Deletion of MGF505-2R Gene Activates the cGAS-STING Pathway Leading to Attenuation and Protection against Virulent African Swine Fever Virus.

African swine fever virus (ASFV) is the etiological agent causing African swine fever (ASF), affecting domestic pigs and wild boar, which is currently the biggest animal epidemic in the world and a major threat to the swine sector. At present, some safety concerns about using LAVs against ASFV still exist despite a commercial vaccine licensed in Vietnam. Therefore, the efforts to identify virulence factors and their mechanisms, as well as to generate new vaccine prototypes, are of major interest. In this work, we have identified the MGF505-2R gene product as an inhibitor of the cGAS/STING pathway, specifically through its interaction with STING protein, controlling IFN-ß production. In addition, immunization of a recombinant virus lacking this gene, Arm/07-ΔMGF505-2R, resulted in complete attenuation, demonstrating its involvement in ASFV virulence. Finally, immunization with Arm/07-ΔMGF505-2R induced the generation of antibodies and proved to be partially protective against virulent ASFV strains. These results identify MGF505-2R, as well as its mechanism of action, as a gene contributing to understanding the molecular mechanisms of ASFV virulence, which will be of great value in the design of future vaccine prototypes.

Difference in Intraspecies Transmissibility of Severe Fever with Thrombocytopenia Syndrome Virus Depending on Abrogating Type 1 Interferon Signaling in Mice.

Severe fever with thrombocytopenia syndrome (SFTS), a tick-borne zoonotic disease, is caused by infection with SFTS virus (SFTSV). A previous study reported that human-to-human direct transmission of SFTSV can occur. However, potential animal-to-animal transmission of SFTSV without ticks has not been fully clarified. Thus, the objective of this study was to investigate potential mice-to-mice transmission of SFTSV by co-housing three groups of mice [i.e., wild-type mice (WT), mice injected with an anti-type I interferon-α receptor-blocking antibody (IFNAR Ab), and mice with knockout of type I interferon-α receptor (IFNAR KO)] as spreaders or recipients with different immune competence. As a result, co-housed IFNAR Ab and IFNAR KO mice showed body weight loss with SFTS viral antigens detected in their sera, extracorporeal secretions, and various organs. Based on histopathology, white pulp atrophy in the spleen was observed in all co-housed mice except WT mice. These results obviously show that IFNAR Ab and IFNAR KO mice, as spreaders, exhibited higher transmissibility to co-housed mice than WT mice. Moreover, IFNAR KO mice, as recipients, were more susceptible to SFTSV infection than WT mice. These findings suggest that type I interferon signaling is a pivotal factor in mice intraspecies transmissibility of SFTSV in the absence of vectors such as ticks.

Genome characterization of a Korean isolate of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea (PED) outbreaks occur annually in the Republic of Korea. The complete genome sequence of the PED virus isolate CKK1-1 obtained from an infected pig was determined. The genome is 28,037 nt long, excluding the poly(A) tail, and contains seven open reading frames flanked by two untranslated regions.

Demonstration of SARS-CoV-2 Exposure in Korean Native Cattle and Korean Native Black Goats in Korea.

The COVID-19 pandemic is caused by the zoonotic SARS-CoV-2 virus. A wide range of animals that interact with humans have been investigated to identify potential infections. As the extent of infection became more apparent, extensive animal monitoring became necessary to assess their susceptibility. This study analyzed nasal swabs and blood samples collected from randomly selected Korean native cattle and Korean native black goats. The tests conducted included real-time qPCR to detect SARS-CoV-2 antigens, an ELISA to detect antibodies, and a plaque reduction neutralization test (PRNT) to determine the presence of neutralizing antibodies. Among the 1798 animals tested (consisting of 1174 Korean native cattle and 624 Korean native black goats), SARS-CoV-2 viral RNA was detected in one Korean native cattle and one Korean native black goat. ELISA testing revealed positive results for antibodies in 54 Korean native cattle (4.60%) and 16 Korean native black goats (2.56%), while PRNTs yielded positive results in 51 Korean native cattle (4.34%) and 14 Korean native black goats (2.24%). The presence of SARS-CoV-2 antigens and/or antibodies was identified in animals on farms where farmworkers were already infected. It is challenging to completely rule out the possibility of reverse zoonotic transmission from humans to livestock in Korea, although the transmission is not to the same extent as it is in highly susceptible animal species like minks, cats, and dogs. This is due to the limited geographical area and the dense, intensive farming practices implemented in these regions. In conclusion, continuous viral circulation between humans and animals is inevitable, necessitating ongoing animal monitoring to ensure public health and safety.

Ultraviolet-C light at 222 nm has a high disinfecting spectrum in environments contaminated by infectious pathogens, including SARS-CoV-2.

Ultraviolet light (UV) acts as a powerful disinfectant and can prevent contamination of personal hygiene from various contaminated environments. The 222-nm wavelength of UV-C has a highly effective sterilization activity and is safer than 275-nm UV-C. We investigated the irradiation efficacy of 222-nm UV-C against contaminating bacteria and viruses in liquid and fabric environments. We conducted colony-forming unit assays to determine the number of viable cells and a 50% tissue culture infectious dose assay to evaluate the virus titration. A minimum dose of 27 mJ/cm2 of 222-nm UV-C was required for >95% germicidal activity for gram-negative and -positive bacteria. A 25.1 mJ/cm2 dose could ensure >95% virucidal activity against low-pathogenic avian influenza virus and severe acute respiratory syndrome coronavirus (SARS-CoV-2). In addition, this energy dose of 222-nm UV-C effectively inactivated SARS-CoV-2 variants, Delta and Omicron. These results provide valuable information on the disinfection efficiency of 222-nm UV-C in bacterial and virus-contaminated environments and can also develop into a powerful tool for individual hygiene.

Development of Effective PEDV Vaccine Candidates Based on Viral Culture and Protease Activity.

Porcine epidemic diarrhea (PED) is a highly contagious disease that has been reported annually in several Asian countries, causing significant economic losses to the swine livestock industry. Although vaccines against the porcine epidemic diarrhea virus (PEDV) are available, their efficacy remains questionable due to limitations such as viral genome mutation and insufficient intestinal mucosal immunity. Therefore, the development of a safe and effective vaccine is necessary. In this study, a virulent Korean strain of PEDV, CKT-7, was isolated from a piglet with severe diarrhea, and six different conditions were employed for serial passage of the strain in a cell culture system to generate effective live attenuated vaccine (LAV) candidates. The characteristics of these strains were analyzed in vitro and in vivo, and the CKT-7 N strain was identified as the most effective vaccine candidate, with a viral titer peak of 8.67 ± 0.29 log10TCID50/mL, and no mortality or diarrhea symptoms were observed in five-day-old piglets. These results indicate that LAV candidates can be generated through serial passage with different culture conditions and provide valuable insights into the development of a highly effective LAV against PEDV.

A Plant-Derived Maternal Vaccine against Porcine Epidemic Diarrhea Protects Piglets through Maternally Derived Immunity.

Newborn piglets are susceptible to a highly contagious enteritis caused by the porcine epidemic diarrhea virus (PEDV), associated with high levels of mortality worldwide. There is pressing need for a rapid, safe, and cost-effective vaccine to safeguard pigs from getting infected by PEDV. PEDV belongs to the coronavirus family and is characterized by high levels of mutability. The primary goal of a PEDV vaccine is to provide immunity to newborn piglets through vaccination of sows. Plant-based vaccines are becoming more popular because they have low manufacturing costs, are easily scalable, have high thermostability, and a long shelf life. This is in contrast to conventional vaccines which include inactivated, live, and/or recombinant types that can be expensive and have limited ability to respond to rapidly mutating viruses. The binding of the virus to host cell receptors is primarily facilitated by the N-terminal subunit of the viral spike protein (S1), which also contains several epitopes that are recognized by virus-neutralizing antibodies. As a result, we generated a recombinant S1 protein using a plant-based vaccine platform. We found that the recombinant protein was highly glycosylated, comparable to the native viral antigen. Vaccination of pregnant sows at four and two weeks before farrowing led to the development of humoral immunity specific to S1 in the suckling piglets. In addition, we noted significant viral neutralization titers in both vaccinated sows and piglets. When challenged with PEDV, piglets born from vaccinated sows displayed less severe clinical symptoms and significantly lower mortality rates compared to piglets born from non-vaccinated sows.

Alteration of the Gut Microbiota in Pigs Infected with African Swine Fever Virus.

The factors that influence the pathogenicity of African swine fever (ASF) are still poorly understood, and the host's immune response has been indicated as crucial. Although an increasing number of studies have shown that gut microbiota can control the progression of diseases caused by viral infections, it has not been characterized how the ASF virus (ASFV) changes a pig's gut microbiome. This study analyzed the dynamic changes in the intestinal microbiome of pigs experimentally infected with the high-virulence ASFV genotype II strain (N = 4) or mock strain (N = 3). Daily fecal samples were collected from the pigs and distributed into the four phases (before infection, primary phase, clinical phase, and terminal phase) of ASF based on the individual clinical features of the pigs. The total DNA was extracted and the V4 region of the 16 s rRNA gene was amplified and sequenced on the Illumina platform. Richness indices (ACE and Chao1) were significantly decreased in the terminal phase of ASF infection. The relative abundances of short-chain-fatty-acids-producing bacteria, such as Ruminococcaceae, Roseburia, and Blautia, were decreased during ASFV infection. On the other hand, the abundance of Proteobacteria and Spirochaetes increased. Furthermore, predicted functional analysis using PICRUSt resulted in a significantly reduced abundance of 15 immune-related pathways in the ASFV-infected pigs. This study provides evidence for further understanding the ASFV-pig interaction and suggests that changes in gut microbiome composition during ASFV infection may be associated with the status of immunosuppression.

Pseudoknot-targeting Cas13b combats SARS-CoV-2 infection by suppressing viral replication.

CRISPR-Cas13-mediated viral genome targeting is a novel strategy for defending against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Here, we generated mRNA-encoded Cas13b targeting the open reading frame 1b (ORF1b) region to effectively degrade the RNA-dependent RNA polymerase gene. Of the 12 designed CRISPR RNAs (crRNAs), those targeting the pseudoknot site upstream of ORF1b were found to be the most effective in suppressing SARS-CoV-2 propagation. Pseudoknot-targeting Cas13b reduced expression of the spike protein and attenuated viral replication by 99%. It also inhibited the replication of multiple SARS-CoV-2 variants, exhibiting broad potency. We validated the therapeutic efficacy of this system in SARS-CoV-2-infected hACE2 transgenic mice, demonstrating that crRNA treatment significantly reduced viral titers. Our findings suggest that the pseudoknot region is a strategic site for targeted genomic degradation of SARS-CoV-2. Hence, pseudoknot-targeting Cas13b could be a breakthrough therapy for overcoming infections by SARS-CoV-2 or other RNA viruses.

Amuvatinib Blocks SARS-CoV-2 Infection at the Entry Step of the Viral Life Cycle.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). SARS-CoV-2 propagation is mediated by the protein interaction between viral proteins and host cells. Tyrosine kinase has been implicated in viral replication, and hence, it has become a target for developing antiviral drugs. We have previously reported that receptor tyrosine kinase inhibitor blocks the replication of hepatitis C virus (HCV). In the present study, we investigated two receptor tyrosine kinase-specific inhibitors, amuvatinib and imatinib, for their potential antiviral efficacies against SARS-CoV-2. Treatment with either amuvatinib or imatinib displays an effective inhibitory activity against SARS-CoV-2 propagation without an obvious cytopathic effect in Vero E6 cells. Notably, amuvatinib exerts a stronger antiviral activity than imatinib against SARS-CoV-2 infection. Amuvatinib blocks SARS-CoV-2 infection with a 50% effective concentration (EC50) value ranging from ~0.36 to 0.45 µM in Vero E6 cells. We further demonstrate that amuvatinib inhibits SARS-CoV-2 propagation in human lung Calu-3 cells. Using pseudoparticle infection assay, we verify that amuvatinib blocks SARS-CoV-2 at the entry step of the viral life cycle. More specifically, amuvatinib inhibits SARS-CoV-2 infection at the binding-attachment step. Moreover, amuvatinib exhibits highly efficient antiviral activity against emerging SARS-CoV-2 variants. Importantly, we demonstrate that amuvatinib inhibits SARS-CoV-2 infection by blocking ACE2 cleavage. Taken together, our data suggest that amuvatinib may provide a potential therapeutic agent for the treatment of COVID-19. IMPORTANCE Tyrosine kinase has been implicated in viral replication and has become an antiviral drug target. Here, we chose two well-known receptor tyrosine kinase inhibitors, amuvatinib and imatinib, and evaluated their drug potencies against SARS-CoV-2. Surprisingly, amuvatinib displays a stronger antiviral activity than imatinib against SARS-CoV-2. Amuvatinib blocks SARS-CoV-2 infection by inhibiting ACE2 cleavage and the subsequent soluble ACE2 receptor. All these data suggest that amuvatinib may be a potential therapeutic agent in SARS-CoV-2 prevention for those experiencing vaccine breakthroughs.

Delta (B1.617.2) variant of SARS-CoV-2 induces severe neurotropic patterns in K18-hACE2 mice.

A highly contagious virus, severe acute respiratory syndrome coronavirus 2, caused the coronavirus disease 19 (COVID-19) pandemic (SARS-CoV-2). SARS-CoV-2 genetic variants have been reported to circulate throughout the COVID-19 pandemic. COVID-19 symptoms include respiratory symptoms, fever, muscle pain, and breathing difficulty. In addition, up to 30% of COVID-19 patients experience neurological complications such as headaches, nausea, stroke, and anosmia. However, the neurotropism of SARS-CoV-2 infection remains largely unknown. This study investigated the neurotropic patterns between the B1.617.2 (Delta) and Hu-1 variants (Wuhan, early strain) in K18-hACE2 mice. Despite both the variants inducing similar pathogenic patterns in various organs, B1.617.2-infected K18-hACE2 mice demonstrated a higher range of disease phenotypes such as weight loss, lethality, and conjunctivitis when compared to those in Hu-1-infected mice. In addition, histopathological analysis revealed that B1.617.2 infects the brain of K18-hACE2 mice more rapidly and effectively than Hu-1. Finally, we discovered that, in B1.617.2-infected mice, the early activation of various signature genes involved innate cytokines and that the necrosis-related response was most pronounced than that in Hu-1-infected mice. The present findings indicate the neuroinvasive properties of SARS-CoV-2 variants in K18-hACE2 mice and link them to fatal neuro-dissemination during the disease onset.

Recombinant African Swine Fever Virus Arm/07/CBM/c2 Lacking CD2v and A238L Is Attenuated and Protects Pigs against Virulent Korean Paju Strain.

African swine fever (ASF) is an obligated declaration swine disease, provoking farm isolation measures and the closing of affected country boarders. ASF virus (ASFV) is currently the cause of a pandemic across China and Eurasia. By the end of 2019, ASF was detected in nine EU Member States: Bulgaria, Romania, Slovakia, Estonia, Hungary, Latvia, Lithuania, Poland and Belgium. The affected area of the EU extended progressively, moving mostly in a southwestern direction (EFSA). Inactivated and/or subunit vaccines have proven to fail since certain virus replication is needed for protection. LAVs are thus the most realistic option, which must be safe, effective and industrially scalable. We here generated a vaccine prototype from the Arm/07/CBM/c2 genotype II strain, in which we have deleted the EP402R (CD2v) and A238L genes by CRISPR/Cas9 in COS-1 cells, without detectable further genetic changes. The successful immunization of pigs has proven this vaccine to be safe and fully protective against the circulating Korean Paju genotype II strain, opening the possibility of a new vaccine on the market in the near future.

Evidence of Exposure to SARS-CoV-2 in Dogs and Cats from Households and Animal Shelters in Korea.

The COVID-19 pandemic was caused by the zoonotic SARS-CoV-2. A variety of animals involved in human life worldwide have been investigated for infection. As the degree of infection increased, extensive monitoring in animals became necessary to determine the degree of infection in animals. The study was conducted on a sample of dogs and cats, which were randomly sampled according to the number of confirmed cases in the region. Animals from both COVID-19-confirmed households and generally disease-negative families and animal shelters were included. Tests included real-time qPCR tests for SARS-CoV-2 antigens, ELISA for antibodies, and plaque reduction neutralization tests (PRNT) for neutralizing antibodies. As a result, SARS-CoV-2 viral RNA was detected in 2 cats out of 1018 pets (672 dogs and 346 cats). A total of 16 dogs (2.38%) and 18 cats (5.20%) tested positive using ELISA, and 14 dogs (2.08%) and 17 cats (4.91%) tested positive using PRNT. Antigens of- and/or antibodies to SARS-CoV-2 were detected in the animals regardless of whether the companion family was infected; this was the case even in animal shelters, which have been regarded as relatively safe from transmission. In conclusion, continuous viral circulation between humans and animals is inevitable; therefore, continuous monitoring in animals is required.

Hepatitis C Virus Nonstructural Protein 5A Interacts with Immunomodulatory Kinase IKKε to Negatively Regulate Innate Antiviral Immunity.

Hepatitis C virus (HCV) infection can lead to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV employs diverse strategies to evade host antiviral innate immune responses to mediate a persistent infection. In the present study, we show that nonstructural protein 5A (NS5A) interacts with an NF-κB inhibitor immunomodulatory kinase, IKKε, and subsequently downregulats beta interferon (IFN-ß) promoter activity. We further demonstrate that NS5A inhibits DDX3-mediated IKKε and interferon regulatory factor 3 (IRF3) phosphorylation. We also note that hyperphosphorylation of NS5A mediats protein interplay between NS5A and IKKε, thereby contributing to NS5A-mediated modulation of IFN-ß signaling. Lastly, NS5A inhibits IKKε-dependent p65 phosphorylation and NF-κB activation. Based on these findings, we propose NS5A as a novel regulator of IFN signaling events, specifically by inhibiting IKKε downstream signaling cascades through its interaction with IKKε. Taken together, these data suggest an additional mechanistic means by which HCV modulates host antiviral innate immune responses to promote persistent viral infection.

Proteomics-based diagnostic peptide discovery for severe fever with thrombocytopenia syndrome virus in patients.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) virus is an emerging infectious virus which causes severe hemorrhage, thrombocytopenia, and leukopenia, with a high fatality rate. Since there is no approved therapeutics or vaccines for SFTS, early diagnosis is essential to manage this infectious disease. METHODS: Here, we tried to detect SFTS virus in serum samples from SFTS patients by proteomic analysis. Firstly, in order to obtain the reference MS/MS spectral data of SFTS virus, medium from infected Vero cell culture was used for shotgun proteomic analysis. Then, tryptic peptides in sera from SFTS patients were confirmed by comparative analysis with the reference MS/MS spectral data of SFTS virus. RESULTS: Proteomic analysis of culture medium successfully discovered tryptic peptides from all the five antigen proteins of SFTS virus. The comparative spectral analysis of sera of SFTS patients revealed that the N-terminal tryptic peptide of the nucleocapsid (N) protein is the major epitope of SFTS virus detected in the patient samples. The prevalence of the peptides was strongly correlated with the viral load in the clinical samples. CONCLUSIONS: Proteomic analysis of SFTS patient samples revealed that nucleocapsid (N) protein is the major antigen proteins in sera of SFTS patients and N-terminal tryptic peptide of the N protein might be a useful proteomic target for direct detection of SFTS virus. These findings suggest that proteomic analysis could be an alternative tool for detection of pathogens in clinical samples and diagnosis of infectious diseases.

Evaluation of Antiviral Effect against SARS-CoV-2 Propagation by Crude Polysaccharides from Seaweed and Abalone Viscera In Vitro.

Crude polysaccharides, extracted from two seaweed species (Hizikia fusiforme and Sargassum horneri) and Haliotis discus hannai (abalone) viscera, were evaluated for their inhibitory effect against SARS-CoV-2 propagation. Plaque titration revealed that these crude polysaccharides efficiently inhibited SARS-CoV-2 propagation with IC50 values ranging from 0.35 to 4.37 µg/mL. The crude polysaccharide of H. fusiforme showed the strongest antiviral effect, with IC50 of 0.35 µg/mL, followed by S. horneri and abalone viscera with IC50 of 0.56 and 4.37 µg/mL, respectively. In addition, immunofluorescence assay, western blot, and quantitative RT-PCR analysis verified that these polysaccharides could inhibit SARS-CoV-2 replication. In Vero E6 cells, treatment with these crude polysaccharides before or after viral infection strongly inhibited the expression level of SARS-CoV-2 spikes, nucleocapsid proteins, and RNA copies of RNA-dependent RNA-polymerase and nucleocapsid. These results show that these crude marine polysaccharides effectively inhibit SARS-CoV-2 propagation by interference with viral entry.

Experimental infection of dogs with severe fever with thrombocytopenia syndrome virus: Pathogenicity and potential for intraspecies transmission.

Severe fever with thrombocytopenia syndrome (SFTS) is caused by infection with Dabie bandavirus [formerly SFTS virus (SFTSV)] and is an emerging zoonotic disease. Dogs can be infected with SFTSV, but its pathogenicity and transmissibility have not been fully elucidated. In experiment 1, immunocompetent dogs were intramuscularly inoculated with SFTSV. In experiment 2, immunosuppressed dogs (immunosuppressed group; oral azathioprine 5 mg/kg/day for 30 days) were intramuscularly inoculated with SFTSV. Both immunosuppressed and immunocompetent contact dogs were co-housed with the SFTSV-inoculated dogs that had been immunosuppressed. Immunocompetent SFTSV-infected dogs did not show any clinical symptom. However, immunosuppressed SFTSV-infected dogs showed high fever and weight loss without lethality. In all SFTSV-infected dogs, viral RNA could be measured in the serum only after 3 days post infection (DPI) and neutralizing antibodies were detected in the serum beginning 9 DPI. SFTSV shedding in the urine and faeces of some infected dogs occurred between 4 and 6 DPI. The immunocompromised SFTSV-infected dogs showed thrombocytopenia beginning 3 DPI to the end of the experiment (24 DPI). We confirmed SFTSV transmission to one of three immunocompetent co-housed dogs. This dog showed a high fever, weight loss, and shed viral RNA by urine. Viral RNA and neutralizing antibodies were also detected in the serum. These results demonstrated that intramuscular inoculation with SFTSV induced minor clinical symptoms in dogs, and intraspecies SFTSV transmission in dogs can occur by contact.

Hepatitis C Virus Nonstructural 5A Protein Interacts with Telomere Length Regulation Protein: Implications for Telomere Shortening in Patients Infected with HCV.

Hepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for viral propagation. Using protein microarray analysis, we identified 90 cellular proteins as HCV nonstructural 5A (NS5A) interacting partners, and selected telomere length regulation protein (TEN1) for further study. TEN1 forms a heterotrimeric complex with CTC and STN1, which is essential for telomere protection and maintenance. Telomere length decreases in patients with active HCV, chronic liver disease, and hepatocellular carcinoma. However, the molecular mechanism of telomere length shortening in HCV-associated disease is largely unknown. In the present study, protein interactions between NS5A and TEN1 were confirmed by immunoprecipitation assays. Silencing of TEN1 reduced both viral RNA and protein expression levels of HCV, while ectopic expression of the siRNA-resistant TEN1 recovered the viral protein level, suggesting that TEN1 was specifically required for HCV propagation. Importantly, we found that TEN1 is re-localized from the nucleus to the cytoplasm in HCV-infected cells. These data suggest that HCV exploits TEN1 to promote viral propagation and that telomere protection is compromised in HCV-infected cells. Overall, our findings provide mechanistic insight into the telomere shortening in HCV-infected cells.

Identification of African Swine Fever Virus Inhibitors through High Performance Virtual Screening Using Machine Learning.

African swine fever virus (ASFV) is a highly contagious virus that causes severe hemorrhagic viral disease resulting in high mortality in domestic and wild pigs, until few antiviral agents can inhibit ASFV infections. Thus, new anti-ASFV drugs need to be urgently identified. Recently, we identified pentagastrin as a potential antiviral drug against ASFVs using molecular docking and machine learning models. However, the scoring functions are easily influenced by properties of protein pockets, resulting in a scoring bias. Here, we employed the 5'-P binding pocket of AsfvPolX as a potential binding site to identify antiviral drugs and classified 13 AsfvPolX structures into three classes based on pocket parameters calculated by the SiteMap module. We then applied principal component analysis to eliminate this scoring bias, which was effective in making the SP Glide score more balanced between 13 AsfvPolX structures in the dataset. As a result, we identified cangrelor and fostamatinib as potential antiviral drugs against ASFVs. Furthermore, the classification of the pocket properties of AsfvPolX protein can provide an alternative approach to identify novel antiviral drugs by optimizing the scoring function of the docking programs. Here, we report a machine learning-based novel approach to generate high binding affinity compounds that are individually matched to the available classification of the pocket properties of AsfvPolX protein.