanism of ASFV DNAPol and will provide a critical structural basis for the development of antiviral drugs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The African swine fever virus (ASFV) is a large and complex DNA virus that causes a highly lethal disease in swine, for which no antiviral drugs or vaccines are currently available. Studying viral-host protein-protein interactions advances our understanding of the molecular mechanisms underlying viral replication and pathogenesis and can facilitate the discovery of antiviral therapeutics. In this study, we employed affinity tagging and purification mass spectrometry to characterize the interactome of VPS39, an important cellular factor during the early phase of ASFV replication. The interaction network of VPS39 revealed associations with mitochondrial proteins involved in membrane contact sites formation and cellular respiration. We show that the ASFV proteins CP204L and A137R target VPS39 by interacting with its clathrin heavy-chain functional domain. Furthermore, we elaborate on the potential mechanisms by which VPS39 may contribute to ASFV replication and prioritize interactions for further investigation into mitochondrial protein function in the context of ASFV infection.

African swine fever virus (ASFV) is endemic to African wild pigs ( Phacochoerus and Potamochoerus ), in which viral infection is asymptomatic, and Ornithodoros soft ticks. However, ASFV causes a lethal disease in Eurasian domestic pigs ( Sus scrofa ). While Sub-Saharan Africa is believed to be the original home of ASFV, publicly available whole-genome ASFV sequences show a strong bias towards p72 Genotypes I and II, which are responsible for domestic pig pandemics outside Africa. To reduce this bias, we hereby describe nine novel East African complete genomes in p72 Genotype IX and present the phylogenetic analysis of all 16 available Genotype IX genomes compared with other ASFV p72 clades. We also document genome-level differences between one specific novel Genotype IX genome sequence (KE/2013/Busia.3) and a wild boar cell-passaged derivative. The Genotype IX genomes clustered with the five available Genotype X genomes. By contrast, Genotype IX and X genomes were strongly phylogenetically differentiated from all other ASFV genomes. The p72 gene region, on which the p72-based virus detection primers are derived, contains consistent SNPs in Genotype IX, potentially resulting in reduced sensitivity of detection. In addition to the abovementioned cell-adapted variant, eight novel ASFV Genotype IX genomes were determined: five from viruses passaged once in primary porcine peripheral blood monocytes and three generated from DNA isolated directly from field-sampled kidney tissues. Based on this methodological simplification, genome sequencing of ASFV field isolates should become increasingly routine and result in a rapid expansion of knowledge pertaining to the diversity of African ASFV at the whole-genome level.

African swine fever (ASF) is an acute infectious disease with a high mortality rate in both domestic and wild boars. Commercial vaccines or antiviral drugs for ASF were not available due to the complex diversity of the structure and genome of its pathogen African swine fever virus (ASFV). In recent years, there have been many reports on candidate strains of attenuated vaccines for ASFV. In this study, we obtained a recombinant virus named SY18ΔL60LΔCD2v by simultaneously deleting the L60L gene and CD2v gene from highly virulent strain SY18. In vitro, SY18ΔL60LΔCD2v displayed a decreased growth kinetic compared to that of parental SY18. In vivo, high doses (10 5 TCID 50 ) of SY18ΔL60LΔCD2v can protect pigs (5/5) from attacks by the parental SY18 strain (10 2 TCID 50 ). Low doses (10 2 TCID 50 ) of SY18ΔL60LΔCD2v only protected 20% of pigs (1/5) from attacks by the parental SY18 strain (10 2 TCID 50 ). The results indicated that the absence of these two genes in SY18 could induce protection against the homologous parental strain, and there were no obvious clinical symptoms or viremia. These results indicate that the SY18ΔL60LΔCD2v strain can serve as a new live attenuated vaccine candidate for the prevention and control of ASFV infection.

African swine fever virus (ASFV) has been spreading through Europe, Asia, and the Caribbean after its introduction in Georgia in 2007 and, due to its particularly high mortality rate, poses a continuous threat to the pig industry. The golden standard to trace back the ASFV is whole genome sequencing, but it is a cost and time-intensive methodology. A more efficient way of tracing the virus is to amplify only specific genomic regions relevant for genotyping. This is mainly accomplished by amplifying single amplicons by PCR followed by Sanger sequencing. To reduce costs and processivity time, we evaluated a multiplex PCR based on the four primer sets routinely used for ASFV genotyping ( B646L , E183L , B602L , and intergenic I73R-I329L ), which was followed by Nanopore ligation-based amplicon sequencing. We show that with this protocol, we can genotype ASFV DNA originating from different biological matrices and correctly classify multiple genotypes and strains using a single PCR reaction. Further optimization of this method can be accomplished by adding or swapping the primer sets used for amplification based on the needs of a specific country or region, making it a versatile tool that can speed up the processing time and lower the costs of genotyping during ASFV outbreaks.

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 10 8 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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The emergence and spread of the African swine fever virus (ASFV) posed a significant threat to the global swine breeding industry, calling for innovative approaches benefiting viral containment and control. A recent study (Z. Zheng, L. Xu, H. Dou, Y. Zhou, X., et al., Microbiol Spectr 12: e02164-23, 2024, https://doi.org/10.1128/spectrum.02164-23) established a multiplexed CRISPR-Cas system targeting the genome of ASFV and tested the consequent antiviral activity both in vitro and in vivo . Application of this system showed a significant reduction of viral replication in vitro , while the germline-edited pigs expressing this system exhibited normal growth with continuous guide RNA expression. Although no survival advantage was observed upon ASFV challenge compared with nonengineered pigs, this marks the first attempt of germline editing to pursue ASFV resistance and paves the way for future disease-resistant animal breeding approaches utilizing CRISPR-Cas technology., Competing Interests: The authors declare no conflict of interest.

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., (© 2024. The Author(s).)

Background: African swine fever (ASF) is a viral disease that affects pigs and wild boars providing economic burden in swine industry., Methods and Results: In this study, we investigated the effect of deleting the ASFV multigene family 110 (MGF110) fragment (1 L-5-6 L) on apoptosis modulation and the expression of proinflammatory cytokines. Gene expression in swine peripheral blood macrophages infected with either the parental "Volgograd/14c" strain or the gene-deleted "Volgograd/D(1L-5-6L) MGF110" strain was analyzed. Caspase-3 activity was 1.15 times higher in macrophages infected with the parental ASFV strain compared to the gene-deleted strain. Gene expression analysis of Caspase-3 (Cas-3), Interferon-A (IFN-A), Tumor Necrosis Factor A (TNF-A), B-cell Lymphoma-2 (Bcl-2), Nuclear Factor Kappa B (NF-kB), Interleukin-12 (IL-12), and Heat Shock Protein-70 (HSP-70) using RT-qPCR at various time points after infection revealed significant differences in expression profiles between the strains. The peak expression of cytokines (except NF-kB) occurred at 24 h post-infection with the "Volgograd/D(1L-5-6L) MGF110" strain. In samples infected with the ASFV "Volgograd/14c" strain, the most intense expression was observed at 72 and 96 h, except for Bcl-2 and NF-kB, which peaked at 6 h post-infection. The cytokine expression trend for the "Volgograd/D(1L-5-6L) MGF110" strain was more stable with higher expression values., Conclusion: The expression trend for the parental strain increased over time, reaching maximum values at 72 and 96 h post-infection, but the overall expression level was lower than that of the gene-deleted strain. These findings suggest that deleting the multigene family 110 members (1 L-5-6 L) contributes to ASFV attenuation without affecting virus replication kinetics., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Viral infection disrupts the normal regulation of the host gene's expression. In order to normalise the expression of dysregulated host genes upon virus infection, analysis of stable reference housekeeping genes using quantitative real-time-PCR (qRT-PCR) is necessary. In the present study, healthy and African swine fever virus (ASFV) infected porcine tissues were assessed for the expression stability of five widely used housekeeping genes (HPRT1, B2M, 18 S rRNA, PGK1 and H3F3A) as reference genes using standard algorithm. Total RNA from each tissue sample (lymph node, spleen, kidney, heart and liver) from healthy and ASFV-infected pigs was extracted and subsequently cDNA was synthesized, and subjected to qRT-PCR. Stability analysis of reference genes expression was performed using the Comparative delta CT, geNorm, BestKeeper and NormFinder algorithm available at RefFinder for the different groups. Direct Cycle threshold (CT) values of samples were used as an input for the web-based tool RefFinder. HPRT1 in spleen, 18 S rRNA in liver and kidney and H3F3A in heart and lymph nodes were found to be stable in the individual healthy tissue group (group A). The majority of the ASFV-infected organs (liver, kidney, heart, lymph node) exhibited H3F3A as stable reference gene with the exception of the ASFV-infected spleen, where HPRT1 was found to be the stable gene (group B). HPRT1 was found to be stable in all combinations of all CT values of both healthy and ASFV-infected porcine tissues (group C). Of five different reference genes investigated for their stability in qPCR analysis, the present study revealed that the 18 S rRNA, H3F3A and HPRT1 genes were optimal reference genes in healthy and ASFV-infected different porcine tissue samples. The study revealed the stable reference genes found in healthy as well as ASF-infected pigs and these reference genes identified through this study will form the baseline data which will be very useful in future investigations on gene expression in ASFV-infected pigs., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

African swine fever virus (ASFV), which was first identified in northern China in 2018, causes high mortality in pigs. Since the I73R protein in ASFV is abundantly expressed during the early phase of virus replication, it can be used as a target protein for early diagnosis. In this study, the I73R protein of ASFV was expressed, and we successfully prepared a novel monoclonal antibody (mAb), 8G11D7, that recognizes this protein. Through both indirect immunofluorescence and Western blotting assays, we demonstrated that 8G11D7 can detect ASFV strains. By evaluating the binding of the antibody to a series of I73R-truncated peptides, the definitive epitope recognized by the monoclonal antibody 8G11D7 was determined to be 58 DKTNTIYPP 66 . Bioinformatic analysis revealed that the antigenic epitope had a high antigenic index and conservatism. This study contributes to a deeper understanding of ASFV protein structure and function, helping establish ASFV-specific detection method., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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 (R 2 = 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., Competing Interests: Declaration of Competing Interest Authors declare that no competing interests exist. Mention of trade names or commercial products in this article is solely for providing specific information and does not imply recommendation or endorsement by the IZSAM., (Copyright © 2024 Elsevier B.V. All rights reserved.)

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), a hemorrhagic illness with high fatality rates in domestic pigs that has resulted in a substantial socio-economic loss and threatens the global pork industry. Very few safe and efficient vaccines or compounds against ASF are commercially available, thus developing new antiviral strategies is urgently required. Targeted protein degradation (TPD) has emerged as one of the most innovative strategies for drug discovery. In this study, we generate Nanobody-based TRIM-aways specifically binding with and targeting ASFV-encoded structural proteins p30, p54, and p72 for degradation. Furthermore, nanobody-based trim-aways exhibit robust viral structural protein degradation capabilities in ASFV-infected iPAM and MA104 cells through both proteasomal and lysosomal pathways, concurrently demonstrating potent anti-ASFV activity with less viral production. Our study highlights the Nanobody-based TRIM-away targeting viral protein degradation as a potential candidate for the development of a novel antiviral strategy against ASF., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

African swine fever virus (ASFV) is a highly complex virus that poses a significant threat to the global swine industry. However, little is known about the mechanisms of ASFV cell entry because ASFV has a multilayered structure and a genome encoding over 150 proteins. This review aims to elucidate the current knowledge on cell entry mechanisms of ASFV and the cellular and viral proteins involved. Experimental evidence suggests that ASFV utilises multiple pathways for entry, which may be cell or tissue type dependent, but the intricate nature of ASFV has hindered the identification of cellular and viral proteins involved in this process. Therefore, further research into the molecular virology of ASFV is essential to advance our understanding of the ASFV entry mechanisms, which will pave the way for innovative strategies to combat this formidable pathogen., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The pS273R protease of the African swine fever virus (ASFV) is responsible for the processing of the viral polyproteins pp220 and pp62, precursors of the internal capsid of the virus. The protease is essential for a productive viral infection and is an attractive target for antiviral therapy. This work presents a method for the production of pS273R in E. coli cells by fusing the protease with the SlyD chaperone. The chimeric protein pS273R protease, during expression, is formed in a soluble form possessing enzymatic activity. Subsequently, pS273R separates from SlyD through autocatalytic cleavage at the TEV protease site in vivo. This work devised a straightforward protocol for chromatographic purification, resulting in the production of a highly purified viral protease. Additionally, we suggest using a fluorescence method to assess the activity of pS273R. This method is predicated on a shift in the chimeric protein thioredoxin-EGFP's electrophoretic mobility following its protease cleavage. It was shown that thioredoxin-EGFP substrate is effectively and selectively cleaved by the pS273R protease, even in complex protein mixtures such as mammalian cell lysates., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

African swine fever (ASF), caused by ASF virus (ASFV), represents one of the most economically important viral infectious diseases in swine industry worldwide. So far there is no vaccine or antiviral drug for controlling ASF pandemics. In the present study, we assessed inhibition of six nucleoside analogues against ASFV replication in ex vivo primary porcine alveolar macrophages (PAMs), including the first approved antiviral drug idoxuridine. Our results showed that, out of the assessed six compounds, 5-Bromo-2'-Deoxyuridine (5-BrdU, an analog of idoxuridine), exhibited the strongest inhibition on the replication of ASFV in PAMs with a 50% inhibitory concentration (IC 50 ) value of 2.9 μM and a low cytotoxicity (CC 50  > 270 μM). Moreover, we showed that 5-BrdU interferes with ASFV DNA replication by incorporating into viral replicating DNA molecules as a competitive substrate for deoxythymidine, ultimately inhibiting the formation of ASFV viral factories. Altogether, our findings suggest that 5-BrdU could serve as a promising therapeutic agent for combating ASFV infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

African swine fever virus (ASFV) recombinant strains pose new challenges for diagnosis and control. This study characterizes genotype I and II recombinant ASFV strains identified in northern Vietnam in 2023 through whole-genome sequencing and comparative genomic analysis. Seven ASFV-positive samples from six provinces were analyzed, with recombinant strains detected in Bac Giang, Phu Tho, and Vinh Phuc provinces. Isolates showed hemadsorption positivity despite having genotype I B646L, indicating their recombinant nature. Genome-wide analysis revealed 19 recombination breakpoints consistent with Chinese recombinant strains. Vietnamese isolates shared 99.86-99.98% nucleotide identity with Chinese recombinants, forming a distinct monophyletic group. Comparative analysis identified 50 SNPs and INDELs, with 39 variations found across Vietnamese strains, distinguishing them from Chinese isolates. Unique genetic markers in C962R, I329L, and MGF 505-11L genes distinguished Vietnamese recombinants from Chinese counterparts, while mutations in C122R and NP1450L differentiated all recombinants from parental genotypes. The central variable region (CVR) of the B602L gene showed diversity among Vietnamese isolates, while the I73R-I329L intergenic regions were recognized as in the IGR2 group. This study enhances understanding of recombinant ASFV evolution through homologous recombination and identifies new genetic markers for improved detection and characterization. The observed genetic diversity highlights challenges for existing diagnostic methods and vaccine development, emphasizing the need for continued surveillance and research into the functional implications of these genetic variations on ASFV pathogenicity and transmissibility.

African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating infectious disease of domestic pigs and wild boar, which threatens the global pig industry. Endoplasmic reticulum (ER) is a multifunctional signaling organelle in eukaryotic cells that is involved in protein synthesis, processing, posttranslational modification and quality control. As intracellular parasitic organisms, viruses have evolved several strategies to modulate ER functions to favor their life cycles. We have previously demonstrated that the differentially expressed genes associated with unfolded protein response (UPR), which represents a response to ER stress, are significantly enriched upon ASFV infection. However, the correlation between the ER stress or UPR and ASFV replication has not been illuminated yet. Here, we demonstrated that ASFV infection induces ER stress both in target cells and in vivo , and subsequently activates the activating transcription factor 6 (ATF6) branch of the UPR to facilitate viral replication. Mechanistically, ASFV infection disrupts intracellular calcium (Ca 2+ ) homeostasis, while the ATF6 pathway facilitates ASFV replication by increasing the cytoplasmic Ca 2+ level. More specifically, we demonstrated that ASFV infection triggers ER-dependent Ca 2+ release via the inositol triphosphate receptor (IP3R) channel. Notably, we showed that the ASFV B117L protein plays crucial roles in ER stress and the downstream activation of the ATF6 branch, as well as the disruption of Ca 2+ homeostasis. Taken together, our findings reveal for the first time that ASFV modulates the ER stress-ATF6-Ca 2+ axis to facilitate viral replication, which provides novel insights into the development of antiviral strategies for ASFV.

African swine fever (ASF) is a rapidly fatal viral haemorrhagic fever in Chinese domestic pigs. Although very high mortality is observed in pig farms after an ASF outbreak, clinically healthy and antibody-positive pigs are found in those farms, and viral detection is rare from these pigs. The ability of pigs to resist ASF viral infection may be modulated by host genetic variations. However, the genetic basis of the resistance of domestic pigs against ASF remains unclear. We generated a comprehensive set of structural variations (SVs) in a Chinese indigenous Xiang pig with ASF-resistant (Xiang-R) and ASF-susceptible (Xiang-S) phenotypes using whole-genome resequencing method. A total of 53,589 nonredundant SVs were identified, with an average of 25,656 SVs per individual in the Xiang pig genome, including insertion, deletion, inversion and duplication variations. The Xiang-R group harboured more SVs than the Xiang-S group. The F-statistics ( F ST ) was carried out to reveal genetic differences between two populations using the resequencing data at each SV locus. We identified 2,414 population-stratified SVs and annotated 1,152 Ensembl genes (including 986 protein-coding genes), in which 1,326 SVs might disturb the structure and expression of the Ensembl genes. Those protein-coding genes were mainly enriched in the Wnt, Hippo, and calcium signalling pathways. Other important pathways associated with the ASF viral infection were also identified, such as the endocytosis, apoptosis, focal adhesion, Fc gamma R-mediated phagocytosis, junction, NOD-like receptor, PI3K-Akt, and c-type lectin receptor signalling pathways. Finally, we identified 135 candidate adaptive genes overlapping 166 SVs that were involved in the virus entry and virus-host cell interactions. The fact that some of population-stratified SVs regions detected as selective sweep signals gave another support for the genetic variations affecting pig resistance against ASF. The research indicates that SVs play an important role in the evolutionary processes of Xiang pig adaptation to ASF infection.

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.

African swine fever virus (ASFV) is a complex DNA virus and the only member of the Asfarviridae family. It causes high mortality and severe economic losses in pigs. The ASFV pB602L protein plays a key role in virus assembly and functions as a molecular chaperone of the major capsid protein p72. In addition, pB602L is an important target for the development of diagnostic tools for African swine fever (ASF) because it is a highly immunogenic antigen against ASFV. In this study, we expressed and purified ASFV pB602L and validated its immunogenicity in serum from naturally infected pigs with ASFV. Furthermore, we successfully generated an IgG2a κ subclass monoclonal antibody (mAb 7E7) against pB602L using hybridoma technology. Using western blot and immunofluorescence assays, mAb 7E7 specifically recognized the ASFV Pig/HLJ/2018/strain and eukaryotic recombinant ASFV pB602L protein in vitro. The 474 SKENLTPDE 482 epitope in the ASFV pB602L C-terminus was identified as the minimal linear epitope for mAb 7E7 binding, with dozens of truncated pB602l fragments characterized by western blot assay. We also showed that this antigenic epitope sequence has a high conservation and antigenic index. Our study contributes to improved vaccine and antiviral development and provides new insights into the serologic diagnosis of ASF. KEY POINTS: • We developed a monoclonal antibody against ASFV pB602L, which can specifically recognize the ASFV Pig/HLJ/2018/ strain. • This study found one novel conserved B-cell epitope 474 SKENLTPDE 482 . • In the 3D structure, 474 SKENLTPDE 482 is exposed on the surface of ASFV pB602L, forming a curved linear structure., (© 2024. The Author(s).)

African swine fever virus (ASFV) is the causative agent of a contagious disease affecting wild and domestic swine. The function of B169L protein, as a potential integral structural membrane protein, remains to be experimentally characterized. Using state-of-the-art bioinformatics tools, we confirm here earlier predictions indicating the presence of an integral membrane helical hairpin, and further suggest anchoring of this protein to the ER membrane, with both terminal ends facing the lumen of the organelle. Our evolutionary analysis confirmed the importance of purifying selection in the preservation of the identified domains during the evolution of B169L in nature. Also, we address the possible function of this hairpin transmembrane domain (HTMD) as a class IIA viroporin. Expression of GFP fusion proteins in the absence of a signal peptide supported B169L insertion into the ER as a Type III membrane protein and the formation of oligomers therein. Overlapping peptides that spanned the B169L HTMD were reconstituted into ER-like membranes and the adopted structures analyzed by infrared spectroscopy. Consistent with the predictions, B169L transmembrane sequences adopted α-helical conformations in lipid bilayers. Moreover, single vesicle permeability assays demonstrated the assembly of lytic pores in ER-like membranes by B169L transmembrane helices, a capacity confirmed by ion-channel activity measurements in planar bilayers. Emphasizing the relevance of these observations, pore-forming activities were not observed in the case of transmembrane helices derived from EP84R, another ASFV protein predicted to anchor to membranes through a α-helical HTMD. Overall, our results support predictions of viroporin-like function for the B169L HTMD.IMPORTANCEAfrican swine fever (ASF), a devastating disease affecting domestic swine, is widely spread in Eurasia, producing significant economic problems in the pork industry. Approaches to prevent/cure the disease are mainly restricted to the limited information concerning the role of most of the genes encoded by the large (160-170 kba) virus genome. In this report, we present the experimental data on the functional characterization of the African swine fever virus (ASFV) gene B169L. Data presented here indicates that the B169L gene encodes for an essential membrane-associated protein with a viroporin function., Competing Interests: The authors declare no conflict of interest.

African swine fever (ASF) is a highly contagious and severe hemorrhagic transboundary swine viral disease with up to a 100% mortality rate, which leads to a tremendous socio-economic loss worldwide. The lack of safe and efficacious ASF vaccines is the greatest challenge in the prevention and control of ASF. In this study, we generated a safe and effective live-attenuated virus (LAV) vaccine candidate VNUA-ASFV-LAVL3 by serially passaging a virulent genotype II strain (VNUA-ASFV-L2) in an immortalized porcine alveolar macrophage cell line (3D4/21, 50 passages). VNUA-ASFV-LAVL3 lost its hemadsorption ability but maintained comparable growth kinetics in 3D4/21 cells to that of the parental strain. Notably, it exhibited significant attenuation of virulence in pigs across different doses (10 3 , 10 4 , and 10 5 TCID 50 ). All vaccinated pigs remained healthy with no clinical signs of African swine fever virus (ASFV) infection throughout the 28-day observation period of immunization. VNUA-ASFV-LAVL3 was efficiently cleared from the blood at 14-17 days post-infection, even at the highest dose (10 5 TCID 50 ). Importantly, the attenuation observed in vivo did not compromise the ability of VNUA-ASFV-LAVL3 to induce protective immunity. Vaccination with VNUA-ASFV-LAVL3 elicited robust humoral and cellular immune responses in pigs, achieving 100% protection against a lethal wild-type ASFV (genotype II) challenge at all tested doses (10 3 , 10 4 , and 10 5 TCID 50 ). Furthermore, a single vaccination (10 4 TCID 50 ) provided protection for up to 2 months. These findings suggest that VNUA-ASFV-LAVL3 can be utilized as a promising safe and efficacious LAV candidate against the contemporary pandemic genotype II ASFV., Competing Interests: The authors declare no conflicts of interest.

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.

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.

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.

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., (© 2024. The Author(s).)

The African swine fever virus (ASFV) is an ancient, structurally complex, double-stranded DNA virus that causes African swine fever. Since its discovery in Kenya and Africa in 1921, no effective vaccine or antiviral strategy has been developed. Therefore, the selection of more suitable vaccines or antiviral targets is the top priority to solve the African swine fever virus problem. B125R , one of the virulence genes of ASFV, encodes a non-structural protein (pB125R), which is important in ASFV infection. However, the epitope of pB125R is not well characterized at present. We observed that pB125R is specifically recognized by inactivated ASFV-positive sera, suggesting that it has the potential to act as a protective antigen against ASFV infection. Elucidation of the antigenic epitope within pB125R could facilitate the development of an epitope-based vaccine targeting ASFV. In this study, two strains of monoclonal antibodies (mAbs) against pB125R were produced by using the B cell hybridoma technique, named 9G11 and 15A9. The antigenic epitope recognized by mAb 9G11 was precisely located by using a series of truncated ASFV pB125R. The 52 DPLASQRDIYY 62 (epitope on ASFV pB125R) was the smallest epitope recognized by mAb 9G11 and this epitope was highly conserved among different strains. The key amino acid sites were identified as D52, Q57, R58, and Y62 by the single-point mutation of 11 amino acids of the epitope by alanine scanning. In addition, the immunological effects of the epitope (pB125R-DY) against 9G11 were evaluated in mice, and the results showed that both full-length pB125R and the epitope pB125R-DY could induce effective humoral and cellular immune responses in mice. The mAbs obtained in this study reacted with the eukaryotic-expressed antigen proteins and the PAM cell samples infected with ASFV, indicating that the mAb can be used as a good tool for the detection of ASFV antigen infection. The B cell epitopes identified in this study provide a fundamental basis for the research and development of epitope-based vaccines against ASFV.